Concise synthesis of novel acyclic analogues of cADPR with an ether chain as the northern moiety

Article abstract of DOI:10.1039/b9nj00595a

To study the properties of hydrolysates of cyclic adenosine diphosphate ribose (cADPR), a series of novel acyclic analogues of cADPR with an ether chain as the northern moiety and 8-substituted adenine or hypoxanthine as the base moiety were synthesized via an N¹ substitution construction, followed by bisphosphorylation, phosphoramidition or pyrophosphorylation. These compounds also provide various precursors for synthesizing cADPR analogues.

Full text of DOI:10.1039/b9nj00595a

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Concise synthesis of novel acyclic analogues of cADPR with an ether
chain as the northern moietyw
Huimin Wu, Zhenjun Yang, Liangren Zhang* and Lihe Zhang
Received (in Montpellier, France) 25th October 2009, Accepted 8th December 2009
First published as an Advance Article on the web 5th February 2010
DOI: 10.1039/b9nj00595a
To study the properties of hydrolysates of cyclic adenosine diphosphate ribose (cADPR),
a series of novel acyclic analogues of cADPR with an ether chain as the northern moiety and
8-substituted adenine or hypoxanthine as the base moiety were synthesized via an N1 substitution
construction, followed by bisphosphorylation, phosphoramidition or pyrophosphorylation.
These compounds also provide various precursors for synthesizing cADPR analogues.
acyclic analogues are resistant to both enzymatic and chemical
Introduction
hydrolysis, since they possess a stable N-alkyl linkage instead
of the N1-glycosidic linkage. These compounds provided a
Cyclic adenosine diphosphate ribose (cADPR, 1, Fig. 1) is a
universal Ca2+-mobilizing second messenger that was first
mini library for in-depth studies of the nature of the metabolite
identified in the sea urchin egg system.1,2 Since its discovery,
and suggest further research on the structure activity relation-
a significant number of cell systems have been described as
ships (SAR) of cADPR analogues. Moreover, they provided
utilizing the cADPR/ryanodine receptor (RyR)/Ca2+ signal-
various precursors for synthesizing cADPR analogues.
ling system to control Ca2+-dependent cellular responses such
as fertilization, secretion, contraction, proliferation and so
on.3,4 Moreover, numerous cADPR analogues have been
Results and discussion
synthesized to further understand its biology. Among these,
Synthesis of bisphosphate analogues
the majority of structural modifications have been applied to
the purine ring and two riboses.5–11
The syntheses of 8-substituted analogues 4a–d are summarized
in Scheme 1. The synthetic route adopted for 4a–d has two
main steps: (i) introduction of the 2-(hydroxyethoxy)ethyl
chain onto N1 of the substituted 20,30-isopropylidene inosines
6a–c and 9, which leads to 7a–d; (ii) bisphosphorylation of
derivatives 7a–d through phosphorus oxychloride (POCl3),
followed by deprotection with aqueous HCOOH to yield the
target compounds.
N1-alkylation of 6a–c was obtained through the N1-(2,4-
dinitrophenyl)inosine intermediate with 2-(hydroxyethoxy)-
ethylamine. 20,30-O-Isopropylideneinosines were converted
into N1-(2,4-dinitrophenyl) derivatives by a reaction with
2,4-dinitrochlorobenzene and K2CO3 in DMF; the latter was
A series of novel cADPR mimics, cIDPRE (2) and cADPRE
(3) (Fig. 1), in which the northern ribose12 (the ribose linked to
the N1 of adenine) was replaced by an ether strand, were
reported by Zhang’s group.13–15 Biological data suggested that
modification of the northern ribose moiety of cADPR could
retain its calcium release activity and increase its membrane
permeability. The results revealed that the greater flexibility of
the whole molecule, introduced by replacing the northern ribose
by an ether strand, continued to allow its fitting into the binding
site, and that the functional groups could support a conforma-
tion very similar to that of cADPR.16,17
cADPR can be hydrolyzed either in vivo or in vitro.18,19
Pyrophosphate is one of the easiest linkages to be hydrolyzed.
It was reported recently that the pyrophosphate linkage of
cADPR could be hydrolyzed by Mn2+-dependent ADP-
ribose/CDP-alcohol pyrophosphatase to afford the bisphos-
phate metabolite.20 Though modification of the northern
ribose by an ether linkage provided relatively stable cADPR
analogues,15 there was no detailed report about the meta-
bolism of these analogues, especially the properties of
their metabolites. In this study, a series of acyclic cADPR
analogues, which may mimic the hydrolysates of the cyclic
pyrophosphate group cleaved in different places, have been
designed and synthesized (Fig. 2). It is hypothesized that these
State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing 100191, China.
E-mail: liangren@bjmu.edu.cn; Fax: +86 10-82805063;
Tel: +86 10-82802567
w This article is part of a themed issue on Biophosphates.
Fig. 1 The structure of cADPR and cADPR analogues.
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by POCl3 in pyridine (Py)/H2O–CH3CN at 0 1C for 4 h to give
10a–d in moderate to high yields. It is noteworthy that when
8-Br intermediate 7b was converted into the bisphosphate by
the bisphosphorylation reaction with POCl3, bromine at the
8-position of inosine was simultaneously replaced by chlorine,
and none of the expected 8-Br bisphosphate was obtained. By
removing the isopropylidene groups in aqueous HCOOH, targets
4a–d were then obtained in a yield of approximately 90%.
Since attempts to introduce similar 8-Br bisphosphates with
corresponding phosphate acids under the above-mentioned
conditions failed, we turned to the phosphoramidite strategy
as a preferred method in ribonucleic acid (RNA) synthesis.21
The phosphoramidition of 7b was achieved with 2-cyanoethyl-
N,N-diisopropylamino-chlorophosphoramidite to afford the
expected 8-Br-substituted phosphate, 11, in high yield (87%)
t
after oxidation of the intermediate phosphite with BuOOH
and deprotection of the cyanoethyl group with NH3/MeOH.
When the deprotection reaction of 11 by aqueous HCOOH
was conducted, it is interesting that debrominated product
4a (15%) was likewise obtained, in addition to the desired
compound 4e (36%), during the deprotection of the isopropyl-
idene group (Scheme 2).
Fig. 2 The general structure of acyclic cADPR analogues.
treated with 2-(hydroxyethoxy)ethylamine for 8 h at 50 1C in
DMF without separation to afford the N1-[(50 0-hydroxy)-
ethoxyethyl]inosine derivatives 7a–c in 70–80% yield. 7d was
obtained in a yield of approximately 80% when intermediate 8
was separated and converted into 9 by treatment with NaOAc/
AcOH. This was followed by the concomitant aminolysis
of the 50-O-acetyl protecting group through stirring with
2-(hydroxyethoxy)ethylamine in DMF under similar condi-
tions to those used in the preparation of 7a–c. The formation
of N1-alkylated derivatives 7a–d proceeded through a rearran-
gement of the pyrimidine ring. Alkylation was induced by
nucleophilic attack of the amine at C2, whose electrophilicity
was enhanced by the presence of the 2,4-dinitrophenyl group
at the N1 position. Compounds 7a–d were bisphosphorylated
For the syntheses of analogues substituted at position 8 in
the base, a different strategy was adopted for the construction
of the N1 substituted adenosine, which was achieved regio-
specifically by condensation reactions of 2-bromo-substituted
5-amino-1-(b-D-ribofuranosyl)-imidazole-4-carboxamide
(AICAR) derivatives and 2-(hydroxyethoxy)ethylamine in the
presence of a catalytic amount of K2CO3 in yields above 90%;
this being similar to the synthesis of compound 14a.14 Sub-
sequently, the 50-O-tert-butyldimethylsilyl (TBDMS) groups
in 14a,b were removed by a tetrabutylammonium fluoride
(TBAF) solution to afford 15a and 15b in a 95% yield.
Compounds 15a,b, underwent phosphorylation procedures,
Scheme 1 Syntheses of compounds 4a–d. Reagents and conditions: (a) DNCB (2,4-dinitrochlorobenzene), K2CO3, DMF, 80 1C, 2 h; (b)
NH2(CH2)2O(CH2)2OH, DMF, 50 1C, 8 h; (c) POCl3, Py, CH3CN, H2O, 0 1C, 4 h; (d) 60% HCOOH/H2O, rt, 8 h; (e) NaOAc, AcOH, reflux, 6 h.
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Scheme 2 Syntheses of compounds 4e. Reagents and conditions: (a) 2-cyanoethyl-N,N-diisopropylamino-chlorophosphoramidite, DIPEA,
t
DCM, rt, 30 min; (b) BuOOH, rt, 1h; (c) NH3/MeOH, rt, 8 h; (d) 60% HCOOH/H2O, rt, 8 h.
leading to bisphosphate derivatives (86–90% yield). After
treatment with 60% aqueous HCOOH at rt for 8 h to facilitate
the removal of the isopropylidene protecting group, 4f,g were
obtained in approximately 90% yield (Scheme 3).
Meanwhile, when compound 19 was protected with a mono-
methoxytrityl (MMTr) group using MMTrCl in dry pyridine,
affording compound 20a, the reaction from 20a to 21a
occurred smoothly in dry CH3CN. The limitation of this
approach was that the active p-toluenesulfonyl (Ts) group
was introduced too early, reducing the overall yield of
precursor 20a for pyrophosphorylation (yield of 21% from
17b to 20a) (Scheme 5).
In order to obtain the 8-oxo-bisphosphate derivative with
adenine as the base, compound 15b was converted into 15c in a
yield of 82% by refluxing with 2-mercaptoethanol in a mixed
solvent solution of CH3CN and water. When 15c was sub-
jected to phosphorylation by POCl3, the expected conversion
of 8-mercaptoethanol to 8-oxo did not take place, though
triphosphate derivative 16c was obtained in 86% yield. By
removing the isopropylidene groups in aqueous HCOOH,
compound 4h was obtained in a yield of 89% (Scheme 4).
Evidently, there is a need for a more efficient method to
synthesize the precursors. Finally, the synthetic route outlined
in Scheme 6 was adopted. Starting from 50-O-TBDMS-20,30-
O-isopropylideneinosine and 50-O-TBDMS-20,30-O-isopropyl-
idene-8-bromoinosine, with the same procedure as that used in
the preparation of 7a, compounds 22a and 22b were obtained
in a yield of 80 and 84%, respectively. After the 500-hydroxyl
group of 22a,b had been protected with an MMTr group
(96 and 92% yield, respectively), it was treated with TBAF in
THF to remove the 50-TBDMS group of 23a,b. A Ts group
was then introduced at the resulting 50-primary hydroxyl
group of 24a,b via the usual method with TsCl/NEt3/
DMAP/DCM to afford precursors 20a,b (approximately
80% yield over two steps). Although two other steps were
required, a significantly improved yield was achieved (64%
from 22a to 20a, compared to 21% from 17b to 20a). Targeted
pyrophosphorylated compounds 5a,b were obtained in almost
90% yield by removing both the MMTr and isopropylidene
groups of compounds 21a,b in one step.
Synthesis of pyrophosphate analogues
For the synthesis of protected pyrophosphate 21a, the method
outlined in Scheme 5 was first attempted. However, compound
18 was not obtained from 17a. This problem was overcome
after modifying the reaction sequence to proceed via compound
17b. Subsequently, compound 18 was converted into 19
using the same reaction conditions as those described earlier.
However, the yield of this step was very low (41%) compared
to similar reactions (above 80%), although various conditions
with different reaction temperatures and times were examined.
During research to find the optimal conditions for converting
compound 19 straight into pyrophosphorylated compound
21a, it was discovered that the unprotected 50 0-hydroxyl group
in 19 indeed affected the pyrophosphorylation, allowing
recovery of 80% of the starting material. The rest of the
material (20%) decomposed into an inseparable mixture.
A similar activation approach to the activation of the
50 0-primary hydroxyl groups of 25a,b by a Ts group was
employed for the synthesis of 26a,b in Scheme 7, which was
Scheme 3 Syntheses of compounds 4f,g. Reagents and conditions: (a) NBS, THF, rt, 10 min; (b) CH(OMe)3, CF3COOH, reflux, 1 h;
(c) NH2(CH2)2O(CH2)2OH, K2CO3, DMF, rt, 8 h; (d) TBAF, AcOH, THF, rt, 2 h; (e) POCl3, Py, CH3CN, H2O, 0 1C, 4 h; (f) 60%
HCOOH/H2O, rt, 8 h.
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Scheme 4 The synthesis of compound 4h. Reagents and conditions: (a) HS(CH2)2OH, NEt3, H2O, reflux, 2 h; (b) POCl3, Py, CH3CN, H2O, 0 1C,
4 h; (c) 60% HCOOH/H2O, rt, 8 h.
Scheme 5 The synthesis of compound 21a. Reagents and conditions: (a) DNCB, K2CO3, DMF, 80 1C, 2 h for 17a; TsCl, Py, rt, 8 h for 17b;
(b) NH2(CH2)2O(CH2)2OH, DMF, 50 1C, 8 h; (c) MMTrCl, Py, rt, 8 h; (d) [(nBu)4N]3P2O5OH, CH3CN, rt, 16 h.
Scheme 6 Syntheses of compounds 5a,b. Reagents and conditions: (a) MMTrCl, Py, rt, 8 h; (b) TBAF, AcOH, THF, rt, 2 h; (c) TsCl, NEt3,
DCM, DMAP, rt, 8 h; (d) [(nBu)4N]3P2O5OH, CH3CN, rt, 16 h; (e) 60% HCOOH/H2O, rt, 8 h.
converted into pyrophosphates 5c,d via pyrophosphorylation
and deprotection. For the pyrophosphorylation of 20a,b and
26a,b, it was observed that when the 8-position of inosine was
substituted with bromine, the reaction yield was roughly 80%
(74% for 20b and 83% for 26b). Meanwhile, in the absence of
substitution, the yield was approximately 20% (20% for 20a
and 21% for 26a). The latter yield was close to that reported
for the pyrophosphorylation of 50-O-Ts nucleosides.22 The
reason for this difference remains unclear.
reactions, followed by bisphosphorylation or phosphoramidition.
Meanwhile, pyrophosphorylated compounds 5a–d were
obtained through pyrophosphorylation. These compounds
provide a mini library, not only for the natural study of
cADPR metabolites, but also as precursors for the develop-
ment of syntheses of cADPR analogues.
Experimental
General synthesis methods
Mass spectra were obtained on either VG-ZAB-HS or Bruker
APEXTM instruments. High resolution FAB (fast atom
bombardment) and HR-ESI (electrospray ionization) mass
spectrometry were performed using a Bruker BIFLEXTM III
Conclusions
In conclusion, novel acyclic metabolites of cADPR analogues,
4a–g, with 8-substituted hypoxanthine or adenine as the base
moiety, were synthesized via N1 substitution construction
1
instrument. H and 13C NMR spectra were recorded using a
Scheme 7 Syntheses of compounds 5c,d. Reagents and conditions: (a) TsCl, NEt3, DCM, DMAP, rt, 8 h; (b) [(nBu)4N]3P2O5OH, CH3CN, rt,
16 h; (c) 60% HCOOH/H2O, rt, 8 h.
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JEOL AL300, Bruker AM/DPX 400 or Varian VXR-500
spectrometer with DMSO-d6 or D2O as the solvent. Chemical
shifts are reported in parts per million downfield from TMS
(1H and 13C). 31P NMR spectra were recorded at room
temperature using a Bruker Avance 300 spectrometer; ortho-
phosphoric acid (85%) was used as an external standard.
Purifications on an Alltech preparative C18 reversed phase
column (2.2 ꢁ 25 cm) were conducted through a Gilson HPLC
with the buffer system MeCN/TEAB (pH 7.5). Analytical
TLC was performed using commercial glass plates coated to
a thickness of 0.25 mm with Kieselgel 60 F254 silica and
visualized under UV light. Flash chromatography was per-
formed using Qingdao (230–400 mesh) silica under a slight
positive pressure of air. Solvents and reagents for anhydrous
reactions were dried prior to use by conventional methods.
Synthesis of 8-substituted N1-[(500-hydroxyl)ethoxyethyl]-20,30-
O- isopropylideneinosines 7a–d
N1-[(50 0-Hydroxyl)ethoxyethyl]-20,30-O-isopropylideneinosine
(7a). A mixture of 6a (308 mg, 1 mmol), K2CO3 (166 mg,
1.2 mmol) and 2,4-dinitrochlorobenzene (243 mg, 1.2 mmol)
was stirred in DMF (3 mL) at 80 1C for 2 h. After the mixture
had cooled to room temperature, the insoluble materials were
filtered off. The filtrate was evaporated and the residue
redissolved in DMF. After 2-(hydroxyethoxy)ethylamine
(0.38 mL, 3.8 mmol) had been added, the resulting solution
was stirred at 50 1C for 8 h and evaporated. The residue was
purified by silica gel column chromatography (4% MeOH in
CH2Cl2) to give compound 7a (276 mg, 70%) as a colorless
fluffy solid. 1H NMR (400 MHz, DMSO-d6): d 8.34, 8.32
(each s, each 1 H), 6.09 (d, J = 2.8 Hz, 1 H), 5.27 (dd, J = 2.8
and 6.0 Hz, 1 H), 5.09 (t, J = 5.6 Hz, 1 H), 4.94 (dd, J = 2.8
and 5.6 Hz, 1 H), 4.57–4.56 (m, 1 H), 4.22 (dd, J = 4.8 and
7.2 Hz, 1 H), 4.18 (t, J = 5.2 Hz, 2 H), 3.66 (t, J = 5.2 Hz, 2 H),
3.54 (t, J = 4.8 Hz, 2 H), 3.48–3.41 (m, 4 H), 1.53 and 1.32
(each s, each 3 H). 13C NMR (125 MHz, DMSO-d6): d 155.8,
149.1, 147.1. 139.2, 123.6, 113.1, 89.5, 86.7, 83.8, 81.2, 72.1,
68.9, 67.8, 61.4, 60.1, 45.4, 27.0 and 25.1. HRMS (ESI,
positive) for C17H24N4O7, calc. 397.1718 [M + 1]+; found
397.1708.
N1-Dinitrobenzene-20,30-O-isopropylidene-8-bromoinosine (8)
A
mixture of 20,30-O-isopropylidene-8-bromoinosine (6b)
(824 mg, 2.1 mmol), 2,4-dinitrochlorobenzene (647 mg,
3.1 mmol) and K2CO3 (442 mg, 3.1 mmol) were suspended
in anhydrous DMF (8 mL) and stirred at 80 1C for 2 h. After
cooling, the mixture was filtered and the filtrate evaporated to
dryness. The residue was purified on a silica gel column eluted
with increasing amounts of MeOH in CH2Cl2 (from 0 to 5%)
to give compound 8 as a pale yellow amorphous solid con-
7b, 7c and 7d were synthesized, respectively, by a procedure
similar to that for 7a.
1
sisting of a 1 : 1 mixture of atropisomers (918 mg, 79%). H
N1-[(50 0-Hydroxyl)ethoxyethyl]-20,30-O-isopropylidene-8-bromo-
inosine (7b). A pale yellow solid, 71% yield. 1H NMR
(400 MHz, DMSO-d6): d 8.36 (s, 1 H), 6.00 (d, J = 2.4 Hz,
2 H), 5.55 (dd, J = 2.4 and 6.4 Hz, 1 H), 4.97 (dd, J = 4.0 and
6.4 Hz, 1 H), 4.94 (t, J = 6.0 Hz, 1 H), 4.57–4.56 (t, J =
4.8 Hz, 1 H), 4.17–4.11 (m, 3 H), 3.65 (t, J = 4.2 Hz, 1 H),
3.52–3.30 (m, 6 H), 1.53 and 1.31 (each s, each 3 H). 13C NMR
(125 MHz, DMSO-d6): d 154.6, 149.4, 148.0, 125.5, 124.1,
113.5, 90.6, 87.3, 82.3, 81.3, 72.1, 67.5, 61.3, 60.1, 45.7, 27.1
and 24.2. HRMS (ESI, positive) for C17H23N4O7Br, calc.
475.0823 [M + 1]+; found 475.0826.
NMR (400 MHz, DMSO-d6): d 8.91 (s, 1 H), 8.77, 8.81 (each t,
each 0.5 H, J = 2.8 Hz), 8.70, 8.68 (each s, each 0.5 H), 8.18,
8.13 (each d, each 0.5 H, J = 8.8 Hz), 6.10 (t, 1 H, J =
4.8 Hz), 5.66, 5.61 (each dd, each 0.5 H, J = 2.4 and 6.4 Hz),
5.08–4.87 (m, 2 H), 4.17 (dd, 1 H, J = 6.0 and 10.0 Hz),
3.62–3.53 (m, 2 H), 1.57 (s, 3 H) and 1.35 (s, 3 H). 13C NMR
(100 MHz, DMSO-d6): d 153.8, 148.3 (d), 147.9, 147.6 (d),
145.5 (d), 134.6 (d), 132.6 (d), 129.4 (d), 126.6 (d), 123.7, 120.8
(d), 113.7 (d), 90.7 (d), 87.4 (d), 82.4 (d), 81.3 (d), 61.4 (d),
54.9, 27.1 (d) and 25.2 (d).
N1-Dinitrobenzene-50-O-acetyl-20,30-O-isopropylidene-8-
N1-[(50 0-Hydroxyl)ethoxyethyl]-20,30-O-isopropylidene-8-amino-
inosine (7c). A pale yellow solid, 76% yield. 1H NMR
(300 MHz, DMSO-d6): d 8.09 (s, 1 H), 6.59 (s, 2 H), 5.99
(d, J = 3.6 Hz, 1 H), 5.38 (dd, J = 3.6 and 4.8 Hz, 1 H),
5.35–5.24 (m, 1 H), 4.96–4.92 (m, 1 H), 4.63–4.57 (m, 3 H),
3.66–3.58 (m, 4 H), 3.46–3.42 (m, 2 H), 1.54 and 1.31 (each s,
each 3 H). 13C NMR (125 MHz, DMSO-d6): d 154.6, 151.0,
145.6, 145.4, 121.3, 113.5, 87.7, 85.0, 81.4, 80.6, 72.1, 67.9,
61.0, 60.1, 45.4, 27.1 and 25.2. HRMS (ESI, negative) for
C17H25N5O7, calc. 410.1681 [M ꢂ 1]ꢂ; found 410.1677.
oxoinosine (9)
A mixture of 8 (730 mg, 1.3 mmol) and NaOAc (326 mg,
3.9 mmol) was suspended in AcOH (8 mL) and refluxed for 6 h.
The mixture was evaporated and the residue was partitioned
between H2O and CHCl3. The organic layer was washed
with brine, dried (Na2SO4) and evaporated. The residue was
purified by silica gel column chromatography (2% MeOH in
CH2Cl2) to give compound 9 as a pale yellow amorphous
solid (685 mg, 99%). 1H NMR (400 MHz, DMSO-d6): d 11.82
(d, 1 H), 8.92 (s, 1 H), 8.80 (d, J = 8.8 Hz, 1 H), 8.58 (s, 1 H),
8.13 (d, J = 8.8 Hz, 1 H), 5.95 (d, J = 3.2 Hz, 1 H), 5.45
(d, J = 6.0 Hz, 1 H), 4.94 (dd, J = 6.0 and 2.4 Hz, 1 H), 4.28
(m, 2 H), 4.17 (m, 1 H), 2.00 (d, 3 H), 1.52 and 1.32 (each s,
each 3 H). 13C NMR (125 MHz, DMSO-d6): d 170.6, 170.6,
151.6, 149.9, 148.4, 146.7, 146.0, 144.0, 143.9, 134.9, 134.9,
132.9, 132.8, 129.9, 121.2, 113.8, 113.7, 108.1, 108.1, 86.7, 85.0,
84.8, 82.8, 82.7, 81.8, 64.3, 64.2, 27.5, 27.4, 26.7, 25.6, 21.0 and
21.0. HRMS (ESI, positive) for C21H20N6O11, calc. 533.1263
[M + 1]+; found 533.1256.
N1-[(50 0-Hydroxyl)ethoxyethyl]-20,30-O-isopropylidene-8-oxo-
inosine (7d). A colorless solid, 73% yield. 1H NMR (400 MHz,
DMSO-d6): d 11.52 (s, 1 H), 8.23 (s, 1 H), 5.82 (d, J = 2.4 Hz,
1 H), 5.38 (dd, J = 2.4 and 6.4 Hz, 1 H), 4.84 (dd, J = 2.8 and
6.4 Hz, 2 H), 4.56 (br s, 1 H), 4.14 (t, J = 4.2 Hz, 1 H),
4.05–4.01 (m, 1 H), 3.65 (t, J = 4.2 Hz, 2 H), 3.44–3.40
(m, 5 H), 3.29 (br s, 1 H), 1.49 and 1.29 (each s, each 3 H). 13
C
NMR (125 MHz, DMSO-d6): d 151.3, 150.3, 148.0, 143.0, 112.
9, 107.9, 86.9, 86.4, 81.7, 81.7, 72.1, 67.5, 61.6, 60.1, 45.8, 27.1
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and 25.2. HRMS (ESI, positive) for C17H24N4O8, calc.
396.1878 [M + 1]+, found 396.1862.
Synthesis of 8-substituted N1-(50 0-O-Phosphonoxyethoxyethyl)-
50-O-(phosphoryl)-inosines 4a–d
N1-(500-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-inosine
(4a). A solution of 10a (40 mg, 72 mmol) in 60% HCOOH
(5 mL) was stirred for 8 h and then evaporated under reduced
pressure. The purification of the residue was performed by
HPLC on a C18 reversed phase column, eluting with a linear
gradient of 0–40% CH3CN in TEAB buffer (0.05 M, pH 7.5)
within 30 min to give target molecule 4a (33 mg, 90%) as a
Synthesis of 8-substituted N1-(50 0-O-phosphonoxyethoxyethyl)-
50-O- (phosphoryl)-20,30-O-isopropylideneinosines 10a–d
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-20,30-
O-isopropylideneinosine (10a). To a mixture of freshly distilled
phosphoryl chloride (358 mL, 3.89 mmol), water (23 mL,
2.48 mmol), pyridine (172 mL, 4.24 mmol) and acetonitrile
(3 mL), which was maintained at 2 1C with stirring, and 7a
(175 mg, 0.44 mmol) were added. After the mixture had stood
for 4 h, ice cooled TEAB (1 M, 10 mL) was added, and the
resulting solution stirred for 1 h at 2 1C. After evaporation,
the residue was dissolved in 2 mL TEAB (0.05 M, pH 7.5). The
solution was purified by a C18 reversed-phase column (2.2 cm ꢁ
25 cm) using a linear gradient of 0–60% CH3CN in TEAB
buffer (0.05 M, pH 7.5) within 30 min to give 10a (205 mg,
84%) as its triethylammonium salt. 1H NMR (400 MHz,
D2O): d 8.32, 8.31 (each s, each 1 H), 6.22 (d, J = 2.8 Hz,
1 H), 5.34 (dd, J = 2.8 and 6.0 Hz, 1 H) 5.11 (dd, J = 2.0 and
6.0 Hz, 1 H), 4.60 (br s, 1 H), 4.27 (dd, J = 4.8 and 10.0 Hz,
2 H), 3.99 (t, J = 4.0 Hz, 2 H), 3.88 (dd, J = 6.0 and 9.2 Hz,
2 H), 3.83 (t, J = 4.8 Hz, 2 H), 3.66–3.64 (m, 2 H), 1.59 and
1.37 (each s, each 3 H). 31P NMR (121.5 MHz, D2O): d 0.64
and 0.16. HRMS (ESI, negative) for C17H26N4O13P2, calc.
555.0899 [M ꢂ 1]ꢂ; found 555.08804.
1
white foam. H NMR (300 MHz, D2O): d 8.38 (s, 1 H), 8.24
(s, 1 H), 5.59 (d, J = 5.7 Hz, 1 H), 4.59 (t, J = 5.7 Hz, 1 H),
4.34–4.31 (m, 1 H), 4.19–4.17 (m, 3 H), 3.86–3.85 (m, 2 H),
3.72–3.68 (m, 4 H) and 3.54–3.51 (m, 2 H). 31P NMR (121.5
MHz, D2O, decoupled with H): d 7.15 and 7.00. HRMS (ESI,
positive) for C14H22N4O13P2, calc. 517.0731 [M + 1]+; found
517.0731.
4b, 4c and 4d were synthesized, respectively, by a procedure
similar to that for 4a.
N1-(500-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-8-
chloroinosine (4b). A white foam, 92% yield. 1H NMR
(500 MHz, D2O): d 8.38 (s, 1 H), 6.11 (d, J = 6.0 Hz, 1 H),
5.25 (dd, J = 6.0 and 5.5 Hz, 1 H), 4.60 (dd, J = 5.5 and
4.5 Hz, 1 H), 4.40 (dd, J = 4.5 and 9.5 Hz, 1 H), 4.25–4.21
(m, 2 H), 4.18–4.10 (m, 2 H), 3.90–3.88 (m, 4 H) and 3.80–3.62
(m, 2 H). 31P NMR (121.5 MHz, D2O, decoupled with H):
d 2.55 and 2.47. HRMS (ESI, negative) for C14H21N4O13P2Cl,
calc. 549.0196 [M ꢂ 1]ꢂ; found 549.0178.
10b, 10c and 10d were synthesized, respectively, by a
procedure similar to that for 10a.
N1-(500-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-8-
aminoinosine (4c). A white foam, 93% yield. 1H NMR
(400 MHz, D2O): d 8.12 (s, 1 H), 5.97 (d, J = 4.4 Hz, 1 H),
5.76 (dd, J = 4.4 and 6.4 Hz, 1 H), 5.46 (dd, J = 3.2 and
6.4 Hz, 1 H), 4.80–4.76 (m, 1 H), 4.37–4.35 (m, 2 H), 4.24–4.19
(m, 2 H), 3.94–3.89 (m, 2 H) and 3.59–3.57 (m, 2 H). 31P NMR
(121.5 MHz, D2O, decoupled with H): d 4.30 and 4.03. HRMS
(ESI, negative) for C14H23N5O13P2, calc. 530.0695 [M ꢂ 1]ꢂ;
found 530.0701.
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-20,30-
O-isopropylidene-8-chloroinosine (10b). A white solid, 88%
1
yield. H NMR (300 MHz, D2O) d 8.37 (s, 1 H), 6.24 (s, 1 H),
5.59 (s, 1 H), 5.14 (s, 1 H), 4.33–4.40 (m, 2 H), 3.86–3.56 (m, 9
H), 1.47 and 1.27 (each s, each 3 H). 31P NMR (121.5 MHz,
D2O, decoupled with H): d 2.15 and 2.08. HRMS (ESI,
negative) for C17H25ClN4O13P2, calc. 589.0504, [M ꢂ 1]ꢂ;
found 589.0539.
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-8-oxo-
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-20,30-
O-isopropylidene-8-aminoinosine (10c). A white foam, 91%
1
inosine (4d). A white foam, 84% yield. H NMR (500 MHz,
D2O): d 8.29 (s, 1 H), 5.88 (d, J = 5.5 Hz, 1 H), 5.15 (dd, J =
5.5 and 5.0 Hz, 1 H), 4.55 (t, J = 5.5 Hz, 1 H), 4.36–4.26
(m, 2 H), 4.21 (dd, J = 5.5 and 8.0 Hz, 1 H), 4.11–4.07
(m, 1 H), 4.03–3.98 (m, 1 H), 3.93–3.87 (m, 4 H) and 3.72–3.74
(m, 2 H). 31P NMR (121.5 MHz, D2O, decoupled with H):
d 3.76. HRMS (ESI, negative) for C14H22N4O14P2, calc.
531.0535 [M ꢂ 1]ꢂ; found 531.0534.
1
yield. H NMR (400 MHz, D2O): d 8.17 (s, 1 H), 6.11 (d, J =
4.4 Hz, 1 H), 5.41 (dd, J = 4.4 and 6.4 Hz, 1 H), 5.15 (dd, J =
3.2 and 6.4 Hz, 1 H), 4.49–4.48 (m, 1 H), 4.28–4.19 (m, 2 H),
4.08–4.07 (m, 2 H), 3.90–3.81 (m, 2 H), 3.84–3.81 (m, 2 H),
1.61 and 1.37 (each s, each 3 H). 31P NMR (121.5 MHz, D2O,
decoupled with H): d 2.89 and 2.43. HRMS (ESI, negative) for
C17H27N5O13P2, calc. 570.1008 [M ꢂ 1]ꢂ; found 570.1014.
N1-50 0-O-[(N,N-diisopropyl)phosphoryl]ethoxyethyl-50-O-[(N,N-
diisopropyl)phosphoryl]-20,30-O-isopropylidene-8-bromoinosine (11)
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-20,30-
O-isopropylidene-8-oxoinosine (10d). A white foam, 76% yield.
1H NMR (400 MHz, D2O): d 8.23 (s, 1 H), 6.06 (d, J = 1.2 Hz,
1 H), 5.57 (dd, J = 1.2 and 6.4 Hz, 1 H), 5.09 (dd, J = 3.6 and
6.4 Hz, 1 H), 4.38–4.34 (m, 1 H), 4.24 (dd, J = 4.8 and 12.8 Hz,
2 H), 3.96–3.86 (m, 4 H), 3.81 (t, J = 4.8 Hz, 2 H), 3.65
(t, J = 4.8 Hz, 2 H), 1.56 and 1.36 (each s, each 3 H). 31P
NMR (121.5 MHz, D2O): d 0.48. HRMS (ESI, negative) for
C17H26N4O14P2, calc. 571.0848 [M ꢂ 1]ꢂ; found 571.0868.
Compound 7b (122 mg, 0.26 mmol) was dissolved in DCM
(5 mL). To the solution was added N,N-diisopropylethylamine
(DIEA) (276 mL, 1.56 mmol) and 2-cyanoethyl-N,N-diiso-
propylamino-chlorophosphoramidite (136 mL, 1.44 mmol).
The mixture was stirred at room temperature for 20 min.
t
After BuOOH (150 mL, 60%) had been added and stirred
for a further 1 h, the solution was diluted with EtOAc/NEt3
(20 mL, 20 : 1, v/v), and then washed with 5% NaHCO3 and a
ꢀc
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saturated aqueous NaCl solution three times. The organic
phase was dried with Na2SO4 and evaporated. The residue was
resolved in saturated NH3/MeOH (5 mL) and stirred at 0 1C
for 8 h. After evaporation, the residue was purified by silica gel
column chromatography (1 : 9 : 90 NEt3/MeOH/CH2Cl2) to
afford compound 11 (179 mg, 87%) as a pale yellow solid.
1H NMR (300 MHz, D2O): d 8.18 (s, 1 H), 6.17 (s, 1 H), 5.59
(s, 1 H), 5.13 (s, 1 H), 4.30–4.22 (m, 2 H), 4.04–3.99 (m, 1 H),
3.68–3.46 (m, 8 H), 1.44, 1.26 (each s, each 3 H), 0.81 (s, 3 H),
0.79 (s, 3 H), 0.65 (s, 3 H) and 0.63 (s, 3 H). 31P NMR (121.5
MHz, D2O, decoupled with H): d 8.98 and 8.35. HRMS (ESI,
negative) for C29H51N6O11P2, calc. 799.2202 [M ꢂ 1]ꢂ; found
799.2225.
N1-[(500-Hydroxyl)ethoxyethyl]-50-O-(tert-butyldimethylsilyl)-
20,30-O-isopropylidene-8-bromoadenosine (14b)
A mixture of 13b (463 mg, 0.90 mmol), 2-(hydroxyethoxy)-
ethylamine (108 mL, 1.08 mmol) and K2CO3 (7 mg, 0.05 mmol)
in DMF (4 mL) was stirred at room temperature for 8 h. The
mixture was evaporated and the residue purified by silica gel
column chromatography (3% MeOH in CH2Cl2) to give
compound 14b (489 mg, 92%) as a white foam. 1H NMR
(300 MHz, DMSO-d6): d 8.00 (s, 1 H), 7.19 (br s, 1 H), 5.97
(s, 1 H), 5.58 (d, J = 6.0 Hz, 1 H), 4.95 (s, 1 H), 4.61 (s, 1 H),
4.22–4.13 (m, 3 H), 3.77–3.66 (m, 3 H), 3.45–3.44 (m, 4 H),
1.52, 1.32 (each s, each 3 H), 0.88 (s, 9 H) and 0.00 (s, 6 H). 13
C
NMR (125 MHz, DMSO-d6): d 151.9, 149.2, 142.1, 123.1,
113.2, 90.6, 87.6, 82.5, 81.3, 72.2, 66.8, 62.9, 60.1, 46.4, 26.9,
25.7, 25.2, 17.9, ꢂ5.5 and ꢂ5.6. HRMS (ESI, positive) for
C23H38N5O6BrSi, calc. 588.1848 [M + 1]+; found 588.1862.
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-8-
bromoinosine (4e)
In a manner similar to that described for the synthesis of 4a, 4e
was obtained from 11 in a yield of 36%. 1H NMR (400 MHz,
D2O): d 8.47 (s, 1 H), 6.19 (d, J = 5.7 Hz, 1 H), 4.73 (t, J =
5.7 Hz, 1 H), 4.53–4.51 (m, 1 H), 4.35–4.28 (m, 3 H), 3.86–3.82
(m, 2 H), 3.80–3.72 (m, 4 H) and 3.58–3.50 (m, 2 H). 31P NMR
(121.5 MHz, D2O): d 0.93. HRMS (ESI, negative) for
C14H21N4O13P2Br, calc. 592.9691 [M ꢂ 1]ꢂ; found 592.9672.
N1-[(50 0-Hydroxyl)ethoxyethyl]-20,30-O-isopropylideneadenosine
(15a)
A mixture of 14a (630 mg, 1.24 mmol), TBAF (1 M in THF,
12.4 mL, 12.4 mmol) and AcOH (45 mL, 0.7 mmol) in THF
(15 mL) was stirred at room temperature for 2 h. The mixture
was evaporated and the residue purified by silica gel column
chromatography (5% MeOH in CH2Cl2) to give compound
1
5-Amino-1-[50-O-(tert-butyldimethylsilyl)-20,30-O-(isopropylidene)-
2-bromo-b-D-ribofuranosyl]imidazole-4-nitrile (12b)
15a (464 mg, 95%) as a colorless solid. H NMR (400 MHz,
DMSO-d6): d 8.13, 7.98 (each s, each 1 H), 6.00 (d, J = 3.2 Hz,
1 H), 5.23 (dd, J = 3.2 and 6.4 Hz, 1 H), 4.91 (dd J = 2.4 and
6.4 Hz, 1 H), 4.18 (t, J = 5.2 Hz, 3 H), 3.70 (t, J = 5.2 Hz,
2 H), 3.52 (dd, J = 2.0 and 5.2 Hz, 2 H), 3.39–3.42 (m, 4 H),
1.53 and 1.32 (each s, each 3 H). 13C NMR (125 MHz, DMSO-d6):
d 153.6, 149.0, 140.9, 137.7, 122.7, 113.1, 89.4, 86.5, 83.6, 81.3,
72.2, 66.9, 61.5, 60.1, 57.5, 46.1, 27.0 and 25.2. HRMS (ESI,
positive) for C17H25N5O6, calc. 396.1878 [M + 1]+; found
396.1865.
To a solution of 12a23 (418 mg, 1.06 mmol) in THF (10 mL)
was added NBS (232 mg, 1.27 mmol). The resulting solution
was stirred at rt for 10 min and then evaporated under reduced
pressure. The residue was purified by silica gel column
chromatography (2% MeOH in CH2Cl2) to give compound
12b (452 mg, 90%) as a pale yellow foam. 1H NMR (300 MHz,
DMSO-d6): d 6.50 (br s, 2 H), 5.83 (d, J = 4.5 Hz, 1 H),
5.03–5.00 (m, 1 H), 4.80–4.77 (m, 1 H), 4.13 (d, J = 3.3 Hz,
1 H), 3.90 (d, J = 3.3 Hz, 2 H), 1.51, 1.31 (each s, each 3 H),
0.89 (9 H) and 0.11 (6 H). 13C NMR (125 MHz, DMSO-d6): d
148.7, 115.6, 114.7, 111. 6, 92.4, 90.2, 83.8, 81.0, 79.1, 62.5,
27.2, 25.9, 25.3, 18.3, ꢂ5.4 and ꢂ5.6. HRMS (ESI, positive)
for C18H29N4O4BrSi, calc. 473.1214 [M + 1]+; found
473.1213.
N1-[(500-Hydroxyl)ethoxyethyl]-20,30-O-isopropylidene-8-
bromoadenosine (15b)
In a manner similar to that described for the synthesis of 10a,
1
15b was synthesized in 95% yield as a pale yellow solid: H
NMR (400 MHz, DMSO-d6): d 8.36 (s, 1 H), 6.01 (d, J = 2.8
Hz, 1 H), 5.67 (dd, J = 4.0 and 6.4 Hz, 1 H), 4.97 (dd, J = 3.6
and 6.4 Hz, 1 H), 4.94 (t, J = 5.6 Hz, 1 H), 4.58–4.55 (m, 1 H),
4.18–4.10 (m, 3 H), 3.67 (t, J = 4.2 Hz, 2 H), 3.52–3.42
(m, 4 H), 1.54 and 1.32 (each s, each 3 H). 13C NMR (125
MHz, DMSO-d6): d 154.6, 149.4, 148.1, 125.5, 124.1, 113.5,
90.6, 87.3, 82.3, 81.3, 72.1, 67.6, 61.3, 60.1, 45.7, 27.1 and 25.2.
HRMS (ESI, positive) for C17H24N5O6, calc. 474.0983
[M + 1]+; found 474.0978.
5-[(Methoxymethylene)amino]-1-[50-O-(tert-butyldimethylsilyl)-
20,30-O-(isopropylidene)-2-bromo-b-D-ribofuranosyl]imidazole-
4-nitrile (13b)
A mixture of 12b (452 mg, 0.96 mmol) and CF3CO2H (10 mL)
in methyl orthoformate (10 mL) was stirred under reflux for
0.5 h. After evaporation, the residue was purified by silica gel
column chromatography (2% MeOH in CH2Cl2) to give
compound 13b (463 mg, 94%) as a pale yellow foam. 1H
NMR (300 MHz, DMSO-d6): d 8.53 (s, 1 H), 5.85 (d, J = 2.7 Hz,
1 H), 5.34–5.32 (m, 1 H), 4.80–4.76 (m, 1 H), 4.05–4.01
(m, 1 H), 3.95 (s, 3 H), 3.74–3.72 (m, 2 H), 1.51, 1.31
(each s, each 3 H), 0.83 (s, 9 H) and 0.00 (s, 6 H). 13C NMR
(125 MHz, DMSO-d6): d 162.9, 146.9, 118.8, 114.6, 114.2,
100.0, 90.4, 86.2, 81.9, 80.5, 62.9, 55.4, 27.0, 25.7, 25.3, 18.0,
ꢂ5.4 and ꢂ5.5. HRMS (ESI, positive) for C20H31N4O5BrSi,
calc. 515.1314 [M + 1]+; found 515.1295.
N1-[(500-Hydroxyl)ethoxyethyl]-20,30-O-isopropylidene-8-
ethylthioadenosine (15c)
To a 1.0 mM suspension of 10b (236 mg, 0.50 mmol) in
H2O/CH3CN (4 mL/4 mL) were added 3 equiv. of 2-mercapto-
ethanol (105 mL, 1.50 mmol) and 10 equiv. of NEt3 (0.70 mL,
5 mmol). The resulting clear solution was then refluxed for 2 h.
Further purification was achieved by silica gel column
chromatography (5% MeOH in CH2Cl2) to give compound
1
15c (193 mg, 82%) as a colorless solid. H NMR (400 MHz,
ꢀc
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962 | New J. Chem., 2010, 34, 956–966

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DMSO-d6): d 8.17 (s, 1 H), 7.63 (t, J = 5.6 Hz, 1 H), 5.98
(d, J = 2.8 Hz, 1 H), 5.58 (dd, J = 2.8 and 5.6 Hz, 1 H), 5.20
(s, 1 H), 5.04 (s, 1 H), 4.99 (dd, J = 2.8 and 6.4 Hz, 1 H), 4.59
(s, 1 H), 4.18–4.14 (m, 1 H), 3.73–3.54 (m, 6 H), 3.49–3.34
(m, 8 H), 1.54 and 1.32 (each s, each 3 H). 13C NMR
(125 MHz, DMSO-d6): d 153.0, 151.6, 149.6, 148.0, 119.6,
113.3, 89.7, 86.5, 81.9, 81.5, 72.1, 68.7, 61.6, 60.2, 59.7, 54.9,
35.3, 27.2 and 25.2. HRMS (ESI, positive) for C19H29N5O7S,
calc. 472.1860 [M + 1]+; found 472.1871.
d 3.88 and ꢂ7.32. HRMS (ESI, negative) for C14H22N5O12P2,
calc. 514.0746 [M ꢂ 1]ꢂ; found 514.0744.
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-8-
chloroadenosine (4g)
In a manner similar to that described for the synthesis of 4a, 4g
was synthesized in 92% yield as a white solid. 1H NMR
(500 MHz, D2O): d 8.54 (s, 1 H), 6.15 (d, J = 5.0 Hz, 1 H),
5.26 (m, 1 H), 4.64 (t, J = 5.0 Hz, 1 H), 4.58–4.55 (m, 2 H),
4.31–4.28 (m, 1 H), 4.11–4.07 (m, 1 H), 4.04–4.01 (m, 3 H),
3.92–3.88 (m, 2 H) and 3.75–3.60 (m, 2 H). 31P NMR
(121.5 MHz, D2O, decoupled with H): d 3.32 and 3.17. HRMS
(ESI, negative) for C14H22N5ClO12P2, calc. 548.0356 [M ꢂ 1]ꢂ;
found 548.0351.
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-20,30-
O-isopropylideneadenosine (16a)
In a manner similar to that described for the synthesis of 10a,
1
16a was synthesized in 86% yield as a white solid. H NMR
(400 MHz, D2O): d 8.46, 8.45 (each s, each 1 H), 6.28 (d, J =
2.4 Hz, 1 H), 5.39 (dd, J = 2.4 and 6.0 Hz, 1 H), 5.12 (dd, J =
6.0 and 1.6 Hz, 1 H), 4.67–4.65 (m, 1 H), 4.51 (t, J = 4.4 Hz,
2 H), 3.96–3.95 (m, 4 H), 3.67 (t, J = 4.4 Hz, 2 H), 1.59 and
1.38 (each s, each 3 H). 31P NMR (121.5 MHz, D2O): d 0.63
and 0.60. HRMS (ESI, negative) for C17H27N5O12P2, calc.
554.1059 [M ꢂ 1]ꢂ; found 554.1084.
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-8-
(phosphorylethylthio)adenosine (4h)
In a manner similar to that described for the synthesis of 4a, 4h
was synthesized in 89% yield as a white solid. 1H NMR
(400 MHz, D2O): d 8.08 (s, 1 H), 5.99 (d, J = 3.0 Hz, 1 H),
5.07 (t, J = 6.4 Hz, 1 H), 4.41 (dd, J = 4.0 and 6.0 Hz, 1 H),
4.14 (dd, J = 4.4 and 10.4 Hz, 1 H), 4.06–3.98 (m, 4 H),
4.12–3.98 (m, 4 H), 3.85 (dd, J = 6.0 and 9.2 Hz, 2 H),
3.74–3.64 (m, 6 H) and 3.52–3.47 (m, 2 H). 31P NMR
(121.5 MHz, D2O, decoupled with H): d 2.04 and 1.87. HRMS
(ESI, negative) for C16H28N5O16P3S, calc. 670.0392 [M ꢂ 1]ꢂ;
found 670.0367.
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-20,30-
O-isopropylidene-8-chloroadenosine (16b)
In a manner similar to that described for the synthesis of 10a,
1
16b was synthesized in 90% yield as a white solid. H NMR
(500 MHz, D2O): d 8.59 (s, 1 H), 6.42 (d, J = 1.5 Hz, 1 H),
5.84 (dd, J = 3.5 and 1.5 Hz, 1 H), 5.22 (dd, J = 3.5 and
2.5 Hz, 1 H), 4.60 (m, 2 H), 4.41 (m, 1 H), 4.08–4.03 (m, 4 H),
3.84–3.74 (m, 4 H), 1.63 and 1.44 (each s, each 3 H). 31P NMR
(121.5 MHz, D2O, decoupled with H): d ꢂ10.05 and ꢂ10.22.
HRMS (ESI, positive) for C17H26N5ClO12P2, calc. 590.0814
[M + 1]+; found 590.0823.
N1-Dinitrobenzene-50-O-para-toluenesulfonyl-20,30-O-
isopropylideneinosine (18)
A mixture of N1-dinitrobenzene-20,30-O-isopropylideneinosine
(17b) (948 mg, 2.0 mmol), TsCl (457 mg, 2.4 mmol), NEt3
(336 mL, 2.4 mmol) and DMAP (catalyst) was suspended in
anhydrous DCM (10 mL) and stirred at rt for 8 h. After the
addition of a few drops of methanol, the mixture was evapo-
rated to dryness and purified on a silica gel column eluted with
increasing amounts of MeOH in CH2Cl2 (from 0 to 3%) to
give compound 18 as a pale yellow amorphous solid consisting
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-(phosphoryl)-20,30-
O-isopropylidene-8-(phosphorylethylthio)adenosine (16c)
In a manner similar to that described for the synthesis of 10a,
except that 14.4 equiv. of POCl3 was used in the synthesis of
13a, 16c was synthesized in 86% yield as a white solid. 1H
NMR (500 MHz, D2O): d 8.22 (s, 1 H), 6.31 (d, J = 3.0 Hz,
1 H), 5.70 (dd, J = 3.0 and 7.0 Hz, 1 H), 5.25 (dd, J = 7.0 and
4.5 Hz, 1 H), 4.44 (dd, J = 5.0 and 11.5 Hz, 1 H), 4.16
(dd, J = 6.5 and 13.5 Hz, 2 H), 4.12–3.98 (m, 4 H), 3.86–3.77
(m, 6 H), 3.61–3.52 (m, 2 H), 1.66 and 1.43 (each s, each 3 H).
31P NMR (121.5 MHz, D2O, decoupled with H): d 3.66, 3.56
and 3.34. HRMS (ESI, negative) for C19H33N5O16P3S, calc.
710.0705 [M ꢂ 1]ꢂ; found 710.0702.
1
of a 1 : 1 mixture of atropisomers (994 mg, 79%). H NMR
(400 MHz, DMSO-d6): d 8.95 (t, J = 2.0 Hz, 1 H), 8.84–8.79
(m, 1 H), 8.60 (s, 1 H), 8.31, 8.30 (each s, each 0.5 H), 8.11
(t, J = 9.6 Hz, 1 H), 7.71–7.68 (m, 2 H), 7.35 (dd, J = 5.6 and
8.0 Hz, 2 H), 6.22, 6.18 (each d, J = 2.4 Hz, each 0.5 H), 5.35,
5.32 (each dd, J = 2.4 and 6.0 Hz, each 0.5 H), 4.97–4.94
(m, 1 H), 4.37–4.30 (m, 2 H), 4.24–4.21 (m, 1 H), 2.35, 2.34
(each s, each 1.5 H), 1.52 and 1.35 (each s, each 3 H). 13C
NMR (125 MHz, DMSO-d6): d 155.4, 155.4, 148.3, 147.8,
147.7, 147.6, 147.4, 146.2, 145.7, 145.6, 140.8, 140.6, 135.4,
135.3, 133.0, 132.9, 132.3, 132.2, 130.5, 130.4, 129.9, 129.8,
128.1, 123.8, 121.2, 114.3, 114.2, 89.6, 89.5, 84.2, 84.0, 80.9,
80.8, 70.2, 70.1, 27.3, 27.3, 25.6, 25.7 and 21.5. HRMS
(ESI, positive) for C26H24N6O11S, calc. 629.1297 [M + 1]+;
found 629.1294.
N1-(50 0-O-Phosphonoxyethoxyethyl)-50-O-
(phosphoryl)adenosine (4f)
In a manner similar to that described for the synthesis of 4a, 4f
was synthesized in 90% yield as a white solid. 1H NMR
(500 MHz, D2O): d 8.77, 8.75 (each s, each 1 H), 6.20
(d, J = 5 Hz, 1 H), 4.47 (dd, J = 5.0 and 4.5 Hz, 1 H), 4.58
(t, J = 5.0 Hz, 2 H), 4.39–4.37 (m, 1 H), 4.08–4.00 (m, 4 H),
3.90 (dd, J = 5.0 and 2.0 Hz, 2 H) and 3.72 (dd, J = 5.0 and
4.0 Hz, 2 H). 31P NMR (121.5 MHz, D2O, decoupled with H):
N1-[(500-Hydroxyl)ethoxyethyl]-50-O-(tert-butyldimethylsilyl)-
20,30-O-isopropylideneinosine (22a)
In a manner similar to that described for the synthesis of 7a, 22a
was synthesized from 50-O-TBDMS-20,30-O-isopropylideneinosine
ꢀc
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in 80% yield as a white solid. 1H NMR (400 MHz, DMSO-d6):
d 8.32, 8.22 (each s, each 1 H), 6.12 (d, J = 2.0 Hz, 1 H), 5.30
(dd, J = 2.0 and 6.0 Hz, 1 H), 4.90 (dd, J = 2.4 and 6.0 Hz,
1 H), 4.56 (t, J = 5.2 Hz, 1 H), 4.24–4.17 (m, 3 H), 3.74–3.64
(m, 3 H), 3.44–3.41 (m, 4 H), 1.52, 1.32 (each s, each 3 H), 0.79
(s, 9 H) and ꢂ0.04 (s, 6 H). HRMS (ESI, positive) for
C23H38N4O7Si, calc. 511.2582 [M + 1]+; found 511.2578.
17.9 and ꢂ5.6. HRMS (ESI, positive) for C43H53N4O8SiBr,
calc. 861.2894 [M + 1]+; found 861.2872.
N1-(50 0-Monomethoxytrityloxyethoxyethyl)-20,30-O-isopropyl-
ideneinosine (24a)
In a manner similar to that described for the synthesis of 14a,
1
24a was synthesized in 91% yield as a pale yellow solid. H
N1-[(50 0-Hydroxyl)ethoxyethyl]-50-O-(tert-butyldimethylsilyl)-
20,30-O-isopropylidene-8-bromoinosine (22b)
NMR (300 MHz, DMSO-d6): d 8.39, 8.34 (each s, each 1 H),
7.36–7.20 (m, 12 H), 6.89–6.86 (m, 2 H), 6.09 (d, J = 2.4 Hz,
1 H), 5.15–5.09 (m, 2 H), 4.91–4.88 (m, 1 H), 4.31–4.20
(m, 3 H), 3.74 (s, 3 H), 3.58–3.51 (m, 5 H), 3.05–3.00 (m, 2 H),
1.52 and 1.24 (each s, each 3 H). 13C NMR (125 MHz, DMSO-d6):
d 158.1, 155.8, 149.1, 147.0, 144.3, 139.2, 135.0, 129.9, 127.9,
127.8, 126.7, 123.7, 113.1, 113.0, 89.4, 86.8, 85.5, 83.8, 81.2,
69.7, 68.0, 62.8, 61.4, 55.0, 45.5, 26.9 and 25.0. HRMS
(ESI, positive) for C37H40N4O8, calc. 669.2919 [M + 1]+;
found 669.2908.
In a manner similar to that described for the synthesis of 7a,
22b was synthesized from 50-O-TBDMS-20,30-O-isopropyl-
1
idene-8-bromoinosine in 84% yield as a pale yellow solid. H
NMR (500 MHz, DMSO-d6): d 8.37 (s, 1 H), 6.05 (d, J = 2.0
Hz, 1 H), 5.62 (dd, J = 2.0 and 6.0 Hz, 1 H), 4.99 (dd, J = 2.5
and 6.0 Hz, 1 H), 4.56 (d, J = 5.5 Hz, 1 H), 4.20–4.15 (m, 3 H),
3.70–3.62 (m, 4 H), 3.46–3.42 (m, 4 H), 1.54, 1.33 (each s, each
3 H), 0.79 (s, 9 H) and 0.00 (s, 6 H). 13C NMR (125 MHz,
DMSO-d6): d 154.5, 149.3, 147.9, 125.8, 124.1, 113.4, 90.6,
87.6, 82.5, 81.2, 72.1, 67.6, 62.9, 60.1, 45.7, 27.0, 25.6, 25.2,
17.9 and ꢂ5.6. Elemental analysis: C23H37BrN4O7Si requires
C, 46.86; H, 6.33; N, 9.50. Found: C, 47.10; H, 6.38; N, 9.51.
N1-(50 0-Monomethoxytrityloxyethoxyethyl)-20,30-O-isopropylidene-
8-bromoinosine (24b)
In a manner similar to that described for the synthesis of 14a,
1
24b was synthesized in 87% yield as a pale yellow solid. H
N1-(50 0-Monomethoxytrityloxyethoxyethyl)-50-O-(tert-butyl-
dimethylsilyl)-20,30-O-isopropylideneinosine (23a)
NMR (400 MHz, DMSO-d6): d 8.41 (s, 1 H), 7.33–7.17
(m, 12 H), 6.84–6.82 (m, 2 H), 5.95 (d, J = 2.0 Hz, 1 H),
5.14 (dd, J = 2.0 and 6.0 Hz, 1 H), 4.89 (t, J = 6.0 Hz, 1 H),
4.84 (dd, J = 4.0 and 6.0 Hz, 1 H), 4.35–4.30 (m, 1 H),
4.21–4.14 (m, 1 H), 4.08–4.04 (m, 1 H), 3.72 (s, 3 H), 3.62–3.38
(m, 4 H), 3.31 (s, 1 H), 3.07–3.02 (m, 1 H), 2.98–2.94 (m, 1 H),
1.48 and 1.09 (each s, each 3 H). 13C NMR (125 MHz,
DMSO-d6): d 158.1, 154.6, 149.5, 147.9, 144.2, 144.2, 135.1,
129.8, 127.9, 127.7, 126.7, 125.6, 124.2, 113.3, 113.1, 90.5, 87.7,
85.4, 82.4, 81.3, 69.7, 67.7, 62.7, 61.4, 55.0, 54.9, 45.9, 27.0 and
25.0. HRMS (ESI, positive) for C37H39N4O8Br, calc. 747.2024
[M + 1]+; found 747.2037.
A mixture of 22a (820 mg, 1.61 mmol) and MMTrCl (743 mg,
2.41 mmol) in pyridine (10 mL) was stirred at room tempera-
ture for 8 h. The mixture was evaporated, and the residue
partitioned between H2O and EtOAc. The organic layer was
washed with brine, dried (Na2SO4) and evaporated. The
residue was purified by silica gel column chromatography
(2% MeOH in CH2Cl2) to give compound 23a (1.21 g, 96%)
as a pale yellow powder. 1H NMR (400 MHz, DMSO-d6):
d 8.34. 8.27 (each s, each 1 H), 7.34–7.28 (m, 12 H), 6.91–6.87
(m, 2 H), 6.09 (s, 1 H), 5.42 (dd, J = 4.0 and 6.4 Hz, 1 H), 5.16
(dd, J = 4.0 and 6.4 Hz, 1 H), 4.48–4.42 (m, 2 H), 4.35–4.31
(m, 1 H), 4.02–3.99 (m, 3 H), 3.78–3.72 (m, 4 H), 3.36–3.28
(m, 1 H), 3.17–3.12 (m, 1 H), 1.57, 1.34 (each s, each 3 H), 1.04
(s, 9 H) and 0.10 (s, 6 H). 13C NMR (125 MHz, DMSO-d6): d
158.0, 154.5, 149.3, 147.2, 145.0, 135.0, 131.5, 129.8, 129.4,
129.0, 127.9, 127.4, 127.3, 126.5, 125.4, 113.4, 113.1, 90.3, 85.3,
85.2, 83.1, 80.4, 69.8, 67.7, 62.6, 61.4, 55.0, 46.0, 27.0, 26.7,
25.0, 21.1 and ꢂ5.3. HRMS (ESI, positive) for C43H54N4O8Si,
calc. 783.3789 [M + 1]+; found 783.3772.
N1-(500-Monomethoxytrityloxyethoxyethyl)-50-O-(para-toluene-
sulfonyl)-20,30-O-isopropylideneinosine (20a)
To a stirred solution of 24a (110 mg, 0.16 mmol) in DCM
(5 mL) were added NEt3 (0.05 mL, 0.32 mmol), TsCl (50 mg,
0.24 mmol) and DMAP (catalyst). After stirring at rt for 8 h,
the mixture was diluted with DCM, washed with brine, dried
(Na2SO4) and evaporated. The residue was purified by
silica gel column chromatography to give compound 20a
N1-(50 0-Monomethoxytrityloxyethoxyethyl)-50-O-(tert-butyl-
dimethylsilyl)-20,30-O-isopropylidene-8-bromoinosine (23b)
(121 mg, 89%) as
a
pale yellow powder. 1H NMR
(400 MHz, DMSO-d6): d 8.38, 8.27 (each s, each 1 H),
7.34–7.18 (m, 12 H), 6.86, 6.84 (each s, each 1 H), 6.12
(d, J = 1.6 Hz, 1 H), 5.17 (dd, J = 2.0 and 6.0 Hz, 1 H),
4.91 (dd, J = 3.6 and 6.0 Hz, 1 H), 4.32–4.06 (m, 5 H),
3.72–3.71 (m, 5 H), 3.56–3.54 (m, 2 H), 3.03–2.98 (m, 2 H),
1.92, 1.50 and 1.23 (each s, each 3 H). 13C NMR (100 MHz,
DMSO-d6): d 170.4, 158.6, 156.3, 149.7, 147.4, 144.8, 140.0,
135.6, 130.3, 128.4, 128.2, 127.2, 124.4, 113.9, 113.6, 89.5,
86.7, 85.9, 84.3, 84.0, 81.3, 70.2, 68.4, 64.1, 63.2, 55.5, 46.1,
27.4, 25.5 and 20.9. Elemental analysis: C44H46N4O10S
requires C, 64.22; H, 5.63; N, 6.81. Found: C, 64.09; H,
5.88; N, 7.02.
In a manner similar to that described for the synthesis of 23a,
23b was synthesized in 92% yield as a pale yellow powder.
1H NMR (400 MHz, DMSO-d6): d 8.70 (s, 1 H), 7.64–7.48
(m, 12 H), 7.14–7.12 (m, 2 H), 6.29 (s, 1 H), 5.52 (dd, J = 4.0
and 6.4 Hz, 1 H), 5.18 (dd, J = 4.0 and 6.4 Hz, 1 H), 4.62–4.51
(m, 2 H), 4.42–4.38 (m, 1 H), 4.02 (s, 3 H), 3.95–3.80 (m, 4 H),
3.38–3.34 (m, 1 H), 3.28–3.25 (m, 1 H), 1.78, 1.40 (each s, each
3 H), 1.05 (s, 9 H) and 0.15 (s, 6 H). 13C NMR (125 MHz,
DMSO-d6): d 158.0, 154.5, 149.4, 147.8, 144.2, 135.0, 129.8,
127.9, 127.7, 126.7, 125.9, 124.1, 113.2, 113.1, 90.5, 87.9, 85.4,
82.5, 81.2, 69.7, 67.8, 63.0, 62.7, 55.0, 45.9, 26.9, 25.6, 25.0,
ꢀc
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N1-(500-Monomethoxytrityloxyethoxyethyl)-50-O-(para-toluene-
sulfonyl)-20,30-O-isopropylidene-8-bromoinosine (20b)
3.85–3.73 (m, 4 H) and 3.70–3.67 (m, 2 H). 31P NMR
(121.5 MHz, D2O): d 3.31 and ꢂ7.12. HRMS (ESI, negative)
for C14H22N4O13P2, calc. 515.0586 [M ꢂ 1]ꢂ; found 515.0586.
In a manner similar to that described for the synthesis of 20a,
20b was synthesized in 85% yield as a pale yellow powder. 1H
NMR (300 MHz, DMSO-d6): d 8.40 (s, 1 H), 7.32–7.21
(m, 16 H), 6.85–6.84 (m, 2 H), 5.98 (s, 1 H), 5.16 (d, J =
3.3 Hz, 1 H), 4.88–4.78 (m, 1 H), 4.36–4.35 (m, 2 H), 3.70
(s, 3 H), 3.58–3.47 (m, 7 H), 3.15–3.09 (m, 1 H), 2.32 (s, 3 H),
1.54 and 1.32 (each s, each 3 H). 13C NMR (100 MHz, DMSO-d6):
d 158.0, 154.5, 149.3, 147.2, 145.0, 144.2, 135.0, 131.5, 129.8,
129.4, 127.9, 127.7, 127.4, 125.6, 124.0, 113.4, 113.1, 90.5, 85.3,
84.0, 80.3, 70.1, 68.4, 62.6, 55.0, 46.0, 26.7, 25.0, 24.8 and 21.1.
HRMS (ESI, positive) for C44H45N4O10S, calc. 901.2118
[M + 1]+; found 901.2134.
N1-[(500-Hydroxyl)ethoxyethyl]-50-O-pyrophosphate-8-
bromoinosine (5b)
In a manner similar to that described for the synthesis of 6a, 5b
was synthesized in 89% yield as a pale yellow foam. 1H NMR
(500 MHz, D2O): d 8.36 (s, 1 H), 6.23 (d, J = 6.5 Hz, 1 H),
5.15 (t, J = 5.5 Hz, 1 H), 4.58–4.56 (m, 1 H), 4.39–4.34 (m, 1 H),
4.28–4.20 (m, 2 H), 4.00–3.98 (m, 2 H), 3.95–3.87 (m, 4 H) and
3.76–3.72 (m, 2 H); 31P NMR (121.5 MHz, D2O): d ꢂ10.12
and ꢂ10.31. HRMS (ESI, negative) for C14H21N4O13P2Br,
calc. 592.9691 [M ꢂ 1]ꢂ; found 592.9678.
N1-[(500-Hydroxyl)ethoxyethyl]-50-O-monomethoxytrityl-20,30-
N1-(50 0-Monomethoxytrityloxyethoxyethyl)-50-O-pyrophosphate-
20,30-O-isopropylideneinosine (21a)
O-isopropylideneinosine (25a)
In a manner similar to that described for the synthesis of 7a,
25a was synthesized from 50-O-monomethoxytrityl-20,30-O-
To a suspension of tris(tetrabutylammonium)hydrogen pyro-
phosphate (263 mg, 0.60 mmol) in 3 mL of acetonitrile was
added dropwise a solution of 20a (121 mg, 0.15 mmol) in 2 mL
of acetonitrile. The reaction mixture was stirred at room
temperature for 16 h and the solvent then removed by rotary
evaporation. The residue was dissolved in 2 mL of TEAB
buffer (1 M, pH 7.5), evaporated to dryness, and partitioned
between H2O and CHCl3. The aqueous layer was washed with
CHCl3 (3 ꢁ 5 mL) and evaporated. The residue was dissolved
in 1 mL of TEAB buffer (0.05M, pH 7.5), applied to a C18
reversed-phase column (2.2 cm ꢁ 25 cm) and developed by a
linear gradient of 0–80% CH3CN in TEAB buffer (0.05 M,
pH 7.5) within 30 min to give 21a (29 mg, 20%) as its
triethylammonium salt. 1H NMR (500 MHz, D2O): d 8.21,
8.10 (each s, each 1 H), 7.28–7.11 (m, 12 H), 6.72, 6.69 (each s,
each 1 H), 6.35 (d, J = 7.5 Hz, 1 H), 5.80–5.75 (m, 1 H), 5.20
(dd, J = 2.0 and 4.5 Hz, 1 H), 4.41–4.36 (m, 1 H), 4.23–4.20
(m, 1 H), 4.03–4.00 (m, 2 H), 3.85–3.84 (m, 2 H), 3.80 (s, 3 H),
3.77–3.72 (m, 2 H), 3.63–3.60 (m, 2 H), 1.52 and 1.30 (each s,
each 3 H). 31P NMR (121.5 MHz, D2O, decoupled with H):
1
isopropylideneinosine in 72% yield as a pale yellow solid. H
NMR (400 MHz, DMSO-d6): d 8.21, 8.11 (each s, each 1 H),
7.32–7.15 (m, 12 H), 6.81–6.81 (d, J = 10.2 Hz, 2 H), 6.19
(d, J = 2.0 Hz, 1 H), 5.75 (s, 1 H), 5.34 (dd, J = 2.0 and
6.4 Hz, 1 H), 4.59 (t, J = 4.2 Hz, 1 H), 4.35–4.31 (m, 1 H),
4.23–4.18 (m, 1 H), 4.15–4.11 (m, 1 H), 3.73 (s, 3 H), 3.67–3.64
(m, 2 H), 3.48–3.42 (m, 3 H), 3.27–3.22 (m, 1 H), 3.11–3.08
(m, 1 H), 1.53 and 1.31 (each s, each 3 H). 13C NMR (100 MHz,
DMSO-d6): d 158.6, 156.3, 149.3, 147.2, 144.5, 144.4, 140.1,
135.1, 130.4, 128.3, 128.3, 128.2, 128.2, 127.3, 127.2, 124.5,
113.8, 113.6, 89.6, 86.4, 86.0, 84.0, 81.6, 72.6, 68.3, 64.4, 60.6,
55.5, 55.3, 45.9, 27.4 and 25.7. HRMS (ESI, positive) for
C37H40N4O8, calc. 669.2919 [M + 1]+; found 669.2943.
N1-[(500-Hydroxyl)ethoxyethyl]-50-O-monomethoxytrityl-20,30-
O-isopropylidene-8-bromoinosine (25b)
In a manner similar to that described for the synthesis of 7a,
25b was synthesized from 50-O-monomethoxytrityloxy-20,30-
O-isopropylidene-8-bromoinosine in 75% yield as a pale
d
ꢂ9.89 and ꢂ10.07. HRMS (ESI, negative) for
C37H42N4O14P2, calc. 827.2095 [M ꢂ 1]ꢂ; found 827.2065.
1
yellow solid. H NMR (300 MHz, DMSO-d6): d 8.11 (s, 1 H),
7.27–6.72 (m, 14 H), 6.08 (d, J = 1.2 Hz, 1 H), 5.54 (d, J =
6.0 Hz, 1 H), 4.94 (dd, J = 1.2 and 4.8 Hz, 1 H), 4.63–4.57
(m, 1 H), 4.38–4.34 (m, 1 H), 4.25–4.23 (m, 1 H), 4.01 (s, 3 H),
N1-(500-Monomethoxytrityloxyethoxyethyl)-50-O-pyrophosphate-
20,30-O-isopropylidene-8-bromoinosine (21b)
3.50–3.38 (m,
5 H), 3.23–3.17 (m, 1 H), 3.02–2.98
In a manner similar to that described for the synthesis of 21a, 21b
1
was synthesized in 74% yield as a pale yellow foam. H NMR
(m, 1 H), 1.54 and 1.30 (each s, each 3 H). 13C NMR
(125 MHz, DMSO-d6): d 158.1, 154.4, 148.9, 147.7, 144.0,
143.9, 134.7, 129.8, 127.8, 127.7, 127.7, 126.8, 126.7, 125.7,
124.0, 113.4, 113.0, 90.6, 86.5, 85.7, 82.6, 81.4, 72.1, 67.6, 63.7,
60.1, 55.0, 45.6, 26.9 and 25.2. HRMS (ESI, positive) for
C37H39BrN4O8, calc. 747.2024 [M + 1]+; found 747.2046.
(400 MHz, D2O): d 8.49 (s, 1 H), 7.18–7.06 (m, 12 H), 6.72
(d, J = 8.8 Hz, 2 H), 5.92 (s, 1 H), 5.13 (t, J = 5.5 Hz, 1 H),
4.73–4.70 (m, 1 H), 4.56–4.53 (m, 1 H), 4.35–4.32 (m, 2 H),
3.96–3.94 (m, 2 H), 3.82–3.78 (m, 4 H), 3.75 (s, 3 H), 3.70–3.64
(m, 2 H), 1.54 and 1.31 (each s, each 3 H). 31P NMR
(121.5 MHz, D2O): d ꢂ13.39 and ꢂ13.90. HRMS (ESI, negative)
for C37H41N4O14P2Br, calc. 905.1202 [M ꢂ 1]ꢂ; found 905.1213.
N1-[(500-para-Toluenesulfonyl)ethoxyethyl]-50-O-monomethoxy-
trityl-20,30-O-isopropylideneinosine (26a)
N1-[(50 0-Hydroxyl)ethoxyethyl]-50-O-pyrophosphateinosine (5a)
In a manner similar to that described for the synthesis of 20a,
1
26a was synthesized in 85% yield as a pale yellow foam. H
In a manner similar to that described for the synthesis of 6a, 5a
was synthesized in 90% yield as a white foam. 1H NMR
(400 MHz, D2O): d 8.26, 8.12 (each s, each 1 H), 6.16 (d, J =
6.5 Hz, 1 H), 5.25 (t, J = 5.5 Hz, 1 H), 4.48–4.45 (m, 1 H),
4.33–4.30 (m, 1 H), 4.24–4.18 (m, 2 H), 4.15–4.09 (m, 2 H),
NMR (300 MHz, DMSO-d6): d 8.23 (s, 1 H), 8.08 (s, 1 H),
7.74–7.72 (m, 2 H), 7.28–7.14 (m, 12 H), 6.82–6.80 (m, 2 H),
6.21 (s, 1 H), 5.34 (d, J = 6.0 Hz, 1 H), 4.92–4.88 (m, 1 H),
4.35–4.30 (m, 1 H), 4.18–4.15 (m, 2 H), 4.08–4.02 (m, 3 H),
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3.72 (s, 3 H), 3.61–3.52 (m, 4 H), 3.25–3.22 (m, 1 H), 3.08–3.03
(m, 1 H), 2.38 (s, 3 H), 1.52 and 1.29 (each s, each 3 H). 13C
NMR (100 MHz, DMSO-d6): d 157.6, 155.1, 149.5, 147.4,
145.2, 144.6, 134.8, 131.3, 129.1, 128.9, 127.8, 127.5, 127.2,
125.8, 124.6, 113.5, 113.0, 90.8, 85.7, 84.2, 79.8, 71.9, 68.2, 62.9,
55.6, 46.3, 26.8, 25.4, 24.6 and 21.0. HRMS (ESI, positive) for
C44H46N4O10S, calc. 823.3007 [M + 1]+; found 823.3009.
HRMS (ESI, negative) for C14H22N4O13P2, calc. 515.0586
[M ꢂ 1]ꢂ; found 515.0612.
N1-[(500-O-Pyrophosphate)ethoxyethyl]-8-bromoinosine (5d)
In a manner similar to that described for the synthesis of 4a, 5d
was synthesized in 91% yield as a white solid. 1H NMR
(500 MHz, D2O): d 8.38 (s, 1 H), 6.11 (d, J = 6.5 Hz, 1 H),
5.17 (t, J = 5.5 Hz, 1 H), 4.65–4.43 (m, 1 H), 4.39–4.32
(m, 1 H), 4.29–4.21 (m, 2 H), 4.05–3.99 (m, 2 H), 3.93–3.84
(m, 4 H) and 3.78–3.69 (m, 2 H). 31P NMR (121.5 MHz, D2O,
decoupled with H): d ꢂ6.99 and ꢂ8.46. HRMS (ESI, negative)
for C14H21N4O13P2Br, calc. 592.9691 [M ꢂ 1]ꢂ; found 592.9684.
N1-[(50 0-para-Toluenesulfonyl)ethoxyethyl]-50-O-monomethoxy-
trityl-20,30-O-isopropylidene-8-bromoinosine (26b)
In a manner similar to that described for the synthesis of 20a,
1
26b was synthesized in 85% yield as a pale yellow foam. H
NMR (300 MHz, DMSO-d6): d 8.00 (s, 1 H), 7.74–6.78 (m, 18 H),
6.08 (s, 1 H), 5.54 (d, J = 3.3 Hz, 1 H), 4.98–4.95 (m, 1 H),
4.41–4.32 (m, 1 H), 4.26–4.20 (m, 1 H), 4.03 (s, 3 H), 3.58–3.37
(m, 7 H), 3.18–3.15 (m, 1 H), 3.03–2.99 (m, 1 H), 2.40
(s, 3 H), 1.54 and 1.31 (each s, each 3 H). 13C NMR (100 MHz,
DMSO-d6): d 158.9, 155.8, 150.7, 148.2, 146.1, 144.7, 135.3,
131.2, 129.5, 129.3, 127.2, 127.0, 126.8, 125.4, 124.2, 113.6,
113.4, 90.7, 85.7, 83.8, 80.2, 70.6, 68.9, 62.7, 55.8, 46.3, 26.9,
25.6 and 24.6. HRMS (ESI, positive) for C44H45N4O10SBr,
calc. 901.2118 [M + 1]+; found 901.2109.
Acknowledgements
This study was supported by the National Natural Sciences
Foundation of China (Grant no. 90713005) and the Ministry
of Education of China (Grant no. 200800010078).
References
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N1-[(500-O-Pyrophosphate)ethoxyethyl]-50-O-monomethoxytrityl-
20,30-O-isopropylideneinosine (27a)
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In a manner similar to that described for the synthesis of 15a,
1
27a was synthesized in 21% yield as a pale yellow solid. H
NMR (500 MHz, D2O): d 8.23, 8.09 (each s, each 1 H),
7.23–7.05 (m, 12 H), 6.72, 6.67 (each s, each 1 H), 6.32
(d, J = 7.5 Hz, 1 H), 5.78–5.64 (m, 1 H), 5.16 (dd, J = 2.0
and 4.5 Hz, 1 H), 4.9–4.28 (m, 1 H), 4.22–4.16 (m, 1 H),
4.05–4.01 (m, 2 H), 3.88–3.84 (m, 2 H), 3.81 (s, 3 H), 3.79–3.75
(m, 2 H), 3.68–3.66 (m, 2 H), 1.62 and 1.40 (each s, each 3 H).
31P NMR (121.5 MHz, D2O, decoupled with H): d ꢂ9.91 and
ꢂ10.09. HRMS (ESI, negative) for C37H42N4O14P2, calc.
827.2095 [M ꢂ 1]ꢂ; found 827.2071.
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N1-[(500-O-Pyrophosphate)ethoxyethyl]-50-O-monomethoxytrityl-
20,30-O-isopropylidene-8-bromoinosine (27b)
12 A. H. Guse, C. C. Kiefer, M. Fukuoka, S. Shuto, K. Weber,
V. C. Bailey, A. Matsuda, G. W. Mayr, N. Oppenheimer,
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J. Med. Chem., 2002, 45, 5340–5352.
In a manner similar to that described for the synthesis of 15a, 27b
1
was synthesized in 83% yield as a pale yellow solid. H NMR
(500 MHz, D2O): d 8.09 (s, 1 H), 7.32–7.17 (m, 12 H), 6.79, 6.77
(each s, each 1 H), 6.36 (d, J = 8.5 Hz, 1 H), 5.95–5.75 (m, 1 H),
5.16 (dd, J = 2.0 and 4.5 Hz, 1 H), 4.42–4.30 (m, 1 H),
4.24–4.16 (m, 1 H), 4.06–4.00 (m, 2 H), 3.88–3.84 (m, 2 H),
3.81 (s, 3 H), 3.79–3.75 (m, 2 H), 3.68–3.66 (m, 2 H), 1.62 and
1.40 (each s, each 3 H). 31P NMR (121.5 MHz, D2O, decoupled
with H): d ꢂ10.10 and ꢂ10.28. HRMS (ESI, negative) for
C37H41N4O14P2Br, calc. 905.1205 [M ꢂ 1]ꢂ; found 905.1205.
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N1-[(50 0-O-Pyrophosphate)ethoxyethyl]inosine (5c)
In a manner similar to that described for the synthesis of 4a, 5c
was synthesized in 82% yield as a white solid. 1H NMR
(500 MHz, D2O): d 8.38, 8.12 (each s, each 1 H), 6.07
(d, J = 6.5 Hz, 1 H), 5.12 (t, J = 5.5 Hz, 1 H), 4.56–4.44
(m, 1 H), 4.37–4.29 (m, 1 H), 4.28–4.16 (m, 2 H), 3.98–3.95
(m, 2 H), 3.90–3.81 (m, 4 H) and 3.62–3.54 (m, 2 H). 31P NMR
(121.5 MHz, D2O, decoupled with H): d ꢂ9.87 and ꢂ10.05.
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ꢀc
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