Synthesis of 7-Deaza-cyclic Adenosine-5′-diphosphate-carbocyclic-ribose and Its 7-Bromo Derivative as Intracellular Ca2+-Mobilizing Agents

Article abstract of DOI:10.1021/acs.joc.5b00723

Cyclic ADP-carbocyclic-ribose (cADPcR, 3) is a biologically and chemically stable equivalent of cyclic ADP-ribose (cADPR, 1), a Ca²⁺-mobilizing second messenger. We became interested in the biological activity of the 7-deaza analogues of cADPcR, i.e., 7-deaza-cADPcR (7) and its 7-bromo derivative, i.e., 7-deaza-7-Br-cADPcR (8), because 7-deazaadenosine is an efficient bioisostere of adenosine. The synthesis of 7 and 8 required us to construct the key N1-carbocyclic-ribosyl-7-deazaadenosine structure. Therefore, we developed a general method for preparing N1-substituted 7-deazaadenosines by condensing a 2,3-disubstituted pyrrole nucleoside with amines. Using this method, we prepared the N1-carbocyclic ribosyl 7-deazaadenosine derivative 10a, from which we then synthesized the target 7-deaza-cADPcR (7) via an Ag⁺-promoted intramolecular condensation to construct the 18-membered pyrophosphate ring structure. The corresponding 7-bromo derivative 8, which was the first analogue of cADPR with a substitution at the 7-position, was similarly synthesized. Biological evaluation for Ca²⁺-mobilizing activity in the sea urchin egg homogenate system indicated that 7-deaza-cADPcR (7) and 7-deaza-7-Br-cADPcR (8) acted as a full agonist and a partial agonist, respectively.

Full text of DOI:10.1021/acs.joc.5b00723

Article
pubs.acs.org/joc
Synthesis of 7‑Deaza-cyclic Adenosine-5′-diphosphate-carbocyclic-
ribose and Its 7‑Bromo Derivative as Intracellular Ca2+-Mobilizing
Agents
Satoshi Takano,† Takayoshi Tsuzuki,† Takashi Murayama,§ Takashi Sakurai,§ Hayato Fukuda,†
Mitsuhiro Arisawa,†,∥ and Satoshi Shuto*,†,‡
‡
†Faculty of Pharmaceutical Sciences, and Center for Research and Education on Drug DiscoveryHokkaido University, Kita-ku,
Sapporo 060-0812, Japan
§Department of Pharmacology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan
S
* Supporting Information
ABSTRACT: Cyclic ADP-carbocyclic-ribose (cADPcR, 3) is a
biologically and chemically stable equivalent of cyclic ADP-ribose
(cADPR, 1), a Ca2+-mobilizing second messenger. We became
interested in the biological activity of the 7-deaza analogues of
cADPcR, i.e., 7-deaza-cADPcR (7) and its 7-bromo derivative, i.e., 7-
deaza-7-Br-cADPcR (8), because 7-deazaadenosine is an efficient
bioisostere of adenosine. The synthesis of 7 and 8 required us to
construct the key N1-carbocyclic-ribosyl-7-deazaadenosine structure.
Therefore, we developed a general method for preparing N1-
substituted 7-deazaadenosines by condensing a 2,3-disubstituted
pyrrole nucleoside with amines. Using this method, we prepared the
N1-carbocyclic ribosyl 7-deazaadenosine derivative 10a, from which we then synthesized the target 7-deaza-cADPcR (7) via an
Ag+-promoted intramolecular condensation to construct the 18-membered pyrophosphate ring structure. The corresponding 7-
bromo derivative 8, which was the first analogue of cADPR with a substitution at the 7-position, was similarly synthesized.
Biological evaluation for Ca2+-mobilizing activity in the sea urchin egg homogenate system indicated that 7-deaza-cADPcR (7)
and 7-deaza-7-Br-cADPcR (8) acted as a full agonist and a partial agonist, respectively.
cADPR mobilizes intracellular Ca2+ in various mammalian
cells, such as pancreatic β-cells, smooth muscle and cardiac
muscle cells, T-lymphocytes, and cerebellar neurons, and
INTRODUCTION
Much attention has been focused on 7-deazaadenosine, which
is also known as the natural product tubercidin, and its
■
therefore cADPR is now considered as a general mediator of
intracellular Ca2+ signaling.4 Analogues of cADPR have been
extensively designed and synthesized due to their potential
usefulness for investigating the mechanism of cADPR-mediated
Ca2+release.5−8 On the basis of the important physiologic roles
of cADPR, cADPR analogues are also expected to be lead
structures for the development of potential drug candidates.4,8
cADPR and its analogues (Figure 2) have been synthesized
by enzymatic or chemo-enzymatic methods using ADP-ribosyl
cyclase from Aplysia californica, which mediates the intra-
molecular ribosylation of NAD+ and some modified NAD+
(prepared chemically or enzymatically) at the N1-position of
the purine moiety, to yield cADPR or the corresponding
analogues.5 These studies disclosed that some 8-substituted
analogues of cADPR, such as 8-NH2-cADPR (2), are
antagonists of cADPR at its intracellular receptor,5a and these
analogues are effective pharmacological tools for studying
cADPR-modulated Ca2+ signaling pathways.4
naturally occurring derivatives such as toyocamycin or
sangivamycin (Figure 1) due to their remarkable biological
Figure 1. 7-Deazaadenosine (tubercidin) and related naturally
occurring compounds.
activities, particularly as antitumor and antiviral agents.1 In
recent years, 7-deazaadenosine and its derivatives have also
been used as base-modified analogues of natural nucleosides in
oligonucleotide chemistry and chemical biology studies.2 The
attractive potency of 7-deazaadenosine as an efficient
bioisostere of adenosine led to our interest in the biological
activity of 7-deaza congeners of cyclic ADP-ribose (cADPR, 1,
Figure 2), a Ca2+-mobilizing second messenger.3
Received: April 2, 2015
© XXXX American Chemical Society
A
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because substitution at the 7-position was possible only in
the 7-deazaadenine motif and not in the adenine itself, and
there have been no 7-substituted cADPR analogues of this type
reported to date. Here, we describe the synthesis of 7-deaza-
cADPcR (7) and 7-Br-7-deaza-cADPcR (8) and their Ca2+-
mobilizing activity. We also developed the first general method
for synthesizing N1-substituted 7-deazaadenosines.
RESULTS AND DISCUSSION
■
Synthetic Plan. As described above, many cADPR
analogues have been synthesized by enzymatic or chemo-
enzymatic methods with ADP-ribosyl cyclase from Aplysia
californica, as described above.5 The analogues obtained by
these methods, however, are limited due to the substrate
specificity of the enzyme.10 Therefore, we have developed an
efficient chemical method for synthesizing cADPR analogues, in
which the key reaction was intramolecular condensation to
form the 18-membered pyrophosphate ring by the Ag+-
promoted activation of phenylthiophosphate-type substrates.9b
This is now a general method for synthesizing these types of
biologically important cyclic nucleotides particularly those
chemically modified in the N-1-linked ribose moiety, which
are not expected to be accessible by an enzymatic route.
The retrosynthetic analysis of the target 7-deaza-cADPcR (7)
and 7-deaza-7-Br-cADPcR (8) is shown in Figure 3. To
Figure 2. cADPR (1) and its analogues 2−8.
Potter and co-workers performed the synthetic studies of 7-
deaza-cADPR (5) and its analogues by enzymatic methods and
identified 7-deaza-cADPR as a partial agonist.7 They also
synthesized its 8-bromo derivative, i.e., 7-deaza-8-Br-cADPR
(6), and demonstrated that it is an antagonist of cADPR with
membrane permeable properties.8 Thus, the 7-deaza mod-
ification appears to enhance the antagonistic function of
cADPR because a partial agonist can be an intermediate form
between an agonist and antagonist.
However, cADPR is readily hydrolyzed at the unstable N1-
ribosyl linkage to produce inactive ADP-ribose (ADPR), even
in neutral aqueous solutions.11 This is due to the fact that
cADPR is in a zwitterionic form positively charged around the
N1−C6−N6 moiety (pKa = 8.3), making the molecule unstable,
where the charged adenine moiety attached to the anomeric
carbon of the N1-linked ribose acts as an efficient leaving
group. Under physiological conditions, cADPR is also hydro-
lyzed at the same N1-ribosyl linkage by cADPR hydrolase to
give the inactive ADP-ribose.11
We previously designed and synthesized cyclic ADP-
carbocyclic-ribose (cADPcR, 3) as a stable mimic of cADPR,
in which the oxygen in the N1-ribose ring of cADPR was
replaced by a methylene group. cADPcR is both chemically and
biologically stable, and effectively mobilizes intracellular Ca2+ in
sea urchin eggs and neuronal cells.9c,f,g On the basis of these
findings, we designed and synthesized the 8-modified analogues
of cADPcR, e.g., 8-NH2-cADPcR (4), expecting that they might
be chemically and biologically stable potent cADPR antago-
nists. To our surprise, however, these analogues acted as
agonists rather than antagonists.9e
The interesting finding mentioned above on the biological
activity of the 7-deaza-cADPR derivatives by Potter and co-
workers that the 7-deaza modification enhances the antago-
nistic function of cADPR led us to synthesize the carbocyclic
analogue of 7-deaza-cADPR, i.e., 7-deaza-cADPcR (7) and its
7-bromo derivative 7-deaza-7-Br-cADPcR (8) as stable
analogues of cADPR of biological interest. We focused our
efforts on the biological activity of 7-deaza-7-Br-cADPcR
Figure 3. Retrosynthetic analysis of 7-deaza-cADPcR (7) and 7-deaza-
7-Br-cADPcR (8).
synthesize the target compounds, although construction of the
18-membered pyrophosphate structure is an important step,
the structure was likely to be constructed using the above-
mentioned Ag+-promoted intramolecular condensation reaction
of an S-phenyl phosphorothioate-type substrates 9.5b,c Sub-
sequent deprotection of the cyclization product would furnish
the desired 7 or 8. The S-phenyl phosphorothioate-type
substrates 9 would be obtained from the N1-β-carbocyclic
ribosyl-7-deazaadenosines 10, which we planned to construct
B
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by condensation between a known β-carbocyclicribosylamine
1212 and the substituted pyrrole nucleosides 11. Both of the
pyrrole nucleosides 11a and 11b were unknown, but 11a was
expected to be prepared from a protected ribose 13 and a
pyrrole derivative 14 via β-selective glycosidation. We planned
to examine the bromination of the pyrrole nucleoside 11a at 4-
position.
Scheme 2. Synthesis of the 2,3-Disubstituted Pyrrole
Nucleosides
Development of a General Procedure for Synthesiz-
ing N1-Substituted 7-Deazaadenosines. Construction of
the N1-carbcyclic ribosyl-7-deazaadenosine structure was a
challenge in the present study, because there are no reported
procedures providing this type of nucleoside structure.
Synthesis of 7-deazaadenosine and its derivatives has been
studied extensively due to their biological importance, and the
Lewis acid-promoted glycosidation between a silylated 6-
chloro-7-deazaadenine base and a tetra-O-acyl ribose, devel-
oped by Seela and co-workers, is thought to be the best
procedure in terms of synthetic usefulness, i.e., excellent yield
and product scope.13 This method, however, was not applicable
to our synthesis of 7-deaza-cADPcR because substitution at the
N1-position of 7-deazaadenosine derivatives is difficult. The
only example of such an N1-substitution of 7-deazaadenosine
derivatives reported to date is the N1-methylation of 7-
deazaadenosine by treating 7-deazaadenosine with a large
excess of MeI.14
We planned to construct the key N1-carbocyclic ribosyl-7-
deazaadenosine structure by condensation between the 2,3-
disubstituted pyrrole nucleoside 11 and the carbocyclic
ribosylamine 12 (Figure 3). Because condensation between
12 and an imidazole nucleoside 15 effectively produced the N1-
carbocyclic-ribosyladenosine derivative 16 (Scheme 1),9c we
Scheme 1. Construction of the N1-Carbocyclic Adenosine
Structure with the Imidazole Nucleoside 15 and Carbocyclic
Amine 12
pyrrole nucleoside 18, it was an inseparable anomeric mixture
(α/β = 1:5). We examined the reaction under other conditions
by changing the solvent and temperature, but the desired
pyrrole β-riboside was not obtained in a pure form. When a
similar glycosidation was performed with an isopropylidene-
protected chlororiboside 13−4,17 however, the desired pyrrole
β-riboside 11a was obtained in pure form. Although the yield
was not high, 11a had the 2,3-O-isopropylidene and 5-O-silyl
protecting groups, which were very suitable for further
transformation, and therefore, we proceeded to the next step,
i.e., condensation of 11a with the carbocyclic ribosylamine 12.
We next investigated the bromination of the pyrrole β-
riboside 11a. In the reaction of pyrroles with electrophiles, an
electrophile usually reacts selectively at the undesired 2-position
(the position adjacent to the nitrogen), Dvarnikova and Trela,
however, found that the 2/3-regioselectivity in pyrrole
bromination could be controlled by changing the reaction
conditions,18 and we therefore applied these conditions to the
pyrrole nucleoside in the present study (Table 1). Thus,
consistent with their report, treatment of the pyrrole nucleoside
11a with only NBS in THF gave 5-bromination product 19
exclusively, while supplementary use of PBr3 as an additive
successfully reversed the regioselectivity to selectively produce
the desired 4-bromination product 11b.
expected that similar condensation with the corresponding
pyrrole nucleoside 11 instead of the imidazole nucleoside 15
would produce the desired N1-carbocyclic ribosyl-7-deazaade-
nosine derivatives. Thus, the pyrrole nucleoside 11 was needed,
but this type of 2-nitrogen substituted 3-cyanopyrrole nucleo-
side was unknown. Although ribosylation of 2-amino-3-
cyanopyrole (14), prepared by Townsend’s procedure,15 was
examined under the Vorbruggen glycosidation conditions with
̈
tetra-O-acyl ribose (13−1), the corresponding pyrrole nucleo-
side was not obtained. We then investigated glycosidation with
the corresponding pyrrole anion (Scheme 2). After conversion
of 14 into the corresponding methoxyimidate 17, it was treated
with NaH in MeCN in the presence of an anomeric mixture of
tri-O-benzoyl-1-chlororibose (13−2),16 which was prepared in
situ from 13−1 (Scheme 2a). Although the reaction gave
We examined the condensation of 11a with various amines in
our attempt to develop a general method for synthesizing 7-
deazaadenosine and its N1-substituted derivatives (Table 2).
Treatment of 11a with NH4OH in the presence of K2CO3 in
MeOH successfully afforded the desired protected 7-
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carbocyclic ribosyl-7-deazaadenosine derivatives 10a and 10b
into S-phenyl phosphorothioate-type substrates 9a and 9b
(Scheme 3). The 5″-hydroxy group of 10a was protected with a
dimethoxytrityl group, and then the 5′-O-TBS group of the
product was removed with TBAF to give 20a. Treatment of
20a with an S,S′-diphenylphosphorodithioate/2,4,6-triisopro-
pylbenzenesulfonyl chloride/pyridine system,19 followed by
removal of the 5″-O-dimethoxytrityl group of the product with
aqueous AcOH, gave the corresponding 5′-bis-S-(phenyl)-
phosphorothioate, which was then treated sequentially with
MeOPOCl2/pyridine20 and with H3PO2/Et3N/triethylammo-
nium acetate.21 This three-step reaction gave S-phenyl
phosphorothioate 9a, which was the substrate for the next
intramolecular condensation reaction. The corresponding 7-
bromo S-phenyl phosphorothioate 9b was similarly obtained.
With the S-phenyl phosphorothioates 9a and 9b in hand, we
next investigated the Ag+-promoted intramolecular cyclization
reaction. Slow addition of a solution of 9a or 9b in pyridine to a
mixture of a large excess of AgNO3 and Et3N in the presence of
MS3A in pyridine at room temperature9b,c led to the
corresponding cyclization products 21a or 21b in 48% or
75% yield, respectively. Finally, removing the isopropylidene
group of 21a and 21b with aqueous HCO2H produced the
target 7-deaza-cADPcR (7) and 7-Br-7-deaza-cADPcR (8),
respectively.
Table 1. Regioselective Bromination of the Pyrrole
Nucleoside 11a
a
entry
additive
temp.
time
yield (11b:19)
1
2
3
4
−78 °C
0 °C
−78 °C
−78 °C
1
3 h
40% (0:1)
89% (0:1)
72% (2,4:1)
85% (3:1)
5 min
3 h
PBr3 (5 mol %)
PBr3 (5 mol %)
1 h
a
The ratio was determined by H NMR.
Table 2. General Method for Preparing N1-Substituted and
Non-substituted 7-Deazaadenosines
Ca2+-Mobilizing Activity in Sea Urchin Egg Homoge-
nate. We examined the Ca2+-mobilizing ability of 7-deaza-
cADPcR (7) and 7-deaza-7-Br-cADPcR (8) as well as cADPcR
(3) by fluorometrically monitoring Ca2+ with Hemicentrotus
pulcherrimus sea urchin egg homogenate (Figure 4).22,23
cADPcR released Ca2+ from the homogenate in a concen-
tration-dependent manner with an EC50 value of 54 nM.
7-Deaza-cADPcR was a full agonist, similar to cADPcR, but
its potency (EC50 = 429 nM) was lower than that of cADPcR.
7-Deaza-7-Br-cADPcR, the first cADPR analogue with a
substituent at the adenine-7-position, was therefore identified
as a weak partial agonist.
entry
substrate
R−NH2
yield
84%
a
1
2
3
4
5
6
7
11a
11a
11a
11a
11a
11a
11b
NH3
MeNH2
EtNH2
quant.
quant.
allyl amine
cyclohexyl amine
12
57%
80%
82% (10a)
79% (10b)
12
a
Aqueous ammonia (28% in H2O) was used.
It is interesting that 7-deaza-cADPR (5) acts as a partial
agonist7 but its carbocyclic congener 7-deaza-cADPcR (7) acts
as a full agonist. Further, we previously demonstrated that
although 8-NH2-cADPR (2) acts as an antagonist, its
carbocyclic congener 8-NH2-cADPcR (4) acts as a full
agonist.9e These findings suggest that replacement of the N1-
ribose with the carbocyclic-ribose in cADPR and its analogues
makes their function more agonistic. Therefore, in cADPR
derivatives, the ring oxygen of the N1-ribose moiety appears to
be essential for showing the antagonistic effect.
In summary, because 7-deazaadenosine is an efficient
bioisostere of adenosine, we designed 7-deaza-cADPcR (7)
and 7-deaza-7-Br-cADPcR (8), which were successfully
synthesized via an Ag+-promoted intramolecular condensation
to construct the 18-membered pyrophosphate ring structure.
We also developed the first general method for preparing N1-
substituted 7-deazaadenosines by the condensation of a 2,3-
disubstituted pyrrole nucleoside with amines, which was
effectively used for the construction of the N1-carbcyclic-
ribosyl 7-deazaadenosine and 7-bromo-7-deazaadenosine struc-
tures, the key structures for the synthesis of the targets
compounds. Biological evaluation of the Ca2+-mobilizing
activity revealed that 7-deaza-cADPcR (7) and 7-deaza-7-Br-
cADPcR (8) act as a full agonist and a partial agonist,
respectively, in the sea urchin egg homogenate system. These
findings provide important structural information on the
deazaadenosine in 84% yield. Similar treatments with a variety
of amines provided the corresponding N1-substituted 7-
deazaadenosines, as summarized in Table 2 (entries 2−5).
Thus, we successfully developed the first general method for
synthesizing N1-substituted 7-deazaadenosine, which is also an
alternative synthetic procedure for 7-deazaadenosine (tuber-
cidin). This method can be applied to the synthesis of a variety
of N1-substituted 7-deazaadenosines of biological interest.
When the carbocyclic ribosylamine 1212 was used as an
amine in the condensation with 11a, the N1-carbocyclicribosyl-
7-deazaadenine ring was effectively constructed to afford the
desired product 10a in 82% yield, and its N1-carbocyclic
structure was confirmed based on its NOE data and HMBC
spectrum (Table 2). Similarly, the reaction of the 4-
bromopyrrole nucleoside 11b and 12 also afforded the
corresponding 7-brominated product 10b in 79% yield.
Synthesis of 7-Deaza-cADPcR (7) and 7-Deaza-7-Br-
cADPcR (8). We next investigated the conversion of N1-β-
D
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Scheme 3. Synthesis of 7-Deaza-cADPcR (7) and 7-Deaza-7-Br-cADPcR (8)
Hz), 6.29 (1 H, d, J = 3.1 Hz) 3.82 (3 H, s); 13C NMR (125 MHz,
CDCl3) δ 158.9, 143.2, 117.4, 116.0, 109.9, 80.3, 54.0; HRMS (ESI-
ion trap, negative) calcd for C7H6N3O 148.05164 [(M − H)−], found
148.05128; mp 75.5−76.0 °C.
3-Cyano-2-(methoxymethyleneamino)-1-(2,3,5-tri-O-benzoyl-β-
D-ribofuranosyl)pyrrole (18). To a solution of 13−1 (504 mg, 1.0
mmol) in CH2Cl2 (16.7 mL), a solution of TiCl4 (109 μL, 1.0 mmol)
in CH2Cl2 (0.9 mL) was added slowly at room temperature, and the
mixture was stirred at the same temperature for 1.5 h. After the
addition of H2O, the resulting mixture was partitioned, and the organic
layer was dried (Na2SO4) and evaporated to give crude 13−2. To a
solution of 17 (74.6 mg, 0.50 mmol) in CH3CN (2 mL) was added
NaH (60% mineral oil, 28 mg, 0.70 mmol) at 0 °C, and the resulting
mixture was stirred at the same temperature for 20 min. To the
resulting mixture, a solution of crude 13−2 in CH3CN (3 mL) was
added at 0 °C, and the mixture was stirred at room temperature for 9 h
and then evaporated. The residue was partitioned between AcOEt and
H2O, and the organic layer was washed with brine, dried (Na2SO4),
and evaporated. The residue was purified by flash column
chromatography (silica gel, hexane/AcOEt = 5/1) to give 18 (α/β
Figure 4. Concentration-dependent Ca2+-mobilizing activity of
cADPcR (3), 7-deaza-cADPcR (7), and 7-deaza-7-Br-cADPcR (8) in
sea urchin egg homogenate. The Ca2+-mobilizing activity of each
compound is expressed as the percent change in the ratio of fura-2
fluorescence (F340/F380) relative to that of 3 μM cADPcR. Data are
the mean SEM of 3 to 6 experiments.
1
= 1:5, 219 mg, 75%, white amorphous solid): H NMR (500 MHz,
CDCl3, α/β mixture) δ 7.99−7.18 (15 H, m), 6.63 (5/6 H, d, J = 3.4
Hz), 6.36 (1/6 H, d, J = 3.4 Hz), 6.26 (1/6 H, d, J = 3.4 Hz), 6.22 (5/
6 H, d, J = 4.5 Hz), 6.21 (5/6 H, d, J = 3.4 Hz), 6.20 (1/6 H, d, J = 5.1
Hz), 5.47 (1/6 H, dd, J = 10.2, 5.1 Hz), 5.26 (5/6 H, dd, J = 9.0, 4.5
Hz), 5.08 (5/6 H, dd, J = 9.0, 5.1 Hz), 5.04 (1/6 H, dd, J = 10.2, 2.8
Hz), 4.71−4.68 (5/6 H, m), 4.63−4.60 (1/6 H, m), 4.46−4.43 (5/6
H, m), 4.41−4.39 (2/6 H, m), 4.06−4.03 (1/6 H, m), 3.49 (15/6 H,
s), 3.14 (3/6 H, s); 13C NMR (125 MHz, CDCl3, α/β mixture) δ
166.0, 166.0, 165.5, 157.7, 157.0, 143.2, 138.7, 137.1, 134.0, 133.8,
133.3, 130.0, 129.9, 129.7, 129.7, 129.6, 129.4, 129.4, 128.7, 128.6,
128.6, 128.5, 128.4, 128.3, 128.2, 125.9, 125.2, 117.4, 117.2, 117.1,
116.9, 116.1, 115.2, 109.0, 106.5, 105.5, 81.7, 81.0, 80.2, 78.2, 73.1,
72.9, 63.0, 62.4, 60.4, 54.0, 53.6, 53.5, 29.7, 21.1, 14.2; HRMS (ESI-ion
trap, positive) calcd for C33H27N3O8Na 616.16904 [(M + Na)+],
found 616.16951
3-Cyano-2-(methoxymethyleneamino)-1-[5-O-(tert-butyldime-
thylsilyl)-2,3-O-(isopropylidene)-β-D-ribofuranosyl]pyrrole (11a). To
a solution of 13−3 (3.04 g, 10.0 mmol) and CCl4 (1.16 mL, 12.0
mmol) in toluene (20 mL) was added HMPT (2.09 mL, 11.5 mmol)
over 20 min at −15 °C, and then, the mixture was stirred at the same
temperature for 1 h. After the addition of brine, the mixture was
partitioned, and the organic layer was dried (Na2SO4) and evaporated
to give crude 13−4. To a solution of 17 (745 mg, 5.0 mmol) in
CH3CN (10 mL) was added NaH (60% mineral oil, 240 mg, 6.0
mmol) at 0 °C, and the resulting mixture was stirred at the same
temperature for 1 h. A solution of the crude 13−4 in CH3CN (30 mL)
was added to the mixture at 0 °C, and the resulting mixture was stirred
agonist−antagonist switching of cADPR-related compounds,
indicating that the ring oxygen of the N1-ribose moiety might
be essential for the antagonistic effect.
EXPERIMENTAL SECTION
■
General Methods and Materials. 1H NMR spectra were
recorded in CDCl3 at ambient temperature unless otherwise noted,
at 400 or 500 MHz, with TMS as an internal standard. 13C NMR
spectra were recorded in CDCl3 at ambient temperature at 100 or 125
MHz. 1H NMR peak assignments were based on H−H COCY
spectrum. Silica gel column chromatography was performed with silica
gel 60 N (spherical, neutral, 63-210 μm). Flash column chromatog-
raphy was performed with silica gel 60 N (spherical, neutral, 40−50
μm). Celite 545 was purchased from a chemical supplier. Analytical
HPLC was performed with YMC J’sphere ODS-M80 (250 × 4.6 mm),
A sol. 5% MeCN in 0.1 M triethylamomonium acetate buffer, and B
sol. 80% MeCN in 0.1 M triethylamomonium acetate buffer; B conc.
0−100% (30 min), 1 mL/min.
3-Cyano-2-(methoxymethyleneamino)pyrrole (17). A solution of
14 (2.30 g, 21.5 mmol) and methyl orthoformate (7.06 mL, 64.5
mmol) in CH3CN (200 mL) was stirred under reflux for 4 h and then
1
evaporated to give 17 (2.98 g, 93%, brown solid): H NMR (400
MHz, DMSO-d6) δ 11.59 (1 H, br), 8.35 (1 H, s), 6.64 (1 H, d, J = 3.1
E
DOI: 10.1021/acs.joc.5b00723
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
at room temperature for 24 h and then evaporated. The residue was
partitioned between AcOEt and H2O, and the organic layer was
washed with brine, dried (Na2SO4), and evaporated. The residue was
purified by flash column chromatography (silica gel, hexane/AcOEt =
General Procedure for the Synthesis of 7-Deazaadenosines
(Table 2). A mixture of 11a or 11b (0.10 mmol), an amine (entries
1−5, 5 equiv; entries 6 and 7, 2.5 equiv), and K2CO3 (0.15 equiv) in
MeOH (0.5 mL, entries 1−6) or MeOH/THF (1:1, 0.5 mL, entry 7)
was stirred at room temperature for 20 h and then evaporated. The
residue was partitioned between CHCl3 and H2O, and the organic
layer was washed with brine, dried (Na2SO4), and evaporated. The
residue was purified by column chromatography (silica gel, hexane/
AcOEt = 4/1 to 1/1).
9-[5-O-(tert-Butyldimethylsilyl)-2,3-O-(isopropylidene)-β-D-ribo-
furanosyl]-7-deazaadenosine (Entry 1). 1H NMR (400 MHz,
CDCl3) δ 8.33 (1 H, s), 7.23 (1 H, d, J = 4.0 Hz), 6.37 (1 H, d, J
= 4.0 Hz), 6.34 (1 H, d, J = 2.7 Hz), 5.12−5.10 (3 H, m), 4.97 (1 H,
dd, J = 6.2, 3.6 Hz), 4.30−4.29 (1 H, m), 3.87 (1 H, dd, J = 10.7, 3.6
Hz), 3.78 (1 H, dd, J = 10.7, 4.1 Hz), 1.63, 1.38 (each 3 H, each s),
0.90 (9 H, s), 0.05, 0.04 (each 3 H, each s); 13C NMR (125 MHz,
CDCl3) δ 156.5, 152.1, 150.5, 122.8, 114.1, 103.7, 98.6, 90.2, 85.9,
84.8, 80.9, 63.3, 27.3, 25.9, 25.5, 18.4, 6.4; HRMS (ESI-ion trap,
positive) calcd for C20H33N4O4Si 421.22656 [(M + H)+], found
421.22690; UV (MeOH) λmax = 273 nm.
9-[5-O-(tert-Butyldimethylsilyl)-2,3-O-(isopropylidene)-β-D-ribo-
furanosyl]-1-methyl-7-deazaadenosine (Entry 2). 1H NMR (500
MHz, CD3OD) δ 7.62 (1 H, s, H-2), 7.00 (1 H, d, J = 3.6 Hz, H-8),
6.44 (1 H, d, J = 3.6 Hz, H-7), 6.19 (1 H, d, J = 3.1 Hz, H-1′), 4.95 (1
H, dd, J = 6.7, 3.1 Hz, H-2′), 4.90 (1 H, dd, J = 6.7 Hz, 2.7 Hz, H-3′),
4.27−4.24 (1 H, m, H-4′), 3.84−3.78 (2 H, m, H-5′a, H-5′b), 3.52 (1
H, s, N−CH3), 1.61, 1.37 (each 3 H, each s, isopropyl CH3), 0.91 (9
H, s, tert-butyl), 0.07, 0.06 (each 3 H, each s, dimethyl); 13C NMR
(125 MHz, CDCl3) δ 156.4, 145.8, 142.5, 119.9, 114.1, 107.0, 102.0,
89.8, 85.7, 85.1, 80.8, 63.3, 35.4, 27.3, 25.9, 25.5, 18.4; HRMS (ESI-ion
trap, positive) calcd for C21H35N4O4Si 435.24221 [(M + H)+], found
435.24207; UV (MeOH) λmax = 273 nm.
9-[5-O-(tert-Butyldimethylsilyl)-2,3-O-(isopropylidene)-β-D-ribo-
furanosyl]-1-ethyl-7-deazaadenosine (Entry 3). 1H NMR (500 MHz,
CDCl3) δ 7.62 (1 H, s), 6.99 (1 H, d, J = 3.4 Hz), 6.43 (1 H, d, J = 3.4
Hz), 6.18 (1 H, d, J = 2.9 Hz), 4.97 (1 H, dd, J = 6.2, 2.8 Hz), 4.91 (1
H, dd, J = 6.2, 3.4 Hz), 4.26−4.24 (1 H, m), 4.06 (2 H, q, J = 6.8 Hz),
3.84−3.76 (2 H, m), 1.62, (3 H, s), 1.38 (3 H, t, J = 6.8 Hz), 1.37 (3
H, s), 0.91 (9 H, s), 0.07, 0.06 (each 3 H, each s); 13C NMR (125
MHz, CDCl3) δ 155.1, 145.4, 142.3, 119.8, 114.1, 107.3, 102.1, 89.9,
85.7, 85.0, 63.3, 42.5, 27.3, 25.9, 25.5, 18.4, 14.5; HRMS (ESI-ion trap,
positive) calcd for C22H37N4O4Si 449.25786 [(M + H)+], found
449.25772; UV (MeOH) λmax = 273 nm.
1
6/1) to give 11a (849 mg, 39%, yellow oil): H NMR (400 MHz,
CDCl3) δ 8.43 (1 H, s), 6.78 (1 H, d, J = 3.2 Hz), 6.28 (1 H, d, J = 3.2
Hz), 6.03 (1 H, d, J = 3.6 Hz), 4.81 (1 H, dd, J = 5.8, 3.2 Hz), 4.68 (1
H, dd, J = 5.8, 3.6 Hz), 4.27−4.25 (1 H, m), 3.92 (1 H, s), 3.87−3.77
(2 H, m), 1.58, 1.35 (each 3 H, each s), 0.91 (9 H, s), 0.09, 0.08 (each
3 H, each s); 13C NMR (125 MHz, CDCl3) δ 158.7, 142.5, 117.6,
115.4, 114.0, 110.7, 89.9, 85.6, 85.4, 80.6, 79.0, 63.4, 54.3, 27.5, 26.0,
25.6, 18.5; HRMS (ESI-ion trap, positive) calcd for C21H33N3O5NaSi
458.20817 [(M + Na)+], found 458.20849.
General Procedure of the Bromination of Pyrrole Nucleo-
side 11a. To a solution of 11a (1 equiv) in THF (0.1 M) was added
NBS (1 equiv) at −78 or 0 °C, then the mixture was stirred at the
same temperature for 3 h or 5 min. After the addition of aqueous
saturated NaHCO3, the resulting mixture was partitioned between
AcOEt and H2O, and the organic layer was washed with brine, dried
(Na2SO4), and evaporated. The residue was purified by column
chromatography (silica gel).
General Procedure of the Bromination of Pyrrole Nucleo-
side 11a Using PBr3. To a solution of 11a (54 mg, 0.12 mol) in THF
(1.5 mL) was added NBS (1 equiv) and BBr3 (5 mo%) at −78 °C,
then the mixture was stirred at the same temperature for 3 or 1 h. After
the addition of Et3N, the resulting mixture was partitioned between
AcOEt and H2O, and the organic layer was washed with aqueous
saturated NaHCO3 and brine, dried (Na2SO4), and evaporated. The
residue was purified by column chromatography (silica gel).
4-Bromo-3-cyano-2-(methoxymethyleneamino)-1-[5-O-(tert-bu-
tyldimethylsilyl)-2,3-O-isopropylidene-β-D-ribofuranosyl]pyrrole
(11b, Table 1/Entry 4). To a solution of 11a (54 mg, 0.12 mmol) in
THF (1.5 mL) was added NBS (22 mg, 0.12 mmol) and PBr3 (10 μL,
0.06 mmol) at −78 °C, then the mixture was stirred at the same
temperature for 1 h. After the addition of Et3N, the resulting mixture
was partitioned between AcOEt and H2O, and the organic layer was
washed with aqueous saturated NaHCO3 and brine, dried (Na2SO4),
and evaporated. The residue was purified by column chromatography
(silica gel, hexane/AcOEt = 6/1) to give 11b (34 mg, 63%, yellow oil)
along with 19 (12 mg, 21%, yellow oil): 1H NMR (400 MHz, CDCl3)
δ 8.43 (1 H, s, NCH), 6.94 (1 H, s, H-5), 6.02 (1 H, d, J = 3.1 Hz,
H-1′), 4.77 (1 H, dd, J = 5.8, 2.2 Hz, H-3′), 4.63 (1 H, dd, J = 5.8, 3.1
Hz, H-2′), 4.32 (1 H, d, J = 2.2 Hz, H-4′), 3.92 (3 H, s, OCH3), 3.81
(1 H, dd, J = 11.3, 2.7 Hz, H-5′a), 3.78 (1 H, dd, J = 11.3, 2.7 Hz, H-
5′b), 1.57, 1.34 (each 3 H, each s, isopropyl CH3), 0.92 (9 H, s, tert-
butyl), 0.12, 0.12 (each 3 H, each s, dimethyl); 13C NMR (125 MHz,
CDCl3) δ 159.1, 142.3, 115.5, 115.2, 113.7, 97.9, 90.4, 85.9, 85.7, 82.6,
80.6, 63.4, 54.4, 27.4, 25.9, 25.9, 25.8, 25.4, 18.4, 6.4; HRMS (ESI-ion
trap, positive) calcd for C21H32N3O5BrNaSi 536.11868 [(M + Na)+],
found 536.11902.
9-[5-O-(tert-Butyldimethylsilyl)-2,3-O-(isopropylidene)-β-D-ribo-
1
furanosyl]-1-allyl-7-deazaadenosine (Entry 4). H NMR (400 MHz,
CDCl3) δ 7.56 (1 H, s), 6.98 (1 H, d, J = 3.6 Hz), 6.53 (1 H, d, J = 3.6
Hz), 6.14 (1 H, d, J = 3.1 Hz), 6.00−5.91 (1 H, m), 5.20−5.14 (2 H,
m), 4.90 (1 H, dd, J = 6.2, 2.6 Hz), 4.83 (1 H, dd, J = 6.2), 4.66−4.64
(2 H, m), 4.22−4.19 (1 H, m), 3.79−3.69 (2 H, m), 1.55, 1.30 (each 3
H, each s), 0.83 (9 H, s), −0.01, −0.07 (each 3 H, each s); 13C NMR
(125 MHz, CDCl3) δ 154.9, 145.3, 142.5, 132.2, 120.5, 117.7, 114.1,
106.7, 102.7, 89.9, 85.8, 85.1, 80.8, 63.3, 49.4, 27.3, 25.9, 25.5, 18.4;
HRMS (ESI-ion trap, positive) calcd for C23H37N4O4Si 461.25786
[(M + H)+], found 461.25797; UV (MeOH) λmax = 273 nm.
5-Bromo-3-cyano-2-(methoxymethyleneamino)-1-[5-O-(tert-bu-
tyldimethylsilyl)-2,3-O-isopropylidene-β-D-ribofuranosyl]pyrrole (19,
Table 1/Entry 2). To a solution of 11a (54 mg, 0.12 mol) in THF (1.5
mL) was added NBS (22 mg, 0.12 mmol) at −0 °C, then the mixture
was stirred at the same temperature for 5 min. After the addition of
Et3N, the resulting mixture was partitioned between AcOEt and H2O,
and the organic layer was washed with aqueous saturated NaHCO3
and brine, dried (Na2SO4), and evaporated. The residue was purified
by column chromatography (silica gel, hexane/AcOEt = 6/1) to give
9-[5-O-(tert-Butyldimethylsilyl)-2,3-O-(isopropylidene)-β-D-ribo-
1
furanosyl]-1-cyclohexyl-7-deazaadenosine (Entry 5). H NMR (400
MHz, CDCl3) δ 7.74 (1 H, s), 6.99 (1 H, d, J = 3.6 Hz), 6.43 (1 H, d, J
= 3.6 Hz), 6.18 (1 H, d, J = 3.2 Hz), 5.00−4.98 (2 H, m), 4.91 (1 H,
dd, J = 6.4, 2.8 Hz), 4.28−4.26 (1 H, m), 3.85−3.76 (2 H, m), 2.07−
2.05 (2 H, m), 1.90−1.89 (2 H, m), 1.79−1.76 (1 H, m), 1.62 (3 H, s),
1.57−1.49 (4 H, m), 1.38 (3 H, s), 1.25−1.22 (1 H, m), 0.91 (9 H, s),
0.07, 0.06 (each 3 H, each s); 13C NMR (125 MHz, CDCl3) δ 155.6,
142.9, 141.7, 119.7, 114.0, 106.9, 102.2, 89.9, 85.7, 85.0, 80.8, 63.3,
52.6, 33.0, 33.0, 27.3, 26.0, 25.9, 25.5, 25.5, 18.4, 6.4, −5.4, −5.5;
HRMS (ESI-ion trap, positive) calcd for C26H43N4O4Si 503.30481
[(M + H)+], found 503.30478; UV (MeOH) λmax = 273 nm.
1
19 (57 mg, 89%, yellow oil): H NMR (400 MHz, CDCl3) δ 8.32 (1
H, s, NCH), 6.35 (1 H, s, H-4), 6.07 (1 H, d, J = 3.6 Hz, H-1′), 5.29
(1 H, dd, J = 6.7, 3.6 Hz, H-2′), 4.79 (1 H, dd, J = 6.7, 4.9 Hz, H-3′),
3.99−3.95 (2 H, m, H-4′, H-5′a), 3.81 (1 H, dd, J = 5.8, 2.7 Hz, H-
5′b), 1.58, 1.36 (each 3 H, each s, isopropyl CH3), 0.89 (9 H, s, tert-
butyl), 0.07, 0.06 (each 3 H, each s, dimethyl); 13C NMR (125 MHz,
CDCl3) δ 159.5, 144.6, 116.0, 115.2, 113.1, 99.3, 89.7, 85.1, 82.8, 80.4,
63.1, 54.8, 29.5, 27.4, 25.9, 25.5, 18.5, 6.4; HRMS (ESI-ion trap,
positive) calcd for C21H32N3O5BrNaSi 536.11868 [(M + Na)+], found
536.11874.
Compound 10a (Entry 6). 1H NMR (500 MHz, CDCl3) δ 7.61 (1
H, s, H-2), 7.04 (1 H, d, J = 3.6 Hz, H-8), 6.45 (1 H, d, J = 3.6 Hz, H-
7), 6.16 (1 H, d, J = 3.1 Hz, H-1′), 5.34 (1 H, dd, J = 10.4, 5.4 Hz, H-
2″), 4.93 (1 H, dd, J = 6.2, 2.7 Hz, H-3′), 4.89 (1 H, dd, J = 6.2, 3.6
F
DOI: 10.1021/acs.joc.5b00723
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The Journal of Organic Chemistry
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Hz, H-2′), 4.77 (1 H, dd, J = 10.4, 5.8 Hz, H- 3″), 4.50 (1 H, m, H-
1″), 4.27 (1 H, dd, J = 6.6, 3.6 Hz), 3.85−3.71 (4 H, m, H-5′a, H-5′b,
H-5″a, H-5″b), 2.58−2.49 (3 H, m, H-4″, H-6″a, H-6″b), 1.61, 1.57,
1.37, 1.32 (each 3 H, each s, isopropyl CH3), 0.90 (9 H, s, tert-butyl),
0.07, −0.06 (each 3 H, each s, dimethyl); 13C NMR (125 MHz,
CDCl3) δ 154.5, 145.9, 141.9, 120.7, 114.1, 111.5, 107.1, 101.9, 90.0,
85.7, 85.1, 83.8, 82.4, 80.8, 70.8, 64.7, 63.3, 44.7, 30.4, 28.1, 27.3, 25.9,
25.4, 25.3, 18.3; HRMS (ESI-ion trap, positive) calcd for
C29H47N4O7Si 591.3208 [(M + H)+], found 591.3215; UV (MeOH)
λmax = 273 nm.
added to 20a (274 mg, 0.352 mmol) at 0 °C, and the mixture was
stirred at the same temperature for 30 min and then at room
temperature for 10.5 h. After the addition of MeOH, the resulting
mixture was evaporated and partitioned between AcOEt and H2O, and
the organic layer was washed with brine, dried (Na2SO4), and
evaporated. The residue was purified by column chromatography
(silica gel, CHCl3/MeOH = 1/0 to 9/1) to give the phosphorothioate
product (colorless amorphous solid). A solution of the product in
aqueous 60% AcOH (2.8 mL) was stirred at room temperature for 20
min, evaporated, and azotropically dried with MeOH. The residue was
purified by column chromatography (silica gel, CHCl3/MeOH = 1/0
to 4/1) to give the 5″-O-DMTr-removed product (white amorphous
solid). A solution of MeOPOCl2 (63 μL, 0.63 mmol) in pyridine (0.4
mL) was stirred at −30 °C for 20 min. To the solution was added a
solution of the 5″-O-DMTr-removed product in pyridine (2.2 mL),
and the mixture was stirred at the same temperature for 3 h. To the
resulting solution was added triethylammonium acetate (TEAA) buffer
(2.0 M, pH 7.0, 2 mL) then H3PO2 (213 μL, 4.2 mmol) and Et3N
(293 μL, 2.1 mmol), and the mixture was stirred at room temperature
for 3.5 h and then evaporated. The residue was purified by column
chromatography (ODS, CH3CN/H2O = 1/1). The product was
lyophilized to give 9a (108 mg, 37% for 3 steps, white powder) as a
Compound 10b (Entry 7). 1H NMR (500 MHz, CDCl3) δ 7.56 (1
H, s, H-2), 7.10 (1 H, s, H-8), 6.16 (1 H, d, J = 2.8 Hz, H-1′), 5.34 (1
H, dd, J = 10.8, 5.6 Hz, H-2″), 4.85 (1 H, dd, J = 5.6, 2.8 Hz, H-2′),
4.80 (1 H, dd, J = 5.6, 3.4 Hz, H-3′), 4.77 (1 H, dd, J = 5.7, 2.8 Hz, H-
3″), 4.47−4.43 (1 H, m, H-1″), 4.30 (1 H, dd, J = 5.6, 2.8 Hz, H-4′),
3.88−3.71 (4 H, m, H-5′a, H-5′b, H-5″a, H-5″b), 2.63−2.58 (2 H, m,
H-6″a, H-6″b), 2.43−2.41 (1 H, m, H-4″), 1.60, 1.55, 1.35, 1.31 (each
3 H, each s, isopropyl CH3), 0.92 (9 H, s, tert-butyl), 0.10, 0.10 (each 3
H, each s, dimethyl); 13C NMR (125 MHz, CDCl3) δ 153.8, 147.0,
142.0, 119.4, 114.0, 111.5, 103.6, 91.4, 90.0, 85.9, 85.6, 83.6, 82.4, 80.7,
70.4, 64.8, 63.4, 45.0, 30.4, 28.0, 27.3, 25.9, 25.8, 25.4, 25.3, 18.4;
HRMS (ESI-ion trap, positive) calcd for C29H46N4O7BrSi 669.23137
[(M + H)+], found 669.23124; UV (MeOH) λmax = 277 nm.
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-[(5-dimethoxy-
trityl)oxymethyl]cyclopentyl]-5′-O-(tert-butyldimethylsilyl)-2′,3′-O-
isopropylidene-7-deazadenosine (20a). A solution of 10a (336 mg,
0.570 mmol) and DMTrCl (290 mg, 0.855 mmol) in pyridine (3.8
mL) was stirred at room temperature for 11 h. After the addition of
MeOH, the resulting mixture was partitioned between AcOEt and
H2O, and the organic layer was washed with brine, dried (Na2SO4),
and evaporated. The residue was purified by column chromatography
(silica gel, hexane/AcOEt = 1/3) to give the 5″-O-DMTr product
(491 mg, 96%, pale yellow amorphous solid). A solution of the
product (486 mg, 0.540 mmol), TBAF (1.0 M in THF, 1.63 mL, 1.63
mmol), and AcOH (30 μL, 0.52 mmol) in THF (5.4 mL) was stirred
at room temperature for 2.5 h and then evaporated. The residue was
purified by column chromatography (silica gel, CHCl3/MeOH = 1/0
1
triethylammonium salt: H NMR (500 MHz, D2O) δ 8.28 (1 H, s),
7.30 (1 H, d, J = 3.4 Hz), 7.10−6.95 (5 H, m), 6.59 (1 H, d, J = 3.4
Hz), 6.20 (1 H, d, J = 2.3 Hz), 5.18 (1 H, dd, J = 6.3, 2.3 Hz), 4.87 (1
H, dd, J = 6.3, 5.7 Hz), 4.71−4.68 (3 H, m), 4.45 (1 H, m), 4.02−3.95
(2 H, m), 3.88−3.86 (2 H, m), 3.03 (6 H, q, J = 7.4 Hz), 2.49−2.38 (2
H, m), 2.29−2.21 (1 H, m), 1.48, 1.46, 1.24, 1.22 (each 3 H, each s),
1.10 (9 H, t, J = 7.4 Hz); 13C NMR (125 MHz, D2O) δ 152.6, 145.8,
142.4, 133.1, 130.0, 129.5, 128.2, 127.3, 116.0, 115.3, 104.1, 103.3,
91.2, 85.8, 84.4, 84.1, 81.8, 81.1, 66.5, 65.8, 64.8, 47.2, 43.9, 33.1, 26.5,
24.9, 24.5, 8.6; 31P NMR (202 MHz, D2O) δ 17.62, 0.94; HRMS (ESI-
ion trap, negative) calcd for C29H37N4O12P2S 727.16094 [(M − H)−],
found 727.16397; UV (H2O) λmax = 273 nm; HPLC purity; column A,
retention time 13.75 min, 90.2%.
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-(phosphonoxy-
methyl)cyclopentyl]-5′-O-[(phenylthio)phosphoryl]-2′,3′-O-isopro-
pylidene-7-bromo-7-deazaadenosine (9b). Title compound 9b (22
mg, 32% for 3 steps, white powder) was prepared from 20b (64 mg,
0.074 mmol) according to the procedure described for the synthesis of
1
to 9/1) to give 20a (420 mg, quant., white amorphous solid): H
NMR (500 MHz, CDCl3) δ 7.64 (1 H, s), 7.43−6.81 (14 H, m), 6.42
(1 H, d, J = 4.1 Hz), 5.08−5.03 (3 H, m), 4.98−4.96 (1 H, m), 4.52−
4.48 (1 H, m), 4.43−4.41 (1 H, m), 3.96−3.93 (1 H, m), 3.78 (3 H, s),
3.78 (3 H, s), 3.76−3.74 (1 H, m), 3.34−3.32 (1 H, m), 3.15−3.12 (1
H, m), 2.45−2.41 (2 H, m), 2.30−2.27 (1 H, m), 1.61, 1.53, 1.35, 1.26
(each 3 H, each s); 13C NMR (125 MHz, CDCl3) δ 158.3, 154.8,
145.0, 144.0, 139.8, 136.2, 136.1, 130.0, 130.0, 128.1, 127.7, 126.7,
123.1, 114.0, 113.4, 113.0, 109.1, 101.7, 95.5, 85.8, 85.2, 83.7, 83.0,
81.5, 81.2, 64.2, 63.2, 63.0, 55.1, 44.7, 34.0, 27.6, 27.5, 25.2; HRMS
(ESI-ion trap, positive) calcd for C44H51N4O9 779.3650 [(M + H)+],
found 779.3648; UV (MeOH) λmax = 273 nm.
1
9a: H NMR (400 MHz, D2O) δ 8.30 (1 H, s), 7.45 (1 H, s), 7.04−
6.88 (5 H, m), 6.14 (1 H, d, J = 2.7 Hz), 5.08 (1 H, dd, J = 6.3, 2.7
Hz), 4.70 (1 H, dd, J = 13.5, 6.3 Hz), 4.69−4.50 (3 H, m), 4.45−4.43
(1 H, m), 4.02 (1 H, dd, J = 10.2, 5.4 Hz), 3.93−3.82 (3 H, m), 3.00
(6 H, q, J = 7.2 Hz), 2.46−2.39 (2 H, m), 2.33−2.26 (1 H, m), 1.45,
1.44, 1.21, 1.21 (each 3 H, each s), 1.07 (9 H, t, J = 7.2 Hz); 13C NMR
(125 MHz, D2O) δ 135.7, 128.6, 126.6, 115.9, 113.5, 113.4, 112.7,
111.2, 110.5, 99.5, 98.6, 85.1, 74.8, 74.3, 69.5, 67.9, 67.2, 65.1, 64.4,
49.6, 48.9, 48.5, 30.5, 27.1, 16.3, 9.8, 8.1, 7.8; 31P NMR (202 MHz,
D2O) δ 17.44, 1.21; HRMS (ESI-ion trap, negative) calcd for
C29H36N4O12BrP2S 805.07145 [(M − H)−], found 805.07145; UV
(H2O) λmax = 277 nm.
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-[(5-dimethoxy-
trityl)oxymethyl]cyclopentyl]-5′-O-(tert-butyldimethylsilyl)-2′,3′-O-
isopropylidene-7-bromo-7-deazadenosine (20b). Title compound
20b (209 mg, quant., white amorphous solid) was prepared from 10b
(163 mg, 0.244 mmol) according to the procedure described for the
7-Deaza-cyclic ADP-Carbocyclic-ribose Diacetonaide (21a). To a
mixture of AgNO3 (96 mg, 57 μmol), Et3N (79 μL, 57 μmol), and MS
3A (powder, 1.0 g) in pyridine (20 mL), a solution of 9a (22 mg, 27
μmol) in pyridine (18 mL) was added slowly over 15 h, using a
syringe-pump, at room temperature under stirring in the dark. To the
mixture was added TEAA buffer (2.0 M, pH 7.0, 2 mL), and the
resulting mixture was filtered with Celite, and the filtrate was
evaporated. The residue was partitioned between AcOEt and H2O,
and the aqueous layer was evaporated. The residue was purified by
column chromatography (ODS, 0−40% CH3CN/0.1 M TEAA buffer
(0.1 M, pH 7.0, 400 mL), linear gradient). The excess TEAA included
in the residue was removed by column chromatography (ODS,
CH3CN/H2O = 1/1). The product was lyophilized to give 21a (9 mg,
48%, white powder) as a triethylammonium salt: 1H NMR (500 MHz,
D2O) δ 8.49 (1 H, s), 7.30 (1 H, d, J = 4.0 Hz), 6.71 (1 H, d, J = 4.0
Hz), 6.05 (1 H, d, J = 1.7 Hz), 5.61 (1 H, dd, J = 6.2, 1.7 Hz), 5.32 (1
H, dd, J = 6.2, 3.4 Hz), 4.68−4.65 (3 H, m), 4.37 (1 H, m), 4.05−3.96
(2 H, m), 3.86−3.82 (2 H, m), 3.03 (6 H, q, J = 7.4 Hz), 2.95−2.94 (1
1
preparation of 20a: H NMR (500 MHz, CDCl3) δ 7.61 (1 H, s),
7.61−7.19 (9 H, m), 6.83−6.81 (5 H, m), 5.59 (1 H, d, J = 4.8 Hz),
5.04−5.00 (4 H, m), 4.53−4.50 (1 H, m), 4.42−4.40 (1 H, m), 3.90 (1
H, dd, J = 12.6, 1.8 Hz), 3.78 (3 H, s), 3.78 (3 H, s), 3.76 (1 H, d, J =
12.6, 2.2 Hz), 3.32 (1 H, dd, J = 9.4, 4.5 Hz), 3.3 (1 H, dd, J = 9.4, 5.8
Hz), 2.43−2.38 (3 H, m), 1.60, 1.52, 1.35, 1.27 (each 3 H, each s); 13C
NMR (125 MHz, CDCl3) δ 158.3, 153.7, 145.6, 145.0, 139.3, 136.2,
136.2, 130.3, 128.1, 127.7, 126.6, 121.9, 114.1, 113.2, 113.0, 105.7,
95.5, 90.9, 85.8, 85.3, 83.6, 82.8, 81.6, 81.1, 64.1, 63.1, 55.2, 44.9, 33.7,
27.7, 27.5, 25.3, 25.2; HRMS (ESI-ion trap, positive) calcd for
C44H50N4O9Br 857.2755 [(M + H)+], found 857.27577; UV (MeOH)
λmax = 277 nm.
N-1-[(1R,2S,3R,4R)-2,3-(Isopropylidenedioxy)-4-(phosphonoxy-
methyl)cyclopentyl]-5′-O- [(phenylthio)phosphoryl]-2′,3′-O-isopro-
pylidene-7-deazaadenosine (9a). A solution of PSS (402 mg, 1.05
mmol) and TPSCl (318 mg, 1.05 mmol) in pyridine (2.5 mL) was
G
DOI: 10.1021/acs.joc.5b00723
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The Journal of Organic Chemistry
Article
H, m), 2.75−2.62 (2 H, m), 1.48, 1.47, 1.28, 1.26 (each 3 H, each s),
1.11 (9 H, t, J = 7.4 Hz); 13C NMR (125 MHz, D2O) δ 162.5, 155.9,
151.7, 140.1, 125.1, 123.1, 114.9, 112.1, 104.0, 97.9, 96.5, 95.0, 94.6,
92.1, 79.3, 77.1, 75.2, 57.3, 54.4, 39.0, 36.8, 35.1, 18.9; 31P NMR (202
MHz, D2O) δ −10.37 (d), −11.08 (d); HRMS (ESI-ion trap,
negative) calcd for C23H31N4O12P2 617.14192 [(M − H)−], found
617.14292; UV (H2O) λmax = 273 nm.
AUTHOR INFORMATION
■
Corresponding Author
*Phone/Fax: +81-11-706-3769. E-mail: shu@pharm.hokudai.
ac.jp.
Present Address
∥M.A.: Graduate School of Pharmaceutical Sciences, Osaka
University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
7-Bromo-7-deaza-cyclic ADP-Carbocyclic-ribose Diacetonaide
(21b). Title compound 21b (10 mg, 75%, brown powder) was
prepared from 9b (15 mg, 0.017 mmol) according to the procedure
Notes
1
described for the synthesis of 21a: H NMR (500 MHz, D2O) δ 8.52
The authors declare no competing financial interest.
(1 H, s), 7.43 (1 H, s), 5.98 (1 H, d, J = 1.7 Hz), 5.57 (1 H, dd, J = 5.7,
1.7 Hz), 5.28 (1 H, dd, J = 5.7, 2.8 Hz), 4.67−4.61 (3 H, m), 4.37−
4.34 (1 H, m), 3.96−3.95 (1 H, m), 3.94−3.92 (1 H, m), 3.89−3.85 (1
H, m), 3.81−3.77 (1 H, m), 3.01 (6 H, q, J = 7.4 Hz), 2.95−2.92 (1 H,
m), 2.74−2.72 (1 H, m), 2.61−2.58 (1 H, m) 1.46, 1.45, 1.26, 1.24
(each 3 H), 1.09 (9 H, t, J = 7.4 Hz); 13C NMR (125 MHz, D2O) δ
152.5, 145.4, 142.6, 129.5, 115.1, 113.0, 102.8, 93.8, 89.5, 87.6, 86.7,
85.1, 84.4, 81.9, 69.4, 67.2, 65.0, 47.2, 44.4, 28.9. 26.7, 24.9, 24.7, 8.8;
31P NMR (202 MHz, D2O) δ −10.46 (d), −11.00 (d); HRMS (ESI-
ion trap, negative) calcd for C23H30N4O12BrP2 695.05243 [(M −
H)−], found 695.05343; UV (H2O) λmax = 277 nm; HPLC purity;
column A, retention time 13.63 min, 98.4%.
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7-Deaza-cyclic ADP-Carbocyclic-ribose (7). A solution of 21a (6.6
mg, 9.1 μmol) in aqueous 60% HCO2H (1.0 mL) was stirred at room
temperature for 4 h and then evaporated. After coevaporation with
H2O, the residue was purified by column chromatography (ODS,
H2O). The eluent was evaporated and lyophilized to give 7 (5.5 mg,
94%, white powder) as a triethylammonium salt: 1H NMR (500 MHz,
D2O) δ 8.32 (1 H, s, H-2), 7.27 (1 H, d, J = 3.6 Hz, H-8), 6.66 (1 H, d,
J = 3.6 Hz, H-7), 5.72 (1 H, d, J = 6.7 Hz, H-1′), 5.09 (1 H, dd, J = 6.2,
4.9 Hz, H-2′), 4.46−4.42 (2 H, m, H-3′, H-5′a), 4.22−4.21 (2 H, m,
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H, m, H-5′b), 3.03 (6 H, q, J = 7.4 Hz, (CH3CH2)3N), 2.92−2.82 (1
H, m, H-6″a), 2.41−2.37 (1 H, m, H-4″), 2.26−2.22 (1 H, m, 6″b),
1.11 (9 H, t, J = 7.4 Hz, (CH3CH2)3N); 13C NMR (125 MHz, CDCl3)
δ 152.6, 145.8, 142.3, 130.2, 105.1, 101.8, 100.6, 93.0, 85.1, 79.5, 74.5,
73.7, 71.4, 65.5, 63.6, 47.4, 28.8, 8.8; 31P NMR (202 MHz, D2O) δ
−9.29 (d), −10.33 (d); HRMS (ESI-ion trap, negative) calcd for
C17H23N4O12P2 537.07932 [(M − H)−], found 537.08012; UV (H2O)
λmax = 275 nm; HPLC purity 97.2% (retention time 3.2 min).
7-Bromo-7-deaza-cyclic ADP-Carbocyclic-ribose (8). Title com-
pound 8 (37.0 OD277 units, white powder) was prepared from 21b
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(59.7 OD277 units) according to the procedure described for 7: H
NMR (500 MHz, D2O) δ 8.84 (1 H, s, H-2), 7.42 (1 H, s, H-8), 5.66
(1 H, d, J = 6.3 Hz, H-1′), 5.02 (1 H, dd, J = 6.3, 2.2 Hz, H-2′), 4.82−
4.78 (1 H, m, H-1″), 4.44 (1 H, dd, J = 5.0, 2.2 Hz, H-3′), 4.39−4.36
(1 H, m, H-5′a), 4.27 (1 H, dd, J = 9.6, 4.5 Hz), 4.21−4.19 (1 H, m,
H-4′), 4.07 (1 H, dd, J = 9.6, 4.0 Hz, H-3″), 4.02−4.01 (2 H, m, H-
5″a, H-5″b), 3.95−3.92 (1 H, m, H-5′b), 3.03 (6 H, q, J = 7.4 Hz,
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(H2O) λmax = 277 nm (ε = 8440, based on the total phosphate
analysis); HPLC purity 99.9% (retention time 5.6 min).
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, 13C NMR, and 31P NMR charts of compounds. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.joc.5b00723.
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DOI: 10.1021/acs.joc.5b00723
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