Synthesis of N7-Alkyl-9-deaza-2′-deoxyguanosines Containing Polar N7 Chains. Examples of Chemically Stable Analogues of N7-Hydroxyethyl and N7-Oxoethyl Adducts of 2′-Deoxyguanosine

Article abstract of DOI:10.1021/acs.joc.6b02110

Development of chemically stable analogues of unstable DNA lesions enables accurate study of polymerase bypass. We report the design and synthesis of N⁷-hydroxyethyl-9-deaza-2′-deoxyguanosine and N⁷-oxoethyl-9-deaza-2′-deoxyguanosine as the analogues of N⁷-hydroxyethyl-2′-deoxyguanosine and N⁷-oxoethyl-2′-deoxyguanosine, respectively. We also developed the synthesis of these two nucleosides whose N⁷ side chains are protected by TBS for the convenience of conversion to phosphoramidites.

Full text of DOI:10.1021/acs.joc.6b02110

Article
pubs.acs.org/joc
Synthesis of N7‑Alkyl-9-deaza-2′-deoxyguanosines Containing
Polar N7 Chains. Examples of Chemically Stable Analogues of
N7‑Hydroxyethyl and N7‑Oxoethyl Adducts of 2′-Deoxyguanosine
Xun Gao† and Haidong Huang*,‡
†Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, New Jersey 07102, United States
‡4Catalyzer, 530 Old Whitfield Street, Guilford, Connecticut 06437, United States
S
* Supporting Information
ABSTRACT: Development of chemically stable analogues of
unstable DNA lesions enables accurate study of polymerase
bypass. We report the design and synthesis of N7-hydroxyethyl-
9-deaza-2′-deoxyguanosine and N7-oxoethyl-9-deaza-2′-deoxy-
guanosine as the analogues of N7-hydroxyethyl-2′-deoxygua-
nosine and N7-oxoethyl-2′-deoxyguanosine, respectively. We
also developed the synthesis of these two nucleosides whose
N7 side chains are protected by TBS for the convenience of conversion to phosphoramidites.
Scheme 1. Compounds of Interest
INTRODUCTION
■
DNA alkylation is one of the most common forms of DNA
damage.1 Many alkylating agents that are found in consumer
products or released from industrial plants may present a threat
to public health. At the molecular level, these agents or their
metabolites are known to react with DNA nucleobases to form
alkyl adducts.2−7 The biological effects of DNA alkyl adducts,
however, are difficult to evaluate at the cellular level due to
the uncontrolled distribution of the adducts within the whole
genome and the involvement of multiple DNA polymerases
and repair enzymes.6,8,9 Furthermore, N7-alkylguanines, the
major reaction products produced by most alkylating agents, are
converted to highly mutagenic abasic sites and weakly mutagenic
N7-alkyl-FAPY-guanines through spontaneous or enzymatic
hydrolysis.10−15 The conversion rate is dependent on the
conditions of the study and the repair activities of the cell line.
Therefore, comprehensive understanding of the biological
picture of N7-alkylguanines at the molecular level, especially
how fast and accurately polymerases bypass the lesions, is
essential for connecting the chemistry of DNA damage to its
biological consequences.
we have used N7-methyl-9-deazaguanine (2a) as the model
of N7-methylguanine (1a) to study its polymerase replication
(Scheme 1).18 We now would like to expand the DNA lesions of
interest to the adducts formed between guanine and epoxides,
particularly those generated in vivo from various olefins.
Specifically we are interested in N7-hydroxyethyl-2′-deoxygua-
nine (1b) and N7-oxoethyl-2′-deoxyguanosine (1c) (Scheme 1).
The former is formed via a reaction with ethylene oxide.19 The
latter can be generated from multiple sources in the chemical
industry, among which the most widely used are vinyl chloride,
vinyl bromide, urethane, and acrylonitrile.20−22 Herein we report
Synthesis of oligonucleotides that contain a single N7-
alkylguanine has become a vital tool to achieve the above goal.
However, the instability of N7-alkyguanines is not compatible
with the phosphoramidite chemistry used by solid-phase
oligonucleotide synthesis. Two studies have been reported in
which a short oligonucleotide containing a single guanine
reacted with alkylating agents to form the desired N7-adducts.16,17
However, depurination may occur if the N7-alkylguanine-
containing oligonucleotide needs to be ligated to a long template
and annealed to a primer for a standard polymerase kinetic assay
where the incorporation accuracy and rate can be determined.
Furthermore, the requirement for a single G site limited the
possibility of investigating different local sequences. In the past,
Received: August 26, 2016
Published: November 2, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.6b02110
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a
Scheme 2. (A) Formation of 9-Deazaguanine Nucleosides through Friedel−Crafts Reactions. (B) Alternative Method To
Introduce N7-Alkyl Groups after C-Glycosidic Bond Formation
a
PG in entry 1 is a p-nitrobenzoyl group. PG in entries 2−7 is a p-toluoyl group. Reaction details are described in Results and Discussion.
the synthesis of N7-hydroxyethyl-9-deaza-2′-deoxyguanosine
to introduce the 7-hydroxyethyl group to 9-deaza-dG caused
serious epimerization, while in the other case, the 7-allyl group
was added to 9-deaza-dG nonselectively at the N2 and N7
positions at the same time. As the last resort, we revisited the
possibility of using alkylated 9-deazaguanines that do not contain
TBS or dmf groups for C-glycosidic bond formation. Here, we
are pleased to report the successful Friedel−Crafts reactions
of N7-hydroxyethyl-9-deazaguanine and N7-allyl-9-deazaguanine
(Scheme 2A, entries 6 and 7). To our surprise, the unprotected
hydroxyl group of N7-hydroxyethyl-9-deazaguanine did not
significantly affect the yield of glycosylation. The free hydroxyl
group was then protected as a TBS ether. Compound 3b was
obtained by selectively removing the O3′ and O5′ toluoyl and
the N1-benzyl groups. The product in entry 7 contained an allyl
group, which was later dihydroxylated to generate a vicinal diol
(Scheme 2A).27−29 The diol was protected by a pair of TBS
groups, yielding compound 3d after removal of the toluoyl and
benzyl groups.
(
(
7HE9CdG, 2b) and N7-oxoethyl-9-deaza-2′-deoxyguanosine
7OE9CdG, 2c) as two N7-alkyl-dG analogues that are resistant
to glycosidic bond cleavage (Scheme 1). We also report the side-
chain-protected nucleosides (3b and 3d) that can be readily
converted to phosphoramidites for oligonucleotide synthesis
(Scheme 1). Notably, 7OE9CdG contains an aldehyde group and
is not compatible with solid-phase oligonucleotide synthesis.
Therefore, its vicinal dihydroxyl precursor (3d) was the focus of
the synthesis.
TBS is an evident choice of protecting group for the side
chains of 3b and 3d, as it is commonly used to protect the
2′-OH of ribonucleosides in RNA synthesis. However, our
preliminary studies showed that TBS is not compatible with the
Friedel−Crafts reaction that is responsible for the formation of
C-glycosidic bonds of 9-deaza-dG (Scheme 2A, entries 4 and 5).18,23
We examined several possible solutions to this problem. The
immediate alternative solution was to directly introduce alkyl group
to the N7 position of fully protected 9-deaza-dG (Scheme 2B).
Using this strategy, 2b, 2d, and 2c were prepared successfully.
Unfortunately, we experienced difficulties when preparing 3b
and 3d. It is well documented that the N,N-dimethylformami-
dine (dmf) group is a mild protecting group for the exocyclic
amine of nucleobases.24,25 However, during the course of our
study of 7Me9CdG (2a), we found that when N1 of 9-deazaguanine
was protected with a benzyl (Bn) group, strong basic conditions
and high temperature were required for complete removal of
N2-dmf.18 Under the same conditions, TBS was also cleaved from
the side chains of 3b and 3d. Therefore, we attempted alkylation
using dmf-free 9-deaza-dG.23,26 The reaction conditions used
RESULTS AND DISCUSSION
■
The N2-dmf-protected 9-deazaguanine 4, previously obtained
from 2-amino-4-hydroxy-6-methylpyrimidine in five steps,23
underwent Lewis-acid-mediated Friedel−Crafts reaction with a
1-O-methyl-3,5-O-ditoluated deoxyribose derivative to yield
C-nucleoside 5 (Scheme 3A). This coupling mainly afforded
the β-anomer in 26% yield. This yield is comparable to the
reaction of N2- and N7-naked 9-deazaguanine (Scheme 2A, entry
1)23 but significantly lower than what we observed for N2-dmf-
N7-methyl-9-deazaguanine (Scheme 2A, entry 3).18 The
hydroxylethyl group was introduced to the deprotonated N7
B
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a
Scheme 3
a
(a) 1-(α,β)-O-methyl-3,5-di-(O-p-toluoyl)-2-deoxy-D-ribose, SnCl4, 1:1 (v/v) dry CH3CN−CH2Cl2, 65 °C, 16 h, 26%; (b) ethylene carbonate,
DBU, dry DMF, 90 °C, 4 h, 49%; (c) 1 M NaOH, 2:1 (v/v) CH3OH-H2O, 70 °C, 16 h, 72%; (d) ammonium formate, Pd/C, CH3OH, 75 °C, 16 h,
48%; (e) allyl bromide, NaH, dry THF, rt, 16 h, 48%; (f) osmium tetraoxide, TBHP, TBAF, 4:1 (v/v) acetone−H2O, rt, 76%; (g) 1 M NaOH, 6:1
(v/v) CH3OH−H2O, 70 °C, 16 h. 54%; (h) ammonium formate, Pd/C, CH3OH, 75 °C, 16 h, 74%; (i) KIO4, 1:1 (v/v) CH3OH−H2O, rt, 30 min, 85%.
position using ethylene carbonate as the alkylating agent, and then
the toluoyl and dmf groups were removed via strong alkaline
hydrolysis to afford 7. Pd/C-catalyzed hydrogenolysis by
ammonium formate was employed to deprotect the N1-Bn
group under mild heating, which was previously demonstrated by
us to be an efficient way to remove the N1-Bn group.18 This
reaction lead to the formation of 7HE9CdG (2b). The synthesis of
7OE9CdG followed the same strategy. Allylation was performed on
5 to generate N7-allyl nucleoside 8. Then the olefin underwent
dihydroxylation to form a vicinal diol (9). After deprotection of
dmf, toluoyl, and benzyl groups successively, 7DHP9CdG (2d) was
obtained. The vicinal diol was cleaved by potassium periodate to
generate 7OE9CdG (2c) (Scheme 3B). Nucleoside 7OE9CdG was
not very stable at room temperature. It was generated and purified
immediately before characterization.
Having completed the synthesis of 7HE9CdG and 7OE9CdG, we
sought to add TBS groups to the 7-alkyl chains of 6 and 9 to
generate side-chain-protected nucleosides. However, efforts
to hydrolyze the toluoyl and dmf groups simultaneously while
keeping the OTBS ether intact were futile (Scheme 4). Different
bases (concentrated ammonia, diluted or 1 M KOH/NaOH),
temperatures (0−75 °C), and solvent ratios (water−ethanol−
THF mixture) were tested. We found that in general, mild
conditions only enabled the removal of the toluoyl groups, while
harsher conditions caused deprotection of toluoyl, dmf, and TBS
at the same time. This was consistent with our past observation
that hydrolysis of N2-dmf in the presence of N1-Bn was difficult.18
Anticipating that removal of the N1-Bn group first should
facilitate the removal of the dmf group, we treated 11 with
Pd/C−ammonium formate in refluxing methanol overnight.
Unfortunately, the same reaction that worked efficiently in the
syntheses of 2b and 2d did not show any effect on 11. Change of
the conditions to refluxing DMF resulted in partial removal of
the N2-dmf group (48 h, less than 10% conversion). The N1-Bn
group, however, remained intact. Therefore, it appears that
N1-Bn and N2-dmf of 11 have remarkable steric effect on each
other, which blocks facile deprotection of either group.
The stabilities of the glycosidic bonds of 7HE9CdG and
7DHP9CdG were examined after incubation under the following
three conditions for 8 h: (i) HCl (pH 2.5), rt, (ii) NaOH
(pH 11.7), rt, and (iii) phosphate buffer (pH 7.2), 70 °C.
The NMR results confirmed that the glycosidic bond was stable
under these conditions, and no sign of epimerization was detected.
This result demonstrated the feasibility of using these structures as
stable analogues of N7-alkyl-dG. 7OE9CdG was excluded from the
study due to the chemical instability of the aldehyde group.
To circumvent this problem, we decided to remove the dmf
group prior to alkylation, on condition that the unprotected
N2-amino group would not compete with the N7 alkylation under
C
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a
Scheme 4
a
(a) TBSCl, imidazole, 4-DMAP, DMF, rt, 16 h, 75%; (b) 5:1 (v/v) CH3OH−H2O, 0.05 M NaOH, rt, 1 h, 75%; (c) 1 M NaOH, 2:1 (v/v)
CH3OH-H2O, 70 °C, 16 h, 72%; (d) different bases (ammonia, KOH, or NaOH), temperatures (0−75 °C), reaction times (1−48 h), and solvent
ratios (water−ethanol−THF mixture).
basic conditions. The dmf-free 9-deaza-dG (14) was treated
with DBU, followed by the addition of ethylene carbonate.
Surprisingly, the reaction did not proceed under the previously
adopted hydroxyethylation temperature (90 °C). This indicated
that removal of N2-dmf group rendered the N7-H more difficult
to dissociate. At an elevated temperature (110 °C), the desired
hydroxyethylated product was obtained (Scheme 5), however, as
in the presence of only 1 mol equiv of DBU, while the same
treatment at 90 °C did not result in detectable epimerization.
Furthermore, the less electron-donating effect exerted by N2-dmf
can attenuate epimerization. This electronic effect is consistent
with the literature record of the C-glycoside of 6-amino-
pyridone.34 In short, the availability of N7-H, alkaline pH, high
temperature, and electron-donating groups on the nucleobase
have in combination created a favorable condition for the
epimerization of 9-deaza-dG.
a
Scheme 5
Nonreacting hydroxyl groups are often protected in a Lewis
acid-promoted glycosylation reaction. However, when the
glycosyl donor is an O-glycoside, attack of the alkoxyl group to
the oxocarbenium intermediate is reversible. As such, forma-
tion of a more stable C-glycosidic bond will be favored over
an O-glycosidic bond. On the basis of this analysis, we treated
the unprotected N7-hydroxyethyl-9-deazaguanine (16) with
the sugar donor successfully with no significantly lower yield
(Scheme 6A). The hydroxyl group was then protected as a TBS
ether, followed by removal of the two toluoyl groups at room
temperature (19). The N1-Bn group of 19 was deprotected
smoothly using Pd/C-catalyzed hydrogenolysis to afford 3b.
The synthesis of 3d was carried out using a similar strategy
(Scheme 6B). As we observed that allylation of 13 produced a
mixture of N2- and N7-allyl products, the N2-dmf-protected
9-deazaguaine (4) was used as the starting material instead. After
the allyl group was specifically introduced to N7, the N2-dmf
group was removed under strong alkaline conditions (21). The
N7-allyl group survived the next Friedel−Crafts reaction. Then
the olefin was dihydroxylated to form a vicinal diol 23, followed
by TBS protection of both hydroxyl groups (24). The two
toluoyl groups and the N1-Bn were successively removed under
the same conditions as described for the synthesis of 3b.
a
(a) 1 M NaOH, 1:4 (v/v) H2O−CH3OH, reflux, 20 h, 75%; (b) 1-(α,β)-
O-methyl-3,5-di-(O-p-toluoyl)-2-deoxy-D-ribose, SnCl4, 1:1 (v/v) dry
CH3CN−CH2Cl2, 65 °C, 16 h, 26%; (c) ethylene carbonate, DBU, dry
DMF, 110 °C, 4 h, 46%, α:β = 1:3.
a mixture of inseparable α/β isomers (1:3, 1H NMR integration).
Therefore, an alternative solution to the synthesis of 3b was then
developed.
It is interesting to compare the epimerization of 14 with
other C-nucleosides reported by us and others.18,23,26,31−33
Hamm et al. first reported epimerization of unalkylated
9-deaza-dG resulting from treatment in concentrated ammonia
at 55 °C for 15 h. The same observation was not found at
room temperature.26 In contrast, we previously observed that
N7-methyl-9-deaza-dG (2a) was not prone to epimerization
under strong alkaline conditions at high temperature.18 There-
fore, we concluded that deprotonating N7-H through either
general base catalysis or specific base catalysis is required for the
epimerization of 9-deazaguanine nucleosides. Here we further
provide evidence to show that 9-deaza-dG epimerizes at 110 °C
CONCLUSION
■
In summary, we have successfully synthesized two polar-chain-
containing N7-alkyl-9-deaza-dGs and expanded the series of
chemically stable analogues of N7-alkyl-dGs. The C-glycosidic
bonds of the two new compounds were proved to be stable under
strong acidic and basic conditions and at high temperatures.
In addition, we overcame the difficulties in stereoselectivity and
regioselectivity when the exocyclic amine of 9-deazaguanine was
D
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a
Scheme 6
a
(a) Ethylene carbonate, DBU, dry DMF, 90 °C, 4 h, 47%; (b) 1-(α,β)-O-methyl-3,5-di-(O-p-toluoyl)-2-deoxy-D-ribose, SnCl4, 1:1 (v/v) dry
CH3CN−CH2Cl2, reflux, 16 h, 28%; (c) TBSCl, imidazole, 4-DMAP, DMF, rt, 16 h, 93%; (d) 0.05 M NaOH, 10:1 (v:v) CH3OH−H2O, rt, 1 h,
74%; (e) ammonium formate, Pd/C, CH3OH, 75 °C, 16 h, 43%; (f) allyl bromide, NaH, dry THF, rt, 4 h, 64%; (g) 1 M NaOH, 1:4 (v/v) H2O−
CH3OH, 70 °C, 16 h, 70%; (h) 1-(α,β)-O-methyl-3,5-di-(O-p-toluoyl)-2-deoxy-D-ribose, SnCl4, 1:1 (v/v) dry CH3CN−CH2Cl2, 65 °C, 16 h, 23%;
(i) osmium tetraoxide, TBHP, TBAF, 4:1 (v/v) acetone−H2O, rt, 16 h, 40%; (j) TBSCl, imidazole, 4-DMAP, DMF, rt, 16 h, 76%; (k) 0.1 M NaOH,
10:1 (v:v) CH3OH−H2O, rt, 1 h, 68%; (l) ammonium formate, Pd/C, CH3OH, 75 °C, 16 h, 56%.
Stability Test. 7HE9CdG and 7DHP9CdG were incubated under the
not protected and developed an efficient synthesis of the two
nucleosides whose N7 polar side chains were protected by TBS
following three conditions for 8 h: (i) HCl (pH 2.5), rt; (ii) NaOH (pH
11.7), rt; (iii) phosphate buffer (pH 7.2), 70 °C. After that, the reaction
groups. These side-chain-protected nucleosides can be readily
mixtures were neutralized, concentrated, and passed through a silica
converted to phosphoramidites for solid-phase oligonucleotide
gel column using a relatively polar eluent (methanol/ethyl acetate 1:2,
synthesis, which will play essential roles in elucidating the
compared to 1:5 for 2b and 2d) to elute possible cleaved products. The
polymerase actions on N7-hydroxyethyl and N7-oxoethyl adducts
of 2′-deoxyguanosine.
NMR spectra were compared with the spectra of 2b and 2d to determine
any possible bond cleavage and epimerization.
(2R,3S,5R)-5-(3-Benzyl-2-(((dimethylamino)methylene)amino)-4-
EXPERIMENTAL SECTION
oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)-2-(((4-
methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-Methylbenzoate
(5). To a suspension of N′-(3-benzyl-4-oxo-4,5-dihydro-3H-pyrrolo-
[3,2-d]pyrimidin-2-yl)-N,N-dimethylformimidamide 4 (3.52 g, 12.0 mmol)
and 1-(α,β)-O-methyl-3,5-di(O-p-toluoyl)-2-deoxy-D-ribose (6.6 g,
17.2 mmol) in a mixture of methylene chloride (20.0 mL) and
acetonitrile (20.0 mL) was added a solution of SnCl4 (23.2 mL,
23.2 mmol, 1 M in CH2Cl2). The reaction mixture was heated at 65 °C
for 16 h. The reaction mixture was diluted with methylene chloride and
washed successively with sat. NaHCO3 and brine. The organic layer was
separated and dried over MgSO4. The solution was concentrated and
purified by column chromatography (SiO2, 0.06−0.20 mm, eluting with
hexane/ethyl acetate 2:1 to 1:2) to give compound 5 as a light yellow
solid (1.96 g, 26%). mp 92−93 °C. 1H NMR (500 MHz, CDCl3) δ ppm
■
General Information. H and 13C NMR spectra were recorded
on a NMR spectrometer operating at 500 or 600 MHz for 1H and 125 or
150 MHz for 13C using the solvent as an internal reference. The coupling
constants (J) for 1H NMR are recorded in hertz. High resolution
mass spectra (HRMS) of compounds 9, 11, 12, 18, and 24 were obtained
using a MALDI-TOF spectrometer and all others with an ICR (ESI)
spectrometer. Melting points were recorded on a microscopic instrument.
THF was distilled freshly from LiAlH4. Acetonitrile, dichloromethane,
and DMF were distilled freshly from CaH2. 1-Benzyl-9-deazaguanine (4)
was prepared following Rana’s protocol, and its characterization was
described previously by Gibson et al.18,23 1-(α,β)-O-Methyl-3,5-di(O-p-
toluoyl)-2-deoxy-D-ribose was prepared according to a reported pro-
cedure.30
1
E
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2.36 (s, 3H), 2.42 (s, 3H), 2.49−2.53 (m, 1H), 2.91−2.95 (m, 1H),
3.05 (s, 3H), 3.12 (s, 3H), 4.50 (s, 1H), 4.58 (dd, J = 10.5, 5.0 Hz, 1H),
4.66 (dd, J = 11.0, 4.5 Hz, 1H), 5.49−5.56 (m, 3H), 5.74 (s, 1H), 7.15−
7.36 (m, 10H), 7.90 (d, J = 7.0 Hz, 2H), 8.01 (s, 2H), 8.58 (s, 1H), 10.36
(s, 1H). 13C NMR (125 MHz, CDCl3) 21.6, 21.7, 35.0, 37.9, 40.8,
45.6, 60.4, 64.8, 73.8, 81.8, 114.4, 116.2, 126.8, 127.0, 127.2, 127.23,
128.0, 128.1, 129.1, 129.14, 129.7, 129.8, 138.9, 143.6, 144.0, 153.7,
156.2, 156.8, 166.2, 166.5. ESI-MS (M + H)+ for C37H37N5O6: expected
648.2822, found 648.2852.
(2R,3S,5R)-5-(3-Benzyl-2-(((dimethylamino)methylene)amino)-5-
(2-hydroxyethyl)-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-
yl)-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-Methyl-
benzoate (6). To a solution of 5 (0.52 g, 0.8 mmol) in dry DMF
(5.0 mL) were added DBU (280.0 μL, 1.8 mmol) and ethylene
carbonate (0.21 g, 2.4 mmol). The reaction was heated under 90 °C for
4 h. After removal of DMF, the reaction mixture was diluted with ethyl
acetate and washed successively with water and brine. The solution was
concentrated and purified by column chromatography (SiO2, 0.06−
0.20 mm, eluting with hexane/ethyl acetate 1:4 to1:8) to give compound
6 as a white solid (0.27 g, 49%). mp 75−76 °C. 1H NMR (500 MHz,
CDCl3) δ ppm 2.40 (s, 3H), 2.44 (s, 3H), 2.52−2.57 (m, 1H), 2.85−
2.89 (m, 1H), 3.05 (s, 3H), 3.15 (s, 3H), 3.90 (s, 2H), 4.46 (s, 2H), 4.51
(s, 1H), 4.59 (dd, J = 11.0, 4.0 Hz, 1H), 4.70 (dd, J = 11.3, 4.5 Hz, 1H),
5.53 (s, 3H), 5.76 (s, 1H), 7.09 (s, 1H), 7.19−7.21 (m, 3H), 7.26−7.29
(m, 4H), 7.35 (d, J = 5.0 Hz, 2H), 7.94 (d, J = 7.5 Hz, 2H), 8.01 (d, J =
7.5 Hz, 2H), 8.60 (s, 1H). 13C NMR (125 MHz, CDCl3) δ ppm 21.5,
21.6, 34.9, 37.8, 40.7, 45.2, 50.8, 63.0, 64.5, 73.5, 81.7, 113.4, 115.2,
126.6, 127.0, 127.04, 127.6, 128.0, 129.0, 129.4, 129.6, 130.7, 138.6,
143.6, 143.8, 153.7, 156.3, 156.6, 166.0, 166.3. ESI-MS (M + H)+ for
C39H41N5O7: expected 692.3084, found 692.3122.
2-Amino-3-benzyl-7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-5-(2-hydroxyethyl)-3,5-dihydro-4H-pyrrolo[3,2-
d]pyrimidin-4-one (7). A suspension of 6 (0.15 g, 0.22 mmol) in
1 M sodium hydroxide in a mixture of methanol (2.0 mL) and water
(1.0 mL) was heated under 70 °C for 16 h and allowed to cool to room
temperature. The reaction mixture was concentrated and purified by
column chromatography (SiO2, 0.06−0.20 mm, eluting with hexane/
ethyl acetate 1:5 to 1:10) to give 7 as a white solid (0.063 g, 72%).
mp 99−100 °C. 1H NMR (500 MHz, CD3OD) δ ppm 2.08 (dd, J = 13.0,
5.0 Hz, 1H), 2.46−2.52 (m, 1H), 3.69 (d, J = 12.0 Hz, 1H), 3.80−3.85
(m, 3H), 4.02 (s, 1H), 4.36−4.41 (m, 3H), 4.45 (d, J = 5.0 Hz, 1H),
5.28−5.32 (m, 3H), 7.22 (d, J = 7.5 Hz, 2H). 7.25 (d, J = 7.5 Hz, 2H),
7.31 (t, J = 8.0 Hz, 2H). 13C NMR (125 MHz, CD3OD) δ ppm 42.1,
43.7, 50.5, 61.7, 63.4, 74.4, 74.7, 87.9, 112.4, 112.8, 126.17, 127.2, 128.4,
131.7, 135.7, 142.6, 151.0, 154.7. ESI-MS (M + H)+ for C20H24N4O5:
expected 401.1825, found 401.1839.
2-Amino-7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-5-(2-hydroxyethyl)-3,5-dihydro-4H-pyrrolo[3,2-
d]pyrimidin-4-one (2b). To a mixture of 7 (0.063 g, 0.16 mmol) and
10% palladium on carbon (0.03 g) in methanol (2.0 mL) was added
ammonium formate (0.10 g, 1.5 mmol) under Ar. The reaction mixture
was heated under 75 °C for 16 h and then filtered through Celite.
The filtrate was concentrated and purified by column chromatography
(SiO2, 0.06−0.20 mm, eluting with methanol/ethyl acetate 1:50 to 1:5)
to give 2b as a white solid (0.024 g, 48%). mp 124−125 °C. 1H NMR
(500 MHz, CD3OD) δ 2.04 (dd, J = 11.0, 4.5 Hz, 1H), 2.43 (ddd, J =
11.0, 9.5, 4.5 Hz, 1H), 3.70 (dd, J = 10.0, 2.0 Hz, 1H), 3.79−3.83 (m,
3H), 4.00 (s, 1H) 4.34−4.37(m, 2H), 4.44 (d, J = 4.5 Hz, 1H), 5.26 (dd,
J = 9.5, 4.5 Hz, 1H). 7.20 (s, 1H). 13C NMR was not available due to the
poor solubility. ESI-MS (M + H)+ for C13H18N4O5: expected 311.1355,
found 311.1377.
was separated, dried over MgSO4, concentrated, and purified by column
chromatography (SiO2, 0.06−0.20 mm, eluting with hexane/ethyl
acetate 5:1 to 3:1) to give compound 8 as a white solid (0.82 g, 48%).
mp 94−95 °C. 1H NMR (500 MHz, CDCl3) δ ppm 2.41 (s, 3H), 2.45 (s,
3H), 2.53−2.57 (m, 1H), 2.89−2.92 (m, 1H), 3.04 (s, 3H), 3.12 (s, 3H),
4.52−4.55 (m, 1H), 4.60 (dd, J = 11.5, 4.0 Hz, 1H), 4.71 (dd, J = 11.5,
5.0 Hz, 1H), 5.01−5.09 (m, 2H), 5.13−5.23 (m, 2H), 5.55 (s, 3H), 5.77
(t, J = 5.0 Hz, 1H), 6.00−6.05 (m, 1H), 7.06 (s, 1H), 7.19−7.23 (m,
3H), 7.26−7.29 (m, 4H), 7.38−7.39 (m, 2H), 7.95 (d, J = 7.5 Hz, 2H),
8.01 (d, J = 7.5 Hz, 2H), 8.59 (s, 1H). 13C NMR (125 MHz, CDCl3)
δ ppm 21.8, 21.83, 35.2, 38.4, 41.0, 45.3, 50.6, 64.8, 68.6, 73.8, 82.1,
114.0, 117.6, 126.8, 127.3, 127.9, 128.3, 129.0, 129.2, 129.23, 129.26,
129.3, 129.85, 129.9, 130.1, 134.5, 143.8, 144.0, 154.2, 166.5. ESI-MS
(M + H)+ for C40H41N5O6: expected 688.3135, found 688.3202.
(2R,3S,5R)-5-(3-Benzyl-5-(2,3-dihydroxypropyl)-2-
(((dimethylamino)methylene)amino)-4-oxo-4,5-dihydro-3H-pyrrolo-
[3,2-d]pyrimidin-7-yl)-2-(((4-methylbenzoyl)oxy)methyl)-
tetrahydrofuran-3-yl 4-Methylbenzoate (9). To the suspension of 8
(0.82 g, 1.2 mmol) in a mixture of acetone (4.0 mL) and water (1.0 mL)
were added TBHP (220 μL, 1.1−1.3 mmol, 5.0−6.0 M in decane),
TBAF (0.028 g, 0.11 mmol), and OsO4 (trace). The reaction was stirred
at room temperature for 16 h. The reaction mixture was diluted with
methylene chloride and washed successively with water and brine. The
organic layer was separated, dried over MgSO4, concentrated, and
purified by column chromatography (SiO2, 0.06−0.20 mm, eluting with
hexane/ethyl acetate 1:1 to 1:3) to give compound 9 as a light yellow
solid (0.66 g, 76%). mp 80−81 °C. 1H NMR (500 MHz, CDCl3) δ ppm
2.40 (s, 3H), 2.44 (s, 3H), 2.52−2.60 (m, 1H), 2.84−2.93 (m, 1H), 3.06
(s, 3H), 3.15 (s, 3H), 3.51 (s, 2H), 3.88−3.94 (m, 2H), 4.30−4.34 (m,
1H), 4.51−4.60 (m, 3H), 4.69−4.72 (m, 1H), 5.54 (s, 3H), 7.11 (s, 1H),
7.19−7.35 (m, 9H), 7.93 (d, J = 8.0 Hz, 2H), 8.01 (s, 2H), 8.58 (s, 1H).
13C NMR for both diastereomers (125 MHz, CDCl3) δ ppm 21.0,
21.6, 35.0, 37.8, 40.8, 45.4, 50.0, 50.1, 60.4, 62.9, 63.0, 64.6, 71.8, 73.5,
73.6, 81.8, 113.8, 126.8, 127.08, 127.1, 127.7, 128.1, 129.0, 129.09, 129.1,
129.2, 129.68, 129.7, 129.72, 131.7, 138.7, 143.7, 143.9, 153.8, 156.8,
166.4. MALDI-MS (M + H)+ for C40H43N5O8: expected 722.3190,
found 722.3193.
2-Amino-3-benzyl-5-(2,3-dihydroxypropyl)-7-((2R,4S,5R)-4-hy-
droxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,5-dihydro-4H-
pyrrolo[3,2-d]pyrimidin-4-one (10). A suspension of 9 (0.66 g,
0.91 mmol) in 1 M sodium hydroxide in a mixture of methanol
(3 mL) and water (0.5 mL) was heated under 70 °C for 16 h and allowed
to cool to room temperature. The reaction mixture was concentrated
and purified by column chromatography (SiO2, 0.06−0.20 mm, eluting
with methanol/ethyl acetate 1:20 to 1:10) to give 10 as a white solid
(0.21 g, 54%). mp 93−94 °C. 1H NMR (500 MHz, CD3OD) δ ppm 2.07
(dd, J = 13.0, 5.5 Hz, 1H), 2.51 (ddd, J = 13.0, 11.5, 5.5 Hz, 1H), 3.48
(ddd, J = 11.5, 5.5, 3.0 Hz, 1H), 3.54 (ddd, J = 11.5, 5.0, 2.0 Hz, 1H), 3.69
(dd, J = 9.5, 2.5 Hz, 1H), 3.82 (dd, J = 9.5, 2.5 Hz, 1H), 3.93−3.97 (m,
1H), 4.00−4.02 (m, 1H), 4.26 (dt, J = 14.0, 5.0 Hz, 1H), 4.45−4.46 (d,
J = 5.0 Hz, 1H), 4.51−4.56 (m, 1H), 5.29−5.35 (m, 3H), 7.23−7.29 (m,
4H), 7.32−7.35 (m, 2H). 13C NMR for both diastereomers (125 MHz,
CD3OD) δ ppm 44.18, 44.2, 45.9, 52.6, 62.2, 65.4, 65.5, 73.9, 76.5,
76.8, 90.1, 114.9, 115.1, 128.3, 129.3, 130.5, 134.3, 137.9, 145.3, 153.1,
157.2. ESI-MS (M + H)+ for C21H26N4O6: expected 431.1931, found
431.1966.
2-Amino-5-(2,3-dihydroxypropyl)-7-((2R,4S,5R)-4-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-3,5-dihydro-4H-pyrrolo[3,2-d]-
pyrimidin-4-one (2d). To a mixture of 10 (0.21 g, 0.49 mmol) and 10%
palladium on carbon (0.1 g) in methanol (5 mL) was added ammonium
formate (0.29 g, 4.5 mmol) under Ar. The reaction mixture was heated
under 75 °C for 16 h and then filtered through Celite. The filtrate was
concentrated and purified by column chromatography (SiO2, 0.06−
0.20 mm, eluting with methanol/ethyl acetate 1:20 to 1:5) to give 2d
as a white solid (0.12 g, 74%). mp 135−136 °C. 1H NMR (500 MHz,
d6-DMSO) δ ppm 1.90 (dd, J = 13.8, 4.8 Hz, 1H), 2.11−2.17 (m, 1H),
3.27 (t, J = 5.0 Hz, 2H), 3.45 (s, 2H), 3.71 (s, 2H), 4.03−4.09 (m, 1H),
4.18 (s, 1H), 4.32−4.35 (m, 1H), 4.66 (s, 1H), 4.91 (s, 2H), 5.07 (dd, J =
11.2, 6.0 Hz, 1H), 5.20 (br, 1 H), 5.72 (s, 2H), 7.15 (s, 1H), 10.53 (s,
1H). 13C NMR for both of the diastereomers (150 MHz, d6-DMSO)
(2R,3S,5R)-5-(5-Allyl-3-benzyl-2-(((dimethylamino)methylene)-
amino)-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)-2-(((4-
methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-Methylbenzoate
(8). To a suspension of 5 (1.61 g, 2.4 mmol) in dry THF (25.0 mL)
was added sodium hydride (0.1 g, 2.3 mmol, 60% in mineral oil). The
mixture was stirred for 15 min before allyl bromide (150 μL, 3.5 mmol)
was added. The mixture was stirred at room temperature for 16 h.
After removal of solvent, the reaction was diluted with methylene
chloride and washed successively with water and brine. The organic layer
F
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Article
δ ppm 42.7, 51.6, 59.9, 63.9, 64.4, 72.3, 73.5, 74.0, 88.4, 113.3, 114.4,
131.3, 132.3, 151.6, 156.3. ESI-MS (M + H)+ for C14H20N4O6: expected
341.1461, found 341.1486.
(0.7 g, 1.9 mmol) in a mixture of methylene chloride (3.0 mL) and
acetonitrile (3.0 mL) was added a solution of SnCl4 (2.5 mL, 2.5 mmol,
1 M in CH2Cl2). The reaction mixture was heated at 65 °C for 16 h.
The reaction mixture was diluted with methylene chloride and
washed successively with sat. NaHCO3 and brine. The organic layer
was separated and dried over MgSO4. The solution was concentrated
and purified by column chromatography (SiO2, 0.06−0.20 mm, eluting
with hexane/ethyl acetate 1:1 to 1:2) to give compound 14 as a light
yellow solid (0.20 g, 26%). mp 84−85 °C. 1H NMR (500 MHz, CDCl3)
δ ppm 2.37 (s, 3H), 2.41 (s, 3H), 2.60−2.63 (m, 2H), 4.53 (s, 1H), 4.64
(dd, J = 12.0, 4.0 Hz, 1H), 4.78 (dd, J = 11.5, 4.5 Hz, 1H), 5.34−5.42 (m,
2H), 5.47−5.50 (m, 1H), 5.65−5.66 (m, 1H), 7.18−7.33 (m, 10H), 7.91
(d, J = 7.5 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 10.54 (s, 1H). 13C NMR
(125 MHz, CDCl3) δ ppm 21.9, 22.0, 30.0, 39.3, 45.1, 65.1, 73.6, 82.9,
113.0, 113.7, 127.0, 127.2, 127.3, 128.5, 129.3, 129.39, 129.4, 129.5,
130.2, 134.8, 143.7, 144.1, 144.2, 151.3, 166.5, 166.8. ESI-MS (M + H)+
for C34H32N4O6: expected 593.2401, found 593.2422.
2-(2-Amino-7-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-
5-yl)acetaldehyde (2c). To compound 2d (0.050 g, 0.15 mmol) in a
mixture of methanol (1 mL) and water (1 mL) was added potassium
periodate (0.035 g, 0.15 mmol). The reaction mixture was stirred under
room temperature for 30 min, concentrated, and purified by column
chromatography (SiO2, 0.06−0.20 mm, eluting with methanol/ethyl
acetate 1:20 to 1:5) to give 2c as a white solid (0.039 g, 85%). mp 117−
1
118 °C. H NMR (600 MHz, d6-DMSO) δ ppm 1.91−1.99 (m, 1H),
2.10−2.13 (m, 1H), 3.44 (s, 2H), 3.72 (s, 1H), 4.20−4.22 (m, 1H),
4.93−4.95 (m, 1H), 5.07 (s, 2H), 5.84−6.01 (m, 2H), 7.16 (s, 1H), 9.60
(s, 1H), 10.66−10.72 (s, 1H). 13C NMR was not available due to the
poor solubility. ESI-MS (M + H)+ for C13H16N4O5: expected 309.1199,
found 309.1220.
(2R,3S,5R,5S)-5-(2-Amino-3-benzyl-5-(2-hydroxyethyl)-4-oxo-4,5-
dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)-2-(((4-methylbenzoyl)-
oxy)methyl)tetrahydrofuran-3-yl 4-Methylbenzoate (15). To com-
pound 14 (0.100 g, 0.17 mmol) in dry DMF (2 mL) were added
DBU (28 μL, 0.18 mmol) and ethylene carbonate (0.04 g, 0.45 mmol).
The reaction was heated under 110 °C for 4 h. After removal of the
solvent, the reaction mixture was diluted with ethyl acetate and washed
with water and brine. The residue was concentrated and purified by
column chromatography (SiO2, 0.06−0.20 mm, eluting with hexane/
ethyl acetate 1:5 to 1:8) to give compound 15 as a white solid (0.050 g,
46%). The ratio of the epimers (α:β = 1:3) was determined by 1H NMR.
1H NMR for both epimers (600 MHz, CDCl3) δ ppm 2.38 (s, 4.78H),
2.41 (s, 3H), 2.50 (dd, J = 11.2, 5.6 Hz, 1.35H), 2.62−2.65 (m, 1.38H),
3.35 (s, 0.66H), 3.67 (s, 2H), 3.89 (t, J = 5.6 Hz, 2.88H), 4.42 (t, J =
5.6 Hz, 2H), 4.46−4.47 (m, 1.59H), 4.51−4.53 (m, 0.53H), 4.55 (dd, J =
11.2, 5.6 Hz, 1H), 4.64−4.65 (m, 0.39H), 4.75 (dd, J = 11.2, 5.6 Hz, 1H),
5.11 (s, 0.61H), 5.20−5.29 (m, 3.21H), 5.42 (dd, J = 9.6, 4.8 Hz, 1H),
5.48 (t, J = 6.6 Hz, 0.38H), 5.52−5.58 (m, 0.34H), 5.64 (d, J = 5.4 Hz,
1H), 7.08 (s, 1H), 7.16−7.36 (m, 13.72H), 7.78 (d, J = 7.2 Hz, 0.62H),
7.93 (d, J = 7.8 Hz, 2.47H), 7.97 (d, J = 8.2 Hz, 2H). 13C NMR for both
epimers (150 MHz, CDCl3) δ ppm 21.80, 21.82, 42.2, 44.6, 51.3,
62.2, 64.7, 73.2, 83.2, 110.6, 110.9, 112.4, 126.9, 127.1, 127.3, 128.6,
129.2, 129.3, 129.8, 129.9, 130.1, 130.2, 131.2, 133.4, 144.0, 144.1, 144.2,
151.36, 151.40, 152.5, 166.5, 166.6, 166.7.
2-Amino-3-benzyl-5-(2-hydroxyethyl)-3,5-dihydro-4H-pyrrolo[3,2-
d]pyrimidin-4-one (16). To compound 13 (1.3 g, 4.4 mmol) in dry
DMF (5 mL) were added DBU (280 μL, 1.83 mmol) and ethylene
carbonate (1.0 g, 11.7 mmol). The reaction was heated under 90 °C for
4 h. After removal of the solvent, the reaction mixture was diluted
with ethyl acetate and washed with water and brine. The residue was
chromatographed by ethyl acetate to give compound 16 as a white solid
(0.6 g, 47%). mp 168−169 °C. 1H NMR (500 MHz, d6-DMSO) δ ppm
3.65 (dd, J = 9.5, 5.0 Hz, 2H), 4.30 (t, J = 4.5 Hz, 2H), 5.23 (s, 2H), 5.90
(s, 1H), 6.25 (s, 1H), 7.21−7.26 (m, 4H), 7.32 (t, J = 6.0 Hz, 2H).
13C NMR (150 MHz, CD3OD) δ ppm 45.1, 51.9, 63.2, 100.3, 112.7,
127.5, 128.5, 129.8, 134.6, 137.3, 146.9, 152.8, 156.2. ESI-MS (M + H)+
for C15H16N4O2: expected 285.1352, found 285.1337.
(2R,3S,5S)-5-(2-Amino-3-benzyl-5-(2-hydroxyethyl)-4-oxo-4,5-di-
hydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)-2-(((4-methylbenzoyl)oxy)-
methyl)tetrahydrofuran-3-yl 4-Methylbenzoate (17). To compound 16
(0.6 g, 2.2 mmol) and 1-(α,β)-O-methyl-3,5-di(O-p-toluoyl)-2-deoxy-D-
ribose (2.4 g, 3.3 mmol) in a mixture of methylene chloride (5.0 mL)
and acetonitrile (5.0 mL) was added a solution of SnCl4 (4.5 mL,
4.5 mmol, 1 M in CH2Cl2). The reaction mixture was heated at 65 °C
for 16 h. The reaction mixture was diluted with methylene chloride and
washed successively with sat. NaHCO3 and brine. The organic layer was
separated and dried over MgSO4. The solution was concentrated and
purified by column chromatography (SiO2, 0.06−0.20 mm, eluting with
hexane/ethyl acetate 1:1 to 1:2) to give compound 17 as a white solid
(0.39 g, 28%). mp 89−90 °C. 1H NMR (500 MHz, CDCl3) δ ppm 2.40
(s, 3H), 2.43 (s, 3H), 2.50 (dd, J = 13.0, 5.0 Hz, 1H), 2.67 (ddd, J =
10.5, 10.5, 4.5 Hz, 1H), 3.60 (s, 1H), 3.90 (s, 2H), 4.42 (s, 3H), 4.55 (dd,
J = 10.5, 4.5 Hz, 1H), 4.73 (dd, J = 10.5, 4.5 Hz, 1H), 5.02 (s, 2H),
(2R,3S,5R)-5-(3-Benzyl-5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2-
(((dimethylamino)methylene)amino)-4-oxo-4,5-dihydro-3H-pyrrolo-
[3,2-d]pyrimidin-7-yl)-2-(((4-methylbenzoyl)oxy)methyl)-
tetrahydrofuran-3-yl 4-Methylbenzoate (11). To a solution of 6 (0.45 g,
0.65 mmol) in DMF were added imidazole (0.13 g, 1.9 mmol), 4-DMAP
(0.002 g, 0.018 mmol), and TBSCl (0.29 g, 1.9 mmol). The reaction was
stirred under room temperature for 16 h. After removal of the solvent,
the mixture was diluted by methylene chloride and washed with
water and brine. The solution was concentrated and purified by
column chromatography (SiO2, 0.06−0.20 mm, eluting with hexane/
ethyl acetate 5:1 to 3:1) to give compound 11 as a light yellow solid
1
(0.43 g, 82%). mp 50−51 °C. H NMR (500 MHz, CDCl3) δ ppm
−0.12 (s, 6H), 0.83 (s, 9H), 2.40 (s, 3H), 2.44 (s, 3H), 2.49−2.52 (m,
1H), 2.87−2.89 (m, 1H), 3.03 (s, 3H), 3.10 (s, 3H), 3.93 (s, 2H), 4.44
(s, 2H), 4.52 (s, 1H), 4.60 (dd, J = 11.3, 4.5 Hz, 1H), 4.66 (dd, J = 11.5,
5.0 Hz, 1H), 5.56 (s, 3H), 5.78 (s, 1H), 7.12 (s, 1H), 7.19−7.21 (m, 3H),
7.25−7.29 (m, 4H), 7.35−7.37 (m, 2H), 7.96 (d, J = 7.5 Hz, 2H), 8.01
(d, J = 6.5 Hz, 2H), 8.59 (s, 1H). 13C NMR (125 MHz, CDCl3) δ ppm
−5.4, 18.4, 21.9, 21.94, 26.1, 31.8, 35.2, 41.0, 45.3, 51.3, 63.5, 65.0,
73.6, 82.1, 113.0, 114.7, 126.8, 127.46, 127.5, 127.9, 128.3, 129.3, 129.4,
129.96, 130.0, 139.3, 143.1, 143.9, 144.1, 154.2, 156.0, 156.8, 166.4,
166.6. MALDI-MS (M + H)+ for C45H55N5O7Si: expected 806.3943,
found 806.3944.
N′-(3-Benzyl-5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-7-
((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-2-yl)-N,N-dimethylfor-
mimidamide (12). To compound 11 (0.43 g, 0.53 mmol) in a mixture
of methanol (5 mL) and water (1 mL) was added sodium hydroxide
(0.05 M). The reaction mixture was stirred under room temperature for
1 h. The reaction mixture was concentrated and purified by column
chromatography (SiO2, 0.06−0.20 mm, eluting with methanol/ethyl
acetate 1:50 to 1:20) to give 12 as a light yellow solid (0.20 g, 72%).
1
mp 65−66 °C. H NMR (500 MHz, CDCl3) δ ppm −0.11 (s, 3H),
−0.09 (s, 3H), 0.83 (s, 9H), 2.04 (dd, J = 11.0, 5.0 Hz, 1H), 2.60−2.66
(m, 1H), 3.03 (s, 3H), 3.14 (s, 3H), 3.72−3.75 (m, 1H), 3.86−3.90 (m,
3H), 4.12 (s, 1H), 4.32−4.35 (m, 1H), 4.56−4.63 (m, 2H), 5.32 (dd, J =
11.0, 5.0 Hz, 1H), 5.49 (s, 2H), 7.06 (s, 1H), 7.19−7.33 (m, 5H), 8.28
(s, 1H). 13C NMR (125 MHz, CDCl3) δ ppm −5.4, 18.4, 26.1, 35.1,
41.0, 43.8, 45.6, 51.1, 63.5, 64.1, 75.7, 88.5, 113.5, 115.1, 127.0, 127.9,
128.4, 131.4, 138.7, 154.8, 155.8, 157.1. MALDI-MS (M + H)+ for
C29H43N5O5Si: expected 570.3112, found 570.3108.
2-Amino-3-benzyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one
(13). A suspension of 6 (3 g, 10.2 mmol) in 1 M sodium hydroxide in a
mixture of methanol (12 mL) and water (6 mL) was heated under 70 °C
for 20 h and allowed to cool to room temperature. The reaction
mixture was concentrated and purified by column chromatography
(SiO2, 0.06−0.20 mm, eluting with ethyl acetate) to give 13 as a
white solid (1.86 g, 75%). The characterization of this compound was
previously described by Gibson et al.23
(2R,3S,5R)-5-(2-Amino-3-benzyl-4-oxo-4,5-dihydro-3H-pyrrolo-
[3,2-d]pyrimidin-7-yl)-2-(((4-methylbenzoyl)oxy)methyl)-
tetrahydrofuran-3-yl 4-Methylbenzoate (14). To compound 13 (0.31 g,
1.3 mmol) and 1-(α,β)-O-methyl-3,5-di(O-p-toluoyl)-2-deoxy-D-ribose
G
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5.19−5.26 (m, 2H), 5.45 (dd, J = 10.5, 5.0 Hz, 1H), 5.65 (d, J = 5.0 Hz,
1H), 7.09 (s, 1H), 7.20−7.32 (m, 9H), 7.95 (d, J = 8.0 Hz, 2H), 7.99 (d, J
= 8.0 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ ppm 21.87, 21.9, 38.6,
44.8, 51.3, 63.0, 65.0, 73.5, 82.6, 112.8, 113.1, 126.7, 127.3, 127.4, 128.2,
129.38, 129.4, 129.9, 130.0, 131.0, 135.3, 144.1, 144.3, 150.9, 155.2,
166.4, 166.7. ESI-MS (M + H)+ for C36H36N4O7: expected 637.2663,
found 637.2697.
solvent, the reaction was diluted with methylene chloride and washed
successively with water and brine. The organic layer was separated, dried
over MgSO4, concentrated, and purified by column chromatography
(SiO2, 0.06−0.20 mm, eluting with hexane/ethyl acetate 5:1 to 3:1) to
give compound 20 as a light yellow solid (1.4 g, 64%). mp 93−94 °C.
1H NMR (500 MHz, CDCl3) δ ppm 3.06 (s, 3H), 3.15 (s, 3H), 5.06−
5.09 (m, 3H), 5.19 (d, J = 10.5 Hz, 1H), 5.52 (s, 2H), 6.05 (ddt, J = 16.5,
10.5, 5.0 Hz, 1H), 6.31 (s, 1H), 7.03 (s, 1H), 7.19−7.21 (m, 1H), 7.27
(t, J = 7.5 Hz, 2H), 7.37 (d, J = 7.5 Hz, 2H), 8.61 (s, 1H). 13C NMR
(125 MHz, CDCl3) δ ppm 35.0, 40.8, 45.1, 50.4, 101.3, 114.3, 116.9,
126.7, 127.9, 128.1, 130.4, 134.8, 139.0, 144.4, 154.3, 155.8, 156.2. ESI-
MS (M + H)+ for C19H21N5O: expected 336.1824, found 336.1832.
5-Allyl-2-amino-3-benzyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-
4-one (21). To compound 20 (1.0 g, 3 mmol) in a mixture of methanol
(20.0 mL) and water (5.0 mL) was added sodium hydroxide (1.0 M).
The reaction mixture was heated under 70 °C for 16 h. The reaction
mixture was concentrated and purified by column chromatography
(SiO2, 0.06−0.20 mm, eluting with ethyl acetate) to give 21 as a white
solid (0.58 g, 70%). mp 121−122 °C. 1H NMR (500 MHz, CD3OD)
δ ppm 4.99−5.05 (m, 3H), 5.16 (dd, J = 10.5, 1.5 Hz, 1H), 5.33 (s, 2H),
6.04−6.12 (m, 2H), 7.21−7.30 (m, 4H), 7.35 (t, J = 7.5 Hz, 2H).
13C NMR (125 MHz, CDCl3) δ ppm 44.5, 50.5, 101.3, 112.3, 117.0,
126.5, 127.9, 129.1, 131.1, 134.6, 135.7, 144.9, 151.1, 154.8. ESI-MS
(M + H)+ for C16H16N4O: expected 281.1403, found 281.1387.
(2R,3S,5R)-5-(5-Allyl-2-amino-3-benzyl-4-oxo-4,5-dihydro-3H-
pyrrolo[3,2-d]pyrimidin-7-yl)-2-(((4-methylbenzoyl)oxy)methyl)-
tetrahydrofuran-3-yl 4-Methylbenzoate (22). To compound 21 (0.58 g,
2.1 mmol) and 1-(α,β)-O-methyl-3,5-di(O-p-toluoyl)-2-deoxy-D-ribose
(2.3 g, 3.1 mmol) in a mixture of methylene chloride (5.0 mL) and
acetonitrile (5.0 mL) was added a solution of SnCl4 (4.2 mL, 4.2 mmol,
1 M in CH2Cl2). The reaction mixture was heated at 65 °C for 16 h.
The reaction mixture was diluted with methylene chloride and
washed successively with sat. NaHCO3 and brine. The organic layer
was separated, dried over MgSO4, concentrated, and purified by column
chromatography (SiO2, 0.06−0.20 mm, eluting with hexane/ethyl
acetate 2:1 to 1:2) to give compound 22 as a light yellow solid (0.30 g,
23%). mp 59−60 °C. 1H NMR (600 MHz, CDCl3) δ ppm 2.40 (s, 3H),
2.41 (s, 3H), 2.56−2.59 (m, 2H), 4.47 (dt, J = 4.2, 1.8 Hz, 1H), 4.57 (dd,
J = 12.0, 4.2 Hz, 1H), 4.76 (dd, J = 12.0, 4.2 Hz, 1H), 4.94−4.96 (m,
2H), 5.09 (d, J = 10.4 Hz, 1H), 5.17 (d, J = 10.4 Hz, 1H), 5.29 (s, 2H),
5.48 (t, J = 8.4 Hz, 1H), 5.64−5.66 (m, 1H), 5.95−6.02 (m, 1H), 7.04 (s,
1H), 7.23−7.29 (m, 7H), 7.31−7.34 (m, 2H), 7.94 (d, J = 8.4 Hz, 2H),
7.97 (d, J = 8.4 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ ppm 21.75,
21.8, 38.6, 44.5, 50.6, 64.8, 73.2, 77.4, 82.5, 112.8, 113.1, 117.6, 126.4,
127.2, 127.3, 128.0, 129.0, 129.2, 129.23, 129.8, 134.2, 135.4, 143.8,
144.1, 151.3, 154.7, 166.2, 166.4. ESI-MS (M + H)+ for C37H36N4O6:
expected 633.2714, found 633.2745.
(2R,3S,5S)-5-(2-Amino-3-benzyl-5-(2-((tert-butyldimethylsilyl)-
oxy)ethyl)-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)-2-
(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl 4-Methylben-
zoate (18). To compound 17 (0.39 g, 0.6 mmol) in DMF were
added imidazole (0.13 g, 1.9 mmol), 4-DMAP (0.002 g, 0.018 mmol),
and TBSCl (0.3 g, 2.0 mmol). The reaction was stirred under room
temperature for 16 h. After removal of the solvent, the mixture was
diluted by methylene chloride and washed with water and brine.
The solution was concentrated and purified by column (SiO2, 0.06−
0.20 mm, eluting with hexane/ethyl acetate 5:1 to 3:1) to give
compound 18 as a gummy solid (0.42 g, 93%). mp 55−56 °C. 1H NMR
(500 MHz, CDCl3) δ ppm −0.11 (d, J = 5.0 Hz, 6H), 0.82 (s, 9H), 2.39
(s, 3H), 2.41 (s, 3H), 2.44 (dd, J = 11.5, 4.5 Hz, 1H), 2.62 (ddd, J = 11.5,
11.5, 4.5 Hz, 1H), 3.90 (t, J = 3.5 Hz, 2H), 4.31−4.37 (m, 2H), 4.44 (dt,
J = 11.5, 3.5 Hz, 1H), 4.51 (dd, J = 9.5, 3.5 Hz, 1H), 4.67 (dd, J = 9.5, 4.0
Hz, 1H), 5.14−5.28 (m, 3H), 5.45 (dd, J = 9.5, 4.5 Hz, 1H), 5.65 (d, J =
4.5 Hz, 1H), 7.08 (s, 1H), 7.19−7.30 (m, 9H), 7.94 (d, J = 7.0 Hz, 2H),
7.97 (d, J = 6.5 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ ppm −5.6,
18.1, 21.6, 21.65, 25.8, 37.8, 44.3, 51.1, 53.5, 63.1, 64.7, 72.6, 82.3, 111.7,
112.2, 126.1, 127.1, 127.3, 127.8, 129.0, 129.1, 129.7, 129.74, 130.9,
135.3, 143.7, 144.0, 151.4, 154.7, 166.0, 166.2. MALDI-MS (M + H)+
for C42H50N4O7Si: expected 751.3528, found 751. 3505.
2-Amino-3-benzyl-5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-7-
((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,5-di-
hydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (19). To compound 18
(0.42 g, 0.56 mmol) in a mixture of methanol (10.0 mL) and water
(1.0 mL) was added sodium hydroxide (0.05 M). The reaction mixture
was stirred under room temperature for 1 h. The reaction mixture was
concentrated and purified by column chromatography (SiO2, 0.06−
0.20 mm, eluting with methanol/ethyl acetate 1:50 to 1:20) to give 19 as
1
a white solid (0.21 g, 74%). mp 57−58 °C. H NMR (500 MHz,
CD3OD) δ ppm −0.09 (d, J = 10.0 Hz, 6H), 0.82 (s, 9H), 2.02 (dd, J =
11.0, 5.0 Hz, 1H), 2.51−2.56 (m, 1H), 3.68 (d, J = 12.0 Hz, 1H), 3.87−
3.90 (m, 2H), 4.08 (s, 1H), 4.31 (dt, J = 14.0, 5.0 Hz, 1H), 4.53−4.58
(m, 2H), 5.13 (d, J = 16.0 Hz, 1H), 5.26 (dd, J = 11.5, 5.5 Hz, 1H), 5.40
(d, J = 16.0 Hz, 1H), 7.06 (s, 1H), 7.22−7.35 (m, 5H). 13C NMR
(125 MHz, CD3OD) δ ppm −4.5, 19.7, 27.2, 44.4, 45.7, 52.6, 64.9, 65.5,
76.7, 89.9, 114.3, 114.7, 128.2, 129.2, 130.4, 134.0, 137.7, 144.9, 152.9,
156.6. ESI-MS (M + H)+ for C26H38N4O5Si: expected 515.2690, found
515.2706.
2-Amino-5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-7-((2S,4S,5R)-4-
hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,5-dihydro-4H-
pyrrolo[3,2-d]pyrimidin-4-one (3b). To compound 19 (0.21 g,
0.41 mmol) and 10% palladium on carbon (0.11 g) in methanol
(5.0 mL) was added ammonium formate (0.28 g, 4.5 mmol) under Ar.
The reaction mixture was heated under 75 °C for 16 h and then filtered
through Celite. The filtrate was concentrate and purified by column
chromatography (SiO2, 0.06−0.20 mm, eluting with methanol/ethyl
acetate 1:20 to 1:5) to give 3b as a white gummy solid (0.075 g, 43%).
mp 49−50 °C. 1H NMR (600 MHz, d6-DMSO) δ ppm −0.13 (s, 6H),
0.79 (s, 9H), 1.88 (dd, J = 12.2, 5.4 Hz, 1H), 2.06−2.11 (m, 1H), 3.42 (s,
2H), 3.72 (s, 1H), 3.81 (t, J = 5.4 Hz, 2H), 4.17 (s, 1H), 4.25 (t, J =
4.8 Hz, 2H), 4.90 (s, 1H), 5.07 (dd, J = 12.0, 5.4 Hz, 1H), 5.71 (s, 1H),
7.12 (s, 1H), 10.78 (s, 1H). 13C NMR (150 MHz, d6-DMSO) δ ppm
−4.7, 18.8, 26.7, 42.8, 51.1, 63.9, 73.4, 74.0, 88.4, 112.8, 114.5, 130.9,
145.8, 151.6, 155.6. ESI-MS (M + H)+ for C19H32N4O5Si: expected
425.2221, found 425.2260.
(2R,3S,5R)-5-(2-Amino-3-benzyl-5-(2,3-dihydroxypropyl)-4-oxo-
4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)-2-(((4-methylbenzoyl)-
oxy)methyl)tetrahydrofuran-3-yl 4-Methylbenzoate (23). To the
suspension of compound 22 (0.30 g, 0.48 mmol) in a mixture of
acetone (4.0 mL) and water (1.0 mL) were added TBHP (100 μL, 0.5−
0.6 mmol, 5.0−6.0 M in decane), TBAF (0.012 g, 0.048 mmol), and
OsO4 (trace). The reaction was stirred at room temperature for 16 h.
The reaction mixture was diluted with methylene chloride and washed
successively with water and brine. The organic layer was separated, dried
over MgSO4, and concentrated. The solution was purified by column
chromatography (SiO2, 0.06−0.20 mm, eluting with hexane/ethyl
acetate 1:1 to 1:3) to give compound 23 as a light yellow solid (0.13 g,
40%). mp 61−62 °C. 1H NMR (500 MHz, CDCl3) δ ppm 2.36 (s, 3H),
2.39 (s, 3H), 2.50−2.58 (m, 2H), 3.46 (dd, J = 12.0, 5.5 Hz, 1H),
3.52 (ddd, J = 11.5, 4.5, 1.5 Hz, 1H), 3.88−3.93 (m, 1H), 4.18 (dt, J =
14.0, 7.0 Hz, 1H), 4.40 (dt, J = 5.0, 2.0 Hz, 1H), 4.44 (dd, J = 14.0,
3.5 Hz, 1H), 4.54 (ddd, J = 12.0, 5.0, 2.0 Hz, 1H), 4.67 (dt, J = 12.0,
5.4 Hz, 1H), 5.26 (s, 2H), 5.43 (dd, J = 10.0, 6.0 Hz, 1H), 5.59−5.60 (m,
1H), 7.19−7.28 (m, 10H), 7.89 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.0 Hz,
2H). 13C NMR for both diastereomers (125 MHz, CDCl3) δ ppm 22.5,
22.56, 46.0, 52.79, 52.8, 65.40, 65.44, 66.7, 73.8, 73.85, 75.5, 79.5, 84.6,
114.1, 114.3, 128.3, 129.0, 129.1, 129.40, 130.6, 131.1, 131.5, 131.53,
N′-(5-Allyl-3-benzyl-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]-
pyrimidin-2-yl)-N,N-dimethylformimidamide (20). To a suspension of
compound 6 (2.0 g, 6.8 mmol) in dry THF (25.0 mL) was added sodium
hydride (0.28 g, 6.7 mmol, 60% in mineral oil). The mixture was stirred
for 15 min before allyl bromide (330.0 μL, 8.0 mmol) was added. The
mixture was stirred at room temperature for 16 h. After removal of
H
DOI: 10.1021/acs.joc.6b02110
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
133.7, 137.7, 146.2, 146.3, 153.4, 156.9, 168.4, 168.6. ESI-MS (M + H)+
for C37H38N4O8: expected 667.2769, found 667.2786.
later stage of the project. We also thank the Rutgers University
(Newark) for granting X. Gao access to NMR and mass spec-
troscopy facilities.
( 2R , 3S , 5R ) -5 -( 2 - Am i n o- 3 - b e n z y l - 5 -( 2 , 3 - b i s ( (t e r t -
butyldimethylsilyl)oxy)propyl)-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]-
pyrimidin-7-yl)-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-
yl 4-Methylbenzoate (24). To compound 23 (0.13 g, 0.19 mmol) in
DMF were added imidazole (0.08 g, 1.1 mmol), 4-DMAP (0.002 g,
0.018 mmol), and TBSCl (0.17 g, 1.1 mmol). The reaction was stirred
under room temperature for 16 h. After removal of the solvent,
the mixture was diluted by methylene chloride and washed with water
and brine. The solution was concentrated and purified by column
chromatography (SiO2, 0.06−0.20 mm, eluting with hexane/ethyl
acetate 5:1 to 3:1) to give compound 24 as a white gummy solid (0.13 g,
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76%). mp 44−45 °C. H NMR (600 MHz, CDCl3) δ ppm −0.31 (s,
3H), −0.07 (d, J = 10.2 Hz, 3H), 0.07 (s, 6H), 0.78 (d, J = 2.4 Hz, 9H),
0.91 (s, 9H), 2.39 (s, 3H), 2.40 (s, 4H), 2.42−2.44 (m, 1H), 3.52−3.55
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(m, 1H), 4.60−4.64 (m, 2H), 4.77 (dd, J = 11.4, 4.8 Hz, 1H), 5.39−5.43
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7.27 (m, 4H), 7.32−7.35 (m, 5H), 7.91 (d, J = 7.8 Hz, 2H), 8.02 (d, J =
7.8 Hz, 2H). 13C NMR for both diastereomers (150 MHz, CDCl3)
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44.6, 52.3, 52.6, 65.2, 65.8, 72.9, 73.0, 73.6, 82.6, 112.2, 112.3, 126.7,
126.8, 127.4, 127.5, 128.3, 129.4, 129.5, 130.0, 130.1, 131.4, 131.6, 135.5,
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0.20 mm, eluting with methanol/ethyl acetate 1:50 to 1:20) to give 25
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(SiO2, 0.06−0.20 mm, eluting with methanol/ethyl acetate 1:20 to 1:5)
to give 3d as a white gummy solid (0.031 g, 56%). mp 53−54 °C.
1H NMR (600 MHz, d6-DMSO) δ ppm −0.28 (d, J = 6.0 Hz, 3H),
−0.08 (s, 3H), 0.05 (s, 6H), 0.79 (s, 9H), 0.89 (s, 9H), 1.88−1.91 (m,
1H), 2.02−2.11 (m, 1H), 3.46−3.50 (m, 2H), 3.70 (d, J = 15.6 Hz, 1H),
3.97−4.03 (m, 2H), 4.18 (d, J = 12.0 Hz, 1H), 4.34 (d, J = 10.2 Hz, 1H),
4.90 (s, 1H), 5.06−5.10 (m, 1H), 5.69 (d, J = 20.0 Hz, 1H), 7.02 (s, 1H),
10.50 (d, J = 15.2 Hz, 1H). 13C NMR (150 MHz, d6-DMSO) δ ppm
−4.5, −3.9, 18.6, 19.0, 26.7, 49.6, 52.1, 52.2, 63.9, 64.0, 66.3, 73.6, 73.9,
74.2, 88.3, 113.2, 114.8, 131.2, 145.8, 151.4, 155.3. ESI-MS (M + H)+ for
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ASSOCIATED CONTENT
* Supporting Information
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S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.6b02110.
1H and 13C NMR spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: haidonghuang2015@gmail.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The financial support of this study was provided by the internal
funds of New Jersey Institute of Technology. We thank
Dr. Edgardo Farinas for providing laboratory space for the
I
DOI: 10.1021/acs.joc.6b02110
J. Org. Chem. XXXX, XXX, XXX−XXX