Syntheses of pyrido[1,2-a][1,3,5]triazin-4-one C-deoxyribonucleosides

Article abstract of DOI:10.1016/j.tet.2007.08.044

We achieved the syntheses of new C-deoxyribonucleosides bearing pyrido[1,2-a][1,3,5]triazin-4-one derivatives using palladium-catalyzed Heck-type coupling. Some of these C-deoxyribonucleosides were able to convert to phosphoramidite reagents, which can be

Full text of DOI:10.1016/j.tet.2007.08.044

Tetrahedron 63 (2007) 11021–11029
Syntheses of pyrido[1,2-a][1,3,5]triazin-4-one
C-deoxyribonucleosides
Hiroshi Oda,a,b, Takeshi Hanami,a,b Takashi Iwashita,a,b Miki Kojima,a,b
*
Masayoshi Itoha,b and Yoshihide Hayashizakia,b
aDiscovery and Research Institute, Genome Science Laboratory, RIKEN Wako Main Campus, 2-1, Hirosawa,
Wako, Saitama 351-0106, Japan
bGenome Exploration Research Group, Genomic Sciences Center (GSC), RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku,
Yokohama, Kanagawa 230-0045, Japan
Received 20 April 2007; revised 14 August 2007; accepted 14 August 2007
Available online 19 August 2007
Abstract—We achieved the syntheses of new C-deoxyribonucleosides bearing pyrido[1,2-a][1,3,5]triazin-4-one derivatives using palladium-
catalyzed Heck-type coupling. Some of these C-deoxyribonucleosides were able to convert to phosphoramidite reagents, which can be used
for DNA synthesizer. DNA oligomers including pyrido[1,2-a][1,3,5]triazin-4-one C-deoxyribonucleoside, which had 2-amino group were
synthesized, and Tm values with a natural nucleoside were measured.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
thought to be difficult to use this type of base pair for a codon,
since the codon consists of only three bases. The binding en-
ergy of the hydrophobic interaction is significantly weak
compared with that of the hydrogen bond. Therefore there
is a possibility that complementary tRNA cannot be accu-
rately selected.
DNA that consists of the four bases A, G, C, and T serves as
one of the most important biomolecules by coding indis-
pensable information for life. An important function of
DNA for the transmittance of genes is replication. A new ap-
tamer that especially interacts with a specific biomolecule
has recently been well investigated.1 However, it seems to
be impossible to prepare all aptamers with satisfactory levels
of specificity. Because an aptamer is a small nucleic acid
compared with a general target such as a protein, there is
a limitation in the diversity. A new nucleoside pair that
can replicate by polymerase chain reaction (PCR) is being
investigated.2 If this succeeds, the genetic alphabet will in-
crease. If the newly added codon can be assigned to unnatu-
ral amino acids, it might be possible to synthesize
a functionalized protein in vitro or in vivo. Of course, several
further issues must be solved before this can be realized.
On the other hand, when designing an artificial nucleoside
utilizing a hydrogen bond similar to the natural nucleoside,
the artificial nucleoside must have the different directions of
the hydrogen bonds from natural four bases. At the same
time, chemical stability against the tautomerization that
breaks down the designed hydrogen bonding pattern is im-
portant. To our knowledge, known artificial base pairs using
hydrogen bonds do not have very high selectivity similar to
the natural bases so far, thus there is no report about replica-
tion of DNA containing these nucleoside pairs under the typ-
ical conditions of PCR.
Unlike natural A–T and G–C pairs, the newly added nucleo-
side includes a base pair utilizing the shape-fitting and hy-
drophobic interaction. Hirao et al. reported a successful
DNA replication using such kind of base pair. However,
when introducing a large amount of this unnatural base
pair, the nucleic acid containing this base pair is expected
to cohere and take an irregular structure, even if it replicates
correctly when using only one base pair. Moreover, it is
We synthesized pyrido[1,2-a][1,3,5]triazin-4-one nucleo-
side derivatives that have the possibility for such purpose.
There are many reports about the construction of the pyr-
ido[1,2-a][1,3,5]triazin-4-one ring that has various substitu-
ents. This heterocycle has a rigid planar structure, and
cannot be subjected to tautomerization. Hitherto, pyr-
ido[1,2-a][1,3,5]triazin-4-one has not be employed as
a base for artificial nucleoside. Because this heterocycle
does not form complemented hydrogen bond with natural
base, it is promising as a counterpart of an artificial base
that can replicate by PCR.
* Corresponding author. Tel.: +81 48 467 9513; fax: +81 48 462 4686;
e-mail: rgscerg@gsc.riken.go.jp
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.08.044

11022
H. Oda et al. / Tetrahedron 63 (2007) 11021–11029
O
R
3-iodopyridine 2 with 10 in the presence of palladium cata-
lyst in moderate yield. The formation of the pyrido[1,2-
a][1,3,5]triazin-4-one ring was carried out using triethyl
orthoformate and trimethylsilyl isocyanate.
6
4
5
N
a
H
7
8
3
N
b
c
d
Me
9a
2
N
R
9
DMTrO
(i-Pr)2N
NH2
NMe2
1
O
2-Amino derivatives of 9-iodopyrido[1,2-a][1,3,5]triazin-
4-one were synthesized as follows. The reaction of amine
2 with ethoxycarbonyl isothiocyanate and the subsequent
one-pot reaction using ammonia in the presence of mercu-
ry(II) salt gave guanidine derivative. 2-Amino-9-iodopyr-
ido[1,2-a][1,3,5]triazin-4-one (3c) was obtained by heating
the DMF solution of the intermediate guanidine derivative
in a microwave instrument. The Heck reaction of 3c with
10 gave a somewhat low yield. However, it is necessary to
introduce a protecting group on the free amino group for
the synthesis of DNA oligomer. Thus, we examined the
Heck reaction using N-protected 2-amino-9-iodopyr-
ido[1,2-a][1,3,5]triazin-4-one (3e) that was easily prepared
through a reaction of 3c with isobutyryl chloride. The yield
of the Heck coupling adduct was significantly increased
using 3e.
e
NHCO(i-Pr)
O
P
CN
O
1a-1e
Figure 1. Target molecule.
We examined the syntheses of the nucleoside amidites 1a–
1e, which had a pyrido[1,2-a][1,3,5]triazin-4-one skeleton,
and synthesized DNA oligomer including 1c or 1d
(Fig. 1). These nucleosides do not only have a different hy-
drogen bond direction compared to the natural bases but also
unlike natural purine bases, attached with the deoxyribose
moiety through the six-membered ring. They are therefore
expected to effectively inhibit the base pair formation of
the pyrimidine bases. The syntheses of the oligomers con-
taining the new artificial C-deoxyribonucleoside and their
properties are described.
The optimization of the reaction condition for the palla-
dium-catalyzed Heck coupling between 3e and 10 was
performed for ligands, bases, solvents, and additives (Ta-
ble 1). Comparably bulky ligands such as tri(o-tolyl)phos-
phine or tri(tert-butyl)phosphine were effective, whereas
the adduct was not obtained using triphenylphosphine, tri-
phenylarsine and tri(2-furyl)phosphine. Diisopropylethyl-
2. Results and discussion
2.1. Syntheses of pyrido[1,2-a][1,3,5]triazin-4-one
nucleoside phosphoramidites
amine gave a higher yield than triethylamine or
dicyclohexylmethylamine. Inorganic bases such as sodium
carbonate, potassium carbonate, cesium carbonate, silver
carbonate, sodium acetate, and potassium phosphate
were not effective in this coupling reaction. The coupling
reaction occurred in acetonitrile and DMF but not in sol-
vents such as THF, 1,4-dioxane, DMSO, and NMP. The
reactions were examined in the presence of several ammo-
nium salts and of lithium chloride as an additive, but none
of the additives could be shown to be effective (data not
shown).
Synthesis of the pyrido[1,2-a][1,3,5]triazin-4-one skeleton
was achieved using 2-amino-3-iodopyridine as the start-
ing material.3a When the substituent on the 2-position of
pyrido[1,2-a][1,3,5]triazin-4-one belonged to the hydro-
gen or alkyl group (compounds 3a and 3b), 2-amino-3-
iodopyridine was reacted with ortho-ester,4 followed
by treatment with trimethylsilyl isocyanate. Moreover,
in the case of 2-amino substituted pyrido[1,2-a]-
[1,3,5]triazin-4-one (compounds 3c and 3d), 2-amino-3-
iodopyridine was converted to thiourea by treatment
with ethoxycarbonyl isothiocyanate. Thiourea was treated
with mercury(II) salt as a scavenger in the presence of
amine, and the annulation of the resulting guanidine
was carried out by heating or by the reaction of hydrogen
chloride.
Similar to the synthesis of 3c, iodide 3d was prepared using
dimethylamine instead of ammonia. The annulation was car-
ried out by refluxing the 1,4-dioxane solution of the interme-
diate guanidine derivative saturated with hydrogen chloride.
The Heck reaction of 3d proceeded smoothly and gave the
corresponding adduct 6d along with the 30-desilylated prod-
uct 5d in good yield.
The synthesis of pyrido[1,2-a][1,3,5]triazin-4-one nucleo-
side phosphoramidite is shown in Scheme 1. At first, we
tried to synthesize the non-substituted C-deoxyribonucleo-
side at the 2-position of the triazine ring. However, the
Heck-type palladium-catalyzed reaction between 9-iodo-
pyrido[1,2-a][1,3,5]triazin-4-one 3a with silyl protected
furanoid glycal 105 gave a complex mixture and the de-
sired coupling product was not detected. On the other
hand, the Heck coupling reaction using 2-methyl deriva-
tive of 9-iodopyrido[1,2-a][1,3,5]triazin-4-one 3b suc-
ceeded to give the adduct 5b, which is a hydrolyzed
product of 30-silyl enol ether. Although the Heck coupling
reaction of iodide 3a was not successful, the synthesis of
the corresponding non-substituted adduct 6a was achieved
by replacing the order of annulation and Heck reaction.
Adduct 4 was obtained through the reaction of 2-amino-
The products of the Heck reaction were subjected to depro-
tection by tetrabutylammonium fluoride in order to obtain
30-keto nucleosides 7a–7e in good to excellent yields.
Compounds 7a–7e were diastereoselectively reduced to
pyrido[1,2-a][1,3,5]triazin-4-one C-deoxyribonucleosides
8a–8e using sodium tri(acetoxy)borohydride.6 The struc-
tures of 8a–8e were determined by various spectra and
was confirmed to have a b-anomer structure through an
NOE experiment.
We have achieved the syntheses of some pyrido[1,2-
a][1,3,5]triazin-4-one nucleoside derivatives (8a–8e) with
2-amino-3-iodopyridine as the starting material. The total
yields of these artificial nucleosides were about 5–30%,

H. Oda et al. / Tetrahedron 63 (2007) 11021–11029
11023
O
N
TBDMSO
O
O
N
N
10
OTBDMS
(a) or (b)
N
N
R
TBDMSO
(c)
O
N
R
I
O
3a R = H (39%)
5b (32%)
3b R = Me (48%)
5d + 6d (57%)
N
3c R = NH2 (58%)
3d R = NMe2 (85%)
3e R = NHCO(i-Pr) (98%)
and / or
(d)
O
N
NH2
I
N
N
2
R
TBDMSO
O
N
NH2
TBDMSO
TBDMSO
10
(a)
R = H
O
6a (41%), 6c (22%)
6e (60%)
(c)
TBDMSO
4 (42%)
O
O
N
O
N
N
N
N
N
N
(e)
(g)
(f)
N
R
N
R
R
HO
DMTrO
HO
O
O
O
OH
OH
O
7a (75%), 7b (80%)
7c (99%), 7d (88%)
7e (96%)
8a (45%), 8b (52%)
8c (81%), 8d (78%)
8e (66%)
9b (59%), 9d (74%)
9e (89%)
O
N
N
N
R
(h)
DMTrO
O
O
P
CN
(i-Pr)2N
O
1b (39%), 1d (48%)
1e (85%)
Scheme 1. Reagents and conditions: (a) (i) RC(OEt)3, reflux, overnight; (ii) Me3SiNCO, rt, two days; (b) (i) EtO2CNCS, DMF, rt, 2 h; (ii) 0.5 M NH3 in 1,4-
dioxane or HNMe2$HCl, HgCl2, 0 ꢀC, 15 min, then rt, 4 h; (iii) DMF, microwave 160 ꢀC, 800 s or satd. HCl in 1,4-dioxane, reflux, 4 h; (c) 10, Pd2dba3–CHCl3,
P(t-Bu)3, i-Pr2NEt, DMF, microwave 160 ꢀC, 10 min; (d) i-PrCOCl, pyridine, 115 ꢀC, 2 h; (e) TBAF, HOAc, THF, rt, overnight; (f) NaBH(OAc)3, HOAc,
MeCN, ꢁ40 ꢀC, 30 min then 0 ꢀC, 2 h; (g) DMTrCl, i-Pr2NEt, pyridine, rt, 1 h; (h) NCCH2CH2OP[N(i-Pr)2]2, 1H-tetrazole, HN(i-Pr)2, CH2Cl2, rt, 3 h.
Table 1. Optimization of the Heck reaction between 3e and 10a
and the key steps were a triazine ring formation and a palla-
dium-catalyzed Heck coupling reaction.
Entry
Ligand
Base
Solvent
Isolated yield
of 6e (%)
To synthesize the DNA oligomers containing these artifi-
cial nucleosides, we induced 8b, 8d, and 8e to a phos-
phoramidite reagent. Unfortunately, for 8a it was proved
that the addition of alcohol occurred gradually at the
1
2
3
4
5
6
7
8
9
10
PPh3
AsPh3
(i-Pr)2NEt
(i-Pr)2NEt
(i-Pr)2NEt
(i-Pr)2NEt
(i-Pr)2NEt
(i-Pr)2NEt
(i-Pr)2NEt
NEt3
DMF
DMF
DMF
DMF
THF
NMP
CH3CN
DMF
DMF
DMF
0
10
60
49
0
P(t-Bu)3
P(o-tolyl)3
P(t-Bu)3
P(t-Bu)3
P(t-Bu)3
P(t-Bu)3
P(t-Bu)3
P(t-Bu)3
1
2,3-CN double bond by measuring of the H NMR spec-
0
trum in methanol-d4. Protic solvent is generally used for
handling DNA, so it is difficult to keep the reactive enone
structure. Thus, tritylation of 8a was not performed. A
reaction with nucleosides 8b, 8d, 8e, and 4,40-dimethoxy-
trityl chloride was carried out to obtain a correspond-
ing tritylated product in good to moderate yields. The
desired phosphoramidite reagents for DNA synthesizers
1b, 1d, and 1e were obtained through a reaction with
38
36
35
0
Cy2NMe
Cs2CO3
a
All reactions were carried out using 3e (1 mmol), 10 (1 mmol), base
(4.5 equiv), tris(dibenzylideneacetone)dipalladium(0) chloroform adduct
(10 mol %), and ligand (20 mol %) in solvent (5 mL) under the irradiation
of microwave at 160 ꢀC for 10 min.

11024
H. Oda et al. / Tetrahedron 63 (2007) 11021–11029
O
N
N
O
N
2-cyanoethyl phosphordiamidite in the presence of
1H-tetrazole.
N
N
H
N
N
H
H
N
N
O
H
N
N
O
dR
dR
2.2. Thermal denaturation studies
H
N Me
H
N
Me
8d
N
dR
dR
Oligonucleotide was synthesized using nucleoside phos-
phoramidites 1b, 1d, and 1e synthesized through the method
described above. All oligonucleotides containing these arti-
ficial nucleosides could be synthesized under the conven-
tional condition of a DNA synthesizer. The trityl monitor
indicated that the extension reaction of 1b, 1d, and 1e pro-
ceeded with almost the same efficiency as the phosphorami-
dite reagents bearing a natural base. The oligonucleotide
attached CPG column was treated with ammonia at 55 ꢀC
overnight, and tritylated oligonucleotide was purified and
deprotected by reverse-phase cartridge. Further purification
was performed by HPLC using ion-exchange resin. To our
regret, it was confirmed through the ESI-TOF mass spectrum
that the oligonucleotide containing nucleoside 8b decom-
posed to 2-aminopyridine at the base moiety. While 8b
was not subjected to the Michael-type addition at the stage
of the nucleoside, it was decomposed under strong basic
condition at the deprotection step of the synthesized oligo-
nucleotide. We assume that the ring-opening reaction and
the following hydrolysis occurred at the triazine ring through
ammonia treatment of CPG. The content of the artificial nu-
cleosides 8c and 8d was confirmed by the ESI-TOF mass
spectra. Pyrido[1,2-a][1,3,5]triazin-4-one possessing the
2-amino group could be synthesized at comparably high
yield and was stable under the basic condition.
8c
dC
dC
Figure 2. Slippage of 8c and steric effect of 8d.
8c as shown in Figure 2. On the other hand, a low Tm value
similar to other bases was obtained in 8d due to a steric hin-
drance of the 2-dimethylamino group introduced to the tri-
azine ring. This result is an evidence for the existence of
miss-annealing by slippage.7
3. Conclusion
We synthesized the artificial nucleosides possessing 6–6 fu-
sion ring with particular hydrogen bond pattern, and also
achieved the syntheses of the oligonucleotide containing
these nucleoside. A five- or six-membered ring having lined
two proton donor sites is a complementary nucleoside to the
pyrido[1,2-a][1,3,5]triazin-4-one nucleoside. The syntheses
of such nucleosides are in progress.
4. Experimental
4.1. General
The measurement of the Tm values between the natural base
and the pyrido[1,2-a][1,3,5]triazin-4-one base are shown in
Table 2. Samples were kept at least 2 min at 10 ꢀC and
were then heated from 10 ꢀC to 70 ꢀC at a rate of 0.5 ꢀC/
min. The absorbance at 260 nm was measured every 10 s.
Each oligonucleotide pair containing 8c and 8d gave low
Tm values compared with the correct natural base pair.
This result shows that these artificial nucleosides do not
efficiently form base pairs with natural bases.
1H NMR spectra were recorded on a JEOL JNM-a400
(400 MHz) instrument. The chemical shifts are reported in
parts per million (d) downfield from tetramethylsilane
(d¼0 ppm). The signal patterns are indicated as s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br s, broad singlet.
The coupling constants (J) are given in hertz. The 13C NMR
spectra were recorded on a JEOL JNM-a400 (100 MHz) in-
strument. The chemical shifts are reported in parts per million
(d) downfield from tetramethylsilane (d¼0 ppm). 31P NMR
spectra were recorded on a JEOL JNM-a400 (160 MHz) in-
strument. The chemical shifts are reported in parts per million
(d) downfield from external 85% phosphoric acid (d¼0 ppm).
FABMS spectra were measured on a JEOL JMS-700 instru-
ment and are reported in the order of the molecular ion peak
and remarkable high peaks (intensity). ESI-TOF mass spectra
weremeasured on a JEOL JMS-T100LCinstrument. Prepara-
tive and analytical HPLC were performed with a Shimadzu
LC-6A instrument using a TOSOH TSK-gel DNA-NPR col-
umn. The reactions involving heating in a microwave were
carried out using a Biotage Initiator Sixty instrument. The
products were purified using the flash chromatography tech-
nique on silica gel 60 (40–100 mm) purchased from Kanto
Chemical Co., Inc. Commercial grade reagents and solvents
were used as supplied. Furanoid glycal 10 was prepared ac-
cording to the method described in the literature.4 All reac-
tions sensitive to oxygen or moisture were carried out under
nitrogen atmosphere.
Although thermal denaturation studies for the duplex DNA
containing 8c or 8d clearly indicate the inefficient base pair-
ing between these artificial nucleosides and all four natural
bases, 8c showed higher Tm value against cytidine in contrast
to 8d. We assume that the miss-annealing occurred in using
Table 2. Thermal denaturation studiesa
X/Y
Tm (ꢀC)
DTm (ꢀC)
T/A
C/G
45.5
47.2
32.7
32.2
40.4
36.8
32.6
32.6
33.9
32.3
1.7
ꢁ12.8
ꢁ13.3
ꢁ5.1
A/8c
G/8c
C/8c
T/8c
A/8d
G/8d
C/8d
T/8d
ꢁ8.7
ꢁ12.9
ꢁ12.9
ꢁ11.6
ꢁ13.2
Sequence pair
4.2. 2-Amino-3-iodopyridine (2)8
50-GGTAAC-X-ATGCG-30
30-CCATTG-Y-TACGC-50.
a
A mixture of 2-amino-3-bromopyridine (17.3 mmol,
3.00 g), sodium iodide (34.6 mmol, 5.19 g), copper(I) iodide
Condition: 0.17 mM purified oligonucleotide, 10 mM Na3PO4, 100 mM
NaCl, and 0.1 mM EDTA at pH 7.0.

H. Oda et al. / Tetrahedron 63 (2007) 11021–11029
11025
(5 mol %, 0.165 g), and trans-N,N0-dimethylcyclohexane-
1,2-diamine (10 mol %, 0.246 g) in 1,4-dioxane (36 mL)
was stirred at 110 ꢀC for 24 h. The mixture was poured
into water and extracted with ether. The combined organic
layer was washed with brine, dried, and concentrated. The
product was isolated by flash column chromatography
(n-hexane–ethyl acetate¼1:1, Rf¼0.45). Yield 86%
250 ꢀC (decomposed). Yield 58% (1.66 g). 1H NMR
(400 MHz, DMSO-d6) d 8.63 (d, J¼6.8 Hz, 1H), 8.47 (d,
J¼7.3 Hz, 1H), 7.45 (br s, 1H), 7.44 (br s, 1H), 6.77 (t,
J¼6.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 164.2,
154.0, 150.7, 150.6, 130.0, 114.4, 93.8; HRMS (FAB
positive): 288.95809 calcd for C7H6IN4O, found 288.9579.
1
(3.29 g, light gray powder). H NMR (400 MHz, CDCl3)
4.6. 2-Dimethylamino-9-iodopyrido[1,2-a][1,3,5]triazin-
4-one (3d)
d 8.03 (dd, J¼1.0, 4.9 Hz, 1H), 7.86 (dd, J¼1.5, 7.8 Hz,
1H), 6.40 (dd, J¼4.9, 7.8 Hz, 1H), 4.97 (br s, 2H).
To a solution of 2 (10 mmol, 2.20 g) in DMF (50 mL) ethoxy-
carbonyl isothiocyanate (10 mmol, 1.31 g) was added. After
being stirred at room temperature for 2 h, the reaction
mixture was cooled down to 0 ꢀC and dimethylamine hydro-
chloride (25 mmol, 0.815 g), triethylamine (40 mmol,
4.05 g), and mercury(II) chloride (10 mmol, 2.72 g) were
added. The mixture was stirred at 0 ꢀC for 15 min, then
warmed up to room temperature. Stirring was continued
for 4 h and then the mixture was poured into ethyl acetate
(75 mL). Precipitation was filtered off using a pad of Celite
and the filtrate was evaporated in vacuo. The concentrated
residue was dissolved in 1,4-dioxane (50 mL) saturated with
hydrogen chloride. After being refluxed for 4 h, saturated po-
tassium bicarbonate solution was poured into the reaction
mixture. The organic layer was separated and the aqueous
layer was extracted with ethyl acetate. The combined organic
layer was dried and concentrated. The product was purified
by flash column chromatography (dichloromethane–ethyl
acetate¼4:1, Rf¼0.35). Yield 85% (2.67 g, white powder).
1H NMR (400 MHz, DMSO-d6) d 8.62 (dd, J¼1.0, 6.8 Hz,
1H), 8.47 (dd, J¼1.5, 7.3 Hz, 1H), 6.78 (t, J¼6.8 Hz, 1H),
2.95 (s, 3H), 3.03 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
d 161.4, 153.1, 150.5, 150.2, 129.9, 114.5. 94.6, 36.4, 36.0;
FABMS (m/e) 316.8 (MH+, 56), 153.8 (100), 136.0 (70),
85.9 (68), 306.9 (25); HRMS(FAB positive): 316.98939 calcd
for C9H10IN4O, found 316.9893.
4.3. 9-Iodopyrido[1,2-a][1,3,5]triazin-4-one (3a)
A mixture of 2 (8 mmol, 1.76 g) and triethyl orthoformate
(5 mL) was stirred at 145 ꢀC overnight, then the excess tri-
ethyl orthoformate was removed in vacuo. To a solution of
the resulted imino ether in dichloromethane (10 mL), trime-
thylsilyl isocyanate was added (8 mmol, 0.92 g) and stirred
at room temperature for two days. Volatiles were removed
in vacuo and ether (20 mL) was added to the residue. The re-
sulting precipitate was collected by filter and washed with
ether. Mp 270 ꢀC (decomposed). Yield 39% (0.858 g, gray
1
powder). H NMR (400 MHz, DMSO-d6) d 9.18 (d, J¼
6.3 Hz, 1H), 9.03 (d, J¼7.3 Hz, 1H), 8.76 (s, 1H), 7.52 (t,
J¼6.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 164.5,
154.1, 152.4, 150.5, 130.4, 120.0, 96.7; FABMS (m/e)
273.9 (MH+, 66), 248.8 (70), 220.5 (100); HRMS (FAB
positive): 273.94719 calcd for C7H5IN3O, found 273.9482.
4.4. 2-Methyl-9-iodopyrido[1,2-a][1,3,5]triazin-4-one
(3b)
This compound was prepared using a similar procedure as
used for the synthesis of 3a with 2 (10 mmol, 2.20 g) and tri-
ethyl orthoacetate (5 mL). Yield 48% (1.37 g, white pow-
1
der). Imino ether: H NMR (400 MHz, CDCl3) d 8.32 (br
d, J¼4.9 Hz, 1H), 8.09 (dd, J¼2.0, 7.8 Hz, 1H), 6.71 (dd,
J¼4.9, 7.8 Hz, 1H), 4.36 (q, J¼7.3 Hz, 2H), 1.89 (s, 3H),
1.39 (t, J¼7.3 Hz, 3H). Compound 3b: 1H NMR
(400 MHz, DMSO-d6) d 8.91 (dd, J¼1.5, 6.8 Hz, 1H),
8.77 (dd, J¼1.5, 7.3 Hz, 1H), 7.23 (t, J¼6.8 Hz, 1H), 2.44
(s, 3H); 13C NMR (100 MHz, DMSO-d6) d 174.6, 153.2,
152.1, 150.6, 130.1, 119.2, 96.2, 26.5; FABMS (m/e) 287.8
(M+, 15), 153.8 (100), 136.0 (66), 107.5 (20), 89.8 (20);
HRMS (FAB positive): 287.96284 calcd for C8H7IN3O,
found 287.9643.
4.7. 2-Isobutyrylamino-9-iodopyrido[1,2-a][1,3,5]tri-
azin-4-one (3e)
To a solution of 3c (0.5 mmol, 144 mg) in pyridine (2.8 mL)
isobutyryl chloride (0.75 mmol, 79 mL) was added drop-
wise. After being stirred at 115 ꢀC for 2 h, the reaction mix-
ture was concentrated in vacuo and then purified by flash
column chromatography (n-hexane–ethyl acetate¼2:1).
Yield 98% (175 mg). 1H NMR (400 MHz, DMSO-d6)
d 10.42 (s, 1H), 8.84 (d, J¼7.8 Hz, 1H), 8.70 (d,
J¼7.3 Hz, 1H), 7.10 (t, J¼7.3 Hz, 1H), 3.51–3.42 (m, 1H),
1.11 (d, J¼6.8 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)
d 176.8, 160.8, 154.5, 152.0, 151.1, 130.3, 117.7, 95.4,
34.3, 19.2; HRMS (FAB positive): 358.99995 calcd for
C11H12IN4O2, found 359.0016.
4.5. 2-Amino-9-iodopyrido[1,2-a][1,3,5]triazin-4-one
(3c)
To a solution of 2 (10 mmol, 2.20 g) in DMF (50 mL) ethoxy-
carbonyl isothiocyanate (10 mmol, 1.31 g) was added. After
stirring at room temperature for 2 h, the reaction mixture was
cooled to 0 ꢀC and 0.5 M ammonia in 1,4-dioxane
(35 mmol, 70 mL) and mercury(II) chloride (10 mmol,
2.72 g) were added. The mixture was stirred at 0 ꢀC for
15 min, then warmed up to room temperature. Stirring was
continued until 4 h had passed, then the mixture was poured
into ethyl acetate (75 mL). Precipitation was filtered off us-
ing a pad of Celite and the filtrate was evaporated in vacuo.
The residue was dissolved in DMF and heated using a micro-
wave at 160 ꢀC for 800 s. The solvent was removed in vacuo
and the residue was recrystallized from acetonitrile. Mp
4.8. General procedure of Heck-type
palladium-catalyzed reaction
A reaction tube containing a mixture of aryl iodide (1 equiv),
tris(dibenzylidene)acetone dipalladium(0) chloroform ad-
duct (5 mol %), tri(tert-butyl)phosphine (20 mol %), fura-
noid glycal 10 (1 equiv), and diisopropylethylamine
(4.5 equiv) in DMF (5 mL/mmol) was sealed. The mixture
was heated using a microwave at 160 ꢀC for 10 min. The
product was isolated by flash column chromatography.

11026
H. Oda et al. / Tetrahedron 63 (2007) 11021–11029
4.8.1. 2R,5R-2-(2-Aminopyridin-3-yl)-4-(tert-butyldime-
thylsiloxy)-5-(tert-butyldimethylsiloxymethyl)-2,5-dihy-
drofuran (4). 1H NMR (400 MHz, CDCl3) d 7.97 (dd,
J¼1.5, 4.9 Hz, 1H), 7.30 (dd, J¼1.5, 7.3 Hz, 1H), 6.55
(dd, J¼5.4, 7.3 Hz, 1H), 5.63 (d, J¼3.4 Hz, 1H), 5.32 (br
s, 2H), 4.86 (s, 1H), 4.53 (dd, J¼2.0, 2.4 Hz, 1H), 3.87
(dd, J¼2.9, 11.7 Hz, 1H), 3.80 (dd, J¼2.9, 11.7 Hz, 1H),
0.95 (s, 9H), 0.86 (s, 9H), 0.24 (s, 3H), 0.23 (s, 3H), 0.01
(s, 3H), 0.00 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 157.7, 152.0, 147.5, 136.9, 119.2, 112.9, 99.9, 85.3,
83.2, 63.2, 26.0, 25.6, 18.7, 18.1, ꢁ4.9, ꢁ5.4, ꢁ5.5; FABMS
(m/e) 437.0 (MH+, 19), 287.0 (10), 246.9 (7), 74.4 (100);
HRMS (FAB positive): 437.26502 calcd for C22H41N2O3Si2,
found 437.2674.
6.99 (t, J¼7.3 Hz, 1H), 6.04 (d, J¼3.9 Hz, 1H), 5.09 (s,
1H), 4.56 (s, 1H), 3.84 (dd, J¼2.0, 11.2 Hz, 1H), 3.72 (dd,
J¼2.9, 11.2 Hz, 1H), 0.91 (s, 9H), 0.83 (s, 9H), 0.22 (s,
3H), 0.16 (s, 3H), 0.04 (s, 3H), 0.01 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 163.9, 153.8, 151.2, 150.3, 138.0,
136.2, 128.1, 113.4, 100.4, 84.1, 78.8, 63.2, 25.8, 25.4,
18.4, 17.9, ꢁ4.9, ꢁ5.2, ꢁ5.4; HRMS (FAB positive):
505.26609 calcd for C24H41N4O4Si2, found 505.2672.
4.8.6. 2-Dimethylamino-9-[2R,5R-4-(tert-butyldimethyl-
siloxy)-5-(tert-butyldimethylsiloxymethyl)-2,5-dihydro-
furan-2-yl]-pyrido[1,2-a][1,3,5]triazin-4-one (6d). 1H
NMR (400 MHz, CDCl3) d 8.68 (d, J¼6.8 Hz, 1H), 8.20
(d, J¼6.8 Hz, 1H), 6.82 (t, J¼6.8 Hz, 1H), 6.12 (s, 1H),
5.11 (s, 1H), 4.64 (br s, 1H), 3.95 (dd, J¼2.4, 11.2 Hz,
1H), 3.79 (dd, J¼3.4, 11.7 Hz, 1H), 3.25 (s, 3H), 3.24 (s,
3H), 0.94 (s, 9H), 0.87 (s, 9H), 0.22 (s, 3H), 0.16 (s, 3H),
0.08 (s, 3H), 0.03 (s, 3H); FABMS (m/e) 532.8 (MH+, 51),
74.2 (100), 216.7 (3), 387.1 (3); HRMS (FAB positive):
533.29739 calcd for C26H45N4O4Si2, found 533.2984.
4.8.2. 2-Methyl-9-[2R,5R-5-(tert-butyldimethylsiloxy-
methyl)-4-oxo-tetrahydrofuran-2-yl]-pyrido[1,2-
a][1,3,5]triazin-4-one (5b). H NMR (400 MHz, CDCl3)
1
d 8.99 (dd, J¼1.0, 6.8 Hz, 1H), 8.48 (dd, J¼1.5, 7.3 Hz,
1H), 7.35 (t, J¼7.3 Hz, 1H), 5.78 (dd, J¼6.3, 10.7 Hz, 1H),
4.15 (t, J¼2.0 Hz, 1H), 4.04 (dd, J¼2.0, 11.2 Hz, 1H), 3.99
(dd, J¼2.9, 11.2 Hz, 1H), 3.31 (dd, J¼6.3, 18.1 Hz, 1H),
2.54 (s, 3H), 2.19 (dd, J¼10.7, 18.1 Hz, 1H), 0.84 (s, 9H),
0.09 (s, 3H), 0.05 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 212.6, 175.0, 152.2, 150.2, 137.2, 136.8, 128.2, 117.1,
82.1, 71.9, 62.4, 44.5, 26.6, 25.5, 18.0, ꢁ5.7, ꢁ5.9; HRMS
(FAB positive): 390.18436 calcd for C19H28N3O4Si, found
390.1867.
4.8.7. 2-Isobutyrylamino-9-[2R,5R-4-(tert-butyldime-
thylsiloxy)-5-(tert-butyldimethylsiloxymethyl)-2,5-dihy-
drofuran-2-yl]-pyrido[1,2-a][1,3,5]triazin-4-one (6e). H
1
NMR (400 MHz, CDCl3) d 8.80 (d, J¼6.8 Hz, 1H), 8.52
(d, J¼7.2 Hz, 1H), 7.70 (s, 1H), 7.10 (t, J¼7.2 Hz, 1H),
6.22 (s, 1H), 5.11 (s, 1H), 4.66 (s, 1H), 3.97–3.79 (m, 2H),
3.61–3.55 (m, 1H), 1.27 (d, J¼6.8 Hz, 6H), 0.93 (s, 9H),
0.89 (s, 9H), 0.24 (s, 3H), 0.17 (s, 3H), 0.09 (s, 3H), 0.03
(s, 3H); 13C NMR (100 MHz, CDCl3) d 177.7, 159.5,
153.2, 150.9, 150.5, 139.8, 138.2, 128.3, 116.6, 100.3,
84.3, 79.2, 63.0, 35.1, 25.9, 25.5, 19.1, 19.0, 18.4, 18.0,
ꢁ4.9, ꢁ5.2, ꢁ5.4; FABMS (m/e) 574.9 (M+, 93), 429.0
(7), 74.2 (100); HRMS (FAB positive): 575.30795 calcd
for C28H47N4O5Si2, found 575.3086.
4.8.3. 2-Dimethylamino-9-[2R,5R-5-(tert-butyldimethyl-
siloxymethyl)-4-oxo-tetrahydrofuran-2-yl]-pyrido[1,2-
a][1,3,5]triazin-4-one (5d). H NMR (400 MHz, CDCl3)
1
d 8.73 (dd, J¼1.5, 7.3 Hz, 1H), 8.15 (d, J¼6.8 Hz, 1H),
7.35 (t, J¼6.8 Hz, 1H), 5.57 (dd, J¼5.9, 10.2 Hz, 1H),
4.12 (t, J¼2.4 Hz, 1H), 4.02 (dd, J¼2.4, 11.2 Hz, 1H), 3.97
(dd, J¼2.4, 11.2 Hz, 1H), 3.27–3.16 (m, 1H), 3.24 (s, 3H),
3.21 (s, 3H), 2.19 (dd, J¼10.2, 18.6 Hz, 1H), 0.83 (s, 9H),
0.08 (s, 3H), 0.04 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 213.7, 161.4, 152.7, 150.8, 135.6, 134.8, 128.6, 112.4,
82.3, 72.3, 62.6, 44.2, 37.05, 36.95, 25.7, 18.2, ꢁ5.5,
ꢁ5.7; FABMS (m/e) 419.0 (MH+, 57), 74.2 (100), 216.7
(19); HRMS (FAB positive): 419.21091 calcd for
C20H31N4O4Si, found 419.2128.
4.9. General procedure of the deprotection of Heck
reaction adduct 6
A mixture of 6 (1 equiv), 75% tetrabutylammonium fluoride
(4 equiv), acetic acid (8 equiv), and THF (10 mL/mmol) was
stirred at room temperature overnight. After concentration in
vacuo, the product was purified by flash column chromato-
graphy.
4.8.4. 9-[2R,5R-4-(tert-butyldimethylsiloxy)-5-(tert-butyl-
dimethylsiloxymethyl)-2,5-dihydrofuran-2-yl]-pyr-
ido[1,2-a][1,3,5]triazin-4-one (6a). 1H NMR (400 MHz,
CDCl3) d 9.01 (dd, J¼1.5, 6.8 Hz, 1H), 8.71 (d, J¼7.3 Hz,
1H), 8.60 (s, 1H), 7.38 (t, J¼6.8 Hz, 1H), 6.34 (s, 1H),
5.04 (s, 1H), 4.66 (dd, J¼2.5, 4.5 Hz, 1H), 3.97 (dd,
J¼2.4, 11.7 Hz, 1H), 3.83 (dd, J¼2.4, 11.2 Hz, 1H), 0.94
(s, 9H), 0.87 (s, 9H), 0.24 (s, 3H), 0.17 (s, 3H), 0.09 (s,
3H), 0.04 (s, 3H); 13C NMR (100 MHz, CDCl3) d 163.8,
153.0, 150.6, 150.5, 139.7, 139.1, 128.1, 118.5, 100.3,
84.3, 79.0, 62.9, 25.9, 25.5, 18.4, 17.9, ꢁ4.9, ꢁ5.2, ꢁ5.4;
HRMS (FAB positive): 490.25519 calcd for C24H40N3O4Si2,
found 490.2556.
4.9.1. 9-(2R,5R-5-Hydroxymethyl-4-oxo-tetrahydro-
furan-2-yl)-pyrido[1,2-a][1,3,5]triazin-4-one (7a). 1H
NMR (400 MHz, DMSO-d6) d 8.96 (d, J¼6.8 Hz, 1H),
8.61 (d, J¼7.3 Hz, 1H), 8.50 (s, 1H), 7.67 (t, J¼6.8 Hz,
1H), 5.77 (dd, J¼6.3, 9.8 Hz, 1H), 5.08 (t, J¼5.4 Hz, 1H),
4.14 (s, 1H), 3.82–3.65 (m, 2H), 3.06 (dd, J¼6.3, 18.1 Hz,
1H), 2.28 (dd, J¼9.8, 18.1 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) d 213.5, 163.4, 152.8, 150.4, 138.4, 136.6,
128.7, 119.0, 82.3, 71.4, 60.4, 44.0; FABMS (m/e) 261.1
(M+, 7), 236.8 (7), 218.8 (10), 190.7 (15), 58.6 (100);
HRMS (FAB positive): 262.08223 calcd for C12H12N3O4,
found 262.0820.
4.8.5. 2-Amino-9-[2R,5R-4-(tert-butyldimethylsiloxy)-5-
(tert-butyldimethylsiloxymethyl)-2,5-dihydrofuran-2-
yl]-pyrido[1,2-a][1,3,5]triazin-4-one (6c). 1H NMR
(400 MHz, DMSO-d6) d 8.58 (dd, J¼1.5, 6.8 Hz, 1H),
8.11 (d, J¼6.8 Hz, 1H), 7.32 (br s, 1H), 7.31 (br s, 1H),
4.9.2. 2-Methyl-9-(2R,5R-5-hydroxymethyl-4-oxo-tetra-
hydrofuran-2-yl)-pyrido[1,2-a][1,3,5]triazin-4-one (7b).
1H NMR (400 MHz, CD3OD) d 8.89 (dd, J¼1.5, 6.8 Hz,
1H), 8.51 (dd, J¼1.0, 6.8 Hz, 1H), 7.47 (t, J¼7.3 Hz, 1H),
5.69 (dd, J¼6.4, 10.3 Hz, 1H), 4.06 (t, J¼3.4 Hz, 1H),

H. Oda et al. / Tetrahedron 63 (2007) 11021–11029
11027
3.86 (dd, J¼2.9, 12.2 Hz, 1H), 3.80 (dd, J¼3.4, 12.2 Hz,
1H), 3.15 (dd, J¼6.3, 18.1 Hz, 1H), 2.22 (dd, J¼10.2,
18.1 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 213.6,
173.5, 152.2, 149.9, 138.1, 135.9, 128.4, 118.0, 82.3, 71.6,
60.5, 44.0, 26.4; HRMS (FAB positive): 276.09788 calcd
for C13H14N3O4, found 276.0979.
J¼5.9, 9.8 Hz, 1H), 4.33 (t, J¼2.9 Hz, 1H), 4.02 (dt,
J¼2.9, 4.9 Hz, 1H), 3.77–3.66 (m, 2H), 2.64 (ddd, J¼2.0,
5.9, 13.2 Hz, 1H), 1.85 (ddd, J¼5.9, 9.8, 13.2 Hz, 1H); 13C
NMR (100 MHz, CDCl3) d 164.4, 154.5, 152.3, 140.1,
139.9, 129.7, 120.4, 89.1, 76.5, 74.2, 63.8, 43.4.
4.10.2. 2-Methyl-9-(20-deoxy-b-D-ribofuranosyl)-pyri-
do[1,2-a][1,3,5]triazin-4-one (8b). 1H NMR (400 MHz,
DMSO-d6) d 8.55 (d, J¼6.8 Hz, 1H), 8.35 (d, J¼7.3 Hz,
1H), 7.51 (t, J¼7.3 Hz, 1H), 5.54 (dd, J¼6.3, 9.7 Hz, 1H),
5.15 (d, J¼3.9 Hz, 1H), 4.84 (t, J¼5.4 Hz, 1H), 4.20 (br s,
1H), 3.89–3.85 (m, 1H), 3.57–3.44 (m, 2H), 2.42 (s, 3H),
1.76–1.68 (m, 1H), 1.61–1.52 (m, 1H); FABMS (m/e)
278.0 (MH+, 10), 242.0 (100), 85.9 (32); HRMS (FAB posi-
tive): 278.11353 calcd for C13H16N3O4, found 278.1148.
4.9.3. 2-Amino-9-(2R,5R-5-hydroxymethyl-4-oxo-tetra-
hydrofuran-2-yl)-pyrido[1,2-a][1,3,5]triazin-4-one (7c).
1H NMR (400 MHz, DMSO-d6) d 8.60 (dd, J¼1.0, 6.8 Hz,
1H), 8.19 (d, J¼6.8 Hz, 1H), 7.34 (br s, 1H), 7.20 (br s, 1H),
7.08 (t, J¼6.8 Hz, 1H), 5.44 (dd, J¼6.3, 10.7 Hz, 1H), 5.03
(br s, 1H), 4.10 (t, J¼2.9 Hz, 1H), 3.78–3.62 (m, 2H), 3.12
(dd, J¼5.9, 18.1 Hz, 1H), 2.25 (dd, J¼10.7, 18.1 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) d 214.5, 163.8, 153.5,
150.6, 136.4, 134.0, 128.3, 113.2, 82.5, 71.8, 60.6, 44.1;
HRMS (FAB positive): 277.09313 calcd for C12H13N4O4,
found 277.0937.
4.10.3. 2-Amino-9-(20-deoxy-b-D-ribofuranosyl)-pyri-
do[1,2-a][1,3,5]triazin-4-one (8c). 1H NMR (400 MHz,
DMSO-d6) d 8.56 (d, J¼6.8 Hz, 1H), 8.02 (d, J¼6.8 Hz,
1H), 7.27 (br s, 2H), 7.02 (t, J¼6.8 Hz, 1H), 5.37 (dd,
J¼5.9, 9.3 Hz, 1H), 5.10 (d, J¼3.9 Hz, 1H), 4.81 (t,
J¼5.9 Hz, 1H), 4.10 (br s, 1H), 3.82 (br s, 1H), 3.53–3.43
(m, 2H), 2.42 (dd, J¼4.9, 11.7 Hz, 1H), 1.67 (ddd, J¼5.9,
9.8, 12.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
d 163.6 153.2, 150.2, 135.8, 135.7, 127.6, 112.8, 87.4,
74.0, 72.0, 62.1, 41.5; FABMS (m/e) 279.0 (MH+, 4),
153.8 (100), 136.0 (64), 253.0 (16); HRMS (FAB positive):
279.10878 calcd for C12H15N4O4, found 279.1167.
4.9.4. 2-Dimethylamino-9-(2R,5R-5-hydroxymethyl-4-
oxo-tetrahydrofuran-2-yl)-pyrido[1,2-a][1,3,5]triazin-4-
one (7d). 1H NMR (400 MHz, DMSO-d6) d 8.60 (d,
J¼6.8 Hz, 1H), 8.22 (d, J¼6.8 Hz, 1H), 7.08 (t, J¼6.8 Hz,
1H), 5.57 (dd, J¼6.4, 10.3 Hz, 1H), 5.01 (t, J¼6.4 Hz,
1H), 4.11 (t, J¼2.9 Hz, 1H), 3.76 (ddd, J¼2.9, 5.4,
12.2 Hz, 1H), 3.68 (ddd, J¼3.42, 6.4, 12.2 Hz, 1H), 3.19
(s, 3H), 3.119 (s, 3H), 3.117 (dd, J¼7.3, 18.1 Hz, 1H),
2.22 (dd, J¼10.2, 18.1 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) d 214.1, 160.9, 152.4, 149.7, 136.0, 134.4,
128.0, 113.1, 82.2, 71.6, 60.4, 43.5, 36.5, 36.1; FABMS
(m/e) 304.9 (MH+, 22), 242.0 (100), 153.8 (46), 136.0
(31), 183.8 (8); HRMS (FAB positive): 305.12443 calcd
for C14H17N4O4, found 305.1260.
4.10.4. 2-Dimethylamino-9-(20-deoxy-b-D-ribofurano-
syl)-pyrido[1,2-a][1,3,5]triazin-4-one (8d). 1H NMR
(400 MHz, DMSO-d6) d 8.54 (d, J¼6.8 Hz, 1H), 8.01 (d,
J¼7.3 Hz, 1H), 7.02 (t, J¼6.8 Hz, 1H), 5.35 (t, J¼6.8 Hz,
1H), 5.09 (t, J¼2.0 Hz, 1H), 4.78 (t, J¼5.9 Hz, 1H), 4.15
(br s, 1H), 3.84 (br s, 1H), 3.51–3.44 (m, 2H), 3.20 (s,
3H), 3.12 (s, 3H), 2.54–2.44 (m, 1H), 1.70–1.62 (m, 1H);
13C NMR (100 MHz, DMSO-d6) d 160.9 152.2, 147.0,
136.1, 135.4, 127.4, 113.1, 87.3, 74.4, 71.9, 62.1, 41.2,
36.4, 36.1; FABMS (m/e) 306.9 (MH+, 46), 153.8 (100),
136.0 (67), 85.9 (48), 288.9 (15), 237.8 (11); HRMS (FAB
positive): 307.14008 calcd for C14H19N4O4, found
307.1409.
4.9.5. 2-Isobutyrylamino-9-(2R,5R-5-hydroxymethyl-4-
oxo-tetrahydrofuran-2-yl)-pyrido[1,2-a][1,3,5]triazin-4-
one (7e). 1H NMR (400 MHz, CDCl3) d 8.95 (d, J¼7.2 Hz,
1H), 8.63 (br s, 1H), 8.36 (d, J¼7.2 Hz, 1H), 7.30 (t,
J¼6.8 Hz, 1H), 5.69 (dd, J¼6.4, 10.7 Hz, 1H), 4.16 (t, J¼
3.4 Hz, 1H), 4.04 (d, J¼3.4 Hz, 2H), 3.59 (dd, J¼6.3,
18.5 Hz, 1H), 3.02–2.90 (m, 1H), 2.37 (dd, J¼10.7,
18.5 Hz, 1H), 1.26 (d, J¼6.8 Hz, 6H); 13C NMR (100 MHz,
DMSO-d6) d 214.2, 168.9, 145.3, 143.5, 129.5, 128.0,
120.2, 108.3, 105.0, 74.4, 64.1, 52.3, 30.3, 27.4, 10.2, 10.1;
FABMS (m/e) 346.9 (MH+, 6), 306.9 (26), 288.9 (14), 242.0
(9), 153.8 (100), 136.0 (63); HRMS (FAB positive):
347.13500 calcd for C16H19N4O5, found 347.1365.
4.10.5. 2-Isobutyrylamino-9-(20-deoxy-b-D-ribofurano-
syl)-pyrido[1,2-a][1,3,5]triazin-4-one (8e). 1H NMR
(400 MHz, DMSO-d6) d 10.44 (s, 1H), 8.79 (d, J¼7.3 Hz,
1H), 8.29 (d, J¼7.3 Hz, 1H), 7.39 (t, J¼7.3 Hz, 1H), 5.48
(dd, J¼6.4, 9.3 Hz, 1H), 5.13 (d, J¼3.9 Hz, 1H), 4.83 (br
s, 1H), 4.19 (br s, 1H), 3.87 (d, J¼2.9 Hz, 1H), 3.55–3.45
(m, 2H), 3.18–3.10 (m, 1H), 2.65–2.58 (m, 1H), 1.77–1.69
(m, 1H), 1.11 (d, J¼6.3 Hz, 6H); 13C NMR (100 MHz,
DMSO-d6) d 176.2, 159.8, 152.9, 150.6, 137.7, 137.3,
128.0, 116.4, 87.5, 74.3, 71.9, 62.1, 41.5, 34.4, 19.1, 19.0;
FABMS (m/e) 348.9 (MH+, 82), 242.0 (100), 186.0 (85),
85.9 (97); HRMS (FAB positive): 349.15065 calcd for
C16H21N4O5, found 349.1526.
4.10. General procedure of the reduction of 30-keto-
nucleoside 7
To a stirring solution of 7 (1 equiv) in acetic acid (20 mL/
mmol) and acetonitrile (60 mL/mmol), sodium tri(acetoxy)-
borohydride (1.1 equiv) was slowly added at ꢁ40 ꢀC. Stir-
ring was continued for 1 h and then the mixture was
allowed to warm to room temperature. The solvent was re-
moved in vacuo and the product was purified by flash column
chromatography.
4.11. General procedure of tritylation of artificial
nucleoside 8
4.10.1. 9-(20-Deoxy-b-D-ribofuranosyl)-pyrido[1,2-
a][1,3,5]triazin-4-one (8a). H NMR (400 MHz, DMSO-
1
A solution of 8 (1 equiv), diisopropylethylamine (3 equiv),
and 4,40-dimethoxytrityl chloride (1.5 equiv) in pyridine
(10 mL/mmol) was stirred at room temperature for 1 h.
d6) d 9.02 (dd, J¼1.5, 6.8 Hz, 1H), 8.53 (dd, J¼1.0,
7.3 Hz, 1H), 8.49 (s, 1H), 7.60 (t, J¼7.3 Hz, 1H), 5.71 (dd,

11028
H. Oda et al. / Tetrahedron 63 (2007) 11021–11029
The mixture was concentrated in vacuo and the residue was
subjected to purification by flash column chromatography.
b-D-ribofuranosyl]-pyrido[1,2-a][1,3,5]triazin-4-one
(1e). 31P NMR (160 MHz, DMSO-d6) d 147.56, 146.98.
FABMS (m/e) 851.3 (M+, 12), 302.1 (97), 363.8 (15),
459.8 (6).
4.11.1. 2-Methyl-9-[20-deoxy-50-(4,40-dimethoxytrityl)-
b-D-ribofuranosyl]-pyrido[1,2-a][1,3,5]triazin-4-one
(9b). 1H NMR (400 MHz, CDCl3) d 8.92 (d, J¼6.8 Hz, 1H),
8.25 (d, J¼6.8 Hz, 1H), 7.46–7.17 (m, 10H), 6.86–6.78
(m, 4H), 5.71 (dd, J¼6.3, 8.9 Hz, 1H), 4.44 (dt, J¼2.9,
5.8 Hz, 1H), 4.17 (dd, J¼4.9, 8.3 Hz, 1H), 3.79 (s, 6H),
3.40 (dd, J¼4.9, 10.3 Hz, 1H), 3.36 (dd, J¼4.9, 10.3 Hz,
1H), 2.74 (ddd, J¼2.4, 6.3, 13.2 Hz, 1H), 2.55 (s, 3H),
1.91 (ddd, J¼6.3, 9.3, 15.6 Hz, 1H); FABMS (m/e) 579.9
(M+, 9), 441.0 (40), 303.0 (40), 146.9 (23), 58.6 (100);
HRMS (FAB positive): 580.24421 calcd for C34H34N3O6,
found 580.2450.
4.13. Synthesis of oligomer
All oligonucleotides were synthesized at 0.2 mmol scale
on a Millipore Expedite 8909 DNA synthesizer using con-
ventional b-cyanoethyl phosphoramidite chemistry. The
standard and the modified bases were dissolved in anhy-
drous acetonitrile (0.1 M final concentration). All oligo-
mers were synthesized ‘trityl on’. After the synthesis,
the solid supports were treated overnight at 55 ꢀC with
concentrated ammonia (1 mL). The crude tritylated oligo-
nucleotide was purified and deprotected by reverse-phase
cartridge (Poly-Pak). Further purification was carried out
using ion-exchange column (TOSOH, TSK-gel DNA-
NPR) with buffer A (Tris–HCl 0.1 M, pH 9.0) and buffer
B (Tris–HCl 0.1 M, sodium chloride 1.0 M, pH 9.0). A
linear gradient of 35–50% buffer B over 20 min at
a flow of 0.75 mL/min was used. The oligonucleotides
were desalted and dried on a Speed-Vac evaporator to
yield colorless solids. ESI-TOF mass spectra are used
for characterization.
4.11.2. 2-Dimethylamino-9-[20-deoxy-50-(4,40-dimethoxy-
trityl)-b-D-ribofuranosyl]-pyrido[1,2-a][1,3,5]triazin-4-
1
one (9d). H NMR (400 MHz, CDCl3) d 8.68 (dd, J¼1.5,
6.8 Hz, 1H), 7.91 (d, J¼6.8 Hz, 1H), 7.47–7.17 (m, 10H),
6.87–6.81 (m, 4H), 6.75 (t, J¼6.8 Hz, 1H), 5.52 (t,
J¼7.8 Hz, 1H), 4.37 (br s, 1H), 4.12–4.09 (m, 1H), 3.79
(s, 6H), 3.40 (dd, J¼4.9, 10.2 Hz, 1H), 3.33 (dd, J¼4.9,
10.2 Hz, 1H), 3.24 (s, 6H), 2.65 (ddd, J¼3.4, 6.8, 13.2 Hz,
1H), 1.91 (ddd, J¼6.3, 8.8, 15.1 Hz, 1H); FABMS (m/e)
609.0 (MH+, 12), 58.6 (100), 302.9 (58), 441.0 (35);
HRMS (FAB positive): 609.27076 calcd for C35H37N4O6,
found 609.2706.
50-GGTAAC-8d-ATGCG-30 (E.M. 3748.4): ESI-mass:
1907.5 (2ꢁ, 3Na+), 1917.5 (2ꢁ, 4Na+), 1929.0 (2ꢁ,
5Na+), 1938.9 (2ꢁ, 6Na+).
50-CGCAT-8d -GTTACC-30 (E.M. 3659.40): ESI-mass:
1219.1 (3ꢁ), 1226.4 (3ꢁ, Na+), 1233.8 (3ꢁ, 2Na+),
1240.9 (3ꢁ, 3Na+), 1248.4 (3ꢁ, 4Na+), 1255.5 (3ꢁ,
5Na+), 1263.1 (3ꢁ, 6Na+), 1270.27 (3ꢁ, 7Na+).
4.11.3. 2-Isobutyrylamino-9-[20-deoxy-50-(4,40-dimethoxy-
trityl)-b-D-ribofuranosyl]-pyrido[1,2-a][1,3,5]triazin-4-
one (9e). H NMR (400 MHz, DMSO-d6) d 10.46 (s, 1H),
1
8.76 (d, J¼6.8 Hz, 1H), 8.11 (d, J¼6.8 Hz, 1H), 7.44–7.18
(m, 10H), 6.93–6.85 (m, 4H), 5.48 (t, J¼7.8 Hz, 1H), 5.20
(d, J¼4.4 Hz, 1H), 4.14 (br s, 1H), 4.04–3.96 (m, 1H),
3.74 (s, 6H), 3.20–3.15 (m, 3H), 2.68–2.61 (m, 1H), 1.82–
1.73 (m, 1H), 1.10 (d, J¼6.8 Hz, 6H); HRMS (FAB posi-
tive): 651.28133 calcd for C37H39N4O7, found 651.2799.
4.14. Tm analysis
Purified oligonucleotides, 0.5 mmol of each, were dissolved
in 3.0 mL of the buffer (10 mM sodium phosphate, 100 mM
sodium chloride, and 0.1 mM EDTA, pH 7.0). Samples were
kept at least 2 min at 10 ꢀC and were then heated from
1070 ꢀC to 70 ꢀC at a rate of 0.5 ꢀC/min. The absorbance
at 260 nm was measured every 10 s.
4.12. General procedure of the preparation of phos-
phoramidite 1
Compound 9 (1 equiv) was dissolved in anhydrous CH2Cl2
(10 mL/mmol). Then, diisopropylethylamine (6 equiv),
1H-tetrazole (6 equiv), and 2-cyanoethyl-N,N,N0,N0-tetra-
isopropylphosphordiamidite (1.5 equiv) were added and
stirred at room temperature for 3 h under nitrogen atmo-
sphere. The reaction mixture was purified by silica gel col-
umn chromatography with CH2Cl2–THF as eluent to give
phosphoramidite.
Acknowledgements
This work has been supported by a Research Grant for the
RIKEN Genome Exploration Research Project from the
Ministry of Education, Culture, Sports, Science and Tech-
nology of the Japanese Government to Y.H.
4.12.1. 2-Methyl-9-[30-(2-cyanoethoxydiisopropyl-
amino)phosphinyl-20-deoxy-50-(4,40-dimethoxytrityl)-b-
D-ribofuranosyl]-pyrido[1,2-a][1,3,5]triazin-4-one (1b).
31P NMR (160 MHz, CDCl3) d 148.8, 148.0.
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