A new synthetic route towards the mono-O-protected anti-conformationally constrained pyrimidine acyclic nucleoside.

Article abstract of DOI:10.1248/cpb.50.1028

Novel synthetic approach to mono-O-protected anti-conformationally constrained pyrimidine acyclic nucleoside was attained from the coupling of lithiated 2,4-dimethoxy-6-methylpyrimidine with 1-benzyloxy-3-(tert-butyldiphenylsilyloxy)propan-2-one, followed by the sequential reactions of methylthiomethylation, cyclization, hydroxylation, and dealkylation.

Full text of DOI:10.1248/cpb.50.1028

1028
Chem. Pharm. Bull. 50(8) 1028—1030 (2002)
Vol. 50, No. 8
A New Synthetic Route towards the Mono-O-protected Anti-
conformationally Constrained Pyrimidine Acyclic Nucleoside
a
b
,a
*
Chi-Tsan LIU, Tsu-Chiang TU, and Ling-Yih HSU
a School of Pharmacy, National Defense Medical Center; P.O. Box 90048-508, Neihu, Taipei, Taiwan, ROC: and
b Department of Pharmacy, Tri-Service General Hospital; Taipei, Taiwan 114, Republic of China.
Received January 18, 2002; accepted March 6, 2002
Novel synthetic approach to mono-O-protected anti-conformationally constrained pyrimidine acyclic nucleo-
side was attained from the coupling of lithiated 2,4-dimethoxy-6-methylpyrimidine with 1-benzyloxy-3-(tert-
butyldiphenylsilyloxy)propan-2-one, followed by the sequential reactions of methylthiomethylation, cyclization,
hydroxylation, and dealkylation.
Key words new synthetic route; pyrimidine acyclic nucleoside; oligonucleotide
Modified oligonucleotides have become an area of in-
1-Benzyloxy-3-(tert-butyldiphenylsilyloxy)propan-2-one
creased activity in the last decade due to their potential use (4) was prepared in four steps from epichlorohydrin. Ring
as antiviral, antitumoral agents and effective tools for many opening reaction of benzyl glycidyl ether (1), prepared fol-
molecular biology applications based on the hybridization lowing the published procedure,7) in the presence of iodine
technique.1—3) However, some problems still remain, which in the acetonitrile and water solution afforded 3-benzyl-
are connected with their stability against enzymatic break- oxypropan-1,2-diol (2) in a yield of 63%. Silylation of 2
down, solubility in biological fluids, ability to penetrate into with tert-butylchlorodiphenylsilane in the presence of cat-
cell membrane and affinity of binding to their duplexes. In alytic amount of 4-N,N-dimethylaminopyridine (DMAP) fur-
order to overcome these problems oligonucleotides are modi- nished 64% of oily 1-benzyloxy-3-(tert-butyldiphenylsilyl-
fied either in the base, sugar or phosphate moiety. Acyclic oxy)propan-2-ol (3). In previous work,6) the hydroxy group
oligonucleotides, one of these modified oligonucleotides, of 1,3-dibenzyloxy-2-propanol was oxidized to its corre-
have been studied for these purpose.4)
sponding ketone with N-chlorosuccinimide and dimethylsul-
Recently, we reported5) the synthesis and nuclease resis- foxide. This procedure resulted in a repulsive odor. However,
tance of dinucleosides monophosphate containing an anti- by using pyridinium chlorochromate (PCC) as an oxidizing
conformationally constrained acyclic thymidine. These dinu- agent this unpleasant odor could be circumvented. Thus, oxi-
cleotides possess potent enzymatic stability toward nuclease dation of the hydroxyl group of 3 with PCC at reflux temper-
S1, bovine spleen phosphodiesterase and snake venom phos- ature in dichloromethane provided 87% of oily 4.
phodiesterase. It therefore seemed reasonable that these
Treatment of 2,4-dimethoxy-6-methylpyrimidine with n-
nucleases stability will still remain if our anti-conformation- butyllithium in dry tetrahydrofuran at Ϫ70 °C gave lithio de-
ally constrained acyclic nucleosides could incorporate into rivative, which reacted with 4 at Ϫ70 °C to afford an oily 6-
oligonucleotide. Thus we initiate the modification of our {2-[1-benzyloxy-3-(tert-butyldiphenylsilyloxy)-2-hydroxy]-
compounds suitable as building units for automated oligonu- propyl}-methyl-2,4-dimethoxypyrimidine (5) in a yield of
cleotide synthesizer. To be good building units for oligonu- 92%. Conversion of the hydroxyl group of 5 to the corre-
cleotide synthesizer, nucleotides require both 5Ј-dimethoxy- sponding methylthiomethyl ether of 6 was accomplished by
trityl and 3Ј-phosphoamidite groups. However, modification treating 5 with a mixture of acetic anhydride and anhydrous
of only one out of two primary hydroxy groups of our anti- dimethyl sulfoxide at room temperature for 33 h. Ring clo-
conformationally constrained pyrimidine acyclic nucleotide sure of 6 was accomplished with iodine in dry tetrahydrofu-
unit to fulfill the requirement of building units for oligonu- ran at room temperature to give 3-benzyloxymethyl-3-(tert-
cleotide synthesizer is difficult. Therefore development of a butyldiphenylsilyloxy)methyl-6-methoxy-(1H,3H,4H)-
new method for the synthesis of mono-O-protected anti-con- pyrimido[1,6-c][1,3]oxazin-8-one (7). In previous work,6) the
formationally constrained pyrimidine acyclic nucleoside as dealkylation of 7 was accomplished by using 2 N sodium hy-
the key intermediate of the building unit for oligonucleotide droxide in dioxane under reflux overnight and the benzyl
synthesizer is an important subject. This report describes the group of 7 was deprotected by hydrogenation in the presence
synthesis of this compound.
of 20% palladium on carbon in methanol at high hydrogen
In previous work,6) 1,3-bis-O-benzylated-1,3-dihydroxy- pressure (50 psi). This method was not practically applicable
acetone prepared from 1,3-dihydroxyacetone served as a for large scale preparation. However, by using different
building block for the synthesis of anti-conformationally equivalents of trimethylsilyl iodide as dealkylating agent,8) 7
constrained pyrimidine acyclic nucleosides. However, draw- was able to demethylate selectively (1.1 eq Me3SiI) to 86% of
back of the identical two primary hydroxy protecting groups 3-benzyloxymethyl-3-(tert-butyldiphenylsilyloxy)methyl-
made this compound being not suitable as building block for (1H,3H,4H,7H)-pyrimido[1,6-c][1,3]oxazin-6,8-dione (8) and
the preparation of title compound. Alternatively, we employ to debenzylate consecutively (3.0 eq Me3SiI) to 71% of
1-benzyloxy-3-(tert-butyl-diphenylsilyloxy)propan-2-one (4) 3-hydroxymethyl-3-(tert-butyldiphenylsilyloxy)methyl-
instead of 1,3-dibenzyloxy-2-propanone as intermediate for (1H,3H,4H,7H)-pyrimido[1,6-c][1,3]oxazin-6,8-dione (9) as
the synthesis of title compound.
a white solid product.
To whom correspondence should be addressed. e-mail: hly@ndmctsgh.edu.tw
© 2002 Pharmaceutical Society of Japan

August 2002
1029
pressure at 78 °C in the presence of P2O5 for at least 12 h unless otherwise
specified. Elemental analyses were obtained from Perkin-Elmer 2400 Ele-
mental Analyzer.
Benzyl Glycidyl Ether7) (1) The solution of sodium (3.2 g, 145 mmol),
benzyl alcohol (10 ml, 97 mmol) in 100 ml of toluene was heated under re-
flux for 1.5 h. After cooling to the r.t. (room temperature), epichlorohydrin
(8 ml, 102 mmol) in 30 ml of toluene was added to the solution. The reaction
mixture was heated under reflux for another 2 h. After cooling, the mixture
was filtered. The filtrate was extracted with dichloromethane and water. The
organic layer was separated, dried over anhydrous sodium sulfate, filtered,
and evaporated under reduced pressure. Column chromatography on silica
gel with 5 : 1 n-hexane/EtOAc as eluent gave 1 (7.6 g, 48%) as a light
brown oil. Rf 0.57 (n-hexane/EtOAcϭ5/1). 1H-NMR (CDCl3) d: 2.58—2.76
(2H, m, CH2), 3.16—3.18 (1H, m, CH), 3.41 (1H , dd, Jϭ11.4, 5.9 Hz,
CHHOBn), 3.76 (1H, dd, Jϭ11.5, 1.7 Hz, CHHOBn), 4.55 and 4.61 (1H
each, d, Jϭ11.9 Hz, OCH2Ph), 7.29—7.36 (5H, m, ArH). 13C-NMR (CDCl3)
d: 44.6, 51.3, 71.4, 73.8, 128.3, 128.9, 138.7. MS m/z: 163 (MϩϪHϩ). Anal.
Calcd for C10H12O2: C, 73.15; H, 7.37. Found: C, 72.96; H, 7.20.
Chart 1
3-Benzyloxypropan-1,2-diol7) (2) Iodine (3.1 g, 24.4 mmol) was added
to a mixture of 1 (2.1 g, 12.8 mmol), 50 ml of acetonitrile and 50 ml of water.
The reaction mixture was heated under reflux for 3 h. The solvent was evap-
orated, ether (50 ml) was added, and the mixture was washed with 10%
aqueous solution of Na2SO3 until disappearance of the iodine color was ob-
served. The aqueous layer was separated and washed with ether (3ϫ20 ml).
The combined organic solution was concentrated to dryness in vacuo. Col-
umn chromatography on silica gel with 1 : 5 n-hexane/EtOAc as eluent gave
2 (1.47 g, 63%) as a colorless oil. Rf 0.37 (n-hexane/EtOAcϭ1/5). 1H-NMR
(CDCl3) d: 3.46—3.61 (2H, br, CH2OBn), 3.88 (2H, br, CH2OH), 4.10 (1H,
m, CH), 4.51 (2H, s, OCH2Ph), 7.32 (5H, s, ArH). 13C-NMR (CDCl3) d:
64.5, 71.5, 72.1, 74.0, 128.4, 129.0, 138.4. MS m/z: 183 (MϩϩHϩ). Anal.
Calcd for C10H14O3: C, 65.91; H, 7.74. Found: C, 65.81; H, 8.01.
1-Benzyloxy-3-(tert-butyldiphenylsilyloxy)propan-2-ol (3) To a chilled
(0 °C) solution of
2 (1.0 g, 5.5 mmol), dimethylaminopyridine (0.2 g,
1.6 mmol), triethylamine (10 ml) in dry dichloromethane (60 ml), tert-
butylchlorodiphenylsilane (1 M; 6 ml, 6 mmol) was added dropwisely. The
resulting mixture was stirred under nitrogen at r.t. for 30 h and then poured
over crushed ice and left overnight. The solution was extracted with
dichloromethane. The organic extract was washed with 1 N HCl and water,
dried over sodium sulfate and evaporated under reduced pressure. The
residue was chromatographed on silica gel with 9 : 1 n-hexane/EtOAc as
eluent to give 3 (1.48 g, 64%) as a colorless oil. Rf 0.24 (n-hexane/
1
EtOAcϭ9/1). H-NMR (CDCl3) d: 1.09 (9H, s, C(CH3)3), 3.59—3.62 (2H,
m, CH2OBn), 3.76 (2H, d, Jϭ5.4 Hz, CH2OSi), 3.92—4.00 (1H, m, CH),
4.57 (2H, s, OCH2Ph), 7.34—7.70 (15H, m, ArH). 13C-NMR (CDCl3) d:
19.9, 27.5, 65.5, 71.4, 71.7, 74.0, 128.2, 128.3, 129.0, 130.3, 133.9, 136.1,
138.7. MS m/z: 421 (MϩϩHϩ). Anal. Calcd for C26H32O3Si: C, 74.24; H,
7.67. Found: C, 74.06; H, 7.82.
Chart 2
1-Benzyloxy-3-(tert-butyldiphenylsilyloxy)propan-2-one (4) To
a
mixture of pyridinium chlorochromate (0.9 g, 4.2 mmol) in dichloromethane
(30 ml) was added 3 (0.9 g, 2.1 mmol) dissolved in dichloromethane (20 ml).
The resulting mixture was heated under reflux for 5 h, cooled and filtered.
The filtrate was concentrated to dryness. The residue was chromatographed
on silica gel with 1 : 1 n-hexane/CH2Cl2 as eluent to give 4 (0.76 g, 87%) as
In conclusion, we have developed a new route for the syn-
thesis of mono-o-protected anti-conformationally con-
strained pyrimidine acyclic nucleoside using 1-benzyloxy-3-
(tert-butyldiphenylsilyloxy)propan-2-one (4) as a building
block in place of 1,3-dibenzyloxypropan-2-ol. This selecting
provided the title compound with only one free hydroxyl
group by selectively deblocking the protected hydroxyl group
with certain reagents, which could be easily modified to the
corresponding 5Ј-dimethoxytrityl and 3Ј-phosphoamidite
acyclic nucleosides suitable for automated oligonucleotide
synthesizer.
1
a colorless oil. Rf 0.38 (n-hexane/CH2Cl2ϭ1 : 1). H-NMR (CDCl3) d: 1.09
(9H, s, C(CH3)3), 4.35 (2H, s, CH2OBn), 4.36 (2H, s, CH2OSi), 4.56 (2H, s,
OCH2Ph), 7.33—7.64 (15H, m, ArH). 13C-NMR (CDCl3) d: 19.8, 27.3,
69.3, 73.8, 74.0, 128.5, 128.6, 129.0, 130.6, 133.1, 136.1, 137.7, 207.1. MS
m/z: 441 (MϩϩNaϩ). Anal. Calcd for C26H30O3Si: C, 74.60; H, 7.22. Found:
C, 74.64; H, 7.38.
6-{2-[1-Benzyloxy-3-(tert-butyldiphenylsilyloxy)-2-hydroxy]propyl}-
methyl-2,4-dimethoxypyrimidine (5) Under nitrogen atmosphere n-
butyllithium (1.6 M; 10.0 ml, 16 mmol) was added dropwisely to a solution
of 2,4-dimethoxy-6-methylpyrimidine (2.0 g, 13.0 mmol) in dry tetrahydro-
furan (150 ml) at Ϫ78 °C. The mixture was raised and stirred at Ϫ50 °C for
Experimental
General Melting points (mp) were taken on a BUCHI 530 apparatus 30 min. Ketone 4 (5.4 g, 13 mmol) was added and the stirring was continued
and are uncorrected. Merck Art No105554 plates precoated with Silica gel for 2 h. The solution was neutralized with acetic acid to pH 7, and the sol-
60 containing fluorescent indicator were used for thin-layer chromatography, vent was removed under reduced pressure. The residue was partitioned be-
and Silica gel 60 (Merck Art No 109385, 230—400 mesh) was employed for tween dichloromethane and water. The organic layer was separated, dried
column chromatography. Evaporations were carried out at Ͻ50 °C using a
over sodium sulfate, and concentrated to dryness. The residue was chro-
rotary evaporator at reduced pressure (water aspirator). 1H- and 13C-NMR matographed on silica gel with 9 : 1 n-hexane/EtOAc as eluent to give 5
spectra were obtained at Varian 300 NMR spectrometer at 300 and 75 MHz, (6.8 g, 92%) as a light brown oil. Rf 0.15 (n-hexane/EtOAcϭ9 : 1). 1H-NMR
respectively. Where necessary, deuterium exchange experiments were used (CDCl3) d: 1.05 (9H, s, C(CH3)3), 2.96 and 3.00 (1H each, d, Jϭ14.9 Hz,
to obtained proton shift assignments. Mass spectra were recorded on a JEOL CH2-6), 3.49 and 3.51 (1H each, d, Jϭ11.3 Hz, CH2OBn), 3.60 and 3.71 (1H
J.M.S-300 spectrophotometer. Analytical samples were dried under reduced each, d, Jϭ10.0 Hz, CH2OSi), 3.91 and 3.97 (3H each, s, OCH3), 4.54 and

1030
Vol. 50, No. 8
4.58 (1H each, d, Jϭ12.0 Hz, OCH2Ph), 6.29 (1H, s, H-5), 7.30—7.64 (m,
15H, ArH). 13C-NMR (CDCl3) d: 19.8, 27.4, 40.3, 54.3, 55.2, 67.2, 74.0,
give 8 (0.92 g, 86%), mp 136—137 °C. Rf 0.45 (n-hexane/EtOAcϭ1 : 1). 1H-
NMR (CDCl3) d: 1.06 (9H, s, C(CH3)3), 2.94 and 2.98 (1H each, d,
74.2, 75.7, 102.8, 128.0, 128.1, 128.2, 128.8, 130.2, 130.2, 133.9, 136.1, Jϭ16.3 Hz, CH2-4), 3.42 and 3.52 (1H each, d, Jϭ9.9 Hz, CH2OBn), 3.62
136.2, 138.9, 165.1, 169.5, 172.5. MS m/z: 573 (MϩϩHϩ). Anal. Calcd for and 3.66 (1H each, d, Jϭ10.8 Hz, CH2OSi), 4.51 (2H, s, OCH2Ph), 5.39 and
C33H40N2O5Si: C, 69.20; H, 7.04; N, 4.89. Found: C, 68.90; H, 6.85; N, 4.77.
5.43 (1H each, d, Jϭ10.0 Hz, OCH2N), 5.55 (1H, s, H-5), 7.24—7.64 (15H,
6-{2-[1-Benzyloxy-3-(tert-butyldiphenylsilyloxy)-2-methylthiomethyl- m, ArH), 10.13 (1H, s, NH). 13C-NMR (CDCl3) d: 19.0, 26.7, 29.4, 66.1,
oxy]propyl}methyl-2,4-dimethoxypyrimidine (6) Acetic anhydride (33 69.2, 72.0, 73.5, 77.6, 100.7, 127.5, 127.7, 127.7, 128.3, 129.9, 129.9, 132.4,
ml) was added to a mixture of 5 (3.3 g, 5.8 mmol) in dry dimethylsulfoxide 132.4, 135.4, 135.4, 137.3, 149.7, 151.4, 163.3. MS m/z: 557 (MϩϩHϩ).
(DMSO) (33 ml). The solution was stirred at r.t. for 33 h. The solution was Anal. Calcd for C32H36N2O5Si: C, 69.04; H, 6.52; N, 5.03. Found: C, 68.89;
extracted with CH2Cl2 and washed with brine and water. The organic layer H, 6.30; N, 4.70.
was dried over MgSO4 and concentrated to an oily residue. This residue was
purified by flash chromatography on silica gel with 9 : 1 n-hexane/EtOAc as
3-Hydroxymethyl-3-(tert-butyldiphenylsilyloxy)methyl-(1H,3H,4H,7H)-pyrim-
ido[1,6-c][1,3]oxazin-6,8-dione (9) To a solution of 7 (10.5 g, 18.9 mmol) in
eluent to give 6 (2.53 g, 69%) as a colorless oil. Rf 0.29 (n-hexane/EtOAcϭ dichloromethane (125 ml) was added trimethylsilyl iodide (8 ml, 56.0 mmol)
1
9 : 1). H-NMR (CDCl3) d: 0.98 (9H, s, C(CH3)3), 2.00 (3H, s, SCH3), 2.91
(2H, s, CH2-6), 3.58, 3.60 (1H each, d, Jϭ10.0 Hz, CH2OBn), 3.69 and 3.77
via a dry syringe. The reaction mixture was stirred at r.t. for 16 h. Water
(150 ml) was then poured into the mixture and the stirring was continued for
(1H each, d, Jϭ10.8 Hz, CH2OSi), 3.75 (3H, s, OCH3), 3.83 (3H, s, OCH3), 10 min. The mixture was concentrated under reduced pressure. The residue
4.38 and 4.42 (1H each, d, Jϭ11.8 Hz, OCH2Ph), 4.75 (2H, s, OCH2S), 6.28 was taken up in dichloromethane, washed with aqueous 5% sodium sulfite,
(1H, s, H-5), 7.18—7.55 (15H, m, ArH). 13C-NMR (CDCl3) d: 15.0, 19.9,
dried over magnesium sulfate and concentrated in vacuo. The residue was
27.5, 39.9, 54.1, 55.1, 65.7, 69.2, 71.6, 74.0, 81.2, 102.9, 128.1, 128.2,
chromatographed on silica gel with 1 : 4 n-hexane/EtOAc as eluent to give 9
1
128.2, 128.4, 128.9, 130.3, 133.7, 136.1, 136.3, 138.7, 165.3, 168.7, 172.3. (6.26 g, 71%), mp 187—188 °C. Rf 0.46 (n-hexane/EtOAcϭ1 : 4). H-NMR
MS m/z: 633 (MϩϩHϩ). Anal. Calcd for C35H44N2O5SSi: C, 66.42; H, 7.01; (DMSO-d6) d: 0.96 (9H, s, C(CH3)3), 2.92 (2H, s, CH2-6), 3.39—3.45 (2H,
N, 4.43. Found: C, 66.44; H, 6.95; N, 4.17.
3-Benzyloxymethyl-3-(tert-butyldiphenylsilyloxy)methyl-6-methoxy-
(1H,3H,4H)-pyrimido[1,6-c][1,3]oxazin-8-one (7) Iodine (1.1 g, 4.3 mmol)
m, CH2OH), 3.54 and 3.60 (1H each, d, Jϭ10.6 Hz, CH2OSi), 5.06 (1H, s,
OH), 5.22 and 5.30 (1H each, d, Jϭ9.9 Hz, OCH2N), 5.57 (1H, s, H-5),
7.41—7.61 (10H, m, ArH), 11.20 (1H, s, NH). 13C-NMR (DMSO-d6) d:
was added to a mixture of 6 (1.7 g, 2.7 mmol) in dry tetrahydrofuran (THF) 18.8, 26.5, 29.4, 63.4, 66.3, 68.1, 78.5, 99.7, 127.9, 130.0, 132.6, 132.6,
(30 ml). The mixture was stirred at r.t. for 21 h. A 5% aq. sodium sulfite so- 135.2, 149.7, 152.2, 162.9. MS m/z: 467 (MϩϩHϩ). Anal. Calcd for
lution was added until the brown color of the mixture disappeared and the C25H30N2O5Si: C, 64.35; H, 6.48; N, 6.00. Found: C, 64.53; H, 6.53; N, 5.71.
resulting solution was extracted with CH2Cl2. The combined extracts were
washed with brine and water, dried over MgSO4 and concentrated in vacuo.
Acknowledgments Financial support from National Science Council
The residue was chromatographed on silica gel with 1 : 1 n-hexane/EtOAc as (NSC 89-2320-B016-100) of the Republic of China is gratefully acknowl-
eluent to give
7 (1.46 g, 95%) as a colorless oil. Rf 0.33 (n- edged.
hexane/EtOAcϭ1 : 1). 1H-NMR (CDCl3) d: 1.05 (9H, s, C(CH3)3), 2.97 (2H,
s, CH2-4), 3.41 and 3.53 (1H each, d, Jϭ9.9 Hz, CH2OBn), 3.60 and 3.67 References
(1H each, d, Jϭ10.7 Hz, CH2OSi), 3.94 (3H, s, OCH3), 4.51 (2H, s,
OCH2Ph), 5.53 (2H, s, OCH2N), 5.68 (1H, s, H-5), 7.31—7.63 (15H, m,
ArH). 13C-NMR (CDCl3) d: 19.8, 30.4, 54.7, 67.0, 71.1, 72.9, 74.3, 78.0,
94.8, 128.2, 128.4, 129.0, 130.5, 133.2, 136.1, 138.1, 154.7, 156.0, 172.0.
MS m/z: 571 (MϩϩHϩ). Anal. Calcd for C33H38N2O5Si: C, 69.44; H, 6.71;
N, 4.91. Found: C, 69.75; H, 6.91; N, 4.69.
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3-Benzyloxymethyl-3-(tert-butyldiphenylsilyloxy)methyl-(1H,3H,4H,7H)-
pyrimido[1,6-c][1,3]oxazin-6,8-dione (8) To a solution of 7 (1.1 g, 1.9
mmol) in dichloromethane (30 ml) was added trimethylsilyl iodide (0.3 ml,
2.1 mmol) via a dry syringe. The reaction mixture was stirred at r.t. for 1 h.
Water (50 ml) was then poured into the mixture and the stirring was contin-
ued for 10 min. The mixture was concentrated under reduced pressure. The
residue was taken up in dichloromethane, washed with aqueous 5% sodium
sulfite, dried over magnesium sulfate and concentrated in vacuo. The residue
was chromatographed on silica gel with 1 : 1 n-hexane/EtOAc as eluent to
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