Design and synthesis of novel dinucleotide analogs

Article abstract of DOI:10.1007/BF03246046

Syntheses of dinucleotide analogs, (S,R) cis-(4-((4-amino-2-oxopyrimidin- 1(2H)-yl)methyl)-1,3-dioxolan-2-yl)methyl (2R,3R,5R)-2-(hydroxymethyl)-5-(5- methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-tetrahydrofuran-3-yl hydrogen phosphate (5a) and (S,R) cis-(5-((4-amino-2-oxopyrimidin-1(2H)-yl)methyl)-1,3- oxathiolan-2-yl)methyl (2R,3R,5R)-2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-tetrahydrofuran-3-yl hydrogen phosphate (5b), were accomplished by the use of a new strategy. The use of phenyldichlorophosphate (Method A) as the coupling reagent was shown to possess superiority relative to the reported use of di(1H-benzo[d][1,2,3]triazol-1-yl)phenyl phosphonate (Method B).

Full text of DOI:10.1007/BF03246046

J. Iran. Chem. Soc., Vol. 7, No. 3, September 2010, pp. 597-602.
JOUIRrNaAnLiaOnF THE
Chemical Society
Design and Synthesis of Novel Dinucleotide Analogs
F. Valiyeva,c,*, V. Abbasovc, H.J. Liua,b and F.Y. Tsaid
aInstitute of Chemistry, Academia Sinica, Taipei 115, Taiwan
bDepartment of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan
cInstitute of Petrochemical Processes of Azerbaijan NAS, Baku, Azerbaijan
dCenter for General Education, Chang Gung University, Tao-Tuan 333, Taiwan
(Received 19 June 2009, Accepted 29 August 2009)
Syntheses of dinucleotide analogs, (S,R) cis-(4-((4-amino-2-oxopyrimidin-1(2H)-yl)methyl)-1,3-dioxolan-2-yl)methyl
(2R,3R,5R)-2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-tetrahydrofuran-3-yl hydrogen phosphate
(5a) and (S,R) cis-(5-((4-amino-2-oxopyrimidin-1(2H)-yl)methyl)-1,3-oxathiolan-2-yl)methyl (2R,3R,5R)-2-(hydroxymethyl)-5-
(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-tetrahydrofuran-3-yl hydrogen phosphate (5b), were accomplished by the
use of a new strategy. The use of phenyldichlorophosphate (Method A) as the coupling reagent was shown to possess superiority
relative to the reported use of di(1H-benzo[d][1,2,3]triazol-1-yl)phenyl phosphonate (Method B).
Keywords: Nucleosides, Dinucleotides, Antivirals, Anti-HIV, Phosphorotriesters
INTRODUCTION
anti-HIV integrase inhibitors by multistep procedures
including N-protection and deprotection steps [18-22].
Our earlier findings [23] prompted us to synthesize
dinucleotide analogues 5a and 5b to examine their antiviral
activities. We used synthetic strategy shown in Scheme 1 to
synthesize the target molecules 5a and 5b via the
corresponding intermediates 3a and 3b, which were prepared
using new synthetic methodology to give 3'-5'-
phosphorotriester linkages with phenyldichlorophosphate as a
coupling reagent.
The gene of HIV encodes three key viral enzymes for the
replication of this virus that can be exploited for the
development of chemotherapeutic agents [1-6]. Two of these
enzymes, HIV reverse transcriptase and HIV protease, have
received much attention in terms of the development of
clinically useful inhibitors [4-10]. On the contrary, the third
enzyme of the pol gene, HIV integrase [2,3,11-13] has
received relatively less attention in terms of inhibitors, in large
part because of the difficulty associated with the discovery of
therapeutically significant inhibitors [14-16].
EXPERIMENTAL
On the other hand, some oligo nucleotides are known to
inhibit HIV-1 integrase [17]. Pommier and coworkers
investigated natural dinucleotides and Nair and coworkers
designed and studied non-natural dinucleotides as potential
General
All starting materials were purchased from Aldrich, TCI
and Across. THF was distilled from sodium benzophenone
1
ketyl. H NMR spectra were recorded with Bruker AMX400,
*Corresponding author. E-mail: famil2002@hotmail.com
proton chemical shifts (1) are reported in parts per million

Valiyev et al.
O
N
O
N
NH2
N
NH
NH
O
Si
O
NH2
N
O
O
N
O
Si
O
O
O
HO
+
PhOPOCl2
NMI, r.t.
N
O
O
P
X
O
PhO
O
OH
O
1
2a,b
X
a. X = O; b. X = S
3a,b
2 % HCl(EtOH)
r.t
O
O
NH
NH
N
O
N
O
HO
NH2
N
HO
NH2
N
O
O
N
O
O
P
N
O
O
P
NH4OH , r.t
O
PhO
O
O
HO
O
O
O
X
X
4a,b
5a,b
Scheme 1
(ppm) relative to the methine singlet at 7.24 ppm for the
residual CHCl3 in the deuteriochloroform, or the methyl pentet
at 2.49 ppm for the residual (CD3)2SO in the DMSO-d6.
Carbon chemical shifts are reported in parts per million
relative to the internal 13C signals in CDCl3 (77.0 ppm) or
DMSO-d6 (39.5 ppm). Mass spectra were obtained with a
FAB JMS-700 double focusing mass spectrometer (JEOL,
Tokyo, Japan).
Purification on silica gel refers to gravity column
chromatography on Merck silica gel 60 (particle size 230-400
Mesh). Analytical TLC was performed on precoated plates
purchased from Merck (Silica gel 60 F254). Compounds were
visualized by the use of UV light.
mixture was stirred for 24 h. Then, pyridine was removed by
co-evaporation with toluene under reduced pressure. The
residue was purified by column chromatography on silica gel
using CH2Cl2-MeOH (20:1), to give 1.17 g, 85% yield of
1
compound 1 as white crystals. m.p.: 193-195 °C. H NMR
(300 MHz, CDCl3) ꢀ (ppm) = 10.06 (s, 1H), 7.53 (s, 1H),
6.38-6.34 (dd, 1H , J = 5.56 and 5.6 Hz), 4.39-4.38 (d, 1H, J =
5.4 Hz), 4.05-4.04 (d, 1H, J = 1.4 Hz), 3.87-3.75 (dddd, 3H,
J = 2.1, 1.8, 2.0 and 2.1 Hz), 2.40-2.35 (dd, 1H, J = 4.6 and 5.2
Hz), 2.07-1.97 (m, 1H), 1.85 (s, 3H ), 0.86 (s, 9H ), 0.05 (s,
6H).
(S,R) cis-4-amino-1-((2-(hydroxymethyl)-1,3-dioxolan-
4-yl)methyl)pyrimidin-2(1H)-one (2a) and (S,R) cis-4-
amino-1-((2-(hydroxymethyl)-1,3-oxathiolan-5-yl)methyl)
pyrimidin-2(1H)-one (2b). Synthesis of compounds 2a and
2b were described before [23].
Synthesis of Compounds (1), 2a and 2b
1-((2R,4R,5R)-5-((tert-Butyldimethylsilyloxy)methyl)-4-
hydroxy-tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4
(1H,3H)-dione (1). To a stirred solution of 0.92 g (4 mmol) of
thymidine in 10 ml dry pyridine was added dropwise a
solution of 0.66 g (4.4 mmol) of tert-butyl-chlorodimethyl
silane in 5 ml dry pyridine at room temperature. The reaction
General Procedures for the Synthesis of 3a,b
Method A. A mixture of compound 1 (1 mmol) and
compound 2 in 5 ml dry N-methylimidazole was placed in a
flame-dried 50 ml two-neck round bottom flask equipped with
598

Design and Synthesis of Novel Dinucleotide Analogs
a stirrer. One equivalent (1 mmol) of phenyldichlorophosphate
J = 7.7 Hz), 5.32 (br, s, 1H), 5.02 (br, 1H), 4.30-3.65 (m, 7H),
3.08 (br, 1H), 2.75- 2.70 (t, 1H, J = 8.9 Hz), 2.48-2.33 (m,
2H), 1.78 (s, 3H), 0.86 (s, 9H), 0.06 (s, 6H). HRMS-FAB: for
C31H44N5O10PSSi (M+H)+ = 738.23.
in anhydrous THF (5 ml) was added dropwise at 0 °C and the
reaction mixture was stirred at room temperature for 3 h.
Water (30 ml) was added and the mixture was stirred for 30
min. Solids were isolated by filtration, dissolved in
dichloromethane and dried over MgSO4. Then, the solvent was
evaporated and the residue was purified by column
chromatography on silica gel using CH2Cl2-MeOH (10:1) as
eluant.
Method B. A solution of phenyldichlorophosphate (1
mmol) in anhydrous THF (2 ml) was added dropwise to a
solution of 1-hydroxybenzotriazole (2 mmol) and anhydrous
pyridine (2 mmol) in dry THF (6 ml) at room temperature. The
reaction mixture was then stirred for 1 h at the same
temperature and the precipitate was filtered. The volume was
reduced to 2 ml and added to a stirred solution of compound 1
(0.22 mmol) in 5 ml dry THF. The reaction mixture was
stirred for 2 h. Then, compound 2 (0.22 mmol) in5 ml dry N-
methylimidazole (NMI) was added and the reaction mixture
was stirred for another 3 h. A few drops of water were added
to the reaction mixture and NMI was removed in vacuum; the
residue was purified by column chromatography on silica gel
using CH2Cl2-MeOH (10:1) as eluant.
(S,R)
cis-(4-((4-Amino-2-oxopyrimidin-1(2H)-yl)
methyl)-1,3-dioxolan-2-yl)methyl (2R,3R,5R)-2-(hydroxy-
methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1
(2H)-yl)-tetrahydrofuran-3-yl phenyl phosphate(4a). To a
solution of 65 mg (0.09 mmol) of compound 3a in 5 ml THF
was added 5 ml solution of 2% HCl in EtOH at room
temperature and the reaction was stirred for 1 h. Reaction
mixture was evaporated to dryness and residue was purified by
column chromatography on silica gel using CH2Cl2-MeOH
(10:1) to give 46.5 mg (85% yield) of compound 4a as white
1
crystals. m.p.: 200 °C (dec.). H NMR (400 MHz, DMSO-d6)
ꢀ = 11.32 (s, 1H), 7.69 (s, 1H), 7.47-7.23 (m, 6H), 7.12 (br,
1H), 6.95 (br, 1H), 621-6.16 (dd, 1H, J = 6.3 and 6.9 Hz),
5.64-5.62 (d, 1H, J = 7.1 Hz), 5.31 (br, 1H), 5.11-5.07 (m,
2H), 4.35-4.28 (m, 1H), 4.19-3.51 (m, 9H), 2.44-2.31 (m, 2H),
1.78 (s, 3H). 13C NMR (125 MHz, DMSO-d6) ꢀ = 166.03;
163.67; 155.95; 150.46; 149.49; 145.6; 135.85; 130.06 (2C);
125.51; 119.99 (2C); 109.75; 101.2; 93.18; 85.10; 83.61;
79.40; 74.25; 67.17; 67.10; 60.89; 50.67; 37.37; 12.25.
HRMS-FAB for C25H30N5O11P (M+H)+ = 608.17.
(S,R) cis-((4-Amino-2-oxopyrimidin-1(2H)-yl)methyl)-
1,3-dioxolan-2-yl)methyl
(2R,3R,5R)-2-((tert-
(S,R)
cis-
(5-((4-Amino-2-oxopyrimidin-1(2H)-yl)
butyldimethylsilyloxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-tetrahydrofuran-3-yl phenyl
phosphate (3a). was obtained in 62% yield as a white crystals
methyl)-1,3-oxathiolan-2-yl)methyl (2R,3R,5R)-2-(hydroxy-
methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1
(2H)-yl)-tetrahydrofuran-3-yl phenyl phosphate (4b). The
procedure was similar to the one used for preparation of
compound 4a. Compound 4b was isolated (53 mg, 85% yield)
1
by using Method A. m.p.: 200 °C (dec.). H NMR (300 MHz,
CDCl3) ꢀ = 7.45-7.42 (d, 1H, J = 8.5 Hz), 7.38-7.17 ( m, 6H),
6,36-6.32 (dd, 1H, J = 4.9 and 4.4 Hz), 5.84 (br. 1H), 5.08 (br,
s, 1H), 5.06-4.95 (m, 1H), 4.5-4.44 (m, 1H), 4.33-3.60 (m,
9H), 2.59- 2.41 (dddd, 1H, J = 4.9, 4.8, 5.1 and 4.4 Hz), 2.12-
1.96 (m, 1H), 1.87 (s, 3H), 0.87 (s, 9H), 0.07 (s, 6H), HRMS-
FAB: for C31H44N5O11PSi (M+H)+ = 722.25.
1
as white crystals. m.p.: 200 °C (dec.). H NMR (400 MHz,
DMSO-d6) ꢀ = 11.34 (s, 1H), 7.69 (s, 1H), 7.51-7.20 (m, 6H),
7.16 (br, 1H), 7.02 (br, 1H), 6.20 (br, 1H), 5.65-5.60 (t, 1H, J
= 6.6 Hz), 5.31 (br, 2H), 5.09 (br, 1H ), 4.30-3.61 (m, 8H),
3.07 (br, 1H), 2.74-2.69 (t, 1H, J = 8.9 Hz), 2.44-2.34 (m, 2H),
1.77 (s, 3H). 13C NMR (100 MHz, DMSO-d6) ꢀ = 165.75;
163.59; 155.49; 150.39; 149.90; 146.45; 135.77; 129.96 (2C );
125.40; 119.93 (2C); 109.68; 93.23; 85.02; 83.58; 82.54;
82.14; 79.14; 69.35; 60.86; 50.38; 37.34; 33.65; 12.19.
HRMS-FAB for C25H30N5O10PS (M+H)+ = 624.15.
(S,R)
cis-(5-((4-Amino-2-oxopyrimidin-1(2H)-yl)
(2R,3R,5R)-2-((tert-
methyl)-1,3-oxathiolan-2-yl)methyl
butyldimethylsilyloxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-tetrahydrofuran-3-yl phenyl
phosphate (3b). was obtained in 46% yield as a white crystals
1
by using Method B. m.p.: 200 °C (dec.). H NMR (400 MHz,
(S,R)
cis-(4-((4-amino-2-oxopyrimidin-1(2H)-yl)
DMSO-d6) ꢀ = 11.4 (s, 1H), 7.46-7.24 (m, 7H), 7.14 (br, 1H),
7.07 (br, 1H), 6,2-6.17 (d, 1H, J = 3.7 Hz), 5.64-5.59 (t, 1H,
methyl)-1,3-dioxolan-2-yl)methyl (2R,3R,5R)-2-(hydroxy-
methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1
599

Valiyev et al.
Fig. 1. 400 MHz 1H NMR, 13C NMR, DEPT 135 and DEPT 90 H NMR spectra of 4a.
600

Design and Synthesis of Novel Dinucleotide Analogs
(2H)-yl)-tetrahydrofuran-3-yl hydrogen phosphate (5a). To
phenyldichlorophosphate as the coupling reagent in the
presence of N-methylimidazole in THF afforded the respective
compounds 3a and 3b in 60-65% yields (Method A). For the
selective introduction of a 3'-5'-phosphorotriester linkage
between 5'-protected deoxyribonucleoside (1) and dioxalane or
a solution of 30.5 mg (0.05 mmol) of compound 4a in 5 ml
THF was added 20 ml of ammonium water (28%), and
reaction mixture was shaken for a few minutes until the
mixture had become almost clear (3 h). The solution was
evaporated to dryness and residue was purified by short
column chromatography on silica gel using CH2Cl2-MeOH
(4:1) to give 20 mg (75% yield) of compound 5a as white
oxathiolane
nucleosides (2a,b), the coupling reagent
dichlorophosphate could be applied, without formation of 3'- ,
or 5'- phosphorodiester intermediates. It should be noted that
the compounds 3a,b were found in 45-50% yield (Method B)
by the use of a bifunctional phosphorylating agent, di(1H-
benzo[d][1,2,3]triazol-1-yl)phenyl phosphonate [25].
The formation of compounds 3a and 3b was confirmed by
their NMR and HRMS data. Deprotections with 2%
hydrochloric acid in ethanol gave 4a and 4b in about 85%
yield, which were characterized by 1H NMR, 13C NMR, DEPT
135, DEPT 90 (Fig. 1) and HRMS data.
1
crystals. m.p.: 200 °C (dec.). H NMR (400 MHz, DMSO-d6)
ꢀ = 11.28 (s, 1H), 7.76 (s, 1H), 7.57 (br, 1H), 7.25 (br, 1H),
6.94 (br, 1H), 6.14 (br, 1H), 5.69 (br, 1H), 5.4 (br, 1H), 4.97
(br,1H), 4.68 (br, 1H), 4.28 (br, 1H), 3.99-3.49 (m, 9H), 2.3-
2.01 ( m, 2H), 1.77 (s, 3H). 13C NMR (125 MHz, DMSO-d6) ꢀ
= 166.13; 162.65; 152.96; 147.46; 141.64; 132.85; 110.75;
98.22; 91.18; 86.10; 81.71; 76.40; 72.28; 66.18; 65.90; 60.52;
51.07; 36.85; 11.96. HRMS-FAB for C19H26N5O11P (M+H)+ =
532.14.
Further deprotection of 4a and 4b with 28% NH4OH
solution in water afforded target molecules 5a and 5b in about
(S,R)
cis-(5-((4-amino-2-oxopyrimidin-1(2H)-yl)
1
methyl)-1,3-oxathiolan-2-yl)methyl (2R,3R,5R)-2-(hydroxy-
methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1
(2H)-yl)-tetrahydrofuran-3-yl hydrogen phosphate (5b).
Compound 5b was prepared like compound 5a (20 mg, 74.7%
75% yield. Structures of 5a and 5b were characterized by H
NMR, 13C NMR, and HRMS data. Compounds 5a and 5b will
be evaluated for their antiviral activities; results will be
reportedelsewhere.
1
yield) as white crystals. m.p.: 200 °C (dec.). H NMR (400
In summary, we described high yield syntheses of novel
nucleotide analogues 5a and 5b via the intermediates 3a and
3b, which in turn were prepared using the coupling reagent,
phenyl dichlorophosphate.
MHz, D2O) ꢀ = 7.52-7.49 (br, 2H), 6.14-6.11 (t, 1H, J = 6.9
Hz), 5.83 (br, 1H), 5.22 (br, 1H), 4.27 (br, 1H), 4.10-3.52 (m,
8H), 3.06-3.02 (dd, 1H, J = 4.7 and 5.2 Hz), 2.70-2.66 (t, 1H, J
= 10.1 Hz), 2.40-2.26 (m, 2H), 1.73(s, 3H). 13C NMR (125
MHz, D2O) ꢀ = 166.36; 151.54; 147.74; 137.39; 137.30;
111.50; 95.42; 85.02; 83.65; 82.00; 81.94; 75.20; 75.00;
67.58; 61.10; 51.50; 37.54; 33.39; 11.53. HRMS-FAB for
C19H26N5O10PS (M+H)+ = 546.11.
ACKNOWLEDGMENTS
The authors thank the National Science Council of Taiwan,
Republic of China, and Academia Sinica for their financial
support.
RESULTS AND DISCUSSION
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