Synthesis of 2-bromomethyl-3-hydroxy-2-hydroxymethyl-propyl pyrimidine and theophylline nucleosides under microwave irradiation. Evaluation of their activity against hepatitis B virus

Article abstract of DOI:10.1080/15257770600793919

Alkylation of 2-methylthiopyrimidin-4(1H)-one (1a) and its 5(6)-alkyl derivatives 1b - d as well as theophylline (7) with 2,2- bis (bromomethyl)-1,3-diacetoxypropane (2) under microwave irradia-tion gave the corresponding acyclonucleosides 1-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl) prop-1- yl ]-2-methyl-thio pyrmidin-4(1H)-ones 3a - d and 7-[(3-acetoxy-2- acetoxymethyl-2-bromomethyl)prop-1- yl]theophylline (8), which upon further irradiation gave the double-headed acyclonucleosides 1,1 ′-[(2,2- diacetoxymethyl)-1,3-propylidene]- bis [(2-(methylthio)-pyrimidin-4(1H)-ones] 4a - c , and 7,7 ′-[(2,2-diacetoxymethyl)-1,3-propylidene]- bis (theophylline) (9). The deacetylated derivatives were obtained by the action of sodium methoxide. The activity of deacetylated nucleosides against Hepatitis B virus was evaluated. Compound 5b showed moderate inhibition activity against HBV with mild cytotoxicity. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770600793919

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Synthesis of 2-Bromomethyl-3-
Hydroxy-2-Hydroxymethyl-Propyl
Pyrimidine and Theophylline Nucleosides
Under Microwave Irradiation. Evaluation
of Their Activity Against Hepatitis B
Virus
a b
a
a
a
a
E. S. H. El Ashry
, N. Rashed , A. Abdel-Rahman , L. F. Awad
&
a
H. A. Rasheed
Department of Chemistry, Faculty of Science, Alexandria
University, Alexandria, Egypt
b
International Centre for Chemical Sciences, H. E. J. Research
Institute, University of Karachi, Karachi, Pakistan
Published online: 16 Feb 2007.
To cite this article: E. S. H. El Ashry , N. Rashed , A. Abdel-Rahman , L. F. Awad & H. A. Rasheed
(2006) Synthesis of 2-Bromomethyl-3-Hydroxy-2-Hydroxymethyl-Propyl Pyrimidine and Theophylline
Nucleosides Under Microwave Irradiation. Evaluation of Their Activity Against Hepatitis B Virus,
Nucleosides, Nucleotides and Nucleic Acids, 25:8, 925-939, DOI: 10.1080/15257770600793919
To link to this article: http://dx.doi.org/10.1080/15257770600793919
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Nucleosides, Nucleotides, and Nucleic Acids, 25:925–939, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600793919
SYNTHESIS OF 2-BROMOMETHYL-3-HYDROXY-2-HYDROXYMETHYL-
PROPYL PYRIMIDINE AND THEOPHYLLINE NUCLEOSIDES UNDER
MICROWAVE IRRADIATION. EVALUATION OF THEIR ACTIVITY
AGAINST HEPATITIS B VIRUS
E. S. H. El Ashry, N. Rashed, A. Abdel-Rahman, L. F. Awad, and
H. A. Rasheed
2
Department of Chemistry, Faculty of Science, Alexandria University,
Alexandria, Egypt
2
Alkylation of 2-methylthiopyrimidin-4(1H)-one (1a) and its 5(6)-alkyl derivatives 1b–d as well
as theophylline (7) with 2,2-bis(bromomethyl)-1,3-diacetoxypropane (2) under microwave irradia-
tion gave the corresponding acyclonucleosides 1-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-
1-yl]-2-methyl-thio pyrmidin-4(1H)-ones 3a–d and 7-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)
prop-1-yl]theophylline (8), which upon further irradiation gave the double-headed acyclonucleosides
ꢁ
ꢁ
1
7
,1 -[(2,2-diacetoxymethyl)-1,3-propylidene]-bis[(2-(methylthio)-pyrimidin-4(1H)-ones] 4a–c, and
,7 -[(2,2-diacetoxymethyl)-1,3-propylidene]-bis(theophylline) (9). The deacetylated derivatives
were obtained by the action of sodium methoxide. The activity of deacetylated nucleosides against
Hepatitis B virus was evaluated. Compound 5b showed moderate inhibition activity against HBV
with mild cytotoxicity.
Keywords Acyclonucleosides; Double-headed acyclonucleosides; Theophylline; Micro-
wave alkylation reactions; Hepatitis B virus
INTRODUCTION
Acyclic nucleosides[1–4] and their chemotherapeutic value as well as
denderimers have attracted the attention toward the synthesis of vari-
ous analogues with variant chemical modifications. Development has pro-
gressed in the synthesis of acyclic nucleosides whose glycons and agly-
cons are modified.[
5–7]
Acyclic nucleosides of the tetraseco type showed
Received 26 September 2005; accepted 5 May 2006.
The authors thank DANIDA for the support of the biological testing. The partial support from
Alexander von Humboldt Stiftung and DFG are highly appreciated.
E. S. H. El Ashry is currently on leave to the International Centre for Chemical Sciences, H. E. J.
Research Institute, University of Karachi, Karachi, Pakistan.
Address correspondence to E. S. H. El Ashry, Department of Chemistry, Faculty of Science,
Alexandria University, Alexandria, Egypt. E-mail: eelashry60@hotmail.com; eelashry 60@link.net
9
25

9
26
E. S. H. El Ashry et al.
FIGURE 1 Target acyclonucleosides.
interesting antiviral activities.[8–12] Although many acyclonucleoside ana-
logues have been synthesized and their activity against Hepatitis B[8,9] virus
were evaluated, an urgent need for new classes of compounds with high
activity and low toxicity is still required. Thus, we report here a fast and
simple procedure for the alkylation of the modified pyrimidine bases and
theophylline with the objective of synthesizing branched chain tetraseco
nucleosides of type I and II (Figure 1).
RESULTS AND DISCUSSION
Microwave irradiation is now widely approved as a safe, convenient,
[
13–19]
and economical way for small-scale work in organic synthesis.
Recent
reports demonstrated that a variety of organic reactions have been con-
ducted efficiently by using selected organic solvents; other reports described
accelerated organic reactions in nonaqueous media. The acceleration in re-
action rates is ascribed to elevated temperatures and high pressure reached
rapidly in a microwave oven. The application of microwave irradiation in
[
18,19]
the synthesis of heterocyclic compounds were reviewed.
our work
Continuing
[
18–23]
on the use of microwave irradiation in organic synthesis,
we have selected this technique for the synthesis of our targets. Thus, the
starting methylthiopyrimidinones 1a–d were synthesized in better yields and
shorter times than using conventional heating, by methylation of substi-
tuted 2-thiouracils with methyl iodide in presence of sodium hydroxide
under microwave irradiation for 2 min. When the alkylation of 1a with

Microwave Alkylation Reactions
927
2,2-bis(bromomethyl)-1,3-diacetoxypropane (2) (2:1 molar equivalent) un-
der conventional heating was done, the product 3a was formed in a very
poor yield even upon prolongation of the time of heating and much of the
base was recovered unchanged. On the other hand, when the reaction of 2.5
molar equivalents of 1a–d with 1 molar equivalent of 2 was carried out under
the microwave irradiation, it gave within 3 min the monoacyclo- nucleosides
3
a–d as the main products (Scheme 1). When the irradiation was continued
SCHEME 1

9
28
E. S. H. El Ashry et al.
for 10 min, the double-headed acyclonucleosides 4a–c were formed in
addition to the monoacyclonucleoside 3. Chromatographic purification of
the reaction mixture on silica gel column afforded 3a–d in 35–61% yield
1
and 4a–c in 22–26% yield. The H NMR spectra for compounds 3a–d
confirmed the presence of two acetyl and four methylene groups in addition
to the protons of the pyrimidine ring. Deacetylation of 3a–d with sodium
methoxide in methanol gave the corresponding acyclonucleoside analogues
SCHEME 2

Microwave Alkylation Reactions
929
SCHEME 3
5
a–d in 76–96% yield. The possible susceptibility of the methylthio group
to nucleophilic substitution reaction by the methoxide ion to give the
respective methoxy derivatives was not observed. This can be due to the
electron donating effect of the alkyl substituent group on C-5 or C-6 which
[
23]
1
retard the nucleophilic substitution reaction.
The H NMR data were
in satisfactory agreement with the assigned structure 5a–d, where it showed
the absence of the two acetyl groups and the presence of the thiomethyl
group in addition to the other protons of the deacetylated products. Mass
spectra of 5a and 5c confirmed the presence of the bromine atom by
showing their molecular ion peaks at m/z 322, 324 and 336, 338, respectively
(Scheme 3) and their base peaks at m/z 143, 157 corresponding to
the respective pyrimidinonium ions. The structure of the double-headed
nucleosides 4a–c and the symmetry in the molecule was deduced based on
1
their H NMR spectra, which showed four methylene protons as two singlets
at δ 4.26, 4.39 and δ 4.47, 4.57 ppm, the two acetyl and the two thiomethyl
protons as two singlets at δ 2.05, 2.09 and δ 2.52, 2.56, respectively, in
addition to the protons of the pyrimidine base. The mass spectrum of
4b confirmed that two pyrimidine bases were alkylated with 2 to give the
respective bisacyclonucleoside. Although the formation of such double-
headed acyclic nucleoside was successful for a variety of substituents on the
pyrimidine ring, the 2-methylthio-6-(n-propyl)-pyrimidin–4(1H)-one (1d)
failed to form the respective acyclonucleoside 4d. This may be attributed
to the bulkness of the substituent on C-6, which hindered the nucleophilic
attack of the N-1 in 1d, on the C-Br in 2 to form the C−N bond. Treatment

9
30
E. S. H. El Ashry et al.
SCHEME 4
of 4a,b with sodium methoxide in methanol furnished the deacetylated
products 6a,b. The structures of 6 were established and confirmed by their
1
spectral data (MS and H NMR). The mass spectrum of 6a revealed a peak
at m/z 384 and 143 corresponding to the molecular ion and base peaks,
1
respectively (Scheme 4). The H NMR spectrum showed the absence of
acetyl groups and presence of two hydroxyl groups in addition to the other
protons of the base and the methylene groups.
On the other hand, when a mixture of theophylline (7) and 2,2-
bis(bromomethyl)-1,3-diacetoxypropane (2) was irradiated in the mi-
crowave oven for 10 min, a mixture of mono- and ditheophyllinyl acyclonu-
cleosides 8 and 9, respectively, was obtained whose ratio was found to be
varied based on the ratio of the reactants. Thus, increasing the amount of

Microwave Alkylation Reactions
931
TABLE 1 Inhibitory Concentration (IC50) Cytotoxic Effect
(
CC50) and Selective Index (SI) of Compounds 4, 5, and 11
Compound
IC50(µM)
CC50(µM)
SI
Lamivudine
<0.1
1.9
3.3
7.5
3.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>1000
>52.6
4
4
4
5
5
5
5
1
a
b
c
a
b
c
>30.3
>13.3
>28.6
>454.5
>8.0
0.2
12.5
11.7
7.7
d
1
>8.5
13.0
the theophylline led to a higher ratio of the double headed analogue. The
two products could be readily separated by column chromatography. The
1
H NMR spectrum of 8 showed the presence of two acetyl, four methylene,
and two N-methyl groups in addition to the aromatic methine proton at
7
.70 ppm. Both of the four methylene and four N-methyl groups in
compound 9 were observed as two singlets at 4.27, 4.49 and 3.35, 3.54 ppm,
respectively, indicating the symmetry in the structure. Deprotection of 8 and
9
with sodium methoxide in methanol gave the deprotected derivative 10
1
and 11, respectively (Scheme 2). H NMR spectra of 10 and 11 showed the
absence of the acetyl groups that present in their precursors at 2.08 and 2.15
ppm and the presence of broad signals at 4.92 and 5.09 ppm, respectively,
corresponding to their hydroxyl groups.
The results of the viral screening against HBV of selected compounds
indicated that compound 5b showed moderate viral replication inhibition
and mild cytotoxicity with selective index >454.5. On the other hand, the
effective concentration of compounds 4a, 4b, 4c, 5a, 5c, 5d, 11 was 10.0 µM,
which showed very low inhibition and high cytotoxicity with selective index
>
52.6, >30.3, >13.3, >28.6, >8.0, >8.5, >13.0, respectively.
EXPERIMENTAL
Melting points were determined with Mel-Temp apparatus and are
uncorrected. All solvents were distilled and dried before using. IR spectra
were recorded for compounds in a matrix of KBr with Unicam SP1025
Spectrophotometer. The NMR spectra were recorded on a Bruker AC
3
00 MHz spectrometers. Chemical shifts were reported in δ scale (ppm)
relative to TMS as a standard and the coupling constants J values are given
in Hz. EI mass spectra were recorded on a Varian MAT 311A spectrometer.
TLC was performed on 60 F254 precoated plastic plates silica gel (Merck).
Column chromatography was performed on silica gel (Baker, 30–60 µm).
Irradiation was done in a domestic microwave oven EM-230M (800 watt

9
32
E. S. H. El Ashry et al.
TABLE 2 Results of Inhibition of HBV Replication by Compounds
4, 5, and 11
Dreug conc.
HBV DNA in
supernatant
Hep G2 viable
cells
Compound
Lamivudine
(µM)
1.0
0.25
0.18
0.15
0.22
0.20
0.19
0.15
0.14
0.14
0.22
0.14
0.11
0.42
0.40
0.40
0.65
0.50
0.45
0.29
0.28
0.25
0.12
0.11
0.10
0.44
0.40
0.38
1.03
1.01
1.07
0.16
0.12
0.08
0.06
0.05
0.04
0.06
0.05
0.03
1.01
1.01
1.01
1.50
1.40
1.30
1.00
0.07
0.06
0.08
0.07
0.07
2.01
1.90
1.80
10.0
1
1
1
1
1
1
1
1
1
00.0
1.0
4
4
4
5
5
5
5
1
a
b
c
10.0
00.0
1.0
10.0
00.0
1.0
10.0
00.0
1.0
a
b
c
10.0
00.0
1.0
10.0
00.0
1.0
10.0
00.0
1.0
d
1
10.0
00.0
1.0
10.0
00.0
output power). Microanalyses were performed in the unit of Microanalysis
at Faculty of Science, Cairo University.
2
-Methylthio-5(6)-substituted Pyrimidin-4(1H)-ones (1a–d): General Procedure.
A solution of substituted 2-thiouracils (0.01 mol) and sodium hydroxide
(0.4 g, 0.01 mol) in water (10 mL) and ethanol (20 mL) was treated with
methyl iodide (5 mL, 0.07 mol) and irradiated by MW for 2 min in a
closed Teflon vessel. The solid was washed thoroughly with water, dried,
and crystallized from ethanol (yield 88–92%); mps. are identical with the
[
24]
literature.
,2-bis(bromomethyl)-1,3-diacetoxypropane (2). To a stirred solution of 2,2-
2
bis(bromomethyl)-propane-1,3-diol (10 g, 38 mmol) in dry pyridine (10 mL)
and acetic anhydride (10 mL) was added and stirring was continued for 2 h.
◦
The reaction mixture was left overnight at 0 C. Crushed ice was added to the
reaction mixture and the product that separated out was collected, washed
with water, dried, and crystallized from ethanol (11.0 g, 82% yield), mp
◦
[25]
◦
8
5–87 C (Slobodin and Shokhor,
mp 84–86 C).

Microwave Alkylation Reactions
933
Reaction of 2,2-bis(bromomethyl)-1,3-diacetoxypropane (2) with 2-methylthio-
(6)-substituted Pyrimidin-4(1H)-ones (1a–d). (a) General procedure under
5
microwave: To a stirred solution of compounds 1a–d (6.25 mmol) in dry
DMF (5 mL), sodium hydride (0.15 g, 6.25 mmol) was added. After com-
plete evolution of hydrogen gas, the mixture was irradiated by MW for 10
min in a closed Teflon vessel. 2,2-bis(Bromomethyl)-1,3-diacetoxypropane
(
2, 0.86 g, 2.5 mmol) was then added and the mixture was subjected to
microwave irradiation for 3 min TLC, using petroleum ether:ethyl acetate
5:1), showed spots corresponding to the reactants and acyclomononucleo-
(
sides 3a–d. The irradiation was continued for another 7 min, whereby a new
spot was observed corresponding to the double-headed acyclonucleosides
4
a–c. The reaction mixture was evaporated to dryness and the residue was
chromatographed on a silica gel column with petroleum ether:ethyl acetate
(
(
(
15:1) to give compounds 3a–d. Further elution gave compounds 4a–c.
b) Conventional procedure: A solution of 1a (0.71 g, 5 mmol) in dry DMF
10 mL) and sodium hydride (0.12 g, 5 mmol) was heated to complete
◦
evolution of hydrogen gas at 80 C for 1 h. Compound 2 (0.86 g, 2.5 mmol)
was then added and the reaction mixture was stirred for 8 h at 80 C, poured
◦
onto cold water (20 mL), and extracted with ethyl acetate. The organic
layers were collected, dried, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel using petroleum
ether:ethyl acetate (15:1) to give 3a (0.1 g, 10% yield) and the recovered
base (0.52 g).
1
-[(3-Acetoxy-2-acetoxymethyl-2- bromomethyl)prop-1-yl]-2-methylthiopyrmidin-4
(
1H)-one (3a). Colorless syrup (35% yield); R = 0.41(5:1 petroleum
f
1
ether:ethyl acetate); H NMR (CDCl , 300 MHz), δ : 2.07 (s, 6 H, 2 OAc),
3
H
2
.55 (s, 3 H, SMe), 3.56 (s, 2 H, CH Br), 4.22 (s, 4 H, 2 CH O), 4.44 (s, 2
2 2
H, CH N), 6.41 (d, 1 H, J 5.6 Hz, H-5), 8.26 (d, 1 H, J 5.6 Hz, H-6). Anal.
2
calcd. for C H BrN O S: C, 41.29; H, 4.70; N, 6.88. Found: C, 41.03; H,
14
19
2
5
4.99; N, 6.98%.
1
-[(3-Acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]-6-methyl-2-methylthiopyr-
imidin-4(1H)-one (3b). Colorless syrup (44% yield); R = 0.24 (5:1
f
1
petroleumether:ethyl acetate); H NMR (CDCl , 300 MHz), δ : 2.07 (s, 6
3
H
H, 2 OAc), 2.37 (s, 3 H, Me), 2.55 (s, 3 H, SMe), 3.55 (s, 2 H, CH Br), 4.21
2
(
s, 4 H, 2CH O), 4.43 (s, 2 H, CH N), 6.25 (s, 1 H, H-5). Anal. calcd. for
2 2
C H BrN O S: C, 42.76; H, 5.02; N, 6.65. Found: C, 42.92; H, 5.33; N,
15
21
2
5
6
.74%.
1
-[(3-Acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]-5-methyl-2-methylthiopyr-
imidin-4(1H)-one (3c). Colorless syrup (46% yield); R = 0.28 (5:1
f
1
petroleum ether:ethyl acetate); H NMR (CDCl , 300 MHz), δ : 2.07 (s,
3
H
6
4
H, 2 OAc,), 2.43 (s, 3 H, Me), 2.54 (s, 3 H, SMe), 3.58 (s, 2 H, CH Br),
2
.23 (s, 4 H, 2 CH O), 4.44 (s, 2 H, CH N), 8.09 (s, 1H, H-6). Anal. calcd.
2
2
for C H BrN O S: C, 42.76; H, 5.02; N, 6.65. Found: C, 42.49; H, 5.32; N,
15
21
2
5
7
.03%.

9
34
E. S. H. El Ashry et al.
1-[(3-Acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]-2-methylthio-6-n-propylp-
yrimidin-4(1H)-one (3d). Colorless syrup (61% yield); R = 0.24 (5:1
f
1
petroleum ether:ethyl acetate); H NMR (CDCl , 300 MHz), δ : 0.88 (t,
3
H
3
2
4
H, J = 7.2 Hz, Me-pr), 1.65 (m, 2 H, CH -pr), 2.00 (s, 6 H, 2 OAc),
2
.47 (s, 3 H, SMe), 2.51 (t, 2 H, CH - pr), 3.45 (s, 2 H, CH Br), 4.14 (s,
2
2
H, 2 CH O), 4.35 (s, 2 H, CH N), 6.15 (s, 1 H, H-5). Anal. calcd. for
2
2
C H BrN O S: C, 45.44; H, 5.61; N, 6.23. Found: C, 45.88; H, 5.34; N,
17
25
2
5
6
.00%.
ꢁ
1
,1 -[(2,2-Diacetoxymethyl)-1,3-propylidene]-bis[(2-methylthio)pyrimidin-4(1H)
-
one] (4a). Colorless syrup (26% yield); R = 0.14 (5:1 petroleum
f
1
ether:ethyl acetate); H NMR (CDCl , 300 MHz), δ : 2.09 (s, 6 H, 2 OAc),
3
H
2
.56 (s, 6 H, 2 SMe), 4.39 (s, 4 H, 2 CH O), 4.57 (s, 4 H, 2 CH N), 6.42
2 2
(d, 2 H, J 5.7 Hz, 2 H-5), 8.26 (d, 2 H, J 5.7, 2 H-6). Anal. calcd. for
C H N O S : C, 48.70; H, 5.16; N, 11.96. Found: C, 49.08; H, 5.54; N,
19
24
4
6 2
1
2.06%.
ꢁ
1
,1 -[(2,2-Diacetoxymethyl)1,3-propylidene]-bis[6-methyl-2-(methylthio)pyrimi-
din-4(1H)-one] (4b). Colorless syrup (23% yield); R = 0.11 (5:1 petroleum
f
1
ether:ethyl acetate); H NMR (CDCl , 300 MHz), δ : 2.05 (s, 6 H, 2 OAc),
3
H
2
.35 (s, 6 H, 2 Me), 2.54 (s, 6 H, 2 SMe), 4.26 (s, 4 H, 2 CH O), 4.47
2
+
(s, 4 H, 2 CH N), 6.23 (s, 2 H, 2 H-5); MS EI, m/z (%) : 496 (M , 12),
2
+
+
+
4
49 (M -SMe, 16), 341 [M -(MeSH, AcOH, SMe), 99], 267 [M -(2MeSH,
AcOH, 2Me, Ac), 55], 157 (6-methyl-2-methylthio-pyrimidin-4(1H)-onium
ion, 100). Anal. calcd. for C H N O S : C, 50.79; H, 5.68; N, 11.28.
21
28
4
6 2
Found: C, 50.93; H, 5.48; N, 11.44%.
ꢁ
1
,1 -[(2,2-Diacetoxymethyl)-1,3-propylidene]-bis[5-methyl-2-(methylthio)- pyrimi-
din-4(1H)-one] (4c). Colorless syrup (22% yield); R = 0.12 (5:1 petroleum
f
1
ether:ethyl acetate); H NMR (CDCl , 300 MHz), δ : 2.05 (s, 12 H,
3
H
2
4
Me, 2 OAc), 2.52 (s, 6 H, 2 SMe), 4.30 (s, 4 H, 2 CH O),
2
.51 (s, 4 H, 2 CH N), 8.07 (s, 2 H, 2 H-6). Anal. calcd. for
2
C H N O S : C, 50.79; H, 5.68; N, 11.28. Found: C, 50.93; H, 5.79; N,
21
28
4
6 2
11.59%.
Deacetylation of compounds 3a–d and 4a,b: General procedure. A solution
of compounds 3a–d or 4a,b (0.2 mmol) in dry methanol (3 mL) was
treated with sodium methoxide (0.15 mol of sodium in 3 mL dry methanol)
with stirring at room temperature for 2 h. The solution was neutralized
+
by Amberlite IR-120(H ) resin. The mixture was filtered, washed with
methanol, and the combined filtrate was evaporated and crystallized from
ethanol to afford 5a–d and 6a,b, respectively.
1
-[(2-Bromomethyl-3-hydroxy-2-hydroxymethyl)prop-1-yl]-2-methylthio-pyrimidin-
◦
1
4
(1H)-one (5a). Colorless crystals (76% yield); mp. 120–122 C; H NMR
(
3
CD OD+D O, 300 MHz), δ : 2.55 (s, 3 H, SMe), 3.59 (s, 2 H, CH Br),
3
2
H
2
.67 (s, 4 H, 2 CH O), 4.41 (s, 2 H, CH N), 6.56 (d, 1 H, J 5.7 Hz, H-5), 8.23
2 2
+
+
(d, 1 H, J 5.7 Hz, H-6); MS EI, m/z (%): 322, 324 (M ,17), 213[M -(Br,
2
Me), 40], 143 (2-methylthiopyrimidin-4(1H)-onium ion, 100). Anal. calcd.

Microwave Alkylation Reactions
935
for C H BrN O S: C, 37.16; H, 4.68; N, 8.67. Found: C, 37.45; H, 4.96; N,
10
15
2
3
8
.55%.
-[(2-Bromomethyl-3-hydroxy-2-hydroxymethyl)prop-1-yl]-6-methyl-2-methylthiop-
1
1
yrimidin-4(1H)-one (5b). Colorless syrup (96% yield); H NMR (DMSO-d
6
,
3
00 MHz), δ : 2.04 (s, 3 H, Me), 2.50 (s, 3 H, SMe), 3.46, 3.48 (2 d, 4 H, J
H
=
5.2, 5.5 Hz, 2 CH O), 3.58 (s, 2 H, CH Br), 4.26 (s, 2 H, CH N), 4.80 (t,
2
2
2
2
H, 2 OH), 6.83 (s, 1 H, H-5). Anal. calcd. for C H BrN O S: C, 39.18;
11
17
2
3
H, 5.08; N, 8.31. Found: C, 39.42; H, 5.08; N, 8.58%.
1
-[(2-Bromomethyl-3-hydroxy-2-hydroxymethyl)prop-1-yl]-5-meth- yl-2-methylthi-
1
opyrimidin-4(1H)-one (5c). Colorless syrup (84% yield);
H
NMR
(
CD OD+D O, 300 MHz), δ : 2.13 (s, 3 H, Me), 2.56 (s, 3 H,
3
2
H
SMe), 3.64 (s, 2 H, CH Br), 3.71 (s, 4 H, 2 CH O), 4.44 (s, 2 H,
CH N), 8.09 (s, 1 H, H-6); MS EI: m/z (%): 336, 338 (M , 50), 157
2
2
+
2
(5-methyl-2-methylthiopyrimidin-4(1H)-onium ion, 100). Anal. calcd. For
C H Br N O S: C, 39.18; H, 5.08; N, 8.31. Found: C, 39.44; H, 5.25; N,
11
17
2
3
8
.11%.
-[(2-Bromomethyl-3-hydroxy-2-hydroxymethyl)prop-1-yl]-2-methylthio-6-(n)prop-
1
1
ylpyrimidin-4(1H)-one (5d). Colorless syrup (77% yield); H NMR
(
2
DMSO-d , 300 MHz), δ : 0.89 (t, 3 H, J = 7.3 Hz, Me-pr), 1.71 (m,
6
H
H, CH -pr), 2.52 (s, 3 H, SMe), 2.55 (t, 2 H, CH -pr), 3.90 (s, 2 H,
2 2
CH Br), 4.47 (2 d, 4 H, J = 5.8, 5.9 Hz, 2 CH O), 4.55 (s, 2 H, CH N), 5.35
2
2
2
(
br t, 2 H, 2 OH), 6.53 (s, 1 H, H-5). Anal. calcd. for C H BrN O S: C,
13 21 2 3
4
2.74; H, 5.79; N, 7.67. Found: C, 42.60; H, 5.33; N, 7.42%.
ꢁ
1
,1 -[(2,2-Dihydroxymethyl)-1,3-propylidene]-bis[(2-methylthio)pyrimidin-4(1H)
1
-one] (6a). Colorless syrup (87% yield); H NMR (DMSO-d , 300 MHz), δ :
6 H
2
.50 (s, 6 H, 2 SMe), 3.47 (s, 4 H, 2 CH O), 4.49 (s, 4 H, 2 CH N), 5.06 (br
2 2
s, 2 H, 2 OH), 6.65 (d, 2 H, J 5.7 Hz, 2 H-5), 8.35 (d, 2 H, J 5.7 Hz, 2 H-6);
+
MS EI: m/z (%): 384 (M , 27), 143 (2-methylthio-pyrimidin-4(1H)-onium
ion, 100), Anal. calcd. for C H N O S : C, 46.86; H, 5.24; N, 14.57 Found:
15
20
4
4 2
C, 47.24; H, 5.69; N, 14.62%.
ꢁ
1
,1 -[(2,2-Dihydroxymethyl)-1,3-propylidene]-bis[6-methyl-2-(methylthio) pyrim-
1
idin-4(1H)-one] (6b). Colorless syrup (96% yield); H NMR (DMSO-d , 300
6
MHz), δ : 2.29 (s, 6 H, 2 Me), 2.50 (s, 6 H, 2 SMe), 4.21 (s, 4 H, 2 CH O),
H
2
4
.46 (s, 4 H, 2 CH N), 5.02 (br t, 2 H, 2 OH), 6.47 (s, 2 H, 2 H-5). Anal.
2
Calcd. for C H N O S : C, 49.50; H, 5.86; N, 13.58. Found: C, 49.21; H,
17
24
4
4 2
6
.21; N, 13.87%.
-[(3-Acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl)theophylline (8) and 7,7 -
(2,2-diacetoxymethyl)-1,3-propylidene]-bis(theophylline) (9). To a stirred suspen-
ꢁ
7
[
sion of 7 (0.45 g, 2.5 mmol) in dry DMF (5 mL), NaH (0.06 g, 2.5 mmol) was
added. After complete evolution of hydrogen gas the mixture was irradiated
by MW for 10 min in a closed Teflon vessel, then treated with compound
2
(0.346 g, 1 mmol), and the reaction mixture was processed as for 1a–d.
Column chromatography gave compound 8 as colorless syrup (47% yield);
1
TLC (CHCl -MeOH 8:2): R = 0.51; H NMR (CDCl , 300 MHz), δ : 2.08
3
f
3
H

9
36
E. S. H. El Ashry et al.
(
s, 6 H, 2 OAc), 3.39 (s, 3 H, NMe), 3.48 (s, 2 H, CH Br), 3.59 (s, 3 H,
2
NMe), 4.18 (s, 4 H, 2 CH O), 4.65 (s, 2 H, CH N), 7.70 (s, 1 H, H-8). Anal.
2
2
calcd. for C H BrN O : C, 43.16; H, 4.75; N, 12.58. Found: C, 43.42; H,
16
21
4
6
5
.03; N, 12.94%. Further elution gave compound 9 as colorless syrup (22%
1
yield); TLC (CHCl -MeOH 8:2): R = 0.38; H NMR (CDCl , 300 MHz),
3
f
3
δ : 2.15 (s, 6 H, 2 OAc), 3.35 (s, 6 H, 2 NMe), 3.54 (s, 6 H, 2 NMe), 4.27
H
(
s, 4 H, 2 CH O), 4.49 (s,4 H, 2 CH N), 7.86 (s, 2 H, 2 H-8). Anal. calcd.
2 2
for C H N O : C, 50.73; H, 5.18; N, 20.58. Found: C, 50.95; H, 5.54; N,
23
28
8
8
20.86%.
7
-[(2-Bromomethyl-3-hydroxy-2-hydroxymethyl)prop-1-yl]theophylline (10).
Deacetylation of compound 8 was carried out as in the general method to
1
afford 10 as colorless syrup (83% yield); H NMR (DMSO-d , 300 MHz), δ :
6
H
3
.27 (s, 3 H, NMe), 3.33 (br s, 4 H, 2 CH O), 3.40 (s, 3 H, NMe), 3.57 (s, 2
2
H, CH Br), 4.44 (s, 2 H, CH N), 4.92 (br t, 2 H, 2 OH), 7.92(s, 1 H, H-8).
2
2
Anal. calcd. for C H BrN O : C, 39.90; H, 4.74; N, 15.51. Found: C, 40.22;
12
17
4
4
H, 5.10; N, 15.92%.
ꢁ
7
,7 -[(2,2-Dihydroxymethyl)-1,3-propylidene]-bis(theophylline) (11).
Compound 9 (0.1 g) was deacetylated as above to afford 11 as colorless syrup
1
(
(
2
5
76% yield); H NMR (DMSO-d , 300 MHz), δ : 3.29 (s, 6 H, 2 NMe), 3.62
6 H
s, 6 H, 2 NMe), 4.21 (br s, 4 H, 2 CH O), 4.39 (s, 4 H, 2 CH N), 5.09 (br t,
H, 2 OH), 7.96 (s, 2 H, 2 H-8). Anal. calcd. for C H N O : C, 49.56; H,
.25; N, 24.34. Found: C, 50.01; H, 4.77; N, 24.36%.
2
2
19
24
8
6
BIOLOGICAL ACTIVITY STUDIES
Preparation and Culture of Hep G2 2.2.15 Cells
The required cell line was made by transfection of Hep G2-cells with
a plasmid containing multiple tandem copies of HBV genome (subtype
[
26]
ayw).
The Hep G2-2.2.15 cell line was maintained in RPMI-1640 (Gluta-
max) culture media containing 100 IU/mL nystatin and 380 µg/mL G418
(
geneticin). The transferred Hep G2–2.2.15 cell line was kept in tissue
◦
culture flask at 37 C + 5% CO . Subcultures were set up after a week by
2
aspiration of the media from culture flask and washing the cells twice by
PBS. A 10% versene/trypsin was added and the cells were incubated for
◦
1
min at 37 C. The drug Lamivudine, which is a potent selective inhibitor of
HBV replication, has been used as a standard for the comparative studies.
PCR-ELLISA
The PCR reaction mixture contained 14 µL extracted supernatant,
4
mmol/L MgCl , 10 µmol/L DIG-11-dUTP, 190 µmol/L dTTP, 200
2
µmol/L dATP, dGTP, dCTP, 1.5 U Taq polymerase, 20 mmol/L HCI (pH
ꢁ
8
.4), 50 mmol/L KCI, 1 µmol/L HCID-1 primer (5 GGA AAG AAG TCA

Microwave Alkylation Reactions
937
ꢁ
ꢁ
GAA GGC A3 ), and 1 µmol/L HCID-2 (5 TTG GGG GAG GAG ATT AGG
TT3), in total volume 50 µL. PCR reaction conditions were 32 cycles of 1
◦
◦
◦
min at 94 C, 30 s, at 58 C and 30 s, at 72 C +3 s, for each cycle in a thermal
circler as described in the literature.[
27]
Cytotoxicity Assay
A colorimetric assay for living cells utilized the colorless substrate 3-
(3,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) that is
modified to a colored product by any living cells, but not by dead cells
or tissue culture medium is the attractive candidate for this purpose. The
cytotoxic effect of the compounds was accessed by culturing the Hep
[
28]
G2–2.2.15 cells in the presence of compounds using a MTT-assay.
Calculation of IC , CC , and SI
5
0
50
The 50% inhibitory concentration of antiviral drugs (IC ) was deter-
50
mined by interpolation from the plots of amount of DNA copies versus
antiviral drug concentration. The 50% cytotoxic effect (CC ) was calcu-
50
lated from the average viability of the cells with concentration of drugs. The
selective index (SI) could be calculated as CC /IC .
50
50
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