Synthesis and anti-HIV-1 activity of novel 10-thiaisoalloxazines, a structural analog of C-5 and/or C-6 substituted pyrimidine acyclonucleoside

Article abstract of DOI:10.1248/cpb.51.630

A series of novel 10-thiaisoalloxazine derivatives bearing an alkoxymethyl or benzyloxymethyl moiety at the N-1 position has been synthesized through the bromination of 1-substituted-5-hydroxyuracils and subsequent condensation with aminobenzenethiol in a one-pot reaction. Contrary to the previous report, the formation of intermediary 5,6-diethoxy-5-hydroxy-5,6-dihydrouracil seems to be not the necessary factor for the formation of the thiaisoalloxazines, since the reaction proceeds in tetrahydrofuran (THF) or acetonitrile far more smoothly than in ethanol. The anti-human immunodeficiency virus (HIV)-1 activity of the resulted thiaisoalloxazine derivatives was evaluated in lymphocyte cells based on the inhibitory activity against the viral-induced cytopathic activity. Among the derivatives, compounds 6, 7, and 8 bearing an alkoxymethyl moiety at the N-1 position exhibited modest inhibitory activity towards the cytotopathic effect of HIV-1.

Full text of DOI:10.1248/cpb.51.630

630
Chem. Pharm. Bull. 51(6) 630—634 (2003)
Vol. 51, No. 6
Synthesis and Anti-HIV-1 Activity of Novel 10-Thiaisoalloxazines, a
Structural Analog of C-5 and/or C-6 Substituted Pyrimidine
Acyclonucleoside
,c
Takanori MIYASHITA,a Masanori BABA,b,1) Shiro SHIGETA,b Kenya MORI,a,2) and Kazuo SHINOZUKA
*
a Biochemical Division, Yamasa Corporation; 2–10–1 Araoicho, Choshi, Chiba 288–0056, Japan: b Fukushima Medical
University; Fukushima 960–1295, Japan: and c Faculty of Engineering, Gunma University; Kiryu, Gunma 376–0052,
Japan. Received January 14, 2003; accepted April 1, 2003
A series of novel 10-thiaisoalloxazine derivatives bearing an alkoxymethyl or benzyloxymethyl moiety at the
N-1 position has been synthesized through the bromination of 1-substituted-5-hydroxyuracils and subsequent
condensation with aminobenzenethiol in a one-pot reaction. Contrary to the previous report, the formation of in-
termediary 5,6-diethoxy-5-hydroxy-5,6-dihydrouracil seems to be not the necessary factor for the formation of
the thiaisoalloxazines, since the reaction proceeds in tetrahydrofuran (THF) or acetonitrile far more smoothly
than in ethanol. The anti-human immunodeficiency virus (HIV)-1 activity of the resulted thiaisoalloxazine deriv-
atives was evaluated in lymphocyte cells based on the inhibitory activity against the viral-induced cytopathic ac-
tivity. Among the derivatives, compounds 6, 7, and 8 bearing an alkoxymethyl moiety at the N-1 position exhib-
ited modest inhibitory activity towards the cytotopathic effect of HIV-1.
Key words thiaisoalloxazine; human immunodeficiency virus; pyrimidine acyclonucleoside; 1-[(2-hydroxyethoxy)methyl]-6-
(phenylthio)thymine (HEPT)
10-Thiaisoalloxazine, pyrimido[4,5-b][1,4]thiazin-2,4-(1H, position was prepared and their anti-HIV activity was exam-
3H)-dione, is the thia analogue of isoalloxazines in which the ined. The anti-HIV-1 assay using MT-4 cells indicated that
nitrogen atom at the 10th position of isoalloxazine is substi- some of the analogs possess inhibitory effect against the cy-
tuted by a sulfur atom. It is known that some thiaisoalloxazine totoxicity of HIV-1 towards lymphocyte cells. Here, we
derivatives exhibit anti-allergy or anti-inflammation activity as would like to describe the synthesis and the first report of the
lipoxygenase inhibitor.3,4) Meanwhile, Miyasaka et al. re- anti-HIV-1 activity of the 10-thiaisoalloxazine derivatives.
ported that C-5 and/or C-6 substituted pyrimidine acyclonu-
cleoside analogues, such as 1-[(2-hydroxyethoxy)methyl]- Chemistry
6-(phenylthio)thymine (HEPT) and 6-benzyl-1-(ethoxy-
1-Substituted 10-thiaisoalloxazine derivatives 6—14 were
methyl)-5-isopropyluracil (MKC-442), have considerable synthesized by following the procedure of Sako et al.9,10) The
anti-human immunodeficiency virus (HIV) activity (Fig. key intermediates, 1-substituted-5-hydroxy uracils (2—4),
1).5—7) These compounds exhibit the activity as a selective except 5-hydroxyuridine (5), were synthesized from 5-hy-
HIV-1 reverse transcriptase inhibitor. From the structural droxyuracil (1) (Chart 1). Thus, compound 1 was treated
point of view, 10-thiaisoalloxazine can be regarded as the C- with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and tri-
6 phenylthio derivative of uracil and, therefore, it shows methylchlorosilane (TMS-Cl) in acetonitrile (CH3CN), fol-
some structural similarity with HEPT.8) These facts prompted lowed by alkoxy chlorides alone or a mixture of 2-ace-
us to synthesize and evaluate the anti-HIV activity of 10-thi- toxyethyl acetoxymethyl ether and tin(IV) chloride (SnCl4) to
aisoalloxazine derivatives. In this study, a series of novel acy- give 1-substituted-5-hydroxyuracils (2—4) in modest yields.
clonucleoside analogs of 10-thiaisoalloxazines bearing an In order to prepare 1-substituted-10-thiaisoalloxazines (6—
alkoxymethyl moiety or benzyloxymethyl moiety at the N-1 12, 14), 1-substituted-5-hydroxyuracils (2—5) were treated
with N-bromosuccinimide (NBS) in ethanol. Subsequently,
3-substituted or non-substituted analogs of 2-aminoben-
zenethiol10) were added to the mixture, and the mixture was
heated under reflux. This one-pot condensation reaction was
effective for the preparation of 1-ethoxymethyl (6—8), 1-
acetoxyethoxymethyl (12) and 1-ribosyl (14) substituted 10-
thiaisoalloxazines from the corresponding uracil derivatives.
The products were obtained in modest yields (Chart 2a, 40—
Fig. 1
69%) (Table 1, entries 1, 3, 5, 18, 19). Deacetylation of 12
Chart 1
* To whom correspondence should be addressed. e-mail: sinozuka@chem.gunma-u.ac.jp
© 2003 Pharmaceutical Society of Japan

June 2003
Table 1.
631
By-product
(yield)
Entry
Substrate
R
RЈ
Solvent
Condition
Yield (%)
Product
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
4
5
1
1
Me
Me
Me
Me
Me
Me
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
EtOH
THF
EtOH
THF
EtOH
THF
EtOH
THF
CHCl3
CH3CN
DMF
EtOH
BnOH
THF
EtOH
BnOH
THF
EtOH
EtOH
EtOH
THF
reflux
r.t.
reflux
r.t.
reflux
r.t.
reflux
r.t.
62
79
60
64
40
70
27
70
64
57
47
1
6
6
7
7
8
8
9
9
9
Me
Me
Cl
Cl
H
H
H
H
H
Me
Me
Me
Cl
Cl
Cl
H
6 (10%)
r.t.
r.t.
r.t.
9
9
reflux
80 °C
r.t.
reflux
80 °C
r.t.
reflux
reflux
reflux
reflux
10
10
10
11
11
11
12
14
15
15
7 (16%)
8 (22%)
5
63
8
4
65
69
59
75
22
AcOCH2
Chart 2a
Chart 2b
using sodium ethoxide in ethanol gave compound 13, almost
quantitatively (Chart 2a).
The same reaction procedure using 1-benzyloxymethyl
substituted uracil (3) as the starting material, however,
resulted in a considerable amount (10 to 22%) of 1-
ethoxymethyl derivatives (6—8) as by-products while the
yields of the desirable compounds (9—11) were quite low
(Chart 2b), as indicated in Table 1 (entries 7, 12, 15). The
Chart 2c
formation of the by-products may be due to an alcohol ex- ethanol as the reaction solvent to prevent the possible alcohol
change reaction (Chart 2b) since ethanol was used as the re- exchange reaction gave the desirable products in only low
action solvent. Using benzyl alcohol (BnOH) instead of yields (4 to 5%, Table 1, entries 13, 16).

632
Vol. 51, No. 6
Table 2. Inhibitory Effects of the Test Compounds on HIV-1 Induced Cy-
topathicity in MT-4 Cells
In further attempts, we employed other reaction solvents
such as tetrahydrofuran (THF), chloroform (CHCl3), CH3CN,
or N,N-dimethylformamide (DMF). These aprotic solvents
worked far better than ethanol. The reaction proceeded even
at room temperature and the yields of the desirable com-
pounds were generally higher than in the cases using ethanol
(Table 1, entries 8—11). THF was the most effective solvent
among the solvents tested here. Compounds 9, 10 and 11
were successfully obtained in 70, 63 and 65% yield, respec-
tively, from the corresponding 1-benzyloxymethyl hydroxyu-
racil (3) and 2-aminobenzenethiol derivatives in THF (Chart
2b) (Table 1, entries 8, 14, 17). Under this reaction condition,
we could not detect the formation of 1-ethoxymethyl deriva-
tives (6—8) at all. Using THF as the reaction solvent also
gave better results for the preparation of 1-ethoxymethyl thi-
aisoalloxazines (6—8) (Chart 2a) (Table 1, entries 2, 4, 6). In
the same manner, compound 159) was obtained directly from
compound 1 (Chart 2c), although the yield was not very
good (Table 1, entry 21).
EC50
(mg/ml)
CC50
(mg/ml)
Compound
S.I.
6
7
8
6.9
1.4
1.4
50
9.5
28
7.2
6.8
20
9
10
11
13
Ͼ23
Ͼ1.9
Ͼ3
23
Ͼ53
23
1.9
3
276
53
Ͼ1
Ͼ1
Ͼ1
12
14
Ͼ1
3Ј-Azido-3Ј-deoxy-
thymidine (AZT)
0.0022
8.3
3773
50% effective concentration or compound concentration required to inhibit HIV-1-in-
duced cytopathicity in MT-4 cells by 50%. 50% cytotoxic concentration or compound
concentration required to reduce MT-4 cell viability by 50%. Selectivity index or ratio
of CC50 to EC50.
reported by Sako et al.9) In the report, it was suggested that
Biological Activity
5-hydroxyuracil reacts with NBS reagent in the presence of
The newly synthesized 1-substituted-10-thiaisoalloxazines ethanol to form an intermediary 5,6-diethoxy-5-hydroxy-5,6-
were evaluated for their anti-HIV-1 activities in the human dihydrouracil (17) (Chart 3, path B, RϭH). Subsequently, the
lymphoblastoid cell line MT-4 based on the inhibition of intermediate reacts with 2-aminobenzenethiol to result in the
HIV-1-induced cytopathic effect, as previously described.12) corresponding 10-thiaisoalloxazine. In this mechanism, the
Briefly, MT-4 cells were suspended in culture medium at presence of ethanol plays a crucial role to form the interme-
3ϫ104 cells/ml and infected HIV at a multiplicity of infec- diate.
tion (MOI, ratio of CCID50 (50% cell culture infective dose)
In our study, however, the procedure gave good results
to cell number) of 0.02. Immediately after virus infection, only for the cases in which 1-ethoxymethyl-, 1-ace-
100 ml of the cell suspension was brought into each well of a toxymethyl-, and 1-ribosyl-5-hydroxyuracil were used as the
flat-bottomed microtiter tray containing various concentra- starting materials. Modest amounts of the corresponding 1-
tions of the test compounds. After 5 d of incubation at 37 °C, substituted 10-thiaisoalloxazins were obtained by the proce-
the number of viable cells was determined by the 3-(4,5-di- dure. Attempts to prepare 1-benzyloxymethyl-10-thiaisoal-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) loxazines starting from the corresponding 5-hydroxyuracil,
method.13) Cytotoxicity of the 1-substituted 10-thiaisoallox- however, led to the formation of a considerable amount of 1-
azines was also assessed based on the viability of mock-in- ethoxymethyl-10-thiaisoalloxazines as by-products, while the
fected host cells as determined by the MTT method. The ex- yields of the desirable compounds were quite low. An incre-
periments were performed in parallel with an antiviral activ- ment of the formation of the desirable products was achieved
ity experiment undertaken for comparison purposes for com- by switching the reaction solvent from ethanol to an aprotic
parability. The results are summarized in Table 2. As shown solvent such as THF, CHCl3, or CH3CN. Among these, THF
in Table 2, compound 13 having a hydroxyethoxymethyl seems to be the most suitable solvent. The formation of the
moiety at the N-1 position was the least toxic among the test products was observed even without heating and the yields
compounds, whereas it displayed a modest antiviral activity were quite satisfactory. Interestingly, this is also the case in
against HIV-1 replication. Like compound 13, compounds which 1-ethoxymetyl-5-hydroxyuracil was used as the start-
6—8 bearing an ethoxymethyl moiety at the N-1 position in- ing material. These results indicate that the formation of the
hibited HIV-1 replication in MT-4 cells, though their selectiv- 5,6-diethoxy type of intermediate 17 is not the necessary fac-
ity indices were not high enough. On the other hand, com- tor for the formation of the 1-substituted-10-thiaisoallox-
pounds 9—11 bearing a benzyloxymethyl moiety at the N-1 azines (Chart 3, path A). Alternatively, 2-aminobenzenethiol
position tend to show considerable cytotoxicity. Cytotoxic ef- may react with the possible bromonium intermediate of 1-
fect of compounds 6—8 bearing an ethoxymethyl moiety at subsituted 5-hydroxyuracil directly in THF, as was mentioned
the N-1 position was relatively lower than that of the other before (Table 1, entries 2, 4, 6). The fact that 10-thiaisoallox-
compounds. Compounds 6—8 exhibited inhibitory activity azine (15)9) was successfully obtained from 5-hydroxyuracil
towards the cytotopathic effect of HIV-1, although the activ- (1) with 2-aminobenzenethiol in THF would support this
ity was modest. Attaching a substitution group (chloro group mechanism. However, the yield of the product in this case
or methyl group) at the benzene ring of the alloxazine moiety was considerably lower than that of the reaction using
seems to increase the cytotoxicity, although it does not have ethanol as the solvent (Table 1, entries 20, 21). This result
much effect on the anti-HIV activity of the compounds.
and those mentioned above indicate that the substituent of
the N-1 position of 5-hydroxyuracils may affect the stability
and/or the reactivity of the bromonium intermediate (16).9)
In the biological study, the thiaisoalloxazines without the
Discussion
Synthesis of 10-thiaisoalloxazine and related compounds
starting from readily available 5-hydroxyuracil (1) was first terminal hydroxyl group at the N1-substituent (compounds

June 2003
633
Chart 3
1
6—8) exhibited inhibitory activity towards the virus-induced (MϩH)ϩ; H-NMR (DMSO-d6) d: 11.45 (1H, bs, NH), 7.15 (1H, s, 6-H),
4.99 (2H, s, CH2), 3.48 (2H, q, Jϭ7.0 Hz, CH2CH3), 1.11 (3H, t, Jϭ7.0 Hz,
CH2CH3). Anal. Calcd for (C7H10N2O3): C, 45.16; H, 5.41; N, 15.05. Found:
C, 45.06; H, 5.36; N, 15.05.
1-[(Phenylmethoxy)methyl]-5-hydroxyuracil (3) To a suspension of 5-
cytotopathic effect of HIV-1 in human lymphocyte cells. This
precludes the possibility that phosphorylation of the thi-
aisoalloxazines is required for the activity. The results coin-
cided with those of HEPT and other related compounds of
Tanaka’s report.7) Although compounds 6—8 selectively in-
hibited HIV-1-induced cytopahtic effect in MT-4 cells, their
activity was not comparable to that of HEPT derivatives in-
cluding MKC-442. These compounds are classified as non-
nucleoside HIV-1 reverse transcriptase inhibitors, which in-
teract with amino acids located near the catalytic site of the
enzyme.14) In these compounds, the bulky alkyl substituent at
the C-5 position of the pyrimidine ring reportedly forces Tyr
181 of the enzyme to induce some conformational change of
the enzyme so that the amino acid residue interacts with the
6-benzyl group.14) The relatively low anti-HIV activity of our
compounds could be attributed to the fact that the current 10-
thiaisoalloxazines may not be able to interact with Tyr 181
residue to trigger the necessary conformational change of the
hydroxyuracil (1, 10.2 g, 80 mmol) in acetonitrile (40 ml), chlorotrimethylsi-
lane (15.6 ml, 120 mmol) and 1,1,1,3,3,3-hexamethyldisilazane (25.2 ml,
120 mmol) were added, and the mixture was stirred at room temperature for
5 h. Benzyl chloromethyl ether (11.2 ml, 81 mmol) was added to the mixture
and allowed to react at room temperature for 20 h. After the reaction was
completed, sat. NaHCO3 was added and the resulted precipitates were col-
lected by filtration to give 12.0 g (61%) of 3; mp 157—159 °C; MS m/z 248
(Mϩ); 1H-NMR (DMSO-d6) d: 11.45 (1H, bs, NH), 8.71 (1H, bs, OH)
7.36—7.27 (5H, m, Ph) 7.18 (1H, s, 6-H), 5.11 (2H, s, PhCH2), 4.54 (2H, s,
CH2). Anal. Calcd for (C12H12N2O3): C, 58.06; H, 4.87; N, 11.28. Found: C,
58.01; H, 4.80; N, 11.34.
1-[[(2-Acetoxy)ethoxy]methyl]-5-hydroxyuracil (4) A mixture of 5-
hydroxyuracil (3, 1.28 g, 10 mmol), chlorotrimethylsilane (2.6 ml, 20 mmol)
and 1,1,1,3,3,3-hexamethyldisilazane (4.2 ml, 20 mmol) was stirred at room
temperature for 5 h. After cooling to 0 °C, 2-acetoxyethyl acetoxymethyl
ether (1.8 ml, 10 mmol) and SnCl4 (0.47 ml, 4.0 mmol) were added. The
mixture was stirred at room temperature for 20 h, then sat. NaHCO3 was
added and the resulted precipitates were collected by filtration to give 0.70 g
1
enzyme. The possibility that the rigid tricyclic structure of (29%) of 4; mp 195—197 °C (MeOH); FABϩMS 245 (MϩH)ϩ; H-NMR
(DMSO-d6) d: 11.48 (1H, bs, NH), 8.75 (1H, bs, OH) 7.16 (1H, s, 6-H),
5.04 (2H, s, CH2), 4.10—4.08 (2H, m, CH2), 3.68—3.66 (2H, s, CH2), 1.99
(3H, s, OAc). Anal. Calcd for (C9H12N2O6·1/4H2O): C, 43.48; H, 5.07; N,
11.26. Found: C, 43.63; H, 4.82; N, 10.97.
General Procedure for the Preparation of 10-Thiaisoalloxazines.
Procedure A NBS (392 mg, 2.2 mmol) was added to 1-substituted-5-hy-
droxyuracil (2.0 mmol) in THF (5 ml), and the mixture was stirred at room
temperature for 30 min. To this mixture was added 5-subsituted-2-aminoben-
zenethiol (4.0 mmol), and the mixture was stirred at room temperature for
1 h.
Procedure B NBS (196 mg, 1.1 mmol) was added to 1-substituted-5-hy-
droxyuracil (1.0 mmol) in ethanol (10 ml), and the mixture was stirred at
room temperature for 30 min. To this mixture was added 2-aminoben-
zenethiol (250 mg, 2.0 mmol), and the mixture was heated under reflux for
1 h.
the thiaisoalloxazines was not favorable to fit the reverse
transcriptase pocket is, however, not precluded.
Conclusion
We have synthesized a new series of structural analogs of
C-5 and/or C-6 substituted pyrimidine acyclonucleoside,
namely, 1-substituted 10-thiaisoalloxazines, from the corre-
sponding 5-hydroxy uracils. It was found that the formation
of the 5,6-diethoxy type of intermediate is not the necessary
factor for the formation of the thiaisoalloxazines. Some of
the thiaisoalloxazines were found to be active against HIV-1
replication in cell cultures. Thus, this class of compounds
should be further pursued for their antiviral activity and
structure–activity relationship.
1-(Ethoxymethyl)-1,5-dihydro-2H-pyrimido[4,5-b][1,4]benzothiadine-
2,4(3H)-dione (6) 1-Ethoxymethyl-5-hydroxyuracil (2, 372 mg, 2.0 mmol)
was allowed to react with 2-aminobenzenethiol (500 mg, 4.0 mmol) in THF
according to procedure A. The reaction mixture was purified by silica gel
column chromatography and the obtained material was crystallized from
ethanol to give compound 6: yield 460 mg (79%): mp 193 °C (EtOH); MS
Experimental
General Procedures Thin-layer chromatography (TLC) analyses were
carried out on Merck silica gel 60 F254-precoated plates. Column chro-
matography was performed with silica gel 60 (Merk 7734). 1H-NMR spectra
were recorded on JEOL GSX-400 NMR spectrometer at 400 MHz, using
tetramethylsilane as the internal standard. FAB mass spectra were recorded
with JEOL JMS-AX500 spectrometer. Melting temperatures were deter-
mined with Yanagimoto micro melting apparatus and are uncorrected. Ele-
mental analyses were performed on a Yanagimoto MT-5 elemental analyzer.
1-[(Ethoxy)methyl]-5-hydroxyuracil (2) To a suspension of 5-hydroxy-
uracil (1, 1.0 g, 8.0 mmol) in acetonitrile (5 ml), chlorotrimethylsilane
(1.6 ml, 12 mmol) and 1,1,1,3,3,3-hexamethyldisilazane (2.5 ml, 12 mmol)
were added and the mixture was stirred at room temperature for 5 h.
Chloromethyl ethyl ether (0.6 ml, 8.0 mmol) was added to the mixture and
allowed to react at room temperature for 20 h. After the reaction was com-
pleted, sat. NaHCO3 was added and the resulted precipitates were collected
by filtration to give 0.84 g (58%) of 2; mp 187—190 °C; FABϩMS 187
1
m/z 292 (Mϩ); H-NMR (DMSO-d6) d: 11.69 (1H, s), 7.80 (1H, s), 7.09—
6.79 (4H, m), 5.27 (2H, s), 3.52 (2H, q, Jϭ7.0 Hz, CH2CH3), 1.13 (3H, t,
Jϭ7.0 Hz, CH2CH3). Anal. Calcd for (C13H13N3O3S): C, 53.60; H, 4.50; N,
14.42. Found: C, 53.32; H, 4.29; N, 14.34.
1-(Ethoxymethyl)-1,5-dihydro-8-methyl-2H-pyrimido[4,5-b][1,4]ben-
zothiadine-2,4(3H)-dione (7) 1-Ethoxymethyl-5-hydroxyuracil (2, 372
mg, 2.0 mmol) was reacted with 2-amino-5-methylbenzenethiol (556 mg,
4.0 mmol) in the same manner as above. The reaction mixture was purified
by silica gel column chromatography and the obtained material was crystal-
lized from ethanol to give compound 7: yield 391 mg (64%): mp 194—
1
197 °C (EtOH); MS m/z 306 (Mϩ); H-NMR (DMSO-d6) d: 11.67 (1H, s),
7.64 (1H, s), 7.35—6.85 (3H, m), 5.26 (2H, s), 3.52 (2H, q, Jϭ7.0 Hz,
CH2CH3), 2.14 (3H, s, Ph-Me), 1.13 (3H, t, Jϭ7.0 Hz, CH2CH3). Anal.
Calcd for (C14H15N3O3S): C, 55.07; H, 4.95; N, 13.76. Found: C, 55.22; H,

634
Vol. 51, No. 6
4.91; N, 14.01.
column chromatography and the obtained material was crystallized from
8-Chloro-1-(ethoxymethyl)-1,5-dihydro-2H-pyrimido[4,5-b][1,4]ben-
ethanol to give compound 14: yield 223 mg (59%); mp 203—207 °C
zothiadine-2,4(3H)-dione (8) 1-Ethoxymethyl-5-hydroxyuracil (2, 372 (EtOH); FABϩMS 366 (MϩH)ϩ; 1H-NMR (DMSO-d6) d: 11.76 (1H, s),
mg, 2.0 mmol) was reacted with 2-amino-5-chlorobenzenethiol (640 mg, 7.87 (1H, s), 6.80—7.12 (4H, m), 5.73 (1H, d, Jϭ4.0Hz), 4.61 (1H, dd,
2.0 mmol) in the same manner as above. The reaction mixture was purified Jϭ2.0, 5.0 Hz), 4.11 (1H, t, Jϭ7.0 Hz), 3.31—3.82 (5H, m). Anal. Calcd for
by silica gel column chromatography and the obtained material was crystal- (C15H15N3O6S·3/4H2O): C, 47.55; H, 4.39; N, 11.09. Found: C, 47.42; H,
lized from ethanol to give compound 8: yield 456 mg (70%): mp 178— 4.15; N, 11.02.
180 °C (EtOH); FABϩMS 326 (MϩH)ϩ; 1H-NMR (DMSO-d6) d: 11.72
1,5-Dihydro-1-[(2-hydroxyethoxy)methyl]-2H-pyrimido[4,5-b][1,4]-
(1H, s), 8.01 (1H, s), 7.35—7.05 (3H, m), 5.26 (2H, s), 3.52 (2H, q, benzothiadine-2,4(3H)-dione (13) To a solution of sodium ethoxide
Jϭ7.0 Hz, CH2CH3), 1.13 (3H, t, Jϭ7.0 Hz, CH2CH3). Anal. Calcd for (680 mg, 10 mmol) in ethanol (20 ml), compound 12 (349 mg, 1.0 mmol)
(C13H12N3O3ClS): C, 47.93; H, 3.71; N, 12.90. Found: C, 48.02; H, 4.00; N, was added and the mixture was stirred at room temperature for 2 h. Glacial
13.15.
1,5-Dihydro-1-[(phenylmethoxy)methyl]-2H-pyrimido[4,5-b][1,4]ben-
acetic acid was occasionally added to the reaction mixture to maintain the
pH at about 4. After evaporation to dryness, water was added to the mixture
and the insoluble materials were collected by filtration, then crystallized
from ethanol to give 255 mg (83%) of 13: mp 195 °C (EtOH); MS m/z 324
zothiadine-2,4(3H)-dione (9) 1-[(Phenylmethoxy)methyl]-5-hydroxyuracil
(3, 496 mg, 2.0 mmol) was reacted with 2-aminobenzenthiol (500 mg,
4.0 mmol) in the same manner as above. The reaction mixture was purified by
silica gel column chromatography and the obtained material was crystallized
from ethanol to give compound 9: yield 494 mg (70%): mp 173 °C (EtOH);
1
(Mϩ); H-NMR (DMSO-d6) d: 11.68 (1H, s), 7.79 (1H, s), 7.09—6.79 (4H,
m), 5.30 (2H, s), 3.51 (5H, m). Anal. Calcd for (C13H13N3O5S): C, 50.81; H,
4.26; N, 13.67. Found: C, 50.59; H, 4.29; N, 13.38.
1
FABϩMS 353 (Mϩ), 354 (MϩH)ϩ; H-NMR (DMSO-d6) d: 11.68 (1H, s),
7.79 (1H, s), 7.35—6.80 (9H, m), 5.38 (2H, s), 4.57 (2H, s). Anal. Calcd for
(C18H15N3O3S): C, 61.18; H, 4.28; N, 11.89. Found: C, 61.24; H, 4.28; N,
11.92.
Acknowledgments The authors are grateful to Prof. H. Sawai of
Gunma University for helpful suggestions. The authors also thank Drs. A.
Kuninaka, K. Kodama, M. Morozumi, S. Sakata, and H. Hayakawa of Ya-
1,5-Dihydro-8-methyl-1-[(phenylmethoxy)methyl]-2H-pyrimido[4,5- masa Corporation for valuable discussions.
b][1,4]benzothiadine-2,4(3H)-dione (10) 1-[(Phenylmethoxy)methyl]-5-
hydroxyuracil (3, 496 mg, 2.0 mmol) was reacted with 2-amino-5-methyl- References and Notes
benzenthiol (556 mg, 4.0 mmol) in the same manner as above. The reaction
mixture was purified by silica gel column chromatography and the obtained
material was crystallized from ethanol to give compound 10: yield 459 mg
1) Present address: Center for Chronic Viral Diseases, Faculty of Medi-
cine, Kagoshima University, Kagoshima 890–8520, Japan.
2) Present address: GeneACT, Inc. Kurume Research Center Building
3–2432 Aikawacho, Kurume, Fukuoka 839–0861, Japan.
3) J. PAT No. H04-230690
1
(63 %): mp 164—167 °C (EtOH); MS m/z 368 (Mϩ); H-NMR (DMSO-d6)
d: 11.65 (1H, s), 7.65 (1H, s), 7.35—6.85 (8H, m), 5.37 (2H, s), 4.57 (2H,
s), 2.14 (3H, s, Me). Anal. Calcd for (C19H17N3O3S): C, 62.11; H, 4.66; N,
11.44. Found: C, 62.10; H, 4.66; N, 11.16.
4) J. PAT No. H04-247090
5) Miyasaka T., Tanaka H., Baba M., Hayakawa H., Walker R. T.,
Balzarni J., De Clercq E., J. Med. Chem., 32, 2507—2509 (1989).
6) Tanaka H., Baba M., Hayakawa H., Sakamaki T., Miyasaka T., Uba-
sawa M., Takashima H., Sekiya K., Nitta I., Shigeta S., Walker R. T.,
Balzarni J., De Clercq E., J. Med. Chem., 34, 349—357 (1991).
7) Tanaka H., Takashima H., Ubasawa M., Sekiya K., Inouye N., Baba
M., Shigeta S., Walker R. T., De Clercq E., Miyasaka T., J. Med.
Chem., 38, 2860—2865 (1995).
8-Chlro-1,5-dihydro-1-[(phenylmethoxy)methyl]-2H-pyrimido[4,5-
b][1,4]benzothiadine-2,4(3H)-dione (11) 1-[(Phenylmethoxy)methyl]-5-
hydroxyuracil (3, 496 mg, 2.0 mmol) was reacted with 2-amino-5-chloroben-
zenethiol (640 mg, 4.0 mmol) in the same manner as above. The reaction
mixture was purified by silica gel column chromatography and the obtained
material was crystallized from ethanol to give compound 11: yield 505 mg
1
(65%): mp 132—135 °C (EtOH); FABϩMS 387 (Mϩ), 388 (MϩH)ϩ; H-
NMR (DMSO-d6) d: 11.71 (1H, s), 8.01 (1H, s), 7.36—6.87 (8H, m), 5.37
(2H, s), 4.56 (2H, s). Anal. Calcd for (C18H14N3O3ClS): C, 55.74; H, 3.64;
N, 10.83. Found: C, 55.52; H, 3.55; N, 10.88.
8) Larsen J. S., Christensen L., Ludving G., Jørgensen P. T., Pedersen E.
B., Nielsen C., J. Chem. Soc., Perkin Trans. 1, 2000, 3035—3038
(2000).
1-[[(2-Acetoxy)ethoxy]methyl]-1,5-dihydro-2H-pyrimido[4,5-b][1,4]-
benzothiadine-2,4(3H)-dione (12) 1-[[(2-Acetoxy)ethoxy]methyl]-5-hy-
9) Sako M., Totani R., Konohara M., Hirota K., Maki Y., J. Chem. Soc.
Perkin Trans. 1, 1991, 2675—2677 (1991).
droxyuracil (4, 244 mg, 1.0 mmol) was reacted with 2-aminobenzenthiol 10) Sako M., Niwa T., Hirota K., Maki Y., Chem. Pharm. Bull., 32,
(250 mg, 2.0 mmol) in ethanol according to procedure B. The reaction mix- 2474—2476 (1984).
ture was purified by silica gel column chromatography and the obtained ma- 11) Gupta R. R., Kalwania G. S., Kumar M., Heterocycles, 16, 1527—
terial was crystallized from ethanol to give compound 12: yield 241 mg 1535 (1981).
(69%): mp 141 °C (EtOH); MS m/z 350 (Mϩ); 1H-NMR (DMSO-d6) d: 12) Pauwels R., De Clercq E., Desmyter J., Balzarini J., Goubau P.,
11.72 (1H, s), 7.81 (1H, s), 7.09—6.80 (4H, m), 5.30 (2H, s), 4.11 (2H, m),
3.71 (2H, s), 2.01 (3H, s). Anal. Calcd for (C15H15N3O5S): C, 51.57; H, 4.33;
N, 12.03. Found: C, 51.84; H, 4.28; N, 11.78.
1,5-Dihydro-1-b-D-ribosyl-2H-pyrimido[4,5-b][1,4]benzothiadine-
2,4(3H)-dione (14) This compound was synthesized from 5-hydroxyuri- 14) Hopkins A. L., Ren J., Esnouf R. M., Willcox B. E., Jones E. Y., Ross
Herdewijn P., Vanderhaeghe H., Vandeputte M., J. Virol. Methods, 16,
171—185 (1987).
13) Pauwels R., Balzarini J., Baba M., Snoeck R., Schols D., Herdewijn P.,
Desmyter J., De Clercq E., J. Virol. Methods, 20, 309—321 (1988).
dine (5, 260 mg, 1.0 mmol) with 2-aminobenzenthiol (250 mg, 2.0 mmol) in
C., Miyasaka T., Walker, R. T., Tanaka H., Stammers D. K., Stuart D.
the same manner as above. The reaction mixture was purified by silica gel
I., J. Med. Chem., 39, 1589—1600 (1996).