Novel reaction products from thiobarbituric acid of biological interest

Article abstract of DOI:10.1002/ardp.200300802

The reactions of 1,2-dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione (1) with some π-deficients have led to the formation of unexpected heterocyclic products such as anilinomethylenethioxopyrimidinedione, 2,2′- bis(pyrimidinecarbothioamide), allylthioxopyrimidinecarbothioamide, indenopyranopyrimidine and benzofuropyrimidine derivatives. A possible mechanism for the formation of these products is discussed, and the biological activity is determined.

Full text of DOI:10.1002/ardp.200300802

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 133−139
Novel Reaction Products from Thiobarbituric Acid 133
Ashraf A. Alya,
Novel Reaction Products from Thiobarbituric
Acid of Biological Interest
Aboul-Fetouh E. Mourada,
Alaa A. Hassana,
Nasr K. Mohameda,
Bahaa A. Alia,
The reactions of 1,2-dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione (1) with some
π-deficients have led to the formation of unexpected heterocyclic products such
as anilinomethylenethioxopyrimidinedione, 2,2Ј-bis(pyrimidinecarbothioamide),
allylthioxopyrimidinecarbothioamide, indenopyranopyrimidine and benzofuropy-
rimidine derivatives. A possible mechanism for the formation of these products
is discussed, and the biological activity is determined.
Mohamed M. El-Sayedb
a Chemistry Department,
Faculty of Science, El-Minia
University, El-Minia,
A.R. Egypt
b Department of Medicinal
Chemistry, Faculty of
Pharmacy, El-Minia
University, El-Minia,
A.R. Egypt
Keywords: Thiobarbituric acid; π-Deficients; Biological activity
Received: March 25, 2003; Accepted: July 9, 2003 [FP802]
DOI 10.1002/ardp.200300802
Introduction
uric acid (1) and phenylisocyanate (2) stirred in py-
ridine at 75Ϫ80 °C gave pyrimidine carboxanilide (3)
1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione (thio-
barbituric acid, 1) is well known as one of the estab-
lished class of pyrimidinethiols. These compounds and
their derivatives are useful intermediates in pyrimidine
chemistry, since the sulfur substitute can be easily re-
moved to give ring-fused pyrimidines [1Ϫ4], or can be
displaced by a variety of nucleophilies [5-8]. The par-
ticularly interesting thiobarbituric acid (1) is accessible
to nucleophilic attacks at C-5 and/or C-2 [9Ϫ21], con-
densation with aldehydes [22Ϫ24], synthesis of dyes
[25Ϫ29], as well as condensation with benzoin to give
thioxofuro[2,3-d]pyrimidines [15]. Furthermore, the py-
rimidine-2-thiole moiety is present in several com-
pounds of biological and medicinal interest [30-34].
[41, 42].
In the present work, we describe the results obtained
by refluxing equimolar amounts of compound 1 and 2
in DMF; 5-(anilinomethylene)-2-thioxohexahydro-4,6-
pyrimidinedione (4) [43] was obtained in 87 % yield.
The formation of 4 can be rationalized in terms of the
Vilsmeier-Haack reaction as illustrated in Scheme 1.
The structural assignment of 4 was established from
comparison of its IR, NMR, and mass spectra as well
as melting point with those of an authentic sample pre-
pared according to reference [43].
Interestingly, on reacting 1 with N-phenyl-1,2,4-triazo-
line-3,5-dione (9) in DMF, compound 4 was also ob-
tained (Scheme 1). This could be rationalized in terms
of the decomposition of 9 in high boiling solvents to
give 2 in high yield [44], which then reacts with 1 to
give 4 (Scheme 1).
Our long-term and continuing interest in chemical re-
actions induced by donor-acceptor interactions via
charge-transfer (CT)-complexation is part of our sys-
tematic efforts to obtain new heterocyclic systems
[35Ϫ39]. Recently, we described the reaction of 2-
thioxo-1,2,4-tetrahydropyrimidine with tetracyano-
ethylene, dicyanomethyleneindane-1,3-dione as well
as 1,4-benzo-and naphthoquinones to synthesize
several heterocyclic compounds [40].
On the basis of structural features, phenyl isothiocyan-
ate (10) should react with 1 in a similar manner as 2.
However, refluxing equimolar amounts of phenyl iso-
thiocyanate (10) and 1 in DMF afforded an interesting
and unexpected compound; 2,2Ј-bis(N5-phenyl-4,6-di-
hydroxy-5-pyrimidine-carbothioamide) (11). As shown
in the proposed mechanism (Scheme 2), a nucleo-
philic addition of pyrimidine-CH2 on 10 gave 12 fol-
lowed by dimerization with elimination of sulfur and de-
hydro-genationto afford the product 11. Compound 11
Results and discussion
Synthesis and spectroscopic characterization
In this paper, we describe the reaction of thiobarbituric
acid (1) as an active donor with some selective π-de-
ficient compounds. It has been reported that thiobarbit-
displayed characteristic IR absorptions at
ν =
3410Ϫ3100 cmϪ1 (OH and NH groups). The 13C-NMR
spectrum of 11 showed absorption signals at δ = 151.6
for pyrimidine-C-2, δ = 197.0 for C=S, and δ = 172.2
for pyrimidine C-4 and C-6. The 1H-NMR spectrum
clearly shows aromatic protons at the expected posi-
tion. Furthermore, phenyl-NH resonates as broad sin-
Correspondence: Aboul-Fetouh E. Mourad, Chemistry
Department, Faculty of Science, El-Minia University, El-Minia
61519, Egypt. Phone: +20 8 634-0304, Fax: +20 8 634-2601,
e-mail: mouradaboulf@yahoo.com
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134 Mourad et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 133−139
Scheme 3.
In a different manner compound 1 reacted with allyl
isothiocyanate (13) to give N5-allyl-4,6-dihydroxy-2-
thioxo-1,2-dihydro-5-pyrimidinecarbothioamide
(14)
Scheme 1.
(Scheme 2). The IR spectrum of 14 showed character-
istic absorptions at ν = 3444-3132 cmϪ1 for OH and
1
NH groups. The H-NMR spectrum showed signals at
δ = 5.90 due to olefinic-CH, 5.20 for olefinic-CH2, 4.20
for CH2-N and at d = 8.20 for aliphatic-NH in addition
to the pyrimidine-NH at δ = 12.50. The 13C-NMR spec-
trum, analytical data and mass spectrum support the
proposed structure 14.
Continuation of our studies on the reactions of 2-
dicyanomethyleneindane-1,3-dione (15) with com-
pounds containing active methylene groups such as
N-arylisoindolines [45], arylaminomethylbenzimid-
azole-2-thiols [46], thioxopyrimidine derivatives [40],
benzimidazolylacetonitrile [47], and 2-mercaptobenz-
azoles [48] prompted us to investigate the behavior of
2-dicyanomethyleneindane-1,3-dione (15) and its fac-
ile isomer 2,3-dicyano-1,4-naphthoquinone [49, 50]
(16) towards 1. In the present investigation both com-
pounds, 6-hydroxy-4-oxo-2-thioxo-1,2,3,4-tetrahydro-
indeno[2Ј,1Ј:5,6]pyrano[2,3-d]pyrimidine-5-carbonitrile
(17) and 1,4-dihydroxy-3-(6-hydroxy-4-oxo-2-thioxo-
1,2,3,4-tetrahydro-5-pyrimidinyl)-2-naphthonitrile (19),
were rather formed than 18 and 20 (Scheme 3).
Scheme 2.
glet at δ = 9.20. The molecular ion peak in the mass
spectrum and satisfactory elemental analysis are also
a further support for the structure of 11.
1
The H-NMR spectrum of 17 clearly shows the pres-
ence of pyrimidine-NHs at δ 11.50 and 12.15 ppm, in
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 133−139
Novel Reaction Products from Thiobarbituric Acid 135
addition to the aromatic protons. The 13C-NMR of 17
shows signals at 105.20 (C-5), 117.20 (CN), 150.10
(C-6), 148.52, 157.34, 171.36 (C-10b, C-5a, C-11a),
167.20 (C = O) and 178.00 (C = S), in addition to the
aromatic carbons. Compound 18 could be ruled out,
owing to the presence of C = S signal in the 13C-NMR
On the other hand, the interaction of 1 with BRL (29)
afforded 5,7,8-tribromo-4,6-dihydroxy-2,3-dihydroben-
zo[4,5]furo[2,3-d]pyrimidine-2-thione (30) (Scheme 4).
The gross formula, C10H3Br3N2O3S of 30 was con-
firmed by the mass spectrum, which exhibited the
molecular ion at m/z 470 (100 %). The IR spectrum
showed absorptions at 3430Ϫ3250 cmϪ1 (OH and
NH). The 1H-NMR displayed one broad singlet at
11.80 ppm for pyrimidine-NH. In the 13C-NMR spec-
trum, C-6 and C-4 resonate at δ 165.30 and 162.40
ppm respectively, 156.80 (C-8a), 172.14 (C-9a) and
182.20 (C = S).
1
and pyrimidine-NH’s in the H-NMR spectra.
The 1H-NMR spectrum of 19 displayed two broad sing-
lets at 11.80 and 12.15 ppm due to pyrimidine-NH’s,
in addition to the aromatic protons. In its 13C-NMR
spectrum, C-6 and C-5 resonate at δ 166.44 and
105.00 respectively; further peaks at δ 143.20, 149.90
(Ar-C-OH), 167.34 (C = O) and 182.50 (C = S) were
also observed. Consequently, compound 20 could be
eliminated on the basis of 1H-NMR spectrum of 19,
which clearly confirms the presence pyrimidine-NHs
and absence of pyrimidine-CH.
From the above findings it can be concluded that the
pyrimidine-CH2 is the more reactive center in thiobar-
bituric acid (1). Moreover, it is interesting to explore
the different behavior of both isomers 15 and 16
towards 1.
Interestingly, the interaction between 2,3-dichlro-5,6-
dicyano-1,4-benzoquinone (DDQ, 21) and 1 resulted
in formation of benzofuropyrimidine derivative 28, in
addition to DDQ-H2 (26).
The IR spectrum of 28 shows two sharp absorptions at
1690 and 2220 cm-1 assigned to carbonyl and cyano
groups, respectively, as well as hydroxyl group at 3460
1
cmϪ1. The H-NMR spectrum of 28 clearly shows the
Scheme 4.
presence of pyrimidine-CH at d 9.25 and pyrimidine-
NH at 11.88. The 13C-NMR (DMSO-d6) of 28 shows
signals at 125.60, 132.60, 153.80, 155.90 and 156.50
for (C-4a, C-11a, C-10b and C-6), respectively, 137.60
(C-2) and 164.20 (C = O). The molecular formula of
28 was supported by elemental analysis and mass
spectrum which gave the expected molecular ion peak
as base peak.
Biological properties
In vitro test for cytotoxic activity [54]
All compounds were tested against tumor cells and
were found to be inactive with the exception of the fol-
lowing two compounds (Table 1).
A proposed mechanism for the formation of the reac-
tion products is illustrated in Scheme 4. Initially a blue-
colored CT-complex was formed, which gradually
changed into ion pair 22 containing 1 cation radical
and DDQ anion radical. Hydrogen proton transfer from
the cation radical to DDQ anion radical generates 23
and 24. The presence of semiquinone 24 induces the
dehydrogenation of 25, which is followed by dimeriz-
ation to give the intermediate 27 after split off sulphur
[37, 48, 51] (identified experimentally). The latter ab-
stracts a molecule of hydrogen from 1 and eliminates
another from H2O to give 5-chloro-6-hydroxy-4-oxo-
1,4-dihydrobenzo[4,5]furo[2,3-d]pyrimidine-7,8-di-
carbonitriles (28).
In vitro disease-oriented primary antitumor
screen [55]
In the routine stage screening each agent is tested
over a broad concentration range against every cell
line in the current panel. The parameter used is GI50,
which is the log10 concentration at which the PG is
+50. As explained in ‘Experimental’, only the tested
compounds 17 and 28 show activity against cell lines
for leukemia, colon cancer, breast cancer, ovarian can-
cer, renal cancer, and prostate cancer. In addition,
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136 Mourad et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 133−139
Table 1.
Compound
No.
% inhibition of cell viability
Dose
Dose
Dose
Dose
100 µg/0.1 mL
75 µg/0.1 mL
50 µg/0.1 mL
25 µg/0.1 mL
17
28
100
100
100
100
100
75
100
60
IR spectra were recorded on 320 FT. IR and Shimadzu 408
spectrophotometer using KBr pellets. MS: Finnigan MAT
8430 at 70 eV (Finnigan, Bremen, Germany). 1H-NMR (400
MHz) and 13C-NMR (101 MHz): Bruker AM-400 (Bruker,
Billerica, MA, USA) with DMSO-d6 as solvent, with tetrameth-
ylsilane (TMS) as internal standard. Preparative layer chro-
matography (plc) used air-dried 1.00 mm thick layers of slur-
ryly applied silica gel Merck PF254 (Merck, Darmstadt, Ger-
many) on 48 cm wide and 20 cm high glass plates, using the
solvents listed. Zones were detected by quenching of indi-
cator fluorescence upon exposure to 254 nm UV light and
eluted with acetone.
compound 17 shows activity against a CNS cancer
cell line.
Antihyperlipedmic activity [56]
Using triton-induced hyperlipidemia animal design
model, where the systemic administration of triton to
mice results in a biphasic elevation of plasma choles-
terol and triglycerides. The mechanism of triton-in-
duced hypercholesterolemia in phase I is thought to
be due to increased hepatic synthesis of cholesterol
through the ability of triton to interfere with the uptake
of plasma lipids by the tissue. Drugs interfering with
cholesterol biosynthesis were shown to be active in
phase I, while drugs interfering with cholesterol ex-
cretion and metabolism were active in phase II. In the
present investigation, both compounds, 11 and 19,
were found to act in phases I and II to lower the chol-
esterol serum level.
Starting materials
1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione (thiobarbit-
uric acid, 1), phenyl isocyanate (2), phenyl isothiocyanate
(10), allyl isothiocyanate (13), and N-phenyl-1,2,4-triazoline-
3,5-dione (9) were purchased from Aldrich (Aldrich, Munich,
Germany) and Fluka (Switzerland) and used as received. 2,3-
Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 21), 2,3,5,6-
tetrabromo-1,4-benzoquinone (BRL, 29), 2-dicyanomethy-
lene-indane-1,3-dione (15), 2,3-dicyano-1,4-naphthoquinone
(16) were purified and prepared as described before [35].
Chemical Reactions
General hypnotic activity [57]
Reaction of 1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione
On performing the potentiation of thiopental sleeping
time in vivo method for the evaluation of the hypnotic
activity, it was found that compound 4 is a very short
acting general hypnotic at a dose level of 35 mg/kg
and lasted for 10 min, while compound 14 is a short
acting, general hypnotic (dose level 40 mg/kg) and
lasted for 30 min.
1 with phenyl isocyanate 2
A solution of phenyl isocyanate 2 (0.12 g, 1 mmol) in 5 mL
DMF was added to a stirred solution of 1 (0.144 g, 1 mmol)
in 15 mL of DMF. The mixture was refluxed for 12h. A yellow
precipitate was formed which was filtered and washed with
ethanol. Recrystallization from DMF/EtOH afforded pure yel-
low crystals of 5-(anilinomethylene)-2-thioxohexahydro-4,6-
pyrimidinedione 4, 0.215 g (87 %), m.p. 343-45 °C [43].
Reaction of 1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione
1 with N-phenyl-1,2,4-triazoline-3,5-dione 9
Acknowledgments
The authors are indebted to the ‘Alexander von Hum-
boldt Foundation’ for the donation of a Shimadzu 408
IR spectrophotometer.
A mixture of 1 (0.144 g, 1 mmol) and N-phenyl-1,2,4-triazo-
line-3,5-dione (9) (0.18 g, 1 mmol) was refluxed in 20 mL
of DMF for 48h. Compound 4 was precipitated, filtered and
recrystallized from DMF/EtOH (0.15 g, 61 %).
Reaction of 1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione
1 with phenyl isothiocyanate 10
Experimental
Apparatus and chemical methods
To a solution of 1 (0.144 g. 1 mmol) in 15 mL DMF, a solution
of phenyl isothiocyanate (10) (0.135 g, 1 mmol) in 5 mL DMF
was added. The reaction mixture was refluxed for 20 h. An
Melting points are uncorrected. Elemental analyses were
measured in the Microanalytical Center at Cairo University.
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 133−139
Novel Reaction Products from Thiobarbituric Acid 137
orange precipitate was formed and recrystallized from DMF/
EtOH to give orange crystals of 2,2Ј-bis(N5-phenyl-4,6-di-
hydroxy-5-pyrimidinecarbothioamide) 11 (0.25 g, 74 %),
275Ϫ77 °C; IR (KBr): νmax 3410Ϫ3100 cmϪ1 (OH, NH) and
1 (0.14 g, 1 mmol) in 10 mLDMF at room temperature. The
reaction mixture becomes pale blue and later turns into violet
color. It was left standing for 48h, then concentrated and the
residue was purified by plc using toluene/ethyl acetate (2:1)
to give 1,4-dih-ydroxy-3-(6-hydroxy-4-oxo-2-thioxo-1,2,3,4-
tetrahydro-5-pyrimidinyl)-2-naphthonitrile 19 (0.25 g, 77 %) as
violet crystals from acetone, m.p. 323Ϫ25 °C; IR (KBr): νmax
3450-3260 cmϪ1 (OH, NH), 2220 (CN) and 1695 (CO); 1H-
NMR (DMSO-d6, 400 MHz) δ 7.30Ϫ7.52 (m, 4H, Ar-H), 11.80
(br, s, 1H, pyrimidine-NH), 12.15 (br, s, 1H, pyrimidine-NH);
13C-NMR ( DMSO-d6, 100.6 MHz) d 89.90, 115.70, 116.92,
120.82, 121.30, 122.94, 126.32, 128.12, 129.96 (Ph-C and
CN), 143.20, 149.90 (Ar-C-OH), 105.00 (C-5), 166.44 (C-6),
167.34 (C-4) and 182.50 (C-2); MS (70 eV), m/z ( %) 327 (M+,
100), 298 (28), 266 (30), 250 (22), 234 (14), 144 (26), 128
(34), 77 (12) (Found: C, 55.12; H, 2.69; N, 12.91; S, 9.68.
1
1600 (C=N); H-NMR (DMSO-d6, 400 MHz): d 7.42-7.86 (m,
10H, Ar-H), 9.20 (br, s, 1H, Ph-NH); 13C-NMR (DMSO-d6,
100.6 MHz): d 124.5 (Ph-2 C), 124.7 (2 pyrimidine-C-5),
125.3 (Ph-4 C), 128.6 (Ph-2 C), 128.8 (Ph-2 C), 139.4 (2 Ar-
C-NH), 151.6, 172.2, 173.2 (2 pyrimidine-C-2, C-4, C-6),
197.0 (2 CS); MS (70 eV): m/z ( %) 492 (M+,11), 338 (96),
246 (36), 228 (29), 92 (100), 77 (44), 57 (38) (Found: C, 5.81;
H, 3.14; N, 16.94; S, 13.16. C22H16N6O4S2 requires C, 53.65;
H, 3.27; N, 17.06; S, 13.02 %).
Reaction of 1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione
1 with allyl isothiocyanate 13
C15H19N3O4S requires C, 55.04; H, 2.77; N, 12.84; S,
In a stirred solution of 1 (0.144 g, 1mmol) in 15 mL DMF a
solution of allyl isothiocyanate (13) (0.119 g, 1 mmol) in 5 mL
DMF was added. The reaction mixture was refluxed for 8 h.
A reddish-brown precipitate was formed. The precipitate was
collected and purified by dissolving it in the minimum amount
of acetone and then applying it for chromatographic (plc). Elu-
tion with chloroform/methanol (10:1) gives only one zone
which was recrystallized from methanol to give a reddish-
brown crystals of N5-allyl-4,6-dihydroxy-2-thioxo-1,2-dihydro-
5-pyrimidinecarbothio-amide 14 (0.18 g, 75 %), m.p.
238Ϫ240 °C; IR (KBr): νmax 3444-3132 cmϪ1 (OH, NH), 2890
(aliphatic- CH); 1H-NMR (400 MHz, DMSO-d6) d 4.20 (d, 2H,
CH2-N), 5.20 (m, 2H, CH2), 5.90 (m, 1H, CH), 8.20 (br, s, 1H,
NH), 12.50 (br, s, 1H, pyrimidine-NH); 13C-NMR(DMSO-d6,
100.6 MHz) δ 51.10 (CH2-N), 67.60 (pyrimidine-C-5), 114.40
(CH2=), 133.80 (CH), 171.60 (pyrimidine-C-4 and C-6),
181.60 (pyrimidine-C-2), 191.20 (C=S); MS (70 eV), m/z ( %)
243 (M+, 24), 225 (100), 209 (5), 186 (15), 100 (9), 56 (19)
(Found: C, 39.61; H, 3.65; N, 17.17;S, 26.42. C8H9N3O2S2
requires C,39.49; H, 3.73; N, 17.27; S, 26.36 %).
9.80 %).
Reaction of 1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione
1 with 2,3-dicyano-5,6-dichloro-1,4-benzoquinone 21
A solution of 2,3-dicyano-5,6-dichloro-1,4-benzoquinone (21)
(0.454 g, 2 mmol) in 10 mL DMF was added dropwise to a
solution of 1 (0.144 g, 1 mmol) in 10 mL DMF at room tem-
perature. The reaction mixture becomes deeply blue and later
turns into yellow color. It was left standing for 48h. The mix-
ture was concentrated and the residue was separated by plc
using toluene/ethyl acetate (1:1) into three zones. The fastest
migrating zone contained sulphur, the second zone contained
5-chloro-6-hydroxy-4-oxo-1,4-dihydrobenzo[4,5]furo-
[2,3-d]pyrimidine-7,8-dicarbonitrile 28, and finally the slowest
one contained DDQ-H2 (26). Compound 28 was recrys-
tallized from methanol to give a yellow crystals (0.18 g, 63 %),
m.p.280 °C (decomp.); IR (KBr): νmax 3460 cm-1 (OH), 2220
(CN) and 1690 (CO); 1H-NMR (DMSO-d6, 400 MHz); δ 9.25
(s, 1H, pyrimidine-CH), 11.88 (br, s, 1H, pyrimidine-NH);
13C-NMR (DMSO-d6, 100.6 MHz), δ 116.80,119.30 (Ph-C-Cl,
Ph-C-CN and CN), 125.60, 132.60, 153.80, 155.90, 156.50
(C-4a, C-4b, C-11a, C-10b and C-6), 137.60 (C-2) and 164.0
(C-4); MS (70 eV): m/z ( %) 288 (30), 286 (M+, 100), 284 (32),
266 (34), 246 (24), 220 (32), 204 (34), 176 (18), 142 (16),
106 (30) and 90 (18) (Found: C, 50.35; H, 1.12; N, 19.42;
Cl, 12.29. C12H3ClN4O3 requires C, 50.28; H, 1.05; N, 19.55;
Cl, 12.37 %).
Reaction of 1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione
1 with 2-dicyanomethyleneindane-1,3-dione 15
A
solution of 2-dicyanomethyleneindane-1,3-dione (15)
(0.416 g, 2 mmol) in 10 mL DMF was added to a solution of
1 (0.144 g, 1mmol) in 10 mL DMF. The reaction mixture was
refluxed for 4h to give a brown precipitate. The precipitate
was dissolved in 5 mL DMF and then applied on plc using
chloroform/methanol (5:1) as eluent to give only one zone
characterized with a brown color. Recrystallization from DMF
Reaction of 1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione
1 with 2,3,5,6-tetrabromo-1,4-benzoquinone (BRL) 29
gave
6-hydroxy-4-oxo-2-thioxo-1,2,3,4-tetrahydroindeno-
[2Ј,1Ј:5,6]-pyrano[2,3-d]pyrimidinyl-5-carbonitrile 17 (0.21 g,
69 %), m.p. 310Ϫ12 °C; IR (KBr): νmax 3440-3220 cmϪ1 (OH,
NH), 2210 (CN) and 1690 (CO); 1H-NMR (DMSO-d6, 400
MHz) δ 7.00-7.45 (m, 4H, Ar-H), 11.50 (br, s, 1H, pyrimidine-
NH), 12.15 (br, s, 1H, pyrimidine-NH); 13C-NMR (DMSO-d6,
100.6 MHz), δ 105.20 (C-5), 107.90, 108.10, 108.40, 113.10
(Ph-C), 129.50 (2 Ph-C), 117.20 (CN), 150.10 (C-6), 148.52,
157.34, 171.36 (C-10 b, C-5a, C-11 a), 167.20 (C-4) and
178.00 (C-2); MS (70 eV), m/z ( %) 309 (M+, 100), 292 (26),
279 (18), 266 (22), 250 (18), 226 (60), 210 (22), 196 (32),
168 (8), 152 (16), 76 (20) and 44 (62) (Found: C, 58.19; H,
2.33; N, 13.64; S, 10.29. C15H7N3SO3 requires,C, 58.25;
H,,2.28; N, 13.59; S, 10.37 %).
A mixture of 1 (0.144 g, 1 mmol) and 2,3,5,6-tetrabromo-1,4-
benzoquinone (BRL, 29) (0.424 g, 1 mmol) in 20 mL glacial
acetic acid was refluxed for 5h. The reaction mixture was co-
oled and added to ice-cold water (200 mL), to give a yellow
precipitate. The precipitate was washed several times with
water and recrystallized from DMF to afford a brown crystals
of 5,7,8-tribromo-4,6-dihydroxy-2,3-dihydrobenzo[4,5]furo-
[2,3-d]pyrimidine-2-thione 30: (0.37 g, 78 %), m.p. 350 °C; IR
(KBr): νmax 3430-3250 cm-1 (OH, NH); 1H-NMR (DMSO-d6,
400 MHz), δ 11.80 (br, s, 1H, NH); 13C-NMR (DMSO-d6,
100.6 MHz) δ 121.20, 125.44, 127.34 (Ph-C and Ph-C-Br),
156.8 (C-8a), 165.30 (C-6), 162.40 (C-4), 172.14 (C-9a) and
182.20 (C-2); MS (70 eV): m/z ( %) 474 (20), 472 (94), 470
(M+, 100), 468 (20), 465 (35), 390 (22), 388 (44), 350 (36),
270 (18), 210 (40), 150 (30), 82 (65) and 60 (54) (Found: C,
25.63; H, 0.58; N, 6.09; S, 6.75. C10H3Br3N2O3S requires C,
25.51; H, 0.64; N, 5.95; S, 6.81 %).
Reaction of 1,2-Dihydro-2-thioxo-4,6(1H,5H)pyrimidinedione
1 with 2,3-dicyano-1,4-naphtho-quinone 16
A solution of 2,3-dicyano-1,4-naphthoquinone (16) (0.416 g,
2 mmol) in 10 mL DMF was added dropwise to a solution of
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138 Mourad et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 133−139
Biological properties
In vitro test for cytotoxic activity
Table 3.
Agent
A set of sterile test tubes was used where 2.5 ϫ 105 tumor
cells per mL (Ehrlich ascites carcinoma) were suspended in
phosphate buffer saline, then 0.1 mL equivalent to 100 mg of
the compound suspended in water and Tween 80 were ad-
ded. The test was incubated for two hours at 37 °C. Trypan
blue exclusion test was then carried out to calculate the per-
centage of non viable cells after two hours in cubation. Com-
pounds producing more than 70 % non viable cells or less
than 30 % viable cells were considered active.
% of serum cholesterol
to normal control value
0 h
6 h
24 h
48 h
Control
Triton
Clofiberate
Compound 11
Compound 19
100
100
100
100
100
100
228
104
103
106
100
231
98
88
91
100
226
98
82
86
In vitro disease-oriented primary antitumor screen
In the routine stage screening, each agent is tested over a
broad concentration range against every cell line in the cur-
rent panel. All lines are incubated onto a series of standard
96 well microtitre plates on day 0, in the majority of cases at
20,000 cells/well, then pre-incubated in absence of drug for
24 hours. Tested drugs are then added in five to ten fold di-
lutions starting from the highest concentration and incubated
for further 48 hours. Following this, the cell are fixed in situ,
washed and dried, sulforhodanine B (SRB) was added, the
bound stain is solubilized and measured spectrophotometri-
cally on automatic plate readers.
[isooctylpolyoxyethylenephenol]. Serum cholesterol level in-
creases sharply 2-3 times after 24 hours (phase I). The hy-
percholesterolemia decreases nearly to control level within
the next 24 hours (phase II). The tested agent and the gum
accacia (used to solubilize the drug) are administrated simul-
taneously with the triton injection. Serum cholesterol analysis
are made 5, 24, and 48 hours after triton injection.
The parameter used here is GI50 (Table 2) which is the log10
concentration at which the PG is + 50 (Table 3).
Antihyperlipedmic activity
Male mice weighing 20-25 g are starved for 20 hours and
then injected intravenously with 200mg/kg triton WR 1339
General hypnotic activity
On performing the potentiation of thiopental sleeping time in
vivo method for evaluation of the hypnotic activity, that de-
pend on injection of the tested compounds 4 and 14 or the
standard 60 min before i.v. injection of 25 mg/kg thiopental to
mice with a weight between 18 and 22 g . The animals are
placed on their backs and the reappearance of the righting
reflux is observed.
Table 2.
Cell line
Compound
GI50
Leukemia
CCRF-CEM
SR
17
28
17
28
17
28
17
28
17
28
17
28
17
28
17
28
17
28
17
28
5.22E-05
3.38E-05
4.05E-05
3.85E-05
4.70E-05
3.81E-05
9.42E-05
7.16E-05
4.70E-05
9.24E-05
4.97E-05
4.44E-05
7.58E-05
6.18E-05
3.54E-05
2.98E-05
3.81E-05
2.96E-05
6.23E-05
5.31E-05
Table 4.
HL-60
Compound
Dose
MOLT-4
RPMI-8226
ED50
D50
4
14
4
20 mg/kg
23 mg/kg
1130 mg/kg
1210 mg/kg
14
Colon cancer
Colo 201
Breast cancer
T-47 D
Ovarian cancer
IGR OV1
Renal cancer
786-O
Toxicological Studies (Tab. 4)
References
[1] M. G. Marei, M. El-Ghanam, J. Chem. Res. (S), 1993,
292Ϫ293.
Prostate cancer
PC-3
[2] V. J. Ram, R. C. Srimol, D. S. Kushwaha, L. Mishra, J.
Prakt. Chem. 1990, 332, 629Ϫ639.
CNS cancer
SF Ϫ 539
SNB Ϫ 19
SNB Ϫ 57
U 251
[3] A. S. Mikhailov, N. G. Pashkurov, V. S. Reznik, Russ. J.
Chem. 1997, 67, 328Ϫ329.
17
17
17
17
4.36E-05
8.16E-05
8.31E-05
5.17E-05
[4] [Chem. Abstr. 1998, 128, 270582 p].
[5] R. Sharma, S. Kumar, H. K. Pujari, Indian J. Chem.
1991, 30 B, 425Ϫ426.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 133−139
Novel Reaction Products from Thiobarbituric Acid 139
[6] T. P. Selby, B. K. Smith, J. Heterocycl. Chem. 1989,
26, 1237Ϫ1240.
[32] K. Kikugawa, J. Kato, A. Iwata, Anal. Biochem. 1988,
174, 512Ϫ521.
[33] J. A. Knight, R. K. Pieper, L. McClellan, Clin. Chem.
1988, 34, 2433Ϫ2438.
[7] W. Nawrocka, M. Zmecki, Arch. Pharm. 1997, 330,
399Ϫ405.
[34] W. Albro, J. T. Corbett, J. L. Schroeder, J. Biochem. Bi-
ophys. Methods 1986, 13, 185Ϫ194.
[8] M. A. F. Sharaf, F. A. Abdel-Aal, A. M. Abdel-Fattah, A.
M. R. Abdel Khalik, J. Chem. Res. (S), 1996, 354Ϫ355.
[35] A. Hassan, N. K. Mohamed, Y. R. Ibrahim, K. U. Sadek,
A. E. Mourad, Bull. Chem. Soc. Jpn. 1993, 66,
2612Ϫ2661.
[9] S. Nakatsuj, K. Nakashima, M. Iyoda, S. Akiyama, Bull.
Chem. Soc. Jpn. 1988, 61, 2253Ϫ2255.
[10] F. F. Abdel-Latif, E. KH. Ahmed, R. Mekheimer, M. M.
[36] A. Aly, A. A. Hassan, A. E. Mourad, Can. J. Chem. 1993,
71, 1845Ϫ1849.
Mashaly, Arch. Pharmacol Res. 1997, 20, 507Ϫ509.
[11] K. Hirota, Y. Kitade, S. Senda, J. Heterocycl. Chem.
1985, 22, 345Ϫ347.
[37] A. Hassan, N. K. Mohamed, B. A. Ali, A. E. Mourad,
Tetrahedron, 1994, 50, 9997Ϫ10010.
[12] M. K. A. Ibrahim, M. R. H. El-Moghayar, M. A. F. Sharaf,
[38] A. Hassan, N. K. Mohamed, A. A. Aly, A. E. Mourad,
Indian. J. Chem. 1987, 26 B, 216Ϫ219.
Monatsh. Chem. 1997, 128, 61Ϫ70.
[13] V. K. Ahluwalia, R. Aggarwal, M. Alauddin, G. Gill, C. H.
[39] A. Aly, N. K. Mohamed, A. A. Hassan, A. E. Mourad,
Khanduri, Heterocycles 1990, 31, 129Ϫ137.
Tetrahedron, 1999, 55, 1111Ϫ1118.
[14] V. K. Ahluwalia, R. Kumar, M. Aluaddin,C. H. Khauduri,
[40] N. K. Mohamed, A. A. Aly, A. A. Hassan, A. E. Mourad,
N. Mallika, Synth. Commun. 1990, 20, 1265Ϫ1272.
H. Hopf, J. Prakt. Chem. 1996, 338, 745Ϫ749.
[15] V. K. Ahluwalia, R. Tyagi, M. Kanr, Indian. J. Chem.
1990, 29 B, 566Ϫ567.
[41] J. A. Minatell, A. D. Brewe, Ger. Offen. DE. 3, 446, 371
(Cl. C07D239/22), [Chem. Abstr. 1986, 104, 19602k].
[16] M. R. Harnden, D. T. Hurs, Aust. J. Chem. 1990, 43,
55Ϫ62.
[42] S. Jeffrey, M. Louis, Drug Metab Dispos. 1991, 19, 263.
[43] H. Zenno, J. Pharm. Soc. Jpn. 1954, 74, 196Ϫ199 and
[17] F. F. Abdel-Latif, Indian J. Chem. 1991, 30 B, 363Ϫ365.
1236-1240.
[18] G. D. Atri, P. Gomarasca, G. Resnati, G. Tronconi, C.
Scolastico, C. R. Sirtori, Med. Chem. 1984, 27,
1621Ϫ1629.
[44] R. A. Izydore, H. E. Johnson, R. T. Horton, J. Org.
Chem. 1985, 50, 4589Ϫ4595.
[45] A. Hassan, Ph. D. Thesis, El-Minia University, 1988.
[19] V. K. Chadha, V. K. Saxena, J. Indian Chem. Soc. 1980,
LVII, 946Ϫ947.
[46] A. Hassan, Phosphorus, Sulfur and Silicon, 1996, 113,
231Ϫ236.
[20] E. M. Afsah, A. Etman, W. S. Hamama, A. F. Sayed
[47] A. Hassan, A. A. Aly, N. K. Mohamed, A. E. Mourad,
Ahmed, Pharmazie, 1995, 50, 567Ϫ569.
Heterocyclic Commun. 1996, 2, 441Ϫ446.
[21] F. F. Abdel-Latif, M. M. Mashaly, R. Mekheimer, T. B.
[48] A. Hassan, N. K. Mohamed, E. H. El-Tamany, B. A. Ali,
Abdel-Aleem, Z. Naturforsch., 1993, 48b, 817Ϫ820.
A. E. Mourad, Monatsh. Chem. 1995, 126, 653Ϫ662.
[22] G. Witz, N. J. Lawrie, A. Zaccaria, H. E. Jr. Ferran, B. D.
[49] G. J. Ashwell, M. R. Bryce, S. R. Davies, M. Hasan, M,
J. Org. Chem. 1988, 53, 4585Ϫ4587.
Goldstein, J. Free Radicals Biol-Med. 1986, 2, 33Ϫ39.
[23] E. I. Ibrahim, M. El-Badawi, H. Farag, M. M. Abbasi,
[50] M. R. Bryce, S. R. Davies, M. Hasan, G. I. Ashwell, M.
Szablewski, M. G. B. Drew, R. Short, M. B. Hursthouse,
J. Chem. Soc. Perkin Trans. 1989, 2, 12185Ϫ12192.
Alexandria J. Pharm. Sci., 1990, 4, 162Ϫ165.
[24] K. C. Joshi, V. N. Pathak, R. Gupta, J. Indian Chem.
Soc. 1990, 67, 434Ϫ435.
[51] W. M. Abdou, M. M. Sidky, H. Z. Wamhoff, Naturforsch.
1987, 42b, 1153Ϫ1158.
[25] S. Akiyama, K. Nakashima, S. Nakatsuji, M. Suzuki, C.
Umekawa, Dyes Pigm. 1987, 8, 459Ϫ463.
[52] A. A. Hassan, Bull. Soc. Chim. Fr. 1994, 131, 424Ϫ428.
[53] A. A. Hassan, N. K. Mohamed, A. A. Aly, A. E. Mourad,
[26] K. Taniguchi, K. Inoue, Kokai Tokkyo Koho Jp., 61, 236,
Pharmazie 1997, 52, 23Ϫ28.
861 (Cl. C 09 B 29/09), C. A. 106 157956w (1987).
[54] M. M. El-Merzabani, A. A. El-Aaser, A. K. El-Duweini, A.
[27] [Chem. Abstr. 1987, 106, 157956w].
M. El-Masry, Planta Med. 1979, 36, 87Ϫ90.
[28] H. Kosugi, K. Kikugawa, Yukagaku, 1989, 38, 224Ϫ230.
[29] [Chem. Abstr. 1989, 111, 41341r].
[55] V. T. DeVita, S. Hellman, S. A. Rosenberg in The Can-
cer, Principles and Practice of Oncology, 6th ed, Lippin-
cott Williams Wilkins, 1989, pp. 3, 5.
[30] A. Monge, V. Martinez-Merino, C. Sammartin, F. J. Fer-
nandez, M. C. Ochoda, C. Bellver, P. Artigas, E. Fernan-
dez-Alverez, Eur. J. Med. Chem. 1989, 24, 209Ϫ216.
[56] D. Frantz, B. T. Hinkelman, J. Exp. Med. 1955, 101,
225Ϫ232.
[31] M. Gerhard, F. Anglika, M. Z. Beate, Gesamte Inn. Med.
ihre Grenzgeb, 1986, 41, 673Ϫ676.
[57] L. S. Harris, F. C. Uhle, J. Pharmcol. Exp. Ther. 1961,
132, 251Ϫ57.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim