Synthesis, antioxidant, antituomer activities of some new thiazolopyrimidines, pyrrolothiazolopyrimidines and triazolopyrrolothiazolopyrimidines derivatives

Article abstract of DOI:10.1002/jccs.201190056

Reaction of 6-amino-2-thiouracil 1 with ethyl bromoacetate yielded ethyl 2-(7-amino-2,5-dioxo-3,5-dihydro-2H-thiazolo[3,2-a]pyrimidin-6-yl)acetate 2. Reaction of 2 with sodium ethoxide afforded the pyrrolothiazolopyrimidine derivative 3. Compound 2 reacte

Full text of DOI:10.1002/jccs.201190056

Journal of the Chinese Chemical Society, 2011, 58, 41-48
41
Synthesis, Antioxidant, Antituomer Activities of Some New
Thiazolopyrimidines, Pyrrolothiazolopyrimidines and
Triazolopyrrolothiazolopyrimidines Derivatives
Ameen Ali Abu-Hashem,a,* Mohamed M. Youssefb and Hoda A. R. Husseina
aPhotochemistry Department, National Research Center, 12622, Dokki, Giza, Egypt
bDepartment of Chemistry, Faculty of Science, Taif University, Taif, Saudi Arabia, and
Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt
Received July 14, 2010; Accepted December 6, 2010; Published Online December 16, 2010
Reaction of 6-amino-2-thiouracil 1 with ethyl bromoacetate yielded ethyl 2-(7-amino-2,5-dioxo-3,5-
dihydro-2H-thiazolo[3,2-a]pyrimidin-6-yl)acetate 2. Reaction of 2 with sodium ethoxide afforded the
pyrrolothiazolopyrimidine derivative 3. Compound 2 reacted with hydrazine hydrate to give 7-amino-
thiazolopyrimidine-carbohydrazide 4. The latter compound 4 reacted with carbon disulphide to form
7-amino-6-(oxadiazolylmethyl) thiazolopyrimidine 5. Compound 5 was heated in methanol to yield
9-thioxotriazolopyrrolothiazolopyrimidine 6. Also, the reaction of 3 with aromatic aldehydes afforded the
diarylmethylenepyrrolothiazolopyrimidine derivatives 7a-c. The latter compounds 7a-c underwent
cyclocondensation with hydroxylamine to give diaryldioxazolopyrrolothiazolopyrimidine derivatives
8a-c. The new prepared compounds were subjected for antioxidant and antituomer studies, some of these
compounds exhibited promising activity.
Keywords: Pyrrolothiazolopyrimidine; Thiazolopyrimidine; Triazolopyrrolothiazolopyrimidine; An-
tioxidant and antituomer activities.
INTRODUCTION
Thiazolopyrimidines and thiazolidinedionepyrimi-
tinuation, this paper describes our approach to the synthe-
sis of thiazolopyrimidines, pyrrolothiazolopyrimidines and
triazolopyrrolothiazolopyrimidines.
dines have hypoglycemic and hypolipidemic and antidia-
betic activities.1 Triazolopyrimidines have antifungal ac-
tivity2 and pyrimidine derivatives have antileshimanial ac-
tivity.3 Thus, these compounds have attracted our attention
due to the wide range of biological activities associated
with this scaffold. Various related compounds of these py-
rimidine derivatives have biological activities ranging
from kinase inhibitors, treatment of disease states associ-
ated with angiogenesis [plated derived growth factor,
PDGFr, fibroplast growth factor, FGFr, and epiderma
growth factor, (EGFr),4 the analogous mitogen-activated
protein (CSBP/P38) kinase inhibitor,5 telomerase inhibi-
tor6], for treatment of arthritis, adult respiratory distress
syndrome, chronic obstructive pulmonary disease, or Alz-
heimer’s disease.7 Arylazopyrimidine derivatives showed
antitumor activity8-11 and anticancer activity.12 Our re-
search group has the interest in the development of syn-
thetic strategy to polyfunctionalized heterocycles. In con-
RESULTS AND DISCUSSION
6-Amino-2-thiouracil (1), (mp 320 C),13,14 under-
o
went cyclocondensation by heating under reflux with ethyl
bromoacetate,15 in boiling ethanolic potassium hydrox-
ide,16 for long times to afford ethyl(7-amino-3,5-dioxo-
2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidin-6-yl)ace-
tate (2). The latter compound underwent cyclocondensa-
tion in sodium ethoxide to afford 6,8-dihydropyrrolo[2,3-
d]thiazolo[3,2-a]pyrimidine-3,5,7(2H)-trione (3) (Scheme
I). The IR spectrum compound 2 showed absorption bands
at n 1749 cm-1 corresponding to ester CO, as well as the
other expected bands for such structure. The IR spectrum
of compound 3 revealed the absence of the ester carbonyl
band; besides the presence three amidic carbonyl absorp-
tions as well as other expected absorption bands (Experi-
mental section). The 1H NMR spectra of 2 and 3 are given
* Corresponding author. E-mail: aminaliabuhashem@yahoo.com

42 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Abu-Hashem et al.
Scheme I
in the experimental section. The Mass spectra of 2 and 3
showed the molecular ion peaks at m/z 269 (30.2%) and
223 (55.5%), respectively.
one single absorption band in the amino region. The ab-
sence of NH2 absorption indicates that the involvement of
NH2 group in 5 in the formation of the triazole ring in 6.
1
The reaction of the pyrimidinyl acetate derivative 2
with hydrazine hydrate in boiling dioxane produces 2-(7-
amino-3,5-dioxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimi-
din-6-yl)acetohydrazide (4) in good yield.17 The latter
compound reacted with carbon disulphide to give produce
7-amino-6-[(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-
methyl]-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione (5).
The latter compound, in turn, underwent reductive cycliza-
tion upon heating under reflux in methanol where 3-thioxo-
2,11-dihydro-3H,10H-thiazolo[3,2-a][1,2,4]triazolo[4¢,3¢:
1,5]pyrrolo[2,3-d]pyrimidine-8,10(7H)-dione (6) was
formed,18,19 (Scheme II). Formation of 6 from 5 may pro-
ceed through solvolytic opening of the oxadiazole ring fol-
lowed by elimination of water to form the new triazole ring.
The IR spectrum compound 4 displayed three carbonyl ab-
sorption bands, besides the absence of absorption repre-
senting ester carbonyl that indicates the formation of the
hydrazide moiety. The IR spectrum of compound 5 dis-
played, among other expected bands, only two carbonyl ab-
sorptions. The IR spectrum of compound 6 displayed only
The H, 13C NMR and mass spectra supported the struc-
tures (Experimental section).
Furthermore, pyrrolothiazolopyrimidinetrione 3, hav-
ing two active methylene groups, condensed with aromatic
aldehydes to afford the 2,6-diarylidene-6,8-dihydropyr-
rolo[2,3-d]thiazolo[3,2-a]pyrimidine-3,5,7(2H)-triones
7a-c which underwent cyclocondensation with hydroxyl-
amine in boiling acetic acid, in the presence of anhydrous
sodium acetate to furnish 3,9-diaryl-2,3,8,9-tetrahydro-
isoxazolo [4¢,5¢:4,5]pyrrolo[2,3-d]isoxazolo[5¢,4¢:4,5]thi-
azolo[3,2-a]pyrimidin-10(6H)-ones 8a-c; the reaction path
way involves a double nucleophilec addition of the hydrox-
yl amine hydrochloride to the ethylenic double bond fol-
lowed by ring closure via elimination two molecules of wa-
ter,17 (Scheme III). The IR spectra of compounds 7a-c dis-
played absorption bands that correspond to three carbonyl
groups, as well as the other expected peaks. IR spectra of
8a-c showed only one carbonyl absorption band. The
1
benzylic protons of 8a-c are described as doublets in H-
NMR within d = 4.55-4.57 and 5.21-5.23 due to coupling
Scheme II

Biological Activity & Annulations to Thiazolopyrimidine
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 43
Scheme III
with the adjacent NH, these signals could also represent
pairs of singlets from the diastereoisomeric structures.
Coupling of the benzylic protons with the adjacent NH
Table 1. Anti-oxidant assay for the prepared new compounds
Methods
ABTS
Erythrocyte
Hemolysis
Bleomycin-
Dependent
1
DNA damage
groups could be proved by carrying out H-NMR in the
Compounds a
Inhibition (%) Hemolysis (%) Absorbance
presence of D2O.Deuterium exchange with the NH protons
caused the benzylic protons signals to appear as singlets.
Correct values in elemental analysis of all new compounds
are in agreements with the assigned structures.
L-ascorbic acid
88.61
0
0.85
0
0.881
0
Control
2
3.65
4.10
3.40
0.80
1.01
1.90
0.85
1.99
1.10
1.70
1.20
0.858
0.843
0.886
0.886
0.851
0.780
0.880
0.862
0.883
0.850
3
10.97
83.73
73.70
18.60
11.90
15.20
65.20
19.50
55.20
4
5
PHARMACOLOGY
6
7a
7b
7c
8a
8b
Antioxidant activity
All compounds were tested for antioxidant activity as
reflected in the ability to inhibit lipid peroxidation in rat
brain and kidney homogenates and rate erythrocyte hemo-
lysis. The pro-oxidant activities of the formed compounds
were assayed via their effects on bleomycin-induced DNA
damage. Compounds 4 and 5 manifested potent antioxida-
tive activity in the lipid peroxidation assay but no inhibi-
tory activity in the hemolysis assay. On the other hand,
compounds 4, 5, 7b, 7c, 8a and 8b exhibited significant an-
tioxidant activity and protected the DNA from damage
(Table 1).
8c
29.90
1.50
0.847
a For testing 50 mL of 2 mmol L–1 solution in 1 mL methanol/
phosphate buffer (1:1, V/V) was used.
activity (46.91-25.04%) (Table 2).
Structural Activity Relationship (SAR)
· Novel thiazolopyrimidine derivatives incorporated
with substituted carbohydrazide, amino group, oxadiazol,
moieties possesses potential for antioxidant activities.
Therefore more active compounds (4, 5) because it was
give free radical ions these used antioxidant with abnormal
cell compared with another compounds.
Anti-tumor Activity using in Vitro Ehrlich used
Ascites Assay
The newly synthesized compounds were screened for
their antitumor activity. Viability of the cells used in con-
trol experiments exceeded 95%. Compound 6 (80.10%)
followed by compounds 8b, 8c, 8a (65.14-50.10%) and 2
(48.87%). The other tested compounds showed very weak
· Thioxo, triazolo, pyrrolothiazolopyrimidine for ex-
ample (6) showed the highest cytotoxic activity than py-
rimidine and pyrimidine derivatives where thioxo-triazole

44 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Abu-Hashem et al.
NMR spectra (d, ppm) were recorded on JEOL-ECA500
(National Research Center, Egypt) and JEOL JNM-LA-
400 FT NMR Spectrometer (Chemistry Department, Cairo
University) and chemical shifts were expressed as d values
against TMS as internal standard. Mass spectra were re-
corded on GCMS-QP 1000 EX Shimadzu Japan (Gas
Chromatography-Mass spectrometer). Microanalytical
data were obtained at the Microanalytical Center at Cairo
University and National Research Center, Egypt.
Table 2. Results of Ehrlich in vitro assay all compounds
Compounds No. a
Dead Cells (%)
Control (no drugs)
5-Floururacil b
0
99.50
48.87
45.12
40.99
46.91
80.10
27.92
25.04
32.19
50.10
65.14
55.17
2
3
4
5
6
7a
7b
7c
8a
8b
8c
Ethyl 2-(7-amino-2,5-dioxo-3,5-dihydro-2H-thiazolo-
[3,2-a]pyrimidin-6-yl)acetate (2)
A mixture of 6-aminothiouracil (1) (1.43 g, 10 mmol)
and (ethyl bromoacetate, 20 mmol) was refluxed in ethanol
(50 mL) in presence of potassium hydroxide (10 mmol) or
(sodium acetate, acetic acid, acetic anhydride) for 20 h (un-
der TLC control). The reaction mixture was cooled with
water; the deposited precipitate was filtered off, washed
with ethanol, dried, and crystallized from methanol to af-
ford 2 as white crystals (85%), mp 205-207 oC (dec.). IR
(KBr) nmax. 3352, 3177 (NH2), 1750 (CO, ester group),
a 1 mg mL–1 in DMSO/RPMI-1640 (1:10)
b 25 mg mL–1 in DMSO/RPMI-1640 (1:10)
group increasing the blood inter tissue cell. Hence this
compounds for used antitumor agents (cytotoxic activity).
· Diaryl-dioxazolo-pyrrolo-thiazolopyrimidine (ex.
8a-c) was found to be of moderate to antitumor activities
when compared with the pyrimidine derivatives.
· Diarylidene-dihydropyrrolothiazolopyrimidine-
triones (ex. 7a-c) showed the moderate antioxidant activity
than pyrimidine derivatives.
1
1652 (br., 2CO, amidic), 1626 cm-1 (C=N); H NMR
(DMSO-d6) d 1.22 (t, 3H, J = 7.10 Hz, CH3), 3.32 (s, 2H,
CH2), 4.14 (q, 2H, J = 7.15 Hz, CH2), 4.78 (s, 2H, CH2,
thiazole ring), 6.79 (br., 2H, NH2, D2O exchangeable); 13
C
NMR (DMSO-d6) d 15.20 (1C, CH3), 32.25 (CH2), 32.69
(C2), 52.89 (CH2O), 81.92 (C6), 162.57 (C7), 164.19 (C8a),
165.56 (C5), 169.83 (C3), 170.54 (CO, ester group); MS (70
ev, %) m/z 271 (M+ +2, 5.1%), 270 (M+ +1, 45.5%), 269
(M+, 30.2%), 241 (100.0%, M+-CO), 224 (15.0%, M+-
· Our prediction is that these compounds with new
ring systems show even weaken antioxidant and antitumor
activities (Tables 1, 2).
CH3CH2O.), 182 (15.1%, M+-CH3CH2OCOCH2 ). Anal.
.
CONCLUSION
New ring systems are prepared. In continuation, this
paper describes our interest in the development of synthetic
strategy to polyfunctionalized heterocycles such as the syn-
thesis of thiazolopyrimidines, pyrrolothiazolopyrimidines
and triazolopyrrolo-thiazolo pyrimidines. The series of
compounds (4, 5, 7b, 7c, 8a, 8b) exhibited a high antioxi-
dant activity and protect the DNA. The series of com-
pounds (6, 8b, 8c, 8a) proved to have the best cytotoxic ac-
tivity.
Calc. for C10H11N3O4S (269.28): C, 44.60; H, 4.12; N,
15.60; S, 11.91. Found: C, 44.50; H, 4.25; N, 15.53; S,
11.85.
6,8-Dihydropyrrolo[2,3-d]thiazolo[3,2-a]pyrimidine-
3,5,7(2H)-trione (3)
A mixture of compound 2 (2.69 g, 10 mmol) was
refluxed in ethanol (50 mL) in presence sodium metal (0.23
g, 10 mmol) for 10 h. The reaction mixture was cooled; the
deposited precipitate was filtered off, washed with ethanol,
the formed salt was dissolve in water and acidified with
10% HCl, the formed precipitate was filtered, dried and
crystallized from methanol to afford 3 as yellow crystals
(77%); mp >350 oC (dec.). IR (KBr) nmax. 3419 (NH), 1712
(CO, pyrrole ring), 1663 (br., 2CO, amidic), 1620 cm-1
(C=N); 1H NMR (DMSO-d6) d 2.93 (s, 2H, CH2, pyrrole
ring), 4.79 (s, 2H, CH2, thiazole ring), 8.02 (s, 1H, NH,
D2O exchangeable); 13C NMR (DMSO-d6) d 33.01 (C2),
EXPERIMENTAL SECTION
General
All melting points are in degree centigrade and were
determined on Gallenkamp electric melting point appara-
tus. The IR spectra were recorded (KBr) on a Perkin-Elmer
1430 spectrometer (National Research Center and Chemis-
try Department, Cairo University). The 1H NMR and 13
C

Biological Activity & Annulations to Thiazolopyrimidine
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 45
33.20 (C6), 105.11 (5a), 158.41 (C8a), 164.20 (C5), 165.31
(C11), 168.80 (C7), 171.90 (C3); MS (70 ev, %) m/z 225 (M+
+2, 4.5%), 224 (M+ +1, 40.5%), 223 (M+, 55.5%), 195
(6.1%, M+-CO), 181 (25.2%, M+-CH2=C=O), 142 (100.0%,
M+-C4H4NO). Anal. Calc. for C8H5N3O3S (223.21): C,
43.05; H, 2.26; N, 18.83; S, 14.37; Found: C, 43.14; H,
2.30; N, 18.75; S, 14.45.
64.30%), 255 (25.15%, M+-CH2=C=O), 183 (15.5%, M+-
C3H2N2OS), 142 (100.0%, M+-C5H5N3OS). Anal. Calc. for
C9H7N5O3S2 (297.31) C, 36.36; H, 2.37; N, 23.56; S,
21.57. Found: C, 36.41; H, 2.45; N, 23.61; S, 21.49.
3-Thioxo-2,11-dihydro-3H,10H-thiazolo[3,2-a][1,2,4]-
triazolo[4¢,3¢:1,5]pyrrolo-[2,3-d]pyrimidine-8,10(7H)-
dione (6)
2-(7-Amino-3,5-dioxo-2,3-dihydro-5H-thiazolo[3,2-a]py
rimidin-6-yl)aceto-hydrazide (4)
A suspension of 5 (2.97 g, 10 mmol) in methanol (30
mL) and glacial acetic acid (2 mL) was refluxed for 14 h.
The reaction mixture was cooled, the solid so obtained was
filtered off dried and recrystallized from methanol to fur-
nish 6 as yellowish color (80%); mp 230-232 oC (dec.). IR
(KBr) nmax. 3446 (br., NH), 1691 (CO, pyrimidine), 1652
(CO, thiazolidinone), 1346 cm-1 (C=S); 1H NMR (DMSO-
d6) d 3.15 (s, 2H, CH2), 4.47 (s, 2H, CH2, thiazol ring), 8.66
(brs., 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) d
32.90 (C7), 33.20 (C11), 100.40 (C10a), 158.90 (C11a),
161.90 (C4a), 164.10 (C5a), 165.85 (C10), 171.45 (C8),
175.55 (C3); MS (70 ev, %) m/z 281 (M+ +2, 8.40%), 280
(M+1 +1, 10.10%), 297 (M+, 68.50%), 239 (4.70%, M+-
.CH2CN), 220 (7.10%, M+-HN=C=O), 205 (8.6%, M+-
C2H2OS), 70 (100.0%, OC-N=CO). Anal. Calc. for
C9H5N5O2S2 (279. 31) C, 38.70; H, 1.80; N, 25.07; S,
22.96. Found: C, 38.60; H, 1.71; N, 25.18; S, 22.80.
2,6-Diarylidene-6,8-dihydropyrrolo[2,3-d]thiazolo[3,2-
a]pyrimidine-3,5,7(2H)-trione derivatives (7a-c): Gen-
eral procedure
A mixture of compound 2 (2.69 g, 10 mmol) and hy-
drazine hydrate (0.5 mL, 10 mmol) was refluxed in dioxane
(50 mL) for 15 h. The reaction mixture was cooled; the de-
posited precipitate was filtered off, washed with ethanol,
dried, and crystallized from ethanol to afford 4 as white
crystals (83%); mp 180-182 oC (dec.). IR (KBr) nmax. 3442-
3431 (brs., NH, NH2), 1712 (CONH) group, 1662 (CO,
1
amide), 1632 (CO, thiazole ring), 1612 cm-1 (C=N); H
NMR (DMSO-d6) d 1.90 (s, 2H, CH2, thiazolo ring), 3.95
(s, 2H, CH2), 4.48 (br., 2H, NH2), 5.73 (br., 2H, NH2), 9.08
(br., 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) d
31.30 (C, CH2), 32.65 (C2), 81.93 (C6), 162.10 (C7), 164.10
(C8a), 165.75 (C5), 170.40 (C, CO, Carbohydrazide),
171.40 (C3); MS (70 ev, %) m/z 257 (M+ +2, 4.7%), 256
(M+ +1, 9.4%), 255 (M+, 65.5%), 227 (3.5%, M+-CO), 213
(2.7%, M+-CH2=C=O), 142 (100.0%, M+-C4H7N3O). Anal.
Calc. for C8H9N5O3S (255.25): C, 37.64; H, 3.55; N, 27.44;
S, 12.56. Found: C, 37.52; H, 3.60; N, 27.50; S, 12.46.
7-Amino-6-[(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-
methyl]-5H-thiazolo[3,2-a]pyrimidine-3,5(2H)-dione
(5)
A mixture of compound 3 (2.23 g, 10 mmol), alde-
hyde derivatives namely; benzaldehyde (2.12 g, 20 mmol),
4-chlorobenzaldehyde (2.80 g, 20 mmol), or 4-methoxy-
benzaldehyde (2.72 g, 20 mmol), and (0.02 mol) of anhy-
drous sodium acetate was stirred under reflux in a mixture
of glacial acetic acid (30 mL) and acetic anhydride (15 mL)
for 5 h. The reaction mixture was allowed to cool to room
temperature, poured into cold water (100 mL). The depos-
ited precipitate was filtered off, dried, and crystallized
from appropriate solvent to give 7a-c, respectively.
2,6-Dibenzylidene-6,8-dihydropyrrolo[2,3-d]thiazolo-
[3,2-a]pyrimidine-3,5,7(2H)-trione (7a)
To a solution of 4 (2.55 g, 10 mmol) in ethanol (30
mL), KOH (0.5 g) in water (5 mL) and CS2 (10 mmol) were
added. The reaction mixture was heated under reflux till the
evolution of hydrogen sulphide ceased. Thereafter, it was
cooled, diluted with cold water (30 mL) and acidified with
acetic acid. The solid that formed was filtered off, dried and
recrystallized from ethanol to afford 5 as yellow crystals
o
(82%); mp 125-127 C (dec.). IR (KBr) nmax. 3433-3444
(brs., NH, NH2), 1676 (CO, amide), 1646 (CO, thiazole
ring), 1621 (C=N, oxadiazole ring), 1351-1346 cm-1
(C=S); 1H NMR (DMSO-d6) d 2.23 (s, 2H, CH2), 4.15 (s,
2H, CH2, thiazol ring), 6.12 (br., 2H, NH2, D2O exchange-
able), 7.33 (br., 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6) d 31.90 (CH2), 32.60 (C2), 81.91 (C6), 157.80
(C5, oxadiazole ring), 160.90 (C7), 163.90 (C8a), 165.55
(C5), 170.90 (C3), 173.10 (1C, C=S); MS (70 ev, %) m/z
299 (M+ +2, 9.10%), 298 (M+ +1, 14.20%), 297 (M+,
Crystallization from dioxane; yellow powder (80%),
mp 280-282 oC (dec.). IR (KBr) nmax. 3432 (brs., NH), 1742
(CO, pyrrole ring), 1690 (CO, amide), 1630 (CO, thiazole
1
ring), 1615 cm-1 (C=N); H NMR (DMSO-d6) d 6.96 (s,
1H, methine), 7.40-7.80 (m, 10H, phenyl), 8.36 (s, 1H,
methine), 8.66 (br., 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6) d 111.91 (C5a), 118.8 (C2), 126.63 (2C, C2, C6,
Ph), 126.73 (2C, C2, C6, Ph), 127.95 (C4, Ph), 128.12 (C4,

46 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Abu-Hashem et al.
Ph), 128.82 (2C, C3, C5, Ph), 129.13 (2C, C3, C5, Ph),
133.96 (C6), 134.62 (C, methine), 134.70 (C1, Ph), 134.90
(C1, Ph), 142.53 (C, methine), 146.20 (C8a), 158.68 (C9a),
165.55 (C5), 167.90 (C7), 172.89 (C3); MS (70 ev, %) m/z
401 (M+ +2, 6.5%), 400 (M+ +1, 27.5%), 399 (M+, 55.5%),
C24H17N3O5S (459.47): C, 62.74; H, 3.73; N, 9.15. Found:
C, 62.80; H, 3.61; N, 9.23.
3,9-Diaryl-2,3,8,9-tetrahydro-isoxazolo[4¢,5¢:4,5]pyr-
rolo[2,3-d]isoxazolo-[5¢,4¢:4,5]thiazolo[3,2-a]pyrimidin-
10-(6H)-one (8a-c): General procedure
322 (14.5%, M+-C6H5 ), 237 (45.7%, M+-C9H6OS), 224
A mixture of 7a (3.99 g, 10 mmol), 7b (4.68 g, 10
mmol), or 7c (4.59 g, 10 mmol), hydroxylamine hydrochlo-
ride (1.39 g, 20 mmol) and anhydrous sodium acetate (0.82
g, 10 mmol) was stirred under reflux in glacial acetic acid
(30 mL) for 5 h. The reaction mixture was allowed to cool
to room temperature and poured into cold water (100 mL).
The deposited precipitate was filtered off, dried and crys-
tallized from appropriate solvent to give 8a-c, respectively.
3,9-Diphenyl-2,3,8,9-tetrahydro-isoxazolo[4¢,5¢:4,5]pyr-
rolo[2,3-d]isoxazolo[5¢,4¢:4,5]thiazolo[3,2-a]pyrimidin-
10-(6H)-one (8a)
.
(9.10), 209 (35.10%, 237-CO), 145 (100%, C9H7NO).
Anal. Calc. for C22H13N3O3S (399.42): C, 66.15; H, 3.28;
N, 10.52; S, 8.03. Found: C, 66.20; H, 3.30; N, 10.60; S,
8.10.
2,6-Di-(4-chlorobenzylidene)-6,8-dihydropyrrolo[2,3-d]-
thiazolo[3,2-a]pyrimidine-3,5,7(2H)-trione (7b)
Crystallization from dimethylformamide; brown
powder (85%); mp >350 oC (dec.). 1H NMR (DMSO-d6) d
6.95 (s, 1H, methine), 7.40 (d, 2H, J = 8.43 Hz, 4-Cl-phen-
yl), 7.45 (d, 2H, J = 8.40 Hz, 4-Cl-phenyl), 7.48 (d, 2H, J =
8.35 Hz), 7.50 (d, 2H, J = 8.38 Hz), 8.35 (s, 1H, methine),
8.65 (br., NH, D2O exchangeable); 13C NMR (DMSO-d6) d
111.96 (C5a), 118.81 (C2), 126.55 (2C, C2, C6, Ph), 126.80
(2C, C2, C6, Ph), 128.90 (2C, C3, C5, Ph), 129.10 (2C, C3,
C5, Ph), 131.45 (C4, Ph), 131.55 (C4, Ph), 133.96 (C6),
134.52 (C, methine), 136.56 (C1, Ph), 138.20 (C1, Ph),
142.65 (C, methine), 146.50 (C8a), 158.75 (C9a), 165.70
(C5), 167.80 (C7), 172.55 (C3); MS (70 ev, %) m/z 471 (M+
+4, 25.6%), 469 (M+1 +2, 50.5%), 467 (M+, 69.5%), 356
(14.5%, M+-.C6H4Cl), 302 (9.7%, M+-C9H5ClO.), 111
(100%, C6H4Cl). Anal. Calc. for C22H11Cl2N3O3S (468.31):
C, 56.42; H, 2.37; N, 8.97; S, 6.85. Found: C, 56.50; H,
2.45; N, 8.85; S, 6.90.
Crystallization from dimethylformamide, brown
powder (85%); mp 345-347 oC (dec.). 1H NMR (DMSO-
d6) d 4.22 (d, 1H, isoxazolo proton), 5.35 (d, 1H, isoxazolo
proton), 7.42-7.82 (m, 10H, phenyl), 8.09 (brs., NH, pyr-
role proton, D2O exchangeable), 11.15 (brs., NH, iso-
xazolo proton, D2O exchangeable), 11.58 (brs., NH, iso-
xazolo proton, D2O exchangeable); 13C NMR (DMSO-d6)
d 62.60 (C9), 65.32 (C3), 112.38 (C3a), 114.01 (C9b), 114.67
(C6a), 116.98 (9a), 120.30 (C5a), 122.50 (C4, Ar), 122.90
(C4, Ar), 128.20 (2C, C3, C5, Ar), 128.80 (2C, C3, C5, Ar),
129.10 (2C, C2, C6, Ar), 129.35 (2C, C2, C6, Ar), 135.80
(C1, Ar), 136.32 (C1, Ar), 149.90 (C11a), 158.78 (C4a),
166.60 (C10); MS (70 ev, %) m/z 431 (M+ +2, 18.9%), 430
(M+ +1, 22.6%), 429 (M+, 50.3%), 352 (7.3%, M+-C6H5 ),
.
2,6-Di-(4-methoxybenzylidene)-6,8-dihydropyrrolo[2,3-
d]thiazolo[3,2-a]pyrimidine-3,5,7(2H)-trione (7c)
Crystallization from dimethylformamide-ethanol mix-
ture; yellow powder (78%); mp >350 oC (dec.). 1H NMR
(DMSO-d6) d 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.94
(s, 1H, methine), 7.12 (d, 2H, J = 8.42 Hz, 4-methoxy
phenyl), 7.15 (d, 2H, J = 8.37 Hz, 4-methoxyphenyl), 7.22
(d, 2H, J= 8.34 Hz), 7.30 (d, 2H, J = 8.41 Hz), 8.33 (s, 1H,
methine), 8.64 (br., NH, D2O exchangeable); 13C NMR
(DMSO-d6) d 55.95 (C, OCH3), 57.01 (C, OCH3), 111.95
(C5a), 118.7 (C2), 125.95 (2C, C2, C6, Ph), 126.10 (2C, C2,
C6, Ph), 127.95 (2C, C3, C5, Ph), 128.90 (2C, C3, C5, Ph),
130.92 (C4, Ph), 131.40 (C4, Ph), 132.98 (C6), 133.95 (C,
methine), 136.90 (C1, Ph), 137.95 (C1, Ph), 141.97 (C,
methine), 145.96 (C8a), 158.30 (C9a), 165.10 (C5), 167.30
(C7), 171.90 (C3); MS (70 ev, %) m/z 461 (M+ +2, 20.6%),
460 (M++1, 24.5%), 459 (M+, 74.0%), 352 (7.5%, M+-
C7H7O.), 339 (100%, M+-C8H8O.). Anal. Calc. for
324 (12.5%, M+-C7H7N.), 308 (10.2%, M+-C7H7NO.), 121
(100%, C7H7NO). Anal. Calc. for C22H15N5O3S (429.45):
C, 61.53; H, 3.52; N, 16.31; S, 7.47. Found: C, 61.60; H,
3.45; N, 16.25; S, 7.50.
3,9-Di(4-chlorophenyl)-2,3,8,9-tetrahydro-isoxazolo-
[4¢,5¢:4,5]pyrrolo[2,3-d]isoxazolo[5¢,4¢:4,5]thiazolo[3,2-
a]pyrimidin-10-(6H)-one (8b)
Crystallization from dioxane, yellow crystals (83%);
mp >350 oC (dec.). 1H NMR (DMSO-d6) d 4.20 (d, 1H, J =
6.51 Hz, isoxazolo proton), 5.30 (d, 1H, J = 6.55 Hz, iso-
xazolo proton), 7.42 (d, 2H, J = 8.40 Hz, 4-Cl-phenyl),
7.45 (d, 2H, J = 8.42 Hz, 4-Cl-phenyl), 7.48 (d, 2H, J = 8.41
Hz, 4-Cl-phenyl), 7.52 (d, 2H, J = 8.39 Hz, 4-Cl-phenyl),
8.10 (brs., NH pyrrole proton, D2O exchangeable), 11.10
(brs., NH, isoxazolo proton, D2O exchangeable), 11.47
(brs., NH, isoxazolo proton, D2O exchangeable); 13C NMR
(DMSO-d6) d 60.10 (C9), 66.05 (C3), 111.97 (C3a), 114.25

Biological Activity & Annulations to Thiazolopyrimidine
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 47
(C9b), 116.53 (C6a), 118.82 (C9a), 121.06 (C5a), 126.30 (2C,
C2, C6, Ar), 126.38 (2C, C2, C6, Ar), 129.40 (2C, C3, C5,
Ar), 129.49 (2C, C3, C5, Ar), 130.80 (C4, Ar), 131.20 (C4,
Ar), 137.85 (C1, Ar), 138.10 (C1, Ar), 149.20 (C11a), 159.05
(C4a), 164.93 (C10); MS (70 ev, %) m/z 502 (M+ +4, 16.9%),
500 (M+ +2, 30.5%), 498 (M+, 53.1%), 386 (25.6%, M+-
C6H4Cl.), 341 (8.5%, M+-C7H6ClNO.), 111(100%, C6H4Cl).
Anal. Calc. for C22H13Cl2N5O3S (498.34): C, 53.02; H,
2.63; N, 14.05; S, 6.43. Found: C, 53.15; H, 2.70; N, 14.15;
S, 6.50.
rated from plasma and the buffy coat was washed three
times with 10 volumes of 0.15 M NaCl. During the last
wash, the erythrocytes were centrifuged at 2500 rev./min
for 10 min to obtain a constantly packed cell preparation.
Erythrocyte hemolysis was mediated by peroxyl radicals in
this assay system.20,25 A 10% suspension of erythrocytes in
phosphate buffered saline pH 7.4 (PBS) was added to the
same volume of 200 mM AAPH solution in PBS containing
samples to be tested at different concentrations. The reac-
tion mixture was shaken gently while being incubated at 37
ºC for 2 h. The reaction mixture was then removed, diluted
with eight volumes of PBS and centrifuged at 805 ´ g for
10 min. The absorbance of the supernatant was read at 540
nm. Similarly, the reaction mixture was treated with 8 vol-
umes of distilled water to achieve complete hemolysis, and
the absorbance of the supernatant obtained after centrifu-
gation was measured at 540 nm. The data percentage hemo-
lysis was expressed as mean standard deviation. L-ascor-
bic acid was used as a positive control.
3,9-Di(4-methoxyphenyl)-2,3,8,9-tetrahydro-isoxazolo-
[4¢,5¢:4,5]pyrrolo[2,3-d]isoxazolo[5¢,4¢:4,5]thiazolo[3,2-
a]pyrimidin-10-(6H)-one (8c)
Crystallization from dioxane, yellow crystals (81%);
o
1
mp >350 C (dec.). H NMR (DMSO-d6) d 3.83 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 4.21 (d, 1H, J = 6.50 Hz,
isoxazolo proton), 5.33 (d, 1H, J = 6.53 Hz, isoxazolo pro-
ton), 7.12 (d, 2H, J = 8.41 Hz, 4-methoxyphenyl), 7.17 (d,
2H, J = 8.39 Hz, 4-methoxyphenyl), 7.24 (d, 2H, J = 8.35
Hz, 4-methoxyphenyl), 7.29 (d, 2H, J = 8.40 Hz, 4-meth-
oxyphenyl), 8.12 (brs., NH pyrrole proton, D2O exchange-
able), 11.14 (brs., NH, isoxazolo proton, D2O exchange-
able), 11.52 (brs., NH, isoxazolo proton, D2O exchange-
able); 13C NMR (DMSO-d6) d 56.71 (C, OCH3), 57.31 (C,
OCH3), 60.31 (C9), 63.52 (C3), 111.72 (C3a), 113.99 (2C,
C3, C5, Ar), 114.21 (2C, C3, C5, Ar), 115.32 (C9b), 116.27
(C6a), 118.55 (C9a), 119.36 (C5a), 124.61 (2C, C2, C6, Ar),
126.30 (2C, C2, C6, Ar), 128.51 (C1, Ar), 129.45 (C1, Ar),
149.61 (C11a), 159.31 (C4, Ar), 160.03 (C4, Ar), 163.52
(C4a), 165.69 (C10); MS (70 ev, %) m/z 491 (M+ +2, 23.5%),
490 (M+ +1, 24.7%), 489 (M+, 68.5%), 461 (54.6%, M+),
Antioxidant activity screening assay (ABTS) method
For each of the investigated compounds (2 mL) of
ABTS solution (60 mM) was added to 3 M MnO2 solution
(25 mg/mL) all prepared in phosphate buffer (pH 7, 0.1 M).
The mixture was shaken, centrifuged, filtered and the
absorbance of the resulting green-blue solution (ABTS
radical solution) at l 734 nm was adjusted to approx. ca.
0.5. Then, 50 mL of (2 mM) solution of the test compound
in spectroscopic grade MeOH/phosphate buffer (1:1) was
added. The absorbance was measured and the reduction in
color intensity was expressed as inhibition percentage. L-
ascorbic acid was used as standard antioxidant (positive
control). Blank sample was run without ABTS and using
MeOH/phosphate buffer (1:1) instead of sample. Negative
control was run with MeOH/phosphate buffer (1:1) instead
of test compound.17,21,22,25
458 (100%, M+-OCH3 ), 382 (8.5%, M+-C7H7O.). Anal.
.
Calc. for C24H19N5O5S (489.50): C, 58.89; H, 3.91; N,
14.31. Found: C, 58.94; H, 3.85; N, 14.25.
PHARMACOLOGICAL SCREENING
Ehrlich cells
Bleomycin-dependent DNA damage
Ehrlich cells (Ehrlich ascites carcinoma, EAC) were
derived from ascetic fluid from diseased mice (the cells
were purchased from the National Cancer institute, Cairo,
Egypt).
The assay was done according to Aeschlach 23,25 with
minor modifications. The reaction mixture (0.5 mL) con-
tained calf thymus DNA (0.5 mg/mL), bleomycin sulfate
(0.05 mg/mL), MgCl2 (5 mM), FeCl3 (50 µM) and samples
to be tested at different concentrations. L-ascorbic acid was
used as a positive control. The mixture was incubated at 37
ºC for 1 h. The reaction was terminated by addition of 0.05
mL EDTA (0.1 M). The color was developed by adding 0.5
mL TBA(1%, w/v) and 0.5 mL HCl (25%, v/v) followed by
Antioxidant activity screening assay for erythrocyte
hemolysis
The blood was obtained from rats by cardiac puncture
and collected in heparinized tubes. Erythrocytes were sepa-

48 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Abu-Hashem et al.
6. Angiolini, M.; Ballinari, D.; Bassini, D. F.; Bonomini, L.;
Gude, M.; Menichincheri, M.; Moll, J.; Trosset, J. Y. U.S.
Pat. Appl. 2004, US 2004009993.
heating at 37 ºC for 15 min. After centrifugation, the extent
of DNAdamage was measured by increase in absorbance at
532 nm.
7. Chen, J. J.; Dunn, J. P.; Goldstein, D. M.; Stahl, C. M. PCT
Int. Appl. 2002, WO 2002064594.
Antitumor activity using Ehrlich ascites in vitro as-
say
8. Sen, A. B.; Kapoor, R. N. J. Indian Chem. Soc. 1973, 486.
9. Mahesh, V. K.; Goyal, R. N.; Gupta, R. J. Indian Chem. Soc.
1977, 738.
Different concentrations of the tested compounds
were prepared (100, 50 and 25 g/mL DMSO). Ascites fluid
from the peritoneal cavity of the donor animal (contains
Ehrlich cells) was aseptically aspirated. The cells were
grown partly floating and partly attached in a suspension
culture in RPMI 1640 medium, supplemented with 10% fe-
tal bovine serum. They were maintained at 37 oC in a hu-
midified atmosphere with 5% CO2 for 2 hrs. The viability
of the cells determined by the microscopical examination
using a hemocytometer and using trypan blue stain (stains
only the dead cells).24,25
10. Derek, D. T.; Stacey, A. D.; Weerasinghe, D. K. Heterocycles
1980, 14, 1753.
11. Fathalla, O. A.; Radwan, H. H.; Awad, S. M.; Mohamed, M.
S. Indian J. Chem. 2006, 45(B), 980-985.
12. Fabrissin, S.; De Nardo, M.; Nisi, C.; Dolfini, E.; Franchi,
G.; Morasca, L. J. Med. Chem. 1976, 19, 639-642.
13. Ernst, C.; Hans, G. K.; Jochims. D. Ger. (East) 1962, 23, 546
Aug, 10, Cl.12p, Appl. Nov. 1959, 6, 2pp; C. A. 1962, 58,
9103f.
14. Akazuka, K.; Fukutomi, Y. Japan 1953, 849(50); C. A. 1953,
47, 2203b.
15. Wyrzkiewiez, E.; Wybieralska, J.; Lapucha, A. Pol. J. Chem.
1987, 61, 253.
ACKNOWLEDGEMENTS
16. Hussain, S. M.; El-Reedy, A. M.; Rezk, A. M. H.; Sife
El-Dien, Kh. A. J. Heterocycl. Chem. 1987, 24, 1605-1610.
17. El-Gazzar, A. B. A.; Youssef, M. M.; Youssef, A. M. S.;
Abu-Hashem, A. A.; Badria, F. A. Eur. J. Med. Chem. 2009,
44, 609-624.
The author wishes to express his most sincere thanks
to Prof. Dr. Farid Abd-Elraheem Badria (Department of
Pharmacognosy, Faculty of Pharmacy, Mansoura Univer-
sity 35516, Egypt) for carrying out the biological activity
tests. The present work was supported by Department of
Photochemistry (Heterocyclic unit); Chemical Industries
Research Division, National Research Centre in Cairo,
Egypt.
18. Guernelli, S.; Meo, P. L.; Morganti, S.; Noto, R.; Spinelli, D.
Tetrahedron 2007, 63, 10260-10268.
19. Levin, Ya. A.; Skorobogatova, M. S. Khimiya
Geterotsiklicheskikh Soedinenii 1967, 3, 339-341.
20. Morimoto, Y.; Tanaka, K.; Iwakiri, Y.; Tokuhiro, S.;
Fukushima, S.; Takeuchi, Y. Biol. Pharm. Bull. 1995, 18,
1417-1422.
REFERENCES
1. Lee, H. W.; Kim, B. Y.; Ahn, J. B.; Kang, S. K.; Lee, J. H.;
Shin, J. S.; Ahn, S. K.; Lee, S. J.; Yoon, S. S. Eur. J. Med.
Chem. 2005, 40, 862-874.
21. Lissi, E.; Modak, B.; Torres, R.; Escobar, J.; Urzå, A. Free
Radical Res. 1999, 30, 471-477.
22. El-Gazzar, A. B. A.; Hafez, H. N.; Abu-Hashem, A. A.; Aly,
A. S. Phosphorus, Sulfur Silicon Relat. Elem. 2009, 184,
379-405.
2. Chen, Q.; Zhu, X.-L.; Jiang, L.-L.; Liu, Z.-M.; Yang, G.-F.
Eur. J. Med. Chem. 2008, 43, 595-603.
3. Pandey, S.; Suryawanshi, S. N.; Gupta, S.; Srivastava, V. M.
L. Eur. J. Med. Chem. 2004, 39, 969-973.
23. Aeschlach, R.; Loliger, J.; Scott, C. B.; Murcia, A.; Butler,
J.; Halliwell, B.; Aruoma, I. O. Food Chem. Toxicol. 1994,
32, 31-36.
4. Boschelli, D. H.; Wu, Z.; Klutchko, S. R.; Showalter, H. D.
H.; Hamby, J. M.; Lu, G. H.; Major, T. C.; Dahring, T. K.;
Batley, B.; Panek, R. L.; Keiser, J.; Hartl, B. G.; Kraker, A.
J.; Klohs, W. D.; Roberts, B. J.; Patmore, S.; Elliott, W. L.;
Steinkampf, R.; Bradford, L. A.; Hallak, H.; Doherty, A. M.
J. Med. Chem. 1998, 41, 4365-4377.
24. Fujita, T.; Takeda, Y.; Han-Dong, S.; Minami, Y.; Marunaka,
T.; Takeda, S.; Yamada, Y.; Togo, T. Planta Med. 1988, 54,
414-417.
25. Abu-Hashem, A. A.; EL-Shehry, M. F.; Badria, F. A. Acta
Pharm. 2010, 60, 311-323.
5. Boehm, J. C.; Widdowson, K. L.; Callahan, J. F.; Wan, Z.
PCT Int. Appl. 2003, WO 2003088972.