Radioprotective and antitumor activity of some novel amino acids and imidazoles containing thieno[2,3-d]pyrimidine Moiety

Article abstract of DOI:10.1080/10426500903126351

A variety of novel thieno[2,3-d]pyrimidine derivatives, comprising amino acids 3a-l, imidazothieno-pyrimidines 4A, 4b-h, and 7, were obtained via the reaction of 4-chloro-5,6-dimethylthieno[2,3-d]pyrimidine 1 with a variety of reagents. The structures of

Full text of DOI:10.1080/10426500903126351

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Radioprotective and Antitumor
Activity of Some Novel
Amino Acids and Imidazoles
Containing Thieno[2,3-
d]pyrimidine Moiety
Saleh I. Alqasoumi a , Fatma A. Ragab b , Ahmed M.
Alafeefy c , Marwa Galal a & Mostafa M. Ghorab a
a Medicinal, Aromatic and Poisonous Plants Research
Center (MAPPRC) , College of Pharmacy, King Saud
University , Saudi Arabia
b Department of Pharmaceutical Chemistry, Faculty
of Pharmacy , Cairo University , Cairo, Egypt
c Department of Pharmaceutical Chemistry , College
of Pharmacy, King Saud University , Saudi Arabia
d Department of Drug Radiation Research , National
Center for Radiation Research and Technology , Nasr
City, Cairo, Egypt
Published online: 18 Nov 2009.
To cite this article: Saleh I. Alqasoumi , Fatma A. Ragab , Ahmed M. Alafeefy ,
Marwa Galal & Mostafa M. Ghorab (2009) Radioprotective and Antitumor Activity of
Some Novel Amino Acids and Imidazoles Containing Thieno[2,3-d]pyrimidine Moiety,
Phosphorus, Sulfur, and Silicon and the Related Elements, 184:12, 3241-3257, DOI:
10.1080/10426500903126351
To link to this article: http://dx.doi.org/10.1080/10426500903126351
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Phosphorus, Sulfur, and Silicon, 184:3241–3257, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500903126351
Radioprotective and Antitumor Activity of Some Novel
Amino Acids and Imidazoles Containing
Thieno[2,3-d]pyrimidine Moiety
Saleh I. Alqasoumi,1 Fatma A. Ragab,2 Ahmed M.
Alafeefy,3 Marwa Galal,1 and Mostafa M. Ghorab1
1Medicinal, Aromatic and Poisonous Plants Research Center
(MAPPRC), College of Pharmacy, King Saud University, Saudi Arabia
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Cairo University, Cairo, Egypt
3Department of Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, Saudi Arabia
4Department of Drug Radiation Research, National Center for
Radiation Research and Technology, Nasr City, Cairo, Egypt
A variety of novel thieno[2,3-d]pyrimidine derivatives, comprising amino acids
3a–l, imidazothieno-pyrimidines 4A, 4b–h, and 7, were obtained via the reaction
of 4-chloro-5,6-dimethylthieno[2,3-d]pyrimidine 1 with a variety of reagents. The
structures of these compounds were confirmed by microanalysis, IR, 1H NMR, and
mass spectrometry. Some of the obtained compounds showed promising radiopro-
tective and antitumor activities.
Keywords Amino acids; antitumor; radioprotective; thieno[2,3-d]pyrimidines
INTRODUCTION
The pharmacological activities of thieno[2,3-d]pyrimidine derivatives
are of great interest in the field of medicinal chemistry.1 They display
antibacterial,2 antifungal,3 antitumor,4–6 and radioprotective charac-
teristics.7–9 In addition, compounds having amino acid moieties are also
known to possess a wide range of biological and pharmacological activ-
ities, such as antitumor and radioprotective capacity.10 This perception
Received 5 February 2008; accepted 18 June 2009.
Many thanks are due to Prof. Dr. Eman Noaman, Professor of Radiation Biology, De-
partment of Radiation Biology, National Center for Radiation Research and Technology,
Atomic Energy Authority, Egypt, for planning and performing the radioprotective and
anticancer activities.
Address correspondence to Mostafa M. Ghorab, Medicinal, Aromatic and Poisonous
Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Saudi
Arabia. E-mail: mmsghorab@yahoo.com
3241

3242
S. I. Alqasoumi et al.
has inspired us to find an efficient way to synthesize a new class of hete-
rocyclic ring systems containing a thienopyrimidine nucleus pendentto
an amino acid and imidazole moiety for biological screening. We per-
formed a series of reactions using 4-chlorothieno[2,3-d]pyrimidine 1 as
a precursor for the versatile functionalized amino acids and imidazoles
bearing thienopyrimidines as possible new antitumor and radioprotec-
tive agents.
RESULTS AND DISCUSSION
Chemistry
The present work was to design and synthesize some new amino acids
containing biologically active thieno[2,3-d]pyrimidine derivatives ex-
pected to have radioprotective and antitumor activities. When chloro
derivative 111 was treated with the sodium salt of various amino acids
under reflux at pH 9.0–9.5, each reaction afforded a single product iden-
tified as N-(5,6-dimethylthieno[2,3-d]pyrimidin-4-yl)amino acids 3a–l.
(Scheme 1). The imidazopyrimidine derivatives having the thiophene
nucleus (purine analogues) were obtained in good yields via reaction
of the amino acid derivatives 3b–h with acetic anhydride in the pres-
ence of anhydrous sodium acetate12 (Scheme 1). In the case of 3a,
when reacted with acetic anhydride in presence of anhydrous sodium
acetate, the acetyl imidazopyrimidine 4A was obtained instead of the
expected imidazopyrimidine 4a (Scheme 2). Also, the interaction of
chloro compound 1 with ethanolamine or 3-aminopropanol in pyri-
dine yielded the hydroxy compounds 5 and 6, respectively (Scheme
3). Cyclization of compound 5 with thionyl chloride afforded 7 (Scheme
3). 5,6-Dimethylthieno[2,3-d]pyrimidine-4-yl-isothiocyanate 8 was ob-
tained via the reaction of compound 1 with ammonium thiocyanate in
dry acetone (Scheme 3). It was reported that the condensation of isoth-
iocyanate with active methylene compounds such as malononitrile was
exploited in the synthesis of heterocyclic derivatives.2 Thus, interaction
of the isothiocyanate 8 with malononitrile in presence of sodium ethox-
ide gave the pyrimidinethione derivative 10. This reaction proceeded
via the initial formation of intermediate 9 followed by intramolecular
cyclization to give pyrimidinethione derivative 10 (Scheme 3). Physical
data for 3a–10 are shown in Table I.
Antitumor Activity
The results of antitumor activity for the synthesized compounds
(Table II) indicated that compounds 3c, 3d, 3e, 3j, 3k, and 8 showed
a significant in vitro activity toward Ehrlich Ascites Carcinoma

Novel Amino Acids and Imidazoles
3243
R
NH-CH-COONa
H3C
Cl
N
R
H3C
H3C
H2N-CH-COONa
pH 9-9.5
N
N
HCOOH
R
H3C
N
S
S
1
2a-k
NH-CH-COOH
pH 9-9.5
COOH
N
H3C
H3C
H
N
S
N
COONa
N
3a-k
H3C
H3C
N
anhyd.
AcONa
Ac2O
S
N
R
2L
N
H3C
H3C
HCOOH
O
N
S
N
COOH
H
CH3
CH(CH3)2
CH2CH(CH3)2
CH2CH2SCH3
CH2Ph
CH2OH
CH2CH2COOH
CH2SSCH2CH(NH2)COOH
a, R =
b, R =
c, R =
d, R =
e, R =
f, R =
g, R =
h, R =
i, R =
N
H3C
H3C
4b-h
N
S
N
3L
j, R =
CH2
CH2
OH
k, R =
N
H
SCHEME 1 Synthesis of compounds 3a–k, 3L, and 4b–h.
N
NHCH2COOH
CH3
S
CH3
S
O
N
N
N
Ac2O
CH3
CH3
anhyd.
AcONa
N
4a
3a
N
OCOCH3
CH3
N
N
CH3
S
OH
Ac2O
N
N
CH3
CH3
S
4A
N
SCHEME 2 The postulated mechanism for the formation of compound 4A.

3244

Novel Amino Acids and Imidazoles
3245
H2N-(CH2) n -OH
NH4SCN
dry acetone
1
pyridine
NH-(CH2)n-OH
N=C=S
H3C
H3C
H3C
N
N
H3C
S
N
S
N
8
5, n =2
6, n =3
CN
CN
SOCl2
S
N
CN
H3C
HN
N
H3C
H3C
CN
N
H3C
S
N
S
N
7
S
N
9
CN
N
N
H3C
H3C
NH2
S
10
SCHEME 3 Synthesis of compounds 5–8 and 10.
cells (EAC). The active compounds having amino acids L-valine-3c,
L-leucine-3d, DL-methionine-3e, tyrosine-3j, DL-tryptophan-3k, and
isothiocyanate group 8 were all found to have more powerful activity
than the other compounds and displayed a significant percentage of
nonviable tumor cells to about 100%, 80%, 80%, 95%, 90%, and 95%,
respectively, at a concentration of 50 µg/mL. A common factor in these
compounds is the presence of thienopyrimidine moieties. Compounds
3c, 3d, 3e, 3k, and 8 are more potent than doxorubicin. Considering
the effect of the tested compounds on tumor volume (TV), treatment
of the animals with compound 8 caused a marked suppression of the
tumor growth where the TV was significantly decreased compared to
corresponding EAC group. γ-Irradiation to mice bearing tumors and
treated with the same compound showed marked synergistic inhibition
in the TV. Generally, the combined therapy showed better suppression
in TV as compared with single treatment either by compound 8 or
irradiation alone. Compound 3k did not show significant reduction in
TV compared to corresponding EAC group.

3246
S. I. Alqasoumi et al.
TABLE II In Vitro Antitumor Activity of Some Newly Synthesized
Compounds
% of Nonviable cells
Conc. (µg / ml)
Compd. No.
400
200
150
100
90
80
75
50
25
10
Doxorubicin 100
100
100
100
100
100
100
100
100
90
100
100
100
100
80
100
100
100
100
100
100
100
100
100
100
100
100
100
100
80
100
100
100
100
0
100
80
100
100
100
100
100
100
100
100
100
70
100
100
55
100
100
100
100
0
90
50
65
70
100
100
90
10
100
100
100
70
50
50
50
100
100
80
90
0
90
40
0
10
85
10
100
100
100
60
40
40
50
100
100
50
90
0
90
40
0
0
75
0
55
0
20
0
10
0
0
20
20
30
0
0
0
20
0
0
0
0
50
0
0
0
0
70
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
4A
4d
4f
4g
5
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
95
100
60
20
30
50
100
100
30
85
0
85
10
0
0
80
100
100
80
80
50
5
30
30
95
90
20
0
0
75
5
0
0
80
30
40
30
0
30
5
90
10
0
0
0
60
0
0
6
7
8
0
0
90
100
100
100
100
30
95
Radioprotective Activity
EAC and radiation exposure significantly increase lipid peroxide con-
tent (LPx). Such an increase seemed to be due to the result of inactiva-
tion of scavenger enzyme activities induced by reactive oxygen species
(ROS). Oxidative stress occurs in living organisms when the produc-
tion of ROS exceeds their ability to prevent their accumulation.13,14
Such elevation of LPx is accompanied by a decline in glutathione level
(GSH) content and in the activity of related antioxidant enzyme super
oxide dismutase (SOD). Additionally, LPx can be initiated by hydrogen
abstraction from lipid molecules by lipid radiolytic products. This leads
to permeability changes, secondary alteration in membrane proteins,
and other sequences.15,16 Also, it was postulated that the decline in
GSH level accompanied by cancer growth is due to reduction in the
glutathione redox status (GSH/GSSG) in the blood of tumor-bearing
mice, which is mainly due to an increase in blood GSSG levels as a re-
sult of oxidative stress.17 On the other hand, the decline in SOD activity

Novel Amino Acids and Imidazoles
3247
in tumor-bearing mice is worthy of mentioning. SOD activity plays an
important role in the antitumor effects of radiation therapy and active
oxygen-forming anticancer agents. However, when the oxidative dam-
age is extreme as a result of tumor growth and/or irradiation, ROS
scavenging enzymes such as SOD are degraded.18
In view of minimizing the toxicity of ionizing radiation on normal
organs of Ehrlich carcinoma-bearing mice, the present study was ap-
plied. The evaluation of agents regarding antitumor effect alone or in
combination with irradiation was also considered. The current inves-
tigation demonstrated that administration of the tested compounds in
tumor-bearing mice almost prevented LPx in plasma and significantly
normalized GSH content and SOD efficiently up to normal values. In
addition, compounds administrated to irradiated mice groups exhib-
ited general amelioration to a great extent the levels of LPx, GSH,
and related antioxidant scavenger enzymes activity in blood. On the
other hand, our reference drug doxorubicin revealed unexpected de-
terioration of GSH and LPx levels. It also showed disruption of the
antioxidant enzyme system SOD to a lesser extent. Mice injected with
doxorubicin and subjected to irradiation showed slight improvement in
the enzymes’ levels, yet still less than our tested compounds 3k and
8.
Effect of Tested Compounds and/or γ -Irradiation on Lipid
Peroxidation and Antioxidant Status
Glutathione level in blood (GSH). As summarized in Table III, a
very high, significant depletion in GSH level was observed in the blood
of tumor-bearing mice (ER group) as compared to the corresponding
controls. The same pattern of GSH depletion was recorded in irradiated
group and in the ER+Rad group as well. Treatment with compound
3k 10 days after tumor inoculation (ER+compd 3k) showed a very
high, significant depletion of GSH content in blood. Similar trends were
seen in animals pretreated with compound 3k prior irradiation (ER +
Compd 3k + Rad). Treatment with compound 8 10 days after tumor
inoculation ER+compd 8 showed a very high significant elevation of
GSH content in blood. Compound 8 treatment prior irradiation (ER +
compd 8 + Rad) for a total of 3 treatments every other day showed
higher elevation, yet still very highly significant in GSH content in
blood.
Lipid peroxidation content (MDA) in plasma. The effects of com-
pounds 3k and 8 treatment and/or irradiation on the level of
lipid peroxidation measured in terms of malondialdehyde (MDA) in
plasma of tumor-free or tumor-bearing mice are shown in Table III.

3248

Novel Amino Acids and Imidazoles
3249
Tumor-bearing mice (ER group) showed a very high significant el-
evation in MDA level in plasma compared with the control values.
MDA levels in plasma of tumor-bearing mice and exposed to irradi-
ation (ER + Rad group) showed high significant increase from the
control group. Treatment with compound 3k 10 days after tumor in-
oculation (ER + Compd 3k) lowered MDA levels, which was still a
very high, significant elevation with respect to the control group. Sim-
ilar trends were seen in animals pretreated with compound 3k and
prior irradiation (ER + Compd 3k + Rad). Treatment with compound
8 10 days after tumor inoculation (ER + compd 8) lowered MDA lev-
els and showed insignificant differences from control group. Similar
trends were seen in animals pretreated with compound 8 and prior
irradiation (ER + compd 8 + Rad) when compared with the control
group.
Superoxide ismutase (SOD) activity in blood. As shown in Table
III, SOD activity in the ER group showed a significant decline in blood.
Very high significant inhibition of SOD activity was observed in the
blood of animals exposed to γ-irradiation. Treatment with compound
3k 10 days after tumor inoculation (ER + Compd 3k) revealed very
high and significant reduction in SOD activity in blood. Similar trends
were seen in animals pretreated with compound 3k prior to irradi-
ation (ER + Compd 3k + Rad) when compared with control group.
Treatment with compound 8 10 days after tumor inoculation (ER +
Compd 8) revealed insignificant difference from the control group. Sim-
ilar trends were seen in animals pretreated with compound 8 prior to
irradiation (ER + compd 8 + Rad) when compared with the control
group.
CONCLUSIONS
It is clear from these results that compounds 3c, 3d, 3e, 3k, and 8
are more potent than doxorubicin as a reference drug. In addition, the
administration of compounds 3k and 8 to solid tumor-bearing mice
not only protected the animals against γ-irradiation induced toxicity
to normal organs, but also exhibited oncolytic activity and acted in
synergy with γ-irradiation to suppress Ehrlich carcinoma tumor.
MATERIALS AND METHODS
General Considerations
Melting points were uncorrected and were determined on a Stuart
melting point apparatus (Stuart Scientific, Redhill, UK). Elemental

3250
S. I. Alqasoumi et al.
analyses (C, H, N) were performed on Perkin-Elmer 2400 analyser
(Perkin-Elmer, Norwalk, CT, USA) at the Microanalytical Laboratories
of the Faculty of Science, Cairo University. The IR spectra (KBr) were
1
measured on Shimadzu IR 110 spectrophotometer, H NMR spectra
were obtained on a Bruker proton NMR-Avance 300 (300 MHz), in
DMSO-d6 as a solvent, using tetramethylsilane (TMS) as internal
standard. Mass spectra were run on an HP model MS-5988.
Synthesis
2-(5,6-Dimethylthieno[2,3-d]pyrimidin-4-ylamino)acetic Acid
(3a), Propionic Acid (3b), 3-Methylbutyric acid (3c),
4-Methylpentanoic Acid (3d), 4-Methylsulfanylbutyric Acid
(3e), 3-Phenylpropionic Acid (3f), 3-Hydroxy-propionic Acid
(3g), Pentanedioic Acid (3h), 2-Amino-3-disulfide-3,4-
dicarboxylic Acid (3i), 2-(5,6-Dimethyl-thieno[2,3-d]pyrimidin-
4-ylamino)-3-(4-hydroxyphenyl)propionic Acid (3j),
3-(2,3-Dihydro-1H-indol-2-yl)-2-(5,6-dimethylthieno[2,3-d]
pyrimidin-4-ylamino)propionic Acid (3k), and 1-(5,6-Dimethyl-
thieno[2,3-d]pyrimidin-4-yl)-pyrrolidine-2-carboxylic Acid (3l)
An amino acid (0.0096 mol) and sodium carbonate (0.57 g, 0.0054
mol) were dissolved in water (10 mL), and the solution was ad-
justed to pH 9.0–9.5. The chloro derivative 1 (0.95 g, 0.0048 mol)
was then added, and the mixture was stirred at 100◦C for 6 h. The
reaction mixture was left overnight at room temperature and then
treated with formic acid (88%). The solid product obtained was fil-
tered off, washed with water, and crystallized from the proper solvent
(Table I).
IR (KBr, cm−1) 3a: 3420 (NH, OH), 2860 (CH aliph.), 1710 (C = O),
1550 (C=N), 1244 (COOH); δ 3a: 2.4, 2.5 [2s, 6H, 2CH3], 3.4[s, 1H, OH],
4.2 [d, 2H, CH2, J = 7.3 Hz], 7.1 [s, 1H, NH], 8.3 [s, 1H, CH pyrimidine].
IR (KBr, cm−1) 3b: 3423 (NH, OH), 2925 (CH aliph.), 1661 (C =
O), 1600 (C=N), 1248 (COOH); δ 3b: 1.5[d, 3H, CH3, J = 7.3 Hz], 2.3,
2.4[2s, 6H, 2 CH3], 6.6[q, 1H, CH], 8.3[s, 1H, CH pyrimidine].
IR (KBr, cm−1) 3c: 3410 (br, NH, OH), 2965 (CH aliph.), 1711 (C =
O), 1600 (C=N), 1272 (COOH); δ 3c: 1.1[t, 6H, γ-CH3], 2.3[m, 1H, β-
CH], 2.4[s, 6H, 2CH3], 4.3[m, 1H, α-CH], 6.3[s, 1H, NH], 6.4[s, 1H, OH],
8.3[s, 1H, CH pyrimidine]; MS (m/z) 3c: 279 (M+,15.07%), 179 (100%),
280 (3.38%), 281 (1.08%), 234 (32.52%), 220 (44.20%), 163 (59.22%), 136
(8.39%), 92 (10.40%), 55 (18.41%).
IR (KBr, cm−1) 3d: 3400 (NH, OH), 2940, 2840 (CH aliph.), 1700
(C = O), 1241 (COOH); δ 3d: 0.9 [d, 6H, 2CH3, J = 7.4 Hz], 1.8 [t, 2H,
β-CH2], 2.1, 2.4 [2s, 6H, 2CH3],3.3[s, 1H, OH], 4.6 [m, 1H, β-CH], 6.4

Novel Amino Acids and Imidazoles
3251
[m, 1H, α-CH], 8.3 [s, 2H, CH pyrimidine + NH]; MS (m/z) 3d: 293
(M+, 12.57%), 206 (100%).
IR (KBr, cm−1) 3e: 3413 (NH, OH), 2916, 2857 (CH aliph.), 1659
(C = O), 1600 (C=N), 1263 (COOH); δ 3e: 2.2 [m, 2H, β-CH2], 2.35,
2.38 [2s, 6H, 2CH3], 2.4 [s, 3H, SCH3], 4.7[m, 2H, γ-CH2], 6.7 [m, 1H,
α-CH], 8.0 [s, 1H, NH], 8.3 [s, 1H, CH pyrimidine], 12.2 [s, 1H, OH];
MS (m/z) 3e: 311 (M+, 10.31%), 237 (100%), 312 (3.26%), 313 (1.86%),
250 (38.75%), 191 (52.59%), 163 (91.35%), 136 (12.74%), 92 (16.07%),
61 (61.30%).
IR (KBr, cm−1) 3f: 3418 cm−1 (NH, OH), 2926, 2859 (CH aliph.),
1718 (C = O), 1573 (C=N), 1245 (COOH); δ 3f: 2.3, 2.4 [2s, 6H, 2CH3],
4.7 [m, 1H, α-CH], 6.45 [d, 2H, β-CH2, J = 7.2 Hz], 7.2–7.4 [m, 5H,
Ar-H], 8.5 [s, 2H, CH pyrimidine + NH], 8.6 [s, 1H, OH].
IR (KBr, cm−1) 3g: 3430 (OH), 3186 (NH), 2922, 2865 (CH aliph.),
1662 (C = O), 1580 (C=N), 1230 (COOH); δ 3g: 2.3 [s, 6H, 2CH3], 3.9
[s, 2H, 2OH], 4.7 [m, 1H, α-CH], 6.6 [d, 2H, β-CH2, J = 7.3 Hz], 8.3 [s,
2H, NH+CH pyrimidine].
IR (KBr, cm−1) 3h: 3563 (OH), 3412 (NH), 2934 (CH aliph.), 1714 (C
= O), 1581 (C=N), 1247 (COOH). δ 3h: 2.2 [m, 2H, β-CH2], 2.3, 2.4 [2s,
6H, 2CH3], 4.8 [t, 2H, γ-CH2], 6.6 [m, 1H, α-CH], 8.2 [s, 2H, NH+CH
pyrimidine], 8.3 [s, 2H, 2OH].
IR (KBr, cm−1) 3i: 3416 (br, NH, NH2, OH), 2980, 2922 (CH aliph.),
1666 (C = O), 1576 (C=N), 1298 (COOH); δ 3i: 1.4 [m, 1H, β-CH], 2.4
[s, 6H, 2CH3], 2.7 [d, 4H, 2 SCH2, J = 7.4 Hz], 4.9 [m, 1H, α-CH], 6.8
[d, 2H, NH2, J = 7.4 Hz], 8.1 [s, 1H, NH], 8.15 [s, 1H, pyrimidine-CH],
8.2, 8.3 [2s, 2H, 2OH].
IR (KBr, cm−1) 3j: 3429 (OH), 3208 (NH), 2930 (CH aliph.), 1722
(C = O), 1570 (C=N), 1240 (COOH); δ 3j: 2.25, 2.37 [2s, 6H, 2CH3],
4.7 [d, 2H, ß-CH2, J = 7.5 Hz], 6.5 [m, 1H, α-CH], 6.6–7.0[m, 4H,
Ar-H], 8.2[s, 1H, NH], 8.3[s, 1H, pyrimidine-CH], 8.45, 9.3[2s, 2H,
2OH]; MS (m/z) 3j: 342 (M-1, 0.33%), 107 (100%), 323 (2.15%),
295 (1.38%), 236 (0.51%), 219 (19.56%), 163 (48.27%), 132 (13.20%),
77 (37.34%).
IR (KBr, cm−1) 3k: 3444 (OH), 3350 (NH), 3073 (CH arom.), 2922
(CH aliph.), 1725 (C = O), 1556 (C=N), 1269 (COOH); δ 3k: 2.2, 2.3 [2s,
6H, 2CH3], 4.9 [d, 2H, ß-CH2, J = 7.3 Hz], 6.35[m, 1H, α-CH], 6.9–7.3
[m, 4H, Ar-H], 7.47, 7.50 [2s, 2H, 2NH], 8.26 [s, 1H, pyrimidine-CH],
10.9 [s, 1H, OH].
IR (KBr, cm−1) 3l: 3449 (OH), 2966, 2922 (CH aliph.), 1721 (C = O),
1550 (C=N), 1212 (COOH); MS (m/z) 3l: 277 (M+, 3.44%), 70 (100%),
232 (58.74%), 204 (20.72%), 190 (67.42%), 163 (51.76%), 109 (9.94%),
92 (14.58%).

3252
S. I. Alqasoumi et al.
7,8-Dimethyl-4-oxoacetylimidazo[1,5:4,5]thieno[2,3-
d]pyrimidine (4A), 5,7,8-Trimethyl-4-oxo (4b), 5-Isopropyl (4c),
5-Isobutyl (4d), 5-(2-Methylsulfanyl-ethyl) (4e), 5-Benzyl (4f),
5-Hydroxymethyl (4g), 5-Propionic Acid (4h), Imidazo[1,5:4,5]
thieno[2,3-d]pyrimidine
A mixture of 3a–h (0.01 mol) and anhydrous sodium acetate (2 g) in
acetic anhydride (30 mL) was refluxed for 3 h. The reaction mixture was
filtered while hot, the solvent was concentrated, and the solid obtained
was crystallized from the proper solvent (Table I).
IR (KBr, cm−1) 4A: 2930 (CH aliph.), 1710 (C = O), 1640 (C=N); δ
4A: 2.2 [s, 6H, 2 CH3], 2.5 [s, 3H, COCH3], 8.1, 8.4 [2s, 2H, CH imidazole
+ CH pyrimidine]; MS (m/z) 4A: 261 (M+, 92.92%), 179 (100%).
IR (KBr, cm−1) 4b: 2900 (CH aliph.), 1740 (C = O), 1600 (C=N). δ
4b: 2.08 [d, 3H, CH3, J = 7.3 Hz], 2.5 [s, 6H, 2CH3], 3.2 [m, 1H, CH
imidazole], 8.1 [s, 1H, CH pyrimidine].
IR (KBr, cm−1) 4c: 2900 (CH aliph.), 1700 (C = O), 1610 (C=N).
δ 4c: 1.612, 1.621 [2s, 6H, 2CH3], 2.07 [d, 1H, CH imidazole, J = 7.2
Hz], 2.5 [2s, 6H, 2CH3 thiophene], 3.3 [m, 1H, CH], 8.1 [hump, 1H, CH
pyrimidine].
IR (KBr, cm−1) 4d: 2952, 2868 (CH aliph.), 1714 (C = O), 1576
(C=N); MS (m/z) 4d: 275 (M+, 56.33%), 69 (100%).
IR (KBr, cm−1) 4e: 2940 (CH aliph.), 1700 (C = O), 1630 (C=N); MS
(m/z) 4e: 293 (M+, 12.28%), 55 (100%), 287 (10.53%), 278 (31.58%), 240
(28.07%), 213 (42.11%), 180 (26.32%), 161 (38.60%), 127 (28.07%), 102
(38.60%).
IR (KBr, cm−1) 4f: 2920 (CH aliph,), 1720 (C = O), 1640 (C=N). MS
(m/z) 4f: 309 (M+, 12.73%), 163 (100%), 279 (4.97%), 218 (59.47%), 190
(16.14%), 136 (5.19%), 91 (52.29%), 65 (23.69%).
IR (KBr, cm−1) 4g: 3500–3300 (br, OH), 2930 (CH aliph.), 1670 (C
= O), 1620 (C=N).
IR (KBr, cm−1) 4h: 3500–3000 (br, OH), 2900 (CH aliph,), 1720,
1700 (2C = O), 1590 (C=N). δ 4h: 1.9, 2.1 [2s, 6H, 2CH3], 3.4 [t,
2H, CH2], 4.0 [t, 2H, CH2CO], 7.8 [s, 1H, CH pyrimidine], 8.2 [s, 1H,
OH].
2-(5,6-Dimethylthieno[2,3-d]pyrimidin-4-ylamino)ethanol (5)
and 3-(5,6-Dimethylthieno[2,3-d]pyrimidin-4-ylamino)propan-
1-ol (6)
A mixture of 1 (1.99 g, 0.01 mol) and ethanolamine or propanolamine
(0.01 mol) in pyridine (20 mL) was refluxed for 12 h. The reaction
mixture was cooled and acidified with dil. HCl and the solid obtained
was crystallized from proper solvent (Table I).

Novel Amino Acids and Imidazoles
3253
IR (KBr, cm−1) 5: 3390 (OH), 3150 (NH), 2940 (CH aliph.), 1595
(C=N). δ 5: 0.9 [t, 2H, CH2OH], 1.2 [t, 2H, NCH2], 2.3, 2.4 [2s, 6H,
2CH3], 6.7 [s, 1H, NH], 8.3 [s, 1H, CH pyrimidine].
IR (KBr, cm−1) 6: 3500–3000 (br, OH+NH), 2920 (CH aliph.), 1600
(C=N). δ 6: 1.8 [t, 2H, NCH2], 2.3, 2.4 [2s, 6H, 2CH3], 3.6–3.8 [m,
4H, OCH2CH2], 5.7 [hump, 1H, OH], 8.1 [t,1H, NH], 8.7 [s, 1H, CH
pyrimidine].
7,8-Dimethyl-4,5-dihydroimidazo[1,5:4,5]thieno[2,3-d]
pyrimidine (7)
A solution of 5 (2.23 g, 0.01 mol) and thionyl chloride (20 mL) was
refluxed for 5 h. The solvent was evaporated under vacuum, and the
solid obtained was crystallized from the proper solvent (Table I).
IR (KBr, cm−1): 2970 (CH alpih.), 1630 (C=N). MS (m/z): 205 (M+,
77.89%), 57 (100%), 204 (93.54%), 98 (54.42%) 83 (81.97%).
4-Isothiocyanato-5,6-dimethylthieno[2,3-d]pyrimidine (8)
A mixture of 1 (1.99 g, 0.01 mol) and ammonium thiocyanate (0.76
g, 0.01 mol) in dry acetone (20 mL) was refluxed for 1 h. The reaction
mixture was filtered while hot then poured into ice water, and the solid
obtained was crystallized from the proper solvent (Table I). IR (KBr,
cm−1): 2940 (CH aliph.), 2150 (N=C=S), 1620 (C=N). MS (m/z): 221
[M+, 100%, base peak].
4-Amino-5-cyano-8,9-dimethyl-6-thioxopyrimido[1,6:4,5]
thieno[2,3-d]pyrimidine (10)
A mixture of 8 (2.21 g, 0.01 mol), malononitrile (0.66 g, 0.01 mol),
and sodium ethoxide (0.68 g, 0.01 mol) in ethanol (30 mL) was refluxed
for 6 h. The reaction mixture was poured into ice water, and the solid
product was crystallized from the proper solvent (Table I).
IR (KBr, cm−1): 3320, 3210 (NH2), 2940 (CH aliph.), 2210 (C≡N),
1600 (C=N).
Biological Testing
Animals, Chemicals, and Facilities
Female Swiss albino mice weighing 25–30 g (the holding company
for biological products and vaccines, VACSERA, Cairo, Egypt) were
housed at a constant temperature (24
2◦C) with alternating 12 h
light and dark cycles and were fed standard laboratory food and water.
All chemicals and reagents were of the highest commercially available
grade. Facilities including animal house, biochemical equipment,

3254
S. I. Alqasoumi et al.
and γ-irradiation were made available by the National Center for
Radiation Research and Technology (NCRRT), Cairo, Egypt. Whole
body irradiation was performed using Gamma cell-40 (Caesium-137
source). All animal experiments were conducted in and approved by
NCRRT.
Antitumor Activity
Ehrlich Ascites Carcinoma (EAC) cells were obtained by needle aspi-
ration of ascitic fluid from the preinoculated mice under aseptic condi-
tions.19 A suspension of tumor cells (2.5 × 106 per mL) was prepared
in saline. Tested compounds were prepared with various dilutions by
dissolving 1.0, 0.75, 0.5, and 0.25 mg of the test compounds in DMF
(1 mL).
In a set of sterile test tubes, 0.1 mL of tumor cells suspension, 0.8
mL saline, and 0.1 mL of each tested compound (corresponding to 400,
200, 150, 100, 90, 80, 75, 50, 25, and 10 µg) were mixed. The test
tubes were incubated at 37◦C for 2 h. The Trypan blue exclusion test
was carried out to calculate the percentage of nonviable cells after
2 h of incubation.20 Compounds producing more than 70% nonviable
cells are considered active.21 The results of in vitro cytotoxic activity
experiments are presented in Table II.
Radioprotective Activity
This study was conducted to evaluate the potency of some of the
synthesized compounds 3k and 8 as protective agents against γ-
irradiation–induced toxicity, which may extend to affect normal organs
in mice bearing solid Ehrlich tumor. Also, to evaluate their antitu-
mor effect, alone or in combination with irradiation, was accomplished
by measuring the change of tumor volume (TV). Female Swiss albino
mice were injected intraperitoneally with a suspension of the tested
compounds in carboxy methylcellulose at the maximum tolerated dose
of 150 mg/kg body weight 10 days after tumor inoculation, then once
every other day for a total of three injections during 7 days. Each injec-
tion was given 30 min prior to exposure to a single dose of whole body
γ-irradiation at a dose level of 2 Gy delivered at a dose rate of 0.86
Gy/min. Lipid peroxide content (LPx), glutathione level (GSH), and the
activity of the antioxidant scavenger enzyme system super oxide dis-
mutase (SOD) were estimated in blood of animals after the end of the
experiment.

Novel Amino Acids and Imidazoles
3255
Experimental Tumor Cells and Tumor Transplantation
A line of EAC was used in this study. The parent line was kindly sup-
plied by the National Cancer Institute, Cairo University, Egypt. The
tumor cells were maintained by weekly intraperitoneal transplanta-
tion of 2.5 × 106 cells. Solid tumors were produced by intramuscular
inoculation with 0.2 mL of EAC in the right thigh of the lower limb
of each mouse. Mice with a palpable solid tumor mass (100 mm3) that
developed within 10 days after inoculation were used in the study. The
change in tumor volume (TV) was measured at the end of the exper-
iment using a Vernier caliper (Hangzhou Jinnan Tools & Measures,
Hangzhou, China) and calculated by the following formula according
to Osman et al.22: TV (mm3) = 0.52 AB2, where A is the minor axis and
B is the major axis.
Experimental Design
From the beginning of the experiment, mice were divided into 10
groups. All experimental animals were categorized as follows:
1. Control: Animals served as untreated control group.
2. CMC: Animals were treated by i.p. injection of carboxy methylcel-
lulose.
3. Rad: Animals were subjected to 3 doses; every other day of whole
body γ-irradiation at a dose level of 2 Gy starting from day 10.
4. ER: Mice bearing solid Ehrlich tumors without any treatment.
5. ER + Rad: Mice bearing solid Ehrlich tumors and subjected to
whole body γ-irradiation starting from day 10.
6. ER + CMC + Rad: Mice bearing solid Ehrlich tumor were in-
jected intraperitoneally with carboxy methylcellulose and subjected
to whole body γ-irradiation starting from day 10.
7. ER
+
Compound 3k[3-(2,3-Dihydro-1H-indol-2-yl)-2-(5,6-
dimethyl-thieno[2,3-d]pyrimidin-4-ylamino)-propionic acid]: Mice
bearing solid Ehrlich tumor wer injected i.p. with compound 3k.
8. ER + Compound 8 (4-Isothiocyanato-5,6-dimethyl-thieno[2,3-
d]pyrimidine): Mice bearing solid Ehrlich tumor were injected i.p.
with compound 8.
9. ER + Compound 3k + R: Mice bearing solid Ehrlich tumor and
injected i.p. with compound 3k were subjected to whole body γ-
irradiation.
10. ER + Compound 8 + R: Mice bearing solid Ehrlich tumor and
injected i.p. with compound 8 were subjected to whole body γ-
irradiation.

3256
S. I. Alqasoumi et al.
Samples Collection
Animals were fasted for 16 h prior to each sampling. Samples were
collected after 1 day post last irradiation dose. Whole blood was col-
lected by heart puncture after light anesthesia using heparinized sy-
ringes. One part was used for glutathione (GSH) and superoxide dismu-
tase (SOD) estimations. The separated plasma from heparinized blood
was used for the determination of lipid peroxide as malondialdehyde
(MDA).
Analytical Procedures
Lipid peroxide (Lpx) content in plasma was ascertained by the for-
mation of MDA and measured as described by Yoshioka et al.23 GSH
content was determined according to Beutler et al.,24 and SOD was
quantized according to Minami and Yoshikawa.25
Statistical Analysis
ANOVA test26 was used for the evaluation of tumor volume (TV) and
other biochemical parameters.
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