Novel triazolo[4,3-a]quinazolinone and bis-triazolo[4,3-a :4,3'-c]quinazolines: Synthesis and antitoxoplasmosis effect

Article abstract of DOI:10.1016/S0014-827X(99)00038-5

Several quinazoline derivatives containing substituted thiosemicarbazido and S-methylisothiosemicarbazido groups at the 2-position and at both the 2- and 4-positions have been synthesized. Treatment of the S-methylthiosemicarbazides with morpholine or diethylamine did not give the corresponding guanidines. Instead, they underwent cyclodesulfurization ihto the condensed ring systems, [1,2,4]triazolo[4,3-a]quinazolinones and bis-[1,2,4]triazolo[4,3-a:4',3'-c]quinazolines. Evaluation of the products for antitoxoplasmosis effect by studying the ultrastructure morphology of the organisms using scanning electron microscopy (SEM) indicated their efficacy in causing structural deformity of Toxoplasma gondii. Such a deformity plays an important role in obstructing the entry of the organisms into host cells.

Full text of DOI:10.1016/S0014-827X(99)00038-5

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 486–495
Novel triazolo[4,3-a]quinazolinone and
bis-triazolo[4,3-a:4,3%-c]quinazolines: synthesis and
antitoxoplasmosis effect
Alaa A. El-Tombary a, Khadiga A. Ismail a, Omaima M. Aboulwafa a,*,
A.-Mohsen M.E. Omar a, Mervat Z. El-Azzouni b, Salwa T. El-Mansoury b
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Uni6ersity of Alexandria, Alexandria 21215, Egypt
b Department of Parasitology, Faculty of Medicine, Uni6ersity of Alexandria, Alexandria 21215, Egypt
Received 10 May 1998; accepted 24 March 1999
Abstract
Several quinazoline derivatives containing substituted thiosemicarbazido and S-methylisothiosemicarbazido groups at the
2-position and at both the 2- and 4-positions have been synthesized. Treatment of the S-methylthiosemicarbazides with
morpholine or diethylamine did not give the corresponding guanidines. Instead, they underwent cyclodesulfurization into the
condensed ring systems, [1,2,4]triazolo[4,3-a]quinazolinones and bis-[1,2,4]triazolo[4,3-a:4%,3%-c]quinazolines. Evaluation of the
products for antitoxoplasmosis effect by studying the ultrastructure morphology of the organisms using scanning electron
microscopy (SEM) indicated their efficacy in causing structural deformity of Toxoplasma gondii. Such a deformity plays an
important role in obstructing the entry of the organisms into host cells. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Quinazoline; Thiosemicarbazide; S-Methylisothiosemicarbazide; Cyclodesulfurization; Triazolo and bis-triazoloquinazoline; Antitoxo-
plasmosis effect
toxoplasmosis [12]. Replacement of the 6-substituted
arylamino function in the dichlorobenzylaminoquinazo-
line derivative by arylthio, arylsulfinyl or arylsulfonyl
groups was also effective in producing highly active
antimalarial agents [13]. On the other hand, attachment
of an urea or thiourea residue to the 2-position of
pyrimidine led to inactive compounds [14]. In contrast,
substitution by an arylguanidine group gave products
with appreciably enhanced activity against experimental
malarial infection [15]. Introduction of a thiosemicar-
bazide or semicarbazide function in the 2-position of
quinoline also led to potent antimalarials [16]. These
compounds represent analogues of the clinically active
quinoline antimalarials, e.g. Chloroquine and Pri-
maquine.
1. Introduction
Structure–activity relationship (SAR) studies on an-
tiprotozoan compounds revealed that the antimalarial
activity is associated with the attachment of alkylamino
and arylamino groups to various heterocyclic nuclei,
as in the 2-arylamino-4-dialkyl-aminoalkylamino-6-
methylpyrimidines [1] and 2-arylamino-4-dialkylam-
inoalkylamino-quinazolines [1,2]. Other studies demon-
strated that the presence of free amino group at the 2-
and 4-positions of pyrimidine and quinazoline was ef-
fective in inducing strong antimalarial properties
against sensitive and drug-resistant lines of Plasmodia
[3–9]. Among the more potent antimalarial agents of
this class of compounds are 2,4-diamino-6-[(3,4-
dichlorobenzyl)amino]quinazoline [6,10,11] and 2,4-di-
amino-5-p-chlorophenyl-6-ethylpyrimidine (pyrimeth-
amine), which is in current clinical use against murine
These observations aroused interest in the investiga-
tion of this structural class and it seemed desirable to
prepare and test some analogues in which the typical
antimalarial basic diamino groups, which were thought
to play a key role in conferring optimal antiplasmodial
effects among folate antagonists [17], have been re-
* Corresponding author. Tel.: +20-3-483 1317; fax: +20-3-483
3273.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00038-5

A.A. El-Tombary et al. / Il Farmaco 54 (1999) 486–495
487
2. Chemistry
The synthesis of the triazoloquinazolinones 4 and the
ditriazoloquinazolines 9 was carried out as depicted in
Schemes 2 and 4. The 2-hydrazinoquinazolin-4-one (1),
prepared according to the method of Claesen and Van-
derhaeghe [20], was condensed with the appropriate
arylisothiocyanate in boiling ethanol to generate the
1-(4-oxoquinazolin-2-yl)-4-aryl-3-thiosemicarbazides
(2a–d).
The use of iodomethane in absolute methanol to
affect S-methylation [18,21] of these thiosemicarbazides
was unsuccessful. However, reacting 2a–d with
dimethyl sulfate in aqueous sodium hydroxide [22,23]
gave a mixture of the 4-oxoquinazolin-2-yl-S-methyl-
isothiosemicarbazides (3a–d) and the corresponding
N,S-dimethyl derivatives 3e–h which were separated by
preparative TLC. The 1H NMR spectra of the S-
methyl derivatives 3a and 3b showed a singlet assigned
to quinazoline NꢀH at l 11.04 and 11.10, respectively.
This proton signal was absent in the spec-tra of N,S-
dimethyl derivatives 3e and 3h, which showed a singlet
resonating at l 3.48 and 3.47, respectively, assigned to
the three protons of the NꢀCH3. The spectra also
confirmed that 1-(4-oxoquinazolin-2-yl)-4-aryl-3-S-
methylisothiosemicarbazides 3a–h existed as
Scheme 1. Aminoguanidine structure in a flexible chain (A) or rigid
heterocyclic ring (B).
placed by the more basic hydrazino moiety in the
2-position or in the 2- and 4-positions of quinazoline.
In addition, it was also desirable to check the effect of
conversion of such hydrazino group into a thiosemicar-
bazido moiety and an aminoguanidine function
(Scheme 1, A). The synthesis of these compounds by
reacting the intermediate S-methylisothiosemicarbazide
derivative of the appropriate quinazoline with morpho-
line or diethylamine, as reported [18], followed a differ-
ent pathway. The S-methylisothiosemicarbazide
underwent cyclodesulfurization to the corresponding
triazoloquinazoline derivatives (Scheme 1, B). These
condensed heterocyclic products retained the
aminoguanidine structure [19] but in a rigid form (B) if
compared with the designed compounds (A). The prod-
ucts were evaluated in vitro for their antitoxoplasmosis
activity and the results are reported in this paper.
SCH
3
ꢀHNꢀNꢁC-NHR
rather than the isomeric
SCH
ꢀHNNHꢀCꢁN3R
as previously reported [18]. This was evidenced by the
presence of the N4-proton at l 8.38–9.45 and the
disappearance of the hydrazino N2-proton at l 7.82.
Scheme 2. Synthesis of triazoloquinazolinones 4a–h.

488
A.A. El-Tombary et al. / Il Farmaco 54 (1999) 486–495
the morpholine or diethylamine part of the amino-
guanidine moiety 5a–h. In fact, the spectra of 4e–h
showed only one NH proton resonating at l 8.02–9.04.
The 3,7-bis-(substituted amino)[1,2,4]ditriazolo[4,3-a:
4%,3%-c]quinazolines (9a,b) were likewise synthesized
starting from the 2,4-dihydrazinoquinazoline 6 [20]
(Scheme 4).
Compound 6 was reacted with the appropriate iso-
thiocyanate and S-methylated by dimethyl sulfate in
alkaline medium to produce the di-S-methylisothiosem-
icarbazides 8a,b. The products were treated with mor-
pholine or diethylamine to undergo cyclodesulfur-
ization to compounds 9a,b. The structure of the prod-
ucts was assessed by elemental analyses, IR and for
1
some representative examples, by H NMR and mass
spectra.
3. Experimental
3.1. Chemistry
Scheme 3. The proposed cyclodesulfurization mechanism of S-
methylisothiosemicarbazides 3a–h into the triazoloquinazolines 4a–h.
Melting points, in open capillary tubes, were taken
using a Griffin melting point apparatus and are uncor-
rected. IR spectra were recorded on a Shimadzu 408
spectrophotometer as KBr discs or Nujol mulls
(w cm−1). The 1H NMR spectra (DMSO-d6) were
recorded on a Varian Gemini 200 (200 MHz) spec-
trophotometer. The chemical shift values, reported in l
(ppm), are relative to TMS as the internal standard.
Abbreviations used are: s, singlet; d, doublet; t, triplet;
m, multiplet; br, broad; dist., distorted. The mass spec-
tra (MS) were run on a Shimadzu GCMS QP 1000 EX
Heating the S-methylisothiosemicarbazides 3a–h un-
der reflux with morpholine or diethylamine, as reported
[18], did not produce the required aminoguanidines
5a–h. Instead, compounds 3a–h underwent cyclodesul-
furization into the triazoloquinazolines 4a–h according
to the proposed mechanism in Scheme 3.
The H NMR spectra of triazoloquinazolines 4a–h
lacked the singlet for SꢀCH3 present in the S-
methylisothiosemicarbazides 3a–h and the signals for
1
Scheme 4. Synthesis of bis-triazoloquinazolinones 9a,b.

A.A. El-Tombary et al. / Il Farmaco 54 (1999) 486–495
489
gas chromatograph–mass spectrometer (GC–MS). Ho-
3.1.3. 1-(4-Oxoquinazolin-2-yl)-4-aryl-3-S-
mogeneity of the products was determined by ascending
TLC on silica gel coated glass plates visualized by
iodine vapors. Preparative TLC was performed on
20×20 cm plates coated with 30 g silica gel 60 GF 254
for TLC, Adwic laboratory chemicals. A duo-UV lamp,
Desega Heidelberg, Germany, was used for location of
the spots. Elemental analyses were performed at the
Microanalytical Unit, Faculty of Science, Cairo Univer-
sity, Egypt. Analyses (C, H, N) were within 90.4% of
the theoretical values.
methylisothiosemicarbazides (3a–d) and 1-(3-methyl-4-
oxoquinazolin-2-yl)-4-aryl-3-S-methylisothiosemi-
carbazides (3e–h)
3.1.3.1. Using iodomethane in dry methanol. A solution
of the thiosemicarbazide derivative 2a–d (0.5 g) in dry
methanol (20 ml) was treated with an equimolar
amount of iodomethane. The solution was kept
overnight at r.t. and evaporated to dryness under re-
duced pressure. The residue was treated with ethanol
(10 ml) and scratched to deposit into solid. This was
filtered and, after purification by preparative TLC
and developing with benzene:chloroform:methanol
5:2.5:0.5, gave a negligible amount of the desired
product.
3.1.1. 2-Hydrazinoquinazolin-4-one (1)
A mixture of 2-chloroquinazolin-4-one [1] (1.2 g, 6.6
mmol), potassium carbonate (2 g), 85% hydrazine hy-
drate (2 ml) and H2O (6 ml) was heated under reflux for
2 h. The mixture was cooled to room temperature (r.t.),
neutralized with acetic acid to deposit 2-hydrazino-
quinazolin-4-one (1) which was crystallized from
ethanol (0.7 g, yield 60%); m.p.\300°C; reported m.p.
dec. 360°C [20]. IR: w 3000 (br, NH and NH2 mixed
with OH of the tautomeric form), 1680 (CꢁO), 1610
(CꢁN), 1600 and 1505 cm−1 (CꢁC Ar).
3.1.3.2. Using dimethyl sulfate in 1 N NaOH. A solution
of the thiosemicarbazide derivative 2a–d (0.5 g) in 1 N
NaOH (10 ml) was treated with dimethyl sulfate (0.2 g,
1.6 mmol) while stirring in an ice-bath. The reaction
mixture was stirred for a further 1 h at 4–5°C. The
obtained yellow sticky mass was scratched and stirred
at r.t. for another 30 min, filtered and washed with
water. Separation of the mixture into its components by
preparative TLC with benzene:chloroform:methanol
5:2.5:0.5, as developing solvent, gave the 4-oxoquina-
zolin-2-yl-S-methylisothiosemicarbazides (3a–d) and
the corresponding N,S-dimethyl derivatives 3e–h. The
physicochemical properties of compounds 3a–h are
listed in Table 1.
3.1.2. 1-(4-Oxoquinazolin-2-yl)-4-aryl-3-
thiosemicarbazides (2a–d)
A solution of 2-hydrazinoquinazolin-4-one (1) (0.5 g,
2.8 mmol) in ethanol (200 ml) was treated with 1.5
equiv. of the appropriate isothiocyanate. The mixture
was heated under reflux for 2 h, concentrated and
cooled to r.t. The separated product was filtered and
crystallized from ethanol.
IR of 3a: w 3350 (NH), 1675 (CꢁO), 1645 (CꢁN),
1
1590 (CꢁC Ar) and 1380 cm−1 (SꢀCH3). H NMR of
2a: m.p.\300°C; yield 92%. Anal. (C15H13N5OS) C,
H, N. IR: w 3400, 3268 (NH), 1700 (CꢁO), 1660 (CꢁN),
1595 (CꢁC Ar), 1560, 1330, 1050 and 875 cm−1 (NCS
amide I, II, III and IV mixed vibrational bands, respec-
3a: l 2.62 (s, 3H, SꢀCH3) 6.84–7.36 (m, 5H, ArꢀH),
7.41–7.58 (m, 3H, quinazolinone C6ꢀC8ꢀH), 8.03 (dd,
1H, Jo=9.9 Hz, Jm=1.8 Hz, quinazolinone C5ꢀH),
8.38 (s, 1H, N1ꢀH). 9.83 (s, dist., 1H, N4ꢀH), and 11.04
(s, 1H, quinazolinone NH). IR of 3b: w 3320 (NH),
1685 (CꢁO), 1630 (CꢁN), 1590 (CꢁC Ar) and 1375
1
tively). H NMR: l 6.9–7.5 (m, 6H, ArꢀH, N1H), 7.82
(s, 1H, N2ꢀH), 8.01–8.31 (m, 3H, quinazoli-
noneꢀC6ꢀC8ꢀH), 8.48 (dd, 1H, Jo=9.9 Hz, Jm=1.8
Hz, quinazolinone C5ꢀH), 9.66 (s, 1H, N4ꢀH), 13.00 (s,
br, 1H, quinazolinone NH).
1
cm−1 (SꢀCH3). H NMR of 3b: l 2.33 (s, 3H, SꢀCH3)
6.80–7.53 (m, 4H, p-ClC6H4ꢀH), 7.63–7.83 (m, 3H,
quinazolinone C6ꢀC8ꢀH), 8.19 (dd, 1H, Jo=9.9 Hz,
Jm=1.8 Hz, quinazolinone C5ꢀH), 9.54 (s, 1H, N1ꢀH).
2b: m.p.\300°C; yield 94%. Anal. (C15H12ClN5OS)
C, H, N. IR: w 3300 (NH), 1690 (CꢁO), 1660 (CꢁN),
1610 (CꢁC Ar), 1550, 1340, 1075 and 830 cm−1 (NCS
amide I, II, III and IV mixed vibrational bands,
respectively).
Table 1
Physical data of compounds 3a–h
Comp.
M.p. (°C)
Yield (%)
Formula (MW)
2c: m.p.\300°C; yield 90%. Anal. (C16H15N5OS) C,
H, N. IR: w 3255 (NH), 1700 (CꢁO), 1650 (CꢁN), 1590
(CꢁC Ar), 1545, 1340, 1070 and 870 cm−1 (NCS amide
I, II, III and IV mixed vibrational bands, respectively).
2d: m.p. 267–269°C; yield 93%. Anal. (C16H15N5O2S)
C, H, N. IR: w 3250 (NH), 1690 (CꢁO), 1660 (CꢁN),
1590 (CꢁC Ar), 1535, 1335, 1060 and 835 cm−1 (NCS
amide I, II, III and IV mixed vibrational bands,
respectively).
3a
3b
3c
3d
3e
3f
291–293
279–280
265–267
252–255
223–225
275–277
260–262
237–239
24
20
25
21
27
28
26
30
C16H15N5OS (325)
C16H14ClN5OS (359.5)
C17H17N5OS (339)
C17H17N5O2S (355)
C17H17N5OS (339)
C17H16ClN5OS (373.5)
C18H19N5OS (353)
C18H19N5O2S (369)
3g
3h

490
A.A. El-Tombary et al. / Il Farmaco 54 (1999) 486–495
9.80 (s, dist., 1H, N4ꢀH), and 11.10 (s, dist., 1H,
quinazolinone NH). IR of 3c: w 3225 (NH), 1680
(CꢁO), 1635 (CꢁN), 1580 (CꢁC Ar) and 1375 cm−1
(SꢀCH3). IR of 3d: w 3295 (NH), 1685 (CꢁO), 1670
(CꢁN), 1585 (CꢁC Ar) and 1360 cm−1 (SꢀCH3). IR of
3e: w 3410 (NH), 1685 (CꢁO), 1655 (CꢁN), 1605 (CꢁC
8.14 (dd, Jo=9.9 Hz, Jm=1.8 Hz, 1H, quinazolinone
+
’
C5ꢀH), 9.52 (s, 1H, NH phenyl). MS of 4a: M
277
absent, M+ −O (M−16) 261 (23%), 55 (100%). IR of
4b: w 3320 (NH), 1675 (CꢁO), 1620 (CꢁN), 1570 (CꢁC
1
Ar). H NMR of 4b: l 6.59 (d, J=10.9 Hz, 2H, ArꢀH
meta to Cl), 6.84 (d, J=10.9 Hz, 2H, ArꢀH ortho to
Cl), 7.06–7.44 (m, 4H, quinazolinone NH+C6ꢀC8ꢀH),
7.91 (dd, Jo=9.9 Hz, Jm=1.8 Hz, 1H, quinazolinone
C5ꢀH), 8.53 (s, 1H, NHꢀAr). IR of 4c: w 3300 (NH),
1
Ar) and 1360 cm−1 (SꢀCH3). H NMR of 3e: l 2.47 (s,
3H, SꢀCH3) 3.48 (s, 3H, NꢀCH3), 6.90–7.39 (m, 6H,
5ArꢀH+N1ꢀH)), 7.46–7.56 (m, 3H, quinazolinone
C6ꢀC8ꢀH), 8.09 (dd, 1H, Jo=9.9 Hz, Jm=1.8 Hz,
quinazolinone C5ꢀH) and 8.98 (s, 1H, N4ꢀH). IR of 3f:
w 3350 (NH), 1688 (CꢁO), 1635 (CꢁN), 1570 (CꢁC Ar)
and 1375 cm−1 (SꢀCH3). IR of 3g: w 3250 (NH), 1695
(CꢁO), 1645 (CꢁN), 1595 (CꢁC Ar) and 1360 cm−1
(SꢀCH3). IR of 3h: w 3320 (NH), 1680 (CꢁO), 1655
(CꢁN), 1580 (CꢁC Ar) and 1365 cm−1 (SꢀCH3).
1H NMR of 3h: l 2.43 (s, 3H, SꢀCH3), 3.47 (s, 3H,
SꢀCH3), 3.80 (s, 3H, OCH3), 6.77–7.26 (m, 4H,
4ArꢀH), 7.46–7.61 (m, 3H, quinazolinone C6ꢀC8ꢀH),
8.03 (dd, 1H, Jo=9.9 Hz, Jm=1.8 Hz, quinazolinone
C5ꢀH), 8.39 (s, dist., 1H, N1ꢀH) and 8.98 (s, 1H,
N4ꢀH).
1
1690 (CꢁO), 1630 (CꢁN), 1570 (CꢁC Ar). H NMR of
4c: l 6.69 (d, J=10.9 Hz, 2H, ArꢀH ortho to CH3),
7.06 (d, J=10.9 Hz, 2H, ArꢀH meta to CH3), 7.24–
7.69 (m, 4H, quinazolinone NH+C6ꢀC8ꢀH), 8.40
(dd, Jo=9.9 Hz, Jm=1.8 Hz, 1H, quinazolinone
C5ꢀH), 9.22 (s, 1H, NHꢀAr). IR of 4d: w 3390 (NH),
1
1680 (CꢁO), 1620 (CꢁN), 1580 (CꢁC Ar). H NMR of
4d: l 3.75 (s, 3H, OCH3), 6.82 (d, J=10.9 Hz, 2H,
ArꢀH meta to CH3), 7.06 (m, 3H, ArꢀH ortho to
CH3+quinazolinone NH), 7.21–7.60 (m, 4H, quina-
zolinone C5ꢀC8ꢀH), 8.60 (s, 1H, NHꢀAr). IR of 4e: w
3410 (NH), 1680 (CꢁO), 1625 (CꢁN), 1570 (CꢁC Ar).
1H NMR of compound 4e: l 3.60 (s, 3H, NꢀCH3),
6.83–6.93 (m, 3H, ArꢀH), 7.18–7.26 (m, 2H, ArꢀH),
7.53 and 7.78 (2t, J=16.6 Hz, 2H, quinazolinone C6
and C7ꢀH), 8.09 (d, 1H, J=8.6 Hz, quinazolinone
C8ꢀH), 8.22 (dd, Jo=9.9 Hz, Jm=1.8 Hz, 1H, quina-
3.1.4. 1-Substituted amino[1,2,4]triazolo[4,3-
a]quinazolin-5-ones (4a–d) and 1-substituted amino-
4-methyl[1,2,4]triazolo[4,3-a]quinazolin-5-ones (4e–h)
zolinone C5ꢀH), 9.04 (s, 1H, NH). MS of compound 4e:
+
’
M
291 (89%), M + −H 290 (100%). IR of 4f: w 3320
’
3.1.4.1. Reaction of the S-methylisothiosemicarbazides
3a–h with diethylamine or morpholine. A solution of the
appropriate S-methylisothiosemicarbazide 3a–d or the
N3,S-dimethyl derivatives 3e–h (250 mg) in morpholine
or diethylamine (5 ml) was stirred at 100°C for 6–8 h.
The mixture was then evaporated to dryness under
reduced pressure and the residue was purified by
preparative TLC using benzene:ethyl acetate 9:1 as
developing system, to give the triazoloquinazolines 4a–
d and 4e–h.
(NH), 1675 (CꢁO), 1620 (CꢁN), 1570 (CꢁC Ar). 1H
NMR of 4f: l 3.61 (s, 3H, NꢀCH3), 7.27 (d, J=10.9
Hz, 2H, 2 ArꢀH meta to Cl), 7.37 (d, J=10.9 Hz, 2H,
2ArꢀH ortho to Cl), 7.53–7.81 (m, 4H, quinazolinone
C6ꢀC8ꢀH+NH), 8.25 (dd, Jo=9.9 Hz, Jm=1.8 Hz,
1H, quinazolinone C5ꢀH). IR of 4g: w 3345 (NH), 1680
1
(CꢁO), 1620 (CꢁN), 1655 (CꢁC Ar). H NMR of com-
pound 4g: l 2.27 (s, 3H, CH3), 3.81 (s, 3H, NꢀCH3),
6.69 (d, J=10.9 Hz, 2H, ArꢀH ortho to CH3), 7.01
(d, J=10.9 Hz, 2H, ArꢀH meta to CH3), 7.60–8.04
(m, 4H, quinazolinone C6ꢀC8ꢀH+NH), 8.40 (dd, Jo=
9.9 Hz, Jm=1.8 Hz, 1H, quinazolinone C5ꢀH). IR of
4h: w 3300 (NH), 1690 (CꢁO), 1630 (CꢁN), 1570 (CꢁC
The physicochemical properties of these compounds
are reported in Table 2.
IR of 4a: w 3410 (NH), 1675 (CꢁO), 1625 (CꢁN),
1
1570 (CꢁC Ar). H NMR of 4a: l 6.73–7.57 (m, 9H,
1
Ar). H NMR of 4h: l 3.71 (s, 3H, OCH3), 3.78 (s, 3H,
ArꢀH+quinazolinone NH+quinazolinone C6ꢀC8ꢀH),
NꢀCH3), 6.71–6.90 (m, 4H, ArꢀH), 7.46–7.61 (m, 3H,
quinazolinone C6ꢀC8ꢀH), 8.02 (s, dist., 1H, NH), 8.36
(dd, Jo=9.9 Hz, Jm=1.8 Hz, 1H, quinazolinone
C5ꢀH).
Table 2
Physical data of compounds 4a–h
Comp.
M.p. (°C)
Yield (%)
Formula (MW)
3.1.5. 2,4-Dihydrazinoquinazoline (6)
4a
4b
4c
4d
4e
4f
230–231
250–252
252–254
245–247
246–247
270–272
268–270
258–259
20
29
35
25
35
40
45
45
C15H11N5O (277)
C15H10ClN5O (311.5)
C16H13N5O (291)
C16H13N5O2 (307)
C16H13N5O (291)
C16H12ClN5O (325.5)
C17H15N5O (305)
C17H15N5O2 (321)
A mixture of 2,4-dichloroquinazoline [1] (2.5 g, 12.6
mmol) and 50% hydrazine hydrate (20 ml) was heated
under reflux for 2 h. The reaction mixture was cooled
to r.t., filtered and crystallized from methanol, m.p.
224–225°C (reported 226–227°C) [19]; yield 40%. IR: w
3250 and 3050 (NH and NH2), 1620 (CꢁN), 1580 and
1500 cm−1 (CꢁC Ar).
4g
4h

A.A. El-Tombary et al. / Il Farmaco 54 (1999) 486–495
491
1
3.1.6. 2,4-Di-(4-substituted thiosemicarbazido)-
quinazolines (7a,b)
(CꢁC Ar). H NMR: l 6.86–7.25 (m, 10H, 2×C6H5),
7.28–7.65 (m, 3H, quinazolinone C6ꢀC8ꢀH), 8.59 (dd,
Jo=9.9 Hz, Jm=1.8 Hz, 1H, quinazolinone C5ꢀH),
10.21 (s, 2H, 2×NHꢀC6H5).
A solution of 2,4-dihydrazinoquinazoline (6) (0.5 g,
2.6 mmol) in ethanol:chloroform (4:1) was treated
dropwise while stirring with phenyl or p-chlorophenyl
isothiocyanate (10 mmol), the reactants were stirred
overnight at r.t. The obtained yellowish-white precipi-
tate was filtered, washed with light petroleum 60–80°C
and crystallized from ethanol. 7a: m.p. 296–298°C;
yield 90%. Anal. (C22H20N8S2) C, H, N. IR of 7a: w
3450 (NH), 1620 (CꢁN), 1590, 1495 (CꢁC Ar), 1545,
1380, 1080 and 810 cm−1 (NCS amide I, II, III and IV
mixed vibrational bands, respectively). 7b: m.p.\
300°C; yield 85%. Anal. (C22H18Cl2N8S2) C, H, N. IR
of 7b: w 3250 (NH), 1640 (CꢁN), 1615, 1490 (CꢁC Ar),
1530, 1370, 1090 and 828 cm−1 (NCS amide I, II, III
and IV mixed vibrational bands, respectively).
1H NMR of 7b: l 7.39–7.49 (m, 8H, 2C6H4Cl(p)),
7.58–7.80 (m, 3H, quinazolinone C6ꢀC8ꢀH), 8.33 (dd,
Jo=9.9 Hz, Jm=1.8 Hz, 1H, quinazolinone C5ꢀH),
9.84–9.99 (m, 4H, 2N1ꢀH and 2N2ꢀH), 10.24 (s, 2H,
2N4ꢀH).
9b: m.p. 285–287°C; yield 30%. Anal. (C22H14Cl2N8)
C, H, N. IR of 9b: w 3450 (NH), 1615 (CꢁN), 1595,
1
1495 (CꢁC Ar). H NMR: l 7.27–7.70 (m, 8H, 2×
C6H4Cl), 7.90–8.21 (m, 3H, quinazolinone C6ꢀC8ꢀH),
8.40 (dd, Jo=9.9 Hz, Jm=1.8 Hz, 1H, quinazolinone
+
’
C5ꢀH), 9.11 (s, 2H, 2×NHꢀC6H4Cl). MS: M
461
absent, M + −2×p-chlorophenyl, −2H, 236 (37%),
’
55 (100%).
3.2. Antitoxoplasmosis properties
The antitoxoplasmosis property of the synthesized
quinazoline derivatives was studied by examining their
effect on the surface morphology of intact fixed organ-
isms, as viewed by scanning electron microscopy
(SEM).
3.2.1. Materials and methods
An RH virulent strain of Toxoplasma gondii was
maintained in the Parasitology Laboratory, Faculty of
Medicine, University of Alexandria, by several passages
(3–4 day intervals) of tachyzoites intraperitoneally in
Swiss albino mice (6–8 weeks old and about 20 g each).
The tachyzoites were harvested from the peritoneal
exudate on the 4th day of infection, washed three times
and diluted to 103 with phosphate buffer solution (PBS)
pH 7.4 [24]. They were then inoculated intraperi-
toneally at a dose of 0.1 ml/mouse in 50 Swiss albino
mice, and were divided into ten groups (five mice each)
[24].
3.1.7. 2,4-Di-(4-substituted-3-S-methylisothiosemi-
carbazido)quinazolines (8a,b)
A solution of the dithiosemicarbazide derivative 7a,b
(0.5 g) in 1 N NaOH (10 ml) containing a few drops of
ethanol was treated with an equimolar amount of
dimethyl sulfate while stirring in an ice-bath. Stirring
was continued for 1 h and the obtained yellow sticky
mass was scratched, and stirred for another 30 min.
The product was filtered, washed with water and
purified by preparative TLC using benzene:ethyl acetate
8:2 as developing solvent.
8a: m.p. 195–197°C; yield 30%. Anal. (C24H24N8S2)
C, H, N. IR of 8a: w 3350 (NH), 1635 (CꢁN), 1590,
1490 (CꢁC Ar) and 1310 cm−1 (SꢀCH3).
3.2.2. Infected, non-treated control group
48 h post-inoculation, DMSO was injected subcuta-
neously in a dose of 0.1 ml/mouse. The parasite-rich
peritoneal fluid was taken 5 days post-inoculation.
8b:
m.p.
205–207°C;
yield
33%.
Anal.
(C24H22Cl2N8S2) C, H, N. IR of 8b: w 3300 (NH), 1620
(CꢁN), 1575, 1500 (CꢁC Ar) and 1315 cm−1 (SꢀCH3).
1H NMR: l 2.44 (s, 6H, 2SꢀCH3), 7.15–7.42 (m, 8H,
3.2.3. Standard group
2C6H4Cl(p)),
7.86–8.19
(m,
3H,
quinazoli-
Pyrimethamine, obtained by extraction of five pulver-
ized Daraprim® tablets (pyrimethamine tablets BP 25
mg, Wellcome, England) with ethanol (50 ml) followed
by evaporation of the solvent, was given orally as a
single dose of 0.25 mg in 0.1 ml DMSO/mouse [25,26]
48 h after inoculation. The peritoneal fluid containing
tachyzoites was collected 3 days after treatment.
noneꢀC6ꢀC8ꢀH), 8.35 (dd, Jo=9.9 Hz, Jm=1.8 Hz,
1H, quinazolinone C5ꢀH), 9.73–9.93 (m, 2H, 2N1ꢀH)
and 10.38 (s, dist., 2H, 2N4ꢀH).
3.1.8. 3,7-bis-(Substituted amino)[1,2,4]ditriazolo-
[4,3-a:4%,3%-c]quinazolines (9a,b)
A
solution of di-S-methylisothiosemicarbazide
derivative 8a,b (250 mg) in excess morpholine or diethyl-
amine (3 ml) was heated on a water bath for 6 h. The
mixture was then evaporated to dryness under reduced
pressure and the residue purified by preparative TLC
using benzene:ethyl acetate 9:1 as developing solvent.
9a: m.p. 260–262°C; yield 25%. Anal. (C22H16N8) C,
H, N. IR of 9a: w 3400 (NH), 1620 (CꢁN), 1590, 1495
3.2.4. Test groups
An amount of each of the test compounds (1, 2b, 3f,
4f, 6, 7b, 8b and 9b) equivalent to the amount of
pyrimethamine was given as a single dose in 0.1 ml
DMSO/mouse. Peritoneal fluid containing tachyzoites
was collected on the 5th day post inoculation.

492
A.A. El-Tombary et al. / Il Farmaco 54 (1999) 486–495
3.2.5. Preparation of specimens for SEM
The collected suspension of Toxoplasma was washed
twice with PBS (600 ml) then fixed in glutaraldehyde.
Specimens were then washed three times by flooding
with large volumes of sterile distilled water. The speci-
mens were processed according to Klainer and Betsch
method [27] and examined using a Jeol-JSM-25 SII
scanning microscope.
4. Results
The tachyzoites of T. gondii in the control group
appeared in SEM as elongate, often crescent-shaped
with a round pole at one end and a more or less
pointed pole at the other (Fig. 1). The site of the conoid
resembled a compressed spring (Fig. 2). The surface of
the tachyzoites had slender ridges radiating from the
anterior to the posterior end. The cytostome appeared
as indentation in the pellicle (Fig. 1). Each parasite was
enclosed in a membrane with blebs at the periphery
(Fig. 3). This membrane isolated the organism from the
host cell cytoplasm. Figs. 4 and 5 show the entry of the
Toxoplasma into the host cells by penetration through
the conoid.
Fig. 2. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by SEM. See Fig. 1 for
abbreviations.
The tachyzoites of T. gondii in the standard group
appeared smooth, homogeneous, regular with more or
less tapered ends and elongated. They lost their crescent
shape (Fig. 6). Some organisms appeared to have pores
or dimples on their surface (Fig. 7).
In the test groups, treatment with 2-hydrazinoquina-
zolin-4-one (1), the p-chlorophenylthiosemicarbazide
2b, S-methylisothiosemicarbazide 3f and the monotria-
zoloquinazolinone 4f caused some organisms to become
smooth, homogeneous and lose their ridges. Their
membrane became redundant with no blebs (Fig. 8)
Fig. 3. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by SEM. See Fig. 1 for
abbreviations.
Fig. 1. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by scanning electron
microscopy (SEM). Abbreviations: T, tachyzoites; C, conoid; Ct,
cytostome; b, blebs; H, host cell; A, anterior end; P, posterior end; r,
ridges.
Fig. 4. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by SEM. See Fig. 1 for
abbreviations.

A.A. El-Tombary et al. / Il Farmaco 54 (1999) 486–495
493
Fig. 8. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by SEM. See Fig. 1 for
abbreviations.
Fig. 5. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by SEM. See Fig. 1 for
abbreviations.
Fig. 9. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by SEM. See Fig. 1 for
abbreviations.
Fig. 6. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by SEM. See Fig. 1 for
abbreviations.
and appeared as if it has been peeled off leaving a
disorganized tachyzoite with a disorganized conoid
(Fig. 9).
In the infected group treated with 2,4-bis-(hydrazino-
quinazoline) (6), 2,4-bis-(p-chlorophenylthiosemicar-
bazide) (7b), 2,4-bis-(S-methylisothiosemicarbazide)
(8b) and the ditriazoloquinazoline (9b), the tachyzoites
of Toxoplasma gradually lost their crescent shape and
became triangular with a complete disorganized surface
and absence of the conoid (Figs. 10–12).
5. Discussion
SEM allows the study of the surface of T. gondii at
high magnification in three-dimensional perspectives.
The present work examined the details of intact and
treated organisms of T. gondii as well as the effect of
Fig. 7. Effect of the synthesized quinazoline derivatives on the surface
morphology of intact fixed T. gondii as viewed by SEM. See Fig. 1 for
abbreviations.

494
A.A. El-Tombary et al. / Il Farmaco 54 (1999) 486–495
test compounds on the presence or absence of conoid.
The latter is important for entry of the parasite in the
host cell membrane [28].
In the groups treated with pyrimethamine and the
monosubstituted quinazolin-4-ones 1, 2b, 3f and 4f, the
shape of organisms became distorted. In the groups
treated with the disubstituted quinazolines 6, 7b, 8b and
9b, the organisms became triangular in shape. The
changes in conoid whether disorganized or completely
absent, are an indication of the antitoxoplasmosis effi-
cacy of the test compounds on the organisms, a prop-
erty which is important for obstructing the entry of the
organisms into the host cells and, in turn, causing their
ultimate elimination. According to Hammouda et al.
[26],
the
tachyzoites
after
treatment
with
Fig. 10. Effect of the synthesized quinazoline derivatives on the
surface morphology of intact fixed T. gondii as viewed by SEM. See
Fig. 1 for abbreviations.
pyrimethamine or spyramicin, change their shape and
become rounded or triangular with loss of their conoid.
These changes in structure may be secondary to the
changes resulting from interference of the drugs in
DNA synthesis of the parasite or interference with the
folic acid cycle.
The results achieved in this study have also shown
that the replacement of the amino group at the 2- or
2,4-positions of quinazolines by hydrazino (1 and 6),
thiosemicarbazido (2b and 7b) or S-methylisothiosemi-
carbazido (3f and 8b) moieties retained the antiproto-
zoan activity. Conversion of these flexible chains into
the rigid triazoloquinazoline structure, as in compounds
4f and 9b, also retained the antiprotozoan activity.
These successful modifications have shed some light on
new structural requirements for antitoxoplasmosis ac-
tivity of quinazoline derivatives. Similar studies on
pyrimidine ring systems are progressing.
Fig. 11. Effect of the synthesized quinazoline derivatives on the
surface morphology of intact fixed T. gondii as viewed by SEM. See
Fig. 1 for abbreviations.
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