Pyrido[2,3-d]pyrimidines and pyrimido [5′,4′: 5,6]pyrido[2,3-d] pyrimidines as new antiviral agents: Synthesis and biological activity

Article abstract of DOI:10.1002/1521-4184(200208)335:6<289::AID-ARDP289>3.0.CO;2-Z

A series of 7-amino- and 7-oxo-5-aryl-6-cyanopyrido[2,3-d]pyrimidines, 4 and 11, respectively, and pyrimido[5′,4′:5,6]pyrido[2,3-d]pyrimidine derivatives 6 and 7 was synthesized and investigated as antiviral agents. Different synthetic strategies for the preparation of the target compounds were explored. A synthetic procedure for 4 and 11 starting with 6-amino-1,2,3,4-tetrahydro-2,4-dioxopyrimidine, proper aldehyde, and malononitrile or ethyl cyanoacetate, respectively, in a one-pot reaction proved to be the method of choice for preparation of compounds of such type. Construction of another pyrimidine ring on the pyridine nucleus of compound 4 was achieved either by reaction with phenyl iso(thio)cyanate or with formic acid to yield 6 and 7, respectively. The structure of the prepared compounds was confirmed through elemental analysis and spectral investigation. Most of the newly synthesized compounds were subjected to antiviral activity testing against herpes simplex virus (HSV) where some of them show good activities.

Full text of DOI:10.1002/1521-4184(200208)335:6<289::AID-ARDP289>3.0.CO;2-Z

Arch. Pharm. Pharm. Med. Chem. 2002, 6, 289–295
New Antiviral Agents 289
Magda N. Nasr,
Magdy M. Gineinah
Pyrido[2,3-d]pyrimidines and
Pyrimido[5؅,4؅:5,6]pyrido[2,3-d]pyrimidines as New
Antiviral Agents: Synthesis and Biological Activity
Department of Medicinal
Chemistry
Faculty of Pharmacy,
University of Mansoura,
Mansoura 35516, Egypt
A series of 7-amino- and 7-oxo-5-aryl-6-cyanopyrido[2,3-d]pyrimidines, 4 and 11,
respectively, and pyrimido[5Ј,4Ј:5,6]pyrido[2,3-d]pyrimidine derivatives 6 and 7 was
synthesized and investigated as antiviral agents. Different synthetic strategies for
the preparation of the target compounds were explored. A synthetic procedure for 4
and 11 starting with 6-amino-1,2,3,4-tetrahydro-2,4-dioxopyrimidine, proper alde-
hyde, and malononitrile or ethyl cyanoacetate, respectively, in a one-pot reaction
proved to be the method of choice for preparation of compounds of such type. Con-
struction of another pyrimidine ring on the pyridine nucleus of compound 4 was
achieved either by reaction with phenyl iso(thio)cyanate or with formic acid to yield 6
and 7, respectively. The structure of the prepared compounds was confirmed
through elemental analysis and spectral investigation. Most of the newly synthe-
sized compounds were subjected to antiviral activity testing against herpes simplex
virus (HSV) where some of them show good activities.
Keywords: 7-Amino-6-cyanopyrido[2,3-d]pyrimidine; Pyrimido[5Ј,4Ј:5,6]pyrido-
[2,3-d]pyrimidine; Antiviral activity; Cytotoxicity; Aphidicholin
Received: December 6, 2001 [FP654]
order to obtain novel pyrido[2,3-d]pyrimidine derivatives
with antiviral and cytotoxic properties.
Introduction
Antiviral and antimicrobial agents are among the most
commonly prescribed pharmaceuticals worldwide. Ef-
forts to improve the therapeutic significance of these
agents have resulted in identification of more potent
compounds. Pyrido[2,3-d]pyrimidine and some of its de-
rivatives display several potentially useful biological ac-
tivities [1, 2]. They show dihydrofolate reductase inhibi-
tion and antitumor [3, 4] as well as diuretic properties [5].
Moreover, some of them possess antimicrobial [6], anti-
viral, and cytotoxic activities [7, 8]. On the other hand,
4,6-disubstituted 2-amino-3-cyanopyridines have been
prepared and evaluated extensively as antimicrobial
agents [9, 10]. Furthermore, substituted 3-cyano-2-
oxopyrido[2,3-d]pyridine was found to be highly cytotoxic
to the infected T4 cell at a concentration which showed
marginal antiviral activity in the infected culture [7].In the
light of these observations, we have synthesized 7-ami-
no-6-cyanopyrido[2,3-d]pyrimidines in addition to other
derivatives with the 7-amino group being replaced with
an oxo functionality. Furthermore, some pyrimido-
[5Ј,4Ј:5,6]pyrido[2,3-d]pyrimidines were prepared in
Results and discussion
Chemistry
The synthetic pathways to the target compounds are
outlined in Schemes 1 and 2.Preparation of the requisite
7-amino-5-aryl-6-cyanopyrido[2,3-d]pyrimidines 4 for
the construction of pyrimido[5Ј,4Ј:5,6]pyrido[2,3-d]pyr-
imidine derivatives 6 and 7 (Table 1) began with 6-amino-
1,2,3,4-tetrahydro-2,4-dioxopyrimidine (1). Synthesis of
the key intermediate 4 from compound 1 was accom-
plished through two procedures. Firstly, an ethanolic
suspension of compound 1 was allowed to react with
equimolar amount of benzylidene derivatives of
malononitrile 2 [11, 12] as α,β-unsaturated compounds
(Method A).The 5-phenyl analogue of compounds 4 was
previously reported [13].In the second procedure, a mix-
ture of compound 1, the proper aromatic aldehyde 3,
malononitrile, and ethanol was heated under reflux in a
one-pot reaction technique (Method B). However, the
one-pot reaction technique is considered to be superior
over Method A in terms of product yields. Structural as-
signment of 4 through IR spectroscopy gave the charac-
teristic nitrile absorption at 2218–2226 cm–1 while bands
Correspondence: Magdy M. Gineinah, Department of
Medicinal Chemistry, Faculty of Pharmacy, University of
Mansoura, Mansoura 35516, Egypt. Phone: +20 50 2247496,
Fax: +20 50 2247496, e-mail: maggineinah@yahoo.com
© WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002
0365-6233/06/0289 $ 17.50+.50/0

290 Nasr et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 6, 289–295
Scheme 1
Scheme 2

Arch. Pharm. Pharm. Med. Chem. 2002, 6, 289–295
New Antiviral Agents 291
Table 1. Physicochemical data of the new compounds.
Comp.
R
X
Mp (°C)
Recryst.
solventa
Yield
(%)
Molecular
formula
4 a
4 b
4 c
4 d
4 e
4 f
4-Cl
>300
>300
M
M
M
AM
AM
M
M
E
88
90
85
79
93
84
89
79
75
82
77
86
90
74
78
69
71
73
68
81
78
82
83
66
75
85
89
93
82
79
91
C14H8ClN5O2
C14H8BrN5O2
C14H8BrN5O2
C14H9N5O3
2-Br
4-Br
>300
3-OH
>300
3-NO2
>300
C14H8N6O4
2,4-(OCH3)2
3,4-(OCH3)2
4-Cl
>300
C16H13N5O4
4 g
5 a
5 b
5 c
5 d
5 e
5 f
>300
C16H13N5O4
S
S
S
S
O
O
O
O
S
S
S
S
O
O
O
O
285–287
296–298
>300
C21H13ClN6O2S
C21H13BrN6O2S
C23H18N6O4S
C23H18N6O4S
C21H13ClN6O3
C21H13BrN6O3
C23H18N6O5
4-Br
E
2,4-(OCH3)2
3,4-(OCH3)2
4-Cl
E
293–295
>300
AE
AE
AE
E
4-Br
>300
5 g
5 h
6 a
6 b
6 c
6 d
6 e
6 f
2,4-(OCH3)2
3,4-(OCH3)2
4-Cl
>300
284–286
>300
E
C23H18N6O5
E
C21H13ClN6O2S
C21H13BrN6O2S
C23H18N6O4S
C23H18N6O4S
C21H13ClN6O3
C21H13BrN6O3
C23H18N6O5
4-Br
>300
M
M
E
2,4-(OCH3)2
3,4-(OCH3)2
4-Cl
>300
>300
>300
M
E
4-Br
>300
6 g
6 h
7 a
7 b
11 a
11 b
11 c
12 a
12 b
12 c
2,4-(OCH3)2
3,4-(OCH3)2
4-Cl
>300
E
>300
M
E
C23H18N6O5
>300
C15H8ClN5O3
C15H8N6O5
3-NO2
>300
M
M
M
M
E
4-Br
294–296
292–294
>300
C14H7BrN4O3
C14H8N4O4
3-OH
2,4-(OCH3)2
4-Br
C16H12N4O5
238–240
284–286
164–166
C14H4BrCl3N4
C14H5Cl3N4O
C16H9Cl3N4O2
3-OH
M
E
2,4-(OCH3)2
a
Recrystallisation solvent: AE: aqueous ethanol, AM: aqueous methanol, E: ethanol, M: methanol.

292 Nasr et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 6, 289–295
of the NH groups came at 3243–3485 cm–1. The 7-ami-
no-5-aryl-6-cyanopyrido[2,3-d]pyrimidine derivatives 4
were subjected to reaction with phenyl iso(thio)cyanate
neat at room temperature to furnish the corresponding
7-(3-phenyl(thio)ureido) derivatives 5. Cyclization of
these urea and thiourea derivatives was achieved
through the use of ammonia solution to give the tricyclic
structure 6.Efforts for the transformation of 4 into pyrimi-
dopyridopyrimidines 7–9 with formic acid, formamide, or
carbon disulfide, respectively, under reflux were unsuc-
cessful.The IR spectra of the products illustrated the typ-
ical absorption peak at 2217–2223 cm–1 indicative of the
unreacted cyano group.Similarly, trials for preparation of
8 by refluxing 4 with formamide even in the presence of
catalytic amount of formic acid or a small amount of
dimethylformamide did not succeed. However, similar
cyclization reactions were successful when the ring
bearing the α-amino-β-cyano groups is a π-excessive
heterocyclic ring [14, 15]. In contrast, heating of 4 with
formic acid in a sealed tube gave compounds 7 a, b.
Analogous conditions of 4 with formamide or carbon di-
sulfide/pyridine did not give 8 or 9, respectively.However,
formation of 7 might be attributed, in part, to the acidity of
the cyclizing agent that catalyzes the hydrolysis of the
cyano group as an initial step prior to cyclization.
An unequivocal structure proof of the previously report-
ed similar compounds was achieved by examination of
the crystal structure through X-ray analysis [12].
Biological screening
Virtually all of the newly synthesized compounds were
evaluated for their antiviral as well as their cytotoxic ac-
tivities.The goal of the current study is to define the opti-
mum substituents on the pyrido[2,3-d]pyrimidine skele-
ton to identify compounds with high potency.The antiviral
activity was assayed against herpes simplex virus (HSV)
type 1.The activity was determined by plaque reduction
assays in Vero cells using aphidicholine as a reference
standard [16]. Cytotoxicity measurements were based
on the inhibition of cultured mammalian cell growth [17].
The biological screening data for the active compounds
are presented in Table 2.
From the active compounds, 4 with 7-amino-5-aryl-6-
cyanopyrido[2,3-d]pyrimidine structure are among the
least active group of compounds. Significant improve-
ments resulted when modifications were made to the py-
ridine ring.Replacement of the 7-amino group in 4 with a
thioureido functionality, sharply increased the antiviral
activity. It is conceivable that the halo substituent on the
5-aryl group in compounds 5 creates better activity.This
may be attributed to the greater lipophilicity of these
compounds. Thus, compound 5 b with 4-bromo group
was found to be the most potent of the test compounds
while compound 5 a with 4-chloro substituent comes at
the second rank in terms of antiviral and cytotoxic activi-
ties.Furthermore, regarding compound 5 b, the selectiv-
ity index [ratio of the 50 % cytotoxic concentration (CC50)
to the 50 % virus inhibitory concentration (IC50)] is about
12.This indicates a fairly selective antiviral action.Within
the thioureido series, compounds 5 c and 5 d with iso-
meric dimethoxyphenyl moieties are of comparable ac-
tivities and less potent than the halo analogs.On the oth-
er hand, replacement of the thioureido group with a ure-
ido functionality afforded compounds 5 f and 5 g.The re-
sulting compounds retain considerable antiviral poten-
cies. However, these compounds are less potent than
the corresponding thioureido analogs. Cyclization of 5
with ammonium hydroxide gave 6 with one more pyrimi-
dine ring fused to the pyridine nucleus.The antiviral ac-
tivities of these tricyclic compounds are comparable to
The 6-cyano-7-oxopyrido[2,3-d]pyrimidine derivatives
11 were obtained using the procedure described in
Scheme 2.They were prepared by a synthetic approach
similar to that used for preparation of 4. The synthetic
pathways utilized were either reaction of 1 with the ben-
zylidene derivatives of ethyl cyanoacetate 10 [12]
(Method A) or a one-pot reaction procedure using equi-
molar ratios of 1, the proper aldehyde 3, and ethyl cy-
anoacetate (Method B).The same advantage of Method
B over Method A is noted here as well.The phenyl ana-
logue of 11 was synthesized applying a procedure more
or less similar to that described in Method A [13]. How-
ever, compounds 11 were subjected to chlorination us-
ing phosphoryl chloride to give the trichloro derivatives
of pyrido[2,3-d]pyrimidine structure 12.The formation of
compounds 4 and 11 in the current work is assumed to
proceed via a sequence similar to that discussed earlier
[11, 12].These reactions take place through 1,4-addition
of the nucleophilic carbon atom C-5 of the pyrimidine
ring of 1 towards the α,β-unsaturated system of 2 or 10
followed by cyclization and autooxidation (Scheme 3).
Scheme 3

Arch. Pharm. Pharm. Med. Chem. 2002, 6, 289–295
New Antiviral Agents 293
Table 2. Anti-HSV (type 1) activity and cytotoxic proper-
ties of tested compounds.
hope of getting more active compounds. Unexpectedly,
compound 12 a was less active than 11 a. This might
suggest that the halo groups are not tolerated at all the
positions of the fused system.
Comp.
Antiviral
activity
IC50
Cytotoxicity
CC50
Selectivity
index
In conclusion, 7-substituted 5-aryl-6-cyanopyrido[2,3-
d]pyrimidine derivatives exhibit antiviral and cytotoxic
activities. Both electron-withdrawing and donating
groups are tolerated on the 5-phenyl ring. Compounds
possessing moieties with hydrophobic properties show
obviously increased activities.Nevertheless, it should be
stated that most halo derivatives exhibit a tendency for
higher antiviral activity compared to the non-halogenat-
ed compounds.The introduction of a thioureido group at
the 8-position proved to furnish the maximum antiviral
activity. Annelation of another pyrimidine ring at the pyri-
dine side gave compounds with good potency. The iso-
steric replacement of a sulfur atom with oxygen resulted
in a slight decrease in activity of both cyclized and non-
cyclized structures.
(µg/mL)b
(µg/mL)a
4 c
4 e
4 g
5 a
40
50
50
8
200
170
200
40
5
3.4
4
5
5 b
5 c
5 d
5 e
4
47
50
50
80
70
80
50
80
80
50
150
190
50
11.75
4.17
3.57
4
3.89
4
12
14
20
18
20
10
16
30
10
40
50
2.1
5 f
5 g
6 b
6 d
6 f
7 b
11 a
12 a
APCc
5
5
Finally, in view of the promising activities displayed by
various compounds described in this study, further im-
provements on their potency could lead to novel and safe
antiviral agents with potential therapeutic utilities.
2.67
5
3.75
3.8
23.81
Acknowledgment
a
50 % inhibitory concentration, or concentration re-
quired to reduce virus plaque formation by 50 %.Virus
input was 40 plaque forming units (PFU);
50 % cytotoxic concentration, or concentration re-
quired to reduce cell growth by 50 %;
The authors thank Dr. Samy Kheira, Dept. of Micro-
biology, Faculty of Pharmacy, Mansoura University, for
carrying out the biological screening.
b
c
APC: Aphidicholin. Assays were performed in dupli-
cate.
Experimental part
Chemistry
Melting points were determined on a Fischer-Johns apparatus
and are uncorrected.1H-NMR spectra were recorded on aVari-
an EM-360 (90 MHz) instrument using TMS as internal stand-
ard (chemical shifts in ppm, δ units).IR spectra were carried out
on a PYE UNICAM SP 1000 spectrophotometer on KBr8 disk
(ν in cm–1)The results of elemental analyses (C,H,N) were with-
in 0.4 % of the theoretical values.Thin-layer chromatography
was performed on silica gel GLF plates, 250 µm.
activities of compounds 5, which are the most active
ones of the congeneric series studied.The thioxo deriva-
tives of 6 are more potent than the oxo analogs. On the
other hand, replacement of the 4-bromophenyl group in
6 b with 3,4-dimethoxyphenyl in 6 d resulted in attenua-
tion of the antiviral activity and cytotoxicity as well. Com-
pound 7 b, with analogous structure to 6, being tricyclic
with the same ring system, has been found to possess
potent antiviral activity. So we can say that the presence
of another pyrimidine ring fused to the pyridine nucleus
or the presence of a substituent with two nitrogen atoms
separated by one carbon (ureido and thioureido groups)
shifted the antiviral activity toward better potency. More-
over, compound 11 a, being substantially less potent
than compounds 5, 6, and 7 and possessing comparable
activity with 4 (both have the same ring system), may
confirm the previous conclusion. However, chlorination
of the three oxo groups of 11 was achieved to obtain
compounds with greater number of halogen atoms in the
7-Amino-5-aryl-6-cyano-1,2,3,4-tetrahydro-2,4-dioxopyrido-
[2,3-d]pyrimidines (4 a–g)
Method A. A mixture of 6-amino-1,2,3,4-tetrahydro-2,4-di-
oxopyrimidine (1) (0.25 g, 2 mmol), the corresponding aryli-
dene derivative of malononitrile 2 (2 mmol) and absolute etha-
nol (15 mL) was heated under reflux for 2 h. After cooling to
room temperature, the product was collected by filtration,
washed with ethanol, dried, and recrystallized to give the de-
sired product.
Method B. A mixture of 1 (0.25 g, 2 mmol), the corresponding
substituted benzaldehyde 3 (2 mmol), malononitrile (2 mmol),
and absolute ethanol (20 mL) was heated under reflux for 4 h.
The reaction mixture was allowed to cool, the product was col-
lected by filtration, washed with ethanol, dried, and recrystal-

294 Nasr et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 6, 289–295
with ammonium hydroxide.The product was collected by filtra-
tion, washed with water, dried, and recrystallized.IR, 7 a:3460,
3233 (NH), 1715, 1646 (C=O). 1H-NMR (DMSO-d6), 7 a:
7.31–7.85 (m, 4 H, Ar-H), 8.65 (s, 1 H, Ar-H), 10.92 (br s, 1 H,
NH; D2O exchangeable), 12.81 (br s, 2 H, 2 NH; D2O exchang-
eable). 7 b: 7.44–7.93 (m, 4 H, Ar-H), 8.66 (s, 1 H, Ar-H), 10.89
(br s, 1 H, NH; D2O exchangeable), 12.75 (br s, 2 H, 2 NH; D2O
exchangeable).
lized. IR, 4 a: 3415, 3320 (NH), 2218 (CN), 1705, 1640 (C=O).
4 b: 3487, 3352 (NH), 2226 (CN), 1716, 1637 (C=O). 4 d: 3243,
3410 (NH and OH), 2219 (CN), 1720, 1619 (C=O). 4 e: 3518,
3364 (NH), 2224 (CN), 1724, 1639 (C=O), 1345, 1518 (NO2).
4 g: 3424, 3243 (NH), 2221 (CN), 1725, 1642 (C=O). 1H-NMR
(DMSO-d6), 4 a: 5.94 (br s, 2 H, NH2; D2O exchangeable),
7.41–7.63 (m, 4 H, Ar-H), 12.31 (br s, 2 H, 2 NH;D2O exchange-
able), 4 b: 6.03 (br s, 2 H, NH2; D2O exchangeable), 7.47–7.72
(m, 4 H, Ar-H), 12.35 (br s, 2 H, 2 NH; D2O exchangeable). 4 d:
5.02 (s, 1 H, OH; D2O exchangeable), 5.89 (br s, 2 H, NH2; D2O
exchangeable), 6.77–7.61 (m, 4 H, Ar-H), 12.19 (br s, 2 H,
2 NH; D2O exchangeable). 4 e: 6.11 (br s, 2 H, NH2; D2O ex-
changeable), 7.48–7.82 (m, 4 H, Ar-H), 12.51 (br s, 2 H, 2 NH;
D2O exchangeable). 4 f: 4.11 (s, 6 H, 2 OCH3), 5.84 (br s, 2 H,
NH2;D2O exchangeable), 6.62–7.62 (m, 3 H, Ar-H), 12.63 (br s,
2 H, 2 NH; D2O exchangeable).
5-Aryl-6-cyano-1,2,3,4,7,8-hexahydro-2,4,7-trioxopyrido[2,3-
d]pyrimidines (11 a–c)
These compounds were prepared following the same proce-
dures (Methods A and B) as were used for preparation of com-
pounds 4 provided that malononitrile was replaced by ethyl cy-
anoacetate. IR, 11 a: 3465, 3296 (NH), 2223 (CN), 1732, 1645
1
(C=O). H-NMR (DMSO-d6), 11 a: 7.32–7.81 (m, 4 H, Ar-H),
12.42 (br s, 3 H, 3 NH; D2O exchangeable). 11 c: 4.17 (s, 6 H, 2
OCH3), 6.75–7.71 (m, 3 H, Ar-H), 12.72 (s, 3 H, 3 NH; D2O ex-
changeable).
5-Aryl-6-cyano-7-(3-phenyl(thio)ureido)-1,2,3,4-tetrahydro-
2,4-dioxopyrido[2,3-d]pyrimidines (5 a–h)
A mixture of 4 (2 mmol), phenyl iso(thio)cyanate (2 mmol), and
absolute ethanol (20 mL) was heated under reflux for 30–40 h.
The reaction mixture was cooled to room temperature, the
product was collected by filtration, dried, and recrystallized
from the proper solvent. IR, 5 a: 3428, 3291 (NH), 2223 (CN),
1707, 1625 (C=O). 5 d: 3470, 3284 (NH), 2216 (CN), 1715,
1642 (C=O). 5 f: 3392, 3218 (NH), 2221 (CN), 1704, 1631
(C=O).1H-NMR (DMSO-d6), 5 a:7.21–7.68 (m, 9 H, Ar-H), 7.81
(br s, 1 H, NH; D2O exchangeable), 8.32 (br s, 1 H, NH; D2O ex-
changeable), 12.34 (br s, 2 H, 2 NH; D2O exchangeable). 5 c:
4.21 (br s, 6 H, 2 OCH3), 6.64–7.57 (m, 8 H, Ar-H), 7.73 (br s,
1 H, NH; D2O exchangeable), 8.41 (br s, 1 H, NH; D2O ex-
changeable, 12.41 (br s, 2 H, 2 NH; D2O exchangeable). 5 f:
7.14–7.58 (m, 9 H, Ar-H), 8.01 (br s, 1 H, NH; D2O exchange-
able), 8.52 (br s, 1 H, NH; D2O exchangeable), 12.81 (br s, 2 H,
2 NH;D2O exchangeable).5 h:4.33 (s, 6 H, 2 OCH3), 6.79–7.61
(m, 8 H, Ar-H), 7.97 (br s, 1 H, NH;D2O exchangeable), 8.49 (br
s, 1 H, NH; D2O exchangeable), 12.79 (br s, 2 H, NH; D2O ex-
changeable).
5-Aryl-2,4,7-trichloro-6-cyanopyrido[2,3-d]pyrimidines
(12 a–c)
A mixture of 11 (5 mmol), phosphoryl chloride (40 mL), and
N,N-dimethylaniline (0.5 mL) was heated under reflux for 8 h.
The reaction mixture was cooled to room temperature, poured
onto ice, the product was collected by filtration, washed with
water, dried, and extracted with ether.The ethereal extract was
washed with water, dried over sodium sulfate, the solvent was
evaporated, and the product was recrystallized. 1H-NMR
(DMSO-d6), 12 b: 5.12 (s, 1 H, OH; D2O exchangeable),
6.82–7.64 (m, 4 H, Ar-H).12 c:4.21 (s, 6 H, 2 OCH3), 6.68–7.63
(m, 3 H, Ar-H).
Biological screening
1. Antiviral testing
Confluent Vero cells grown in 96-well microtiter trays were in-
fected with 40 plaque forming units (PFU) of Herpes simplex
virus-type 1 (HSV-1) in 100 µL medium containing 10 % (v/v)
calf serum.A serial dilution of the test compounds (in duplicate)
dissolved in the medium was added (100 µL in each well). After
66 h of incubation at 37 °C in 5 % CO2 atmosphere, the trays
were inverted onto a pad of paper towels and remaining cells
were rinsed with the medium and fixed with 3.7 % (v/v) form-
aldehyde in saline solution for 20 min. The cells were then
rinsed with water, stained with 0.5 % (w/v) crystal violet solution
in 20 % ethanol for 30 min and rinsed with water.The trays were
examined visually. The antiviral activity is expressed as IC50
which represents the compound concentration required to re-
duce virus plaque formation by 50 %.
5-Aryl-4-imino-3-phenyl-1,2,3,4,6,7,8,9-octahydro-6,8-dioxo-
2-(thio)oxopyrimido-[5Ј,4Ј:5,6]pyrido[2,3-d]pyrimidines (6 a–h)
A mixture of compound 4 (1 mmol), ethanol (10 mL), and aque-
ous ammonia solution (25 %, 10 mL) was stirred at room tem-
perature for 24 h.The reaction mixture was refluxed for 1 h, the
product was collected by filtration, washed with water, dried,
1
and recrystallized from the proper solvent. H-NMR (DMSO-
d6), 6 a: 7.22–7.74 (m, 9 H, Ar-H), 8.94 (br s, 1 H, NH; D2O ex-
changeable), 11.42 (br s, 1 H, NH; D2O exchangeable), 12.69
(br s, 2 H, 2 NH;D2O exchangeable).6 b:7.25–7.82 (m, 9 H, Ar-
H), 8.99 (br s, 1 H, NH; D2O exchangeable), 11.44 (br s, 1 H,
NH;D2O exchangeable), 12.71 (br s, 2 H, 2 NH;D2O exchange-
able). 6 d: 4.27 (s, 6 H, 2 OCH3), 6.68–7.74 (m, 8 H, Ar-H), 8.89
(br s, 1 H, NH; D2O exchangeable), 11.43 (br s, 1 H, NH; D2O
exchangeable, 12.68 (br s, 2 H, 2 NH; D2O exchangeable). 6 f:
7.31–7.82 (m, 9 H, Ar-H), 9.02 (br s, 1 H, NH; D2O exchange-
able), 11.95 (br s, 1 H, NH; D2O exchangeable, 12.84 (s, 2 H,
2 NH;D2O exchangeable).6 g:4.35 (s, 6 H, 2 OCH3), 6.61–7.76
(m, 8 H, Ar-H), 8.91 (br s, 1 H, NH; D2O exchangeable), 12.02
(br s, 1 H, NH; D2O exchangeable), 12.74 (br s, 2 H, 2 NH; D2O
exchangeable).
2. Cytotoxicity assay
Cytotoxicity measurements were based on the inhibition of 3T3
cell growth. 3T3 fibroblasts were seeded at a rate of 2 × 103
cells/well microtiter plates. Different concentrations of the test
compounds were then added (in duplicate), and after 3–4 days
of incubation at 37 °C in 5 % CO2 atmosphere, the cell number
was determined using a hemocytometer. Cytotoxicity is ex-
pressed as CC50, which represents the compound concentra-
tion required to reduce cell growth by 50%.
5-Aryl-1,2,3,4,6,7-hexahydro-2,4,6-trioxopyrimido[5Ј,4Ј:5,6]-
pyrido[2,3-d]pyrimidines (7 a, b)
References
A mixture of 4 (0.5 mmol) and formic acid (10 mL) was heated in
a sealed tube at 140–150 °C for 5 h. The reaction mixture was
then allowed to cool, poured onto crushed ice, and neutralized
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