Synthesis and antitumor activity of new pyrido[2,3-d]pyrimidine derivatives

Article abstract of DOI:10.1007/s00044-012-0396-0

New series of pyrido[2,3-d]pyrimidines such as; 5-(4-aryl-5-sulfanyl-4H-[1, 2,4]triazol-3-yl) 1H,3H,8H-pyrido[2,3-d]pyrimidine-2,4,7-triones 6, 7; S-[3-(2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidin-5-yl) -4-(4-substituted phenyl)-4H-[1,2,4]triazol-5-yl]-2-(4-phenylpiperazin-1-yl) ethanethioates 10, 11; 2,4,7-trioxo-N′-[(4-substituted piperazin-1-yl)acetyl]-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5- carbohydrazides 13-16 and N′-arylidene-2,4,7-trioxo-1,2,3,4,7,8- hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazides 17-19 was synthesized through the reaction of the key intermediate 2,4,7-trioxo-1,2,3,4,7,8- hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazide 3 with different reagents. The structures of the newly synthesized compounds were elucidated through microanalysis, IR, ¹H NMR, ¹³C NMR, and mass spectroscopy. These compounds have been subjected to in vitro antitumor evaluation by bleomycin-dependant DNA damage assay. The most active antitumor compound 6 was selected for further in vivo evaluation of antineoplastic activity against Ehrlich ascites carcinoma in mice. It was observed that our target compound has a potent antitumor activity.

Full text of DOI:10.1007/s00044-012-0396-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0396-0
ORIGINAL RESEARCH
Synthesis and antitumor activity of new pyrido[2,3-d]pyrimidine
derivatives
•
Magdy M. Gineinah Magda N. A. Nasr
•
•
Sahar M. I. Badr Walaa M. El-Husseiny
Received: 10 February 2012 / Accepted: 5 December 2012
Ó Springer Science+Business Media New York 2012
Abstract New series of pyrido[2,3-d]pyrimidines such as;
5-(4-aryl-5-sulfanyl-4H-[1,2,4]triazol-3-yl) 1H,3H,8H-pyr-
ido[2,3-d]pyrimidine-2,4,7-triones 6, 7; S-[3-(2,4,7-trioxo-1,2,
3,4,7,8-hexahydropyrido[2,3-d]pyrimidin-5-yl)-4-(4-
substituted phenyl)-4H-[1,2,4]triazol-5-yl]-2-(4-phenylpi-
perazin-1-yl)ethanethioates 10, 11; 2,4,7-trioxo-N0-[(4-
substituted piperazin-1-yl)acetyl]-1,2,3,4,7,8-hexahydro-
pyrido[2,3-d]pyrimidine-5-carbohydrazides 13–16 and
N0-arylidene-2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-
d]pyrimidine-5-carbohydrazides 17–19 was synthesized
through the reaction of the key intermediate 2,4,
7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-
carbohydrazide 3 with different reagents. The structures of
the newly synthesized compounds were elucidated through
Introduction
The discovery of new compounds with antitumor activity
has become one of the most important goals in medicinal
chemistry. Several literatures enlightening the anticancer
nature of pyrido[2,3-d]pyrimidines such as piritrexim,
piritrexim analogs, isopiritrexim, and compounds V–VII
(El-Nassan, 2011; Kurumurthy et al., 2011; Shi et al.,
2011; Tu et al., 2006; Mohamed et al., 2007; Cordeu et al.,
2007; Zink et al., 2004; Gangjee et al., 2001; Agrawal
et al., 2002; Joska and Anderson, 2006; Chan et al., 2005;
Sanmartin et al., 2005) (Fig. 1). In addition, as revealed
from the literatures, potential antitumor activity was
reported by many hydrazides (De et al., 2010; Grande
et al., 2007), piperazines (Guo et al., 2004), and [1,2,4]-
triazoles (Al-Soud et al., 2004; Formagio et al., 2008).
From the aforementioned argument, we designed and
synthesized the target compounds having pyrido[2,3-d]
pyrimidine pharmacophore (Fig. 2); this scaffold hybrid-
ized with one or more heterocyclic moieties (piperazines or
triazoles) and some functionalities that possess antitumor
potency; this kind of integration might result in compounds
with high efficacy as antitumor agents.
1
microanalysis, IR, H NMR, 13C NMR, and mass spec-
troscopy. These compounds have been subjected to in vitro
antitumor evaluation by bleomycin-dependant DNA dam-
age assay. The most active antitumor compound 6 was
selected for further in vivo evaluation of antineoplastic
activity against Ehrlich ascites carcinoma in mice. It was
observed that our target compound has a potent antitumor
activity.
Keywords Pyrido[2,3-d]pyrimidines ꢀ Piperazines ꢀ
Triazoles ꢀ Synthesis ꢀ Antitumor activity
Results and discussion
Chemistry
The target compounds were prepared as outlined in
Schemes 1, 2, 3. The key intermediate 2,4,7-trioxo-1,2,3,
4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazide
3 was prepared by refluxing methyl 2,4,7-trioxo-1,2,3,4,
M. M. Gineinah ꢀ M. N. A. Nasr ꢀ S. M. I. Badr (&) ꢀ
W. M. El-Husseiny
Department of Pharmaceutical Organic Chemistry, Faculty
of Pharmacy, Mansoura University, Mansoura 35516, Egypt
e-mail: saharmbadr@yahoo.com
7,8-hexahydropyrido[2,3-d] pyrimidine-5-carboxylate
(Anderson et al., 1977) with hydrazine hydrate in ethanol.
2
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Med Chem Res
Fig. 1 Structures of active antitumor compounds containing pyrido[2,3-d]pyrimidine moiety
Fig. 2 Structure of the
proposed pyrido[2,3-
potassium hydroxide solution underwent cyclization to the
corresponding title compounds 6, 7. While in the second
route, the acid hydrazide 3 was heated under reflux with
aryl isothiocyanates in alcoholic potassium hydroxide fol-
lowed by acidification with hydrochloric acid (Scheme 1).
Furthermore, 2-chloro-S-[3-(2,4,7-trioxo-1,2,3,4,7,8-hexa-
hydropyrido[2,3-d]pyrimidin-5-yl)-4-(4-substituted phenyl)-
4H-[1,2,4]triazol-4-yl]ethanethioates 8, 9 were prepared by
refluxing compounds 6 or 7 with chloroacetyl chloride in
toluene in the presence of triethylamine (TEA) as a base.
Their IR spectroscopy showed the peak at 2,600 cm-1
characteristic for (SH) group disappeared, while peaks at
about 1,658–1,662 and 3,000–3,016 cm-1 attributed to
(C=O) and (CH2), respectively, were observed. Then,
d]pyrimidine pharmacophore in
the present study
Then 5-(4-aryl-5-sulfanyl-4H-[1,2,4]triazol-3-yl) 1H,3H,
8H-pyrido[2,3-d]pyrimidine-2,4,7-triones 6, 7 were pre-
pared through two different routes. In the first one, the acid
hydrazide 3 was reacted with aryl isothiocyanates in
absolute ethanol to yield N-aryl-(2,4,7-trioxo-1,2,3,4,7,8-
hexahydropyrido[2,3-d]pyrimidine)-5-carbohydrazidocarbo
thioamides 4, 5 which on heating under reflux with 5 %
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Med Chem Res
Scheme 1 Synthesis of 5-(4-aryl-5-sulfanyl-4H-[1,2,4]triazol-3-
yl)1H,3H,8H-pyrido[2,3-d]pyrimidine-2,4,7-triones 6,7. Reagent and
condition: a dimethylacetylenedicarboxylate, b hydrazine hydrate,
EtOH, reflux, c appropriate isothiocyanate, EtOH, reflux, d KOH,
reflux, e KOH, appropriate isothiocyanate, reflux
compounds
S-[3-(2,4,7-trioxo-1,2,3,4,7,8-hexahydropyri-
All compounds were spectroscopically characterized
and the spectral data agree with the proposed structures.
do[2,3-d]pyrimidin-5-yl)-4-(4-substituted phenyl)-4H-[1,2,
4]triazol-5-yl]-2-(4-phenylpiperazin-1-yl)ethanethioates 10,
11 were prepared by stirring compounds 8, 9 with 1-phe-
nylpiperazine in dimethylformamide using potassium car-
bonate as a base (Scheme 2). Moreover, the reaction of
pyrido[2,3-d]pyrimidine carbohydrazide 3 with chloroace-
tyl chloride in ethanol afforded N0-chloroacetyl-2,4,7-tri
oxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbo
hydrazide 12, which on stirring with the appropriate
1-substituted piperazine in dimethylformamide in the
presence of potassium carbonate as a base gave 2,4,7-tri-
Biologic screening
The therapeutically active antitumor agents should pos-
sess high and potential efficacy with minimal side
effects. As a result, interaction of antitumor agents with
DNA should produce as little DNA damage as possible.
The newly synthesized compounds had been subjected to
bleomycin-dependant DNA damage assay (Ayyad et al.,
2011a, b) to screen their antitumor activity and the
degree of DNA damage caused by these compounds.
Table 1 shows the effect of the newly synthesized
compounds on the degree of DNA damage described in
terms of sample absorbance (A). As sample absorbance
(A) increases, DNA damage increases and the sample
efficiency as antitumor agent decreases. Compounds 2–4,
6, 7, 9, 11–14, and 18 showed the lowest absorbance,
and hence the highest antitumor activity, while com-
pounds 5 and 19 showed moderate activity. We can
oxo-N0-[(4-substituted
piperazin-1-yl)acetyl]-1,2,3,4,
7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazides
13–16. Finally, the reaction of compound 3 with the
appropriate aromatic aldehyde in ethanol yielded N0-ary-
lidene-2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]
pyrimidine-5-carbohydrazides 17–19. Their IR spec-
troscopy showed the peak characteristic for (NH2) of
the acid hydrazide 3 disappeared while peak at about
1,600 cm-1 characteristic for (C=N) was observed
(Scheme 3).
conclude that compound
6
exhibited the highest
123

Med Chem Res
Scheme 2 Synthesis of S-[3-
(2,4,7-trioxo-1,2,3,4,7,8-
hexahydropyrido[2,3-
d]pyrimidin-5-yl)-4-(aryl)-4H-
[1,2,4]triazol-5-yl]-2-(4-
phenylpiperazin-1-
yl)ethanethioates 10, 11.
Reagent and condition:
a chloroacetyl chloride, toluene,
TEA, reflux, b 1-
phenylpiperazine, K2CO3,
DMF, rt
antitumor activity, and as a result it was subjected for
antineoplastic activity against Ehrlich ascites carcinoma
(EAC) in mice (Ayyad et al., 2011a, b; Qureshi et al.,
2001; Bala et al., 2010). The prolongation of life span of
EAC-bearing hosts, the reduction in viable tumor cell
count, and the recovery of normal biochemical and
hematologic profiles are three important measures that
have been used in this in vivo testing for evaluation of
the antineoplastic activity. Effect on survival time was
the first measure used to detect the antineoplastic
activity of the tested compound where an increase in
survival time for this group over the control group was
detected. The mean survival time (MST) for each group
was calculated by dividing the total survival times for all
the mice in that group by the number of mice in the
same group and then the percent increase in life span for
each group over the control group was calculated as
follows:
% increase in life span over control
MST of treated group
¼
ꢁ 100 ꢂ 100
MST of control group
By comparing the percentage increase in life span over
the control group in the treated group, it was found that
compound 6 had shown increase in the life span higher
than that of 5-fluorouracil (5-FU, Table 2). Effect on EAC
viable cell count, after 5 days of treatment: 100 ll samples
were taken from Ehrlich ascites cells from three mice in
each of the treated groups and from the control group.
20-fold dilution was made for the taken cells in saline. The
cells in the final dilution were stained with Giemsa stain
and the number of the viable cells was counted under the
microscope. As shown in Table 3, compound 6 showed
significant reduction in viable tumor cell count. Effect on
biochemical and hematologic parameters: on the day 14,
the biochemical and hematologic parameters, as regard to
hemoglobin level (Hb/g%), hematocrits (HCT/g%), and
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Med Chem Res
Scheme 3 Synthesis of 2,4,7-
trioxo-N0-[(4-substituted
piperazin-1-yl)acetyl]-
1,2,3,4,7,8-
hexahydropyrido[2,3-
d]pyrimidine-5-carbohydrazides
13–16 and N0-arylidene-2,4,7-
trioxo-1,2,3,4,7,8-
hexahydropyrido[2,3-
d]pyrimidine-5-carbohydrazides
17–19. Reagent and condition:
a chloroacetyl chloride, EtOH,
reflux, b appropriate substituted
piperazine, K2CO3, DMF, rt,
c appropriate aromatic
aldehyde, EtOH, reflux
the group treated with the compound 6 has been nearly
recovered completely to be within the normal values. The
values are better than those obtained from the group treated
with 5-FU.
Table 1 Absorbance degree (A) of tested compounds as estimated by
bleomycin-dependant DNA damage assay
Compound numbers
Sample absorbance (A)
Ascorbic acid
0.068
0.055
0.064
0.054
0.097
0.052
0.057
0.074
0.062
0.060
0.071
0.053
0.057
0.079
Studying structure–activity relationship of the tested
compounds revealed that regardless of changes in the
structure of the starting compound 2, the biologic activity
was retained due to the presence of the main moiety pyr-
idopyrimidine. However, the ester transformation into the
acid hydrazide, compound 3, reduced the activity, intro-
duction of N-phenylthiosemicarbazide moiety, compound
4, at position five of the pyridopyrimidine ring system led
to better activity in comparing to the carbohydrazide 3
itself, while introducing the electron-withdrawing fluoro
group at the four position of the phenyl ring, compound 5,
decreased the activity. Compounds 6 and 7 are more active
in general than their non-cyclized counterparts, compounds
4 and 5 indicating that cyclization step of thiosemicarba-
zide containing compounds into [1,2,4]-triazoles affords
higher and more potent antitumor activity. Moreover,
compound 9 showed lower activity than compounds 6 and
7; thus, we can conclude that free sulfanyl group (–SH)
manifests more potent activity. On the other hand, com-
pound 11 is highly active as antitumor agent comparing to
2
3
4
5
6
7
9
11
12
13
14
18
19
leukocytes counts (WBCs/103 mm3), were compared in the
group treated with the standard drug 5-FU and the group
treated with the newly synthesized drug 6 with the values
obtained from normal and control groups. As shown in
Table 4, the biochemical and hematologic parameters in
123

Med Chem Res
antitumor activity. Our target compound 6 showed potential
antitumor activity.
Table 2 The percentage increase in life span for each treated group
over the control group
Group no.
Treatment (IP)
Percent increase in life span
1
2
3
4
Normal (untreated)
Control (Ehrlich only)
Compound 6
–
Experimental
9.1
18.6
16.7
Chemistry
5-FU (20 mg/kg)
Melting points were determined on Fisher-Johns melting
point apparatus and are uncorrected. Microanalyses were
performed at the microanalytical center, Cairo University,
and the values are found within 0.4 % of theoretic val-
ues. IR spectra were recorded on Mattson 5000 FT-IR
spectrometer (t in cm-1) using KBr disk at the Faculty of
Science, Mansoura University. 1H NMR and 13C NMR
spectra were recorded on FT-NMR spectrometer (200
MHz) using TMS as internal standard (chemical shifts in
ppm, d units) at the microanalytical center, Cairo Univer-
sity. MS analyses were performed on JEOL JMS-600H
spectrometer at Cairo University. TLC analysis was carried
out on silica gel-protected aluminum sheets (Type 60 F
254, Merck) and the spots were detected under UV-Lamp
at k 254 nm. Methyl 2,4,7-trioxo-1,2,3,4,7,8-hexahydro-
pyrido[2,3-d]pyrimidine-5-carboxylate 2 was prepared as
reported method (Anderson et al., 1977).
Table 3 The number of viable cells for each group
Group no.
Group type
Count cells
2
3
4
Control (Ehrlich only)
Compound 6
5-FU
220
153.6
123.0
Table 4 The biochemical and hematologic parameters for each
group
Group type
Hb/g%
HCT/g%
WBCs/103 mm3
Normal
13.9
8.7
53.6
35.5
51.6
42.3
8.4
38.6
11.2
13.8
Control
Compound 6
5-FU
11.7
10.0
Preparation of 2,4,7-trioxo-1,2,3,4,7,8-
hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazide (3)
Hb/g%, hemoglobin level; HCT/g%, hematocrits; WBCs/103 mm3,
leukocytes counts
To a suspension of the ester 2 (2.37 g, 10 mmol) in absolute
ethanol (40 ml), hydrazine hydrate 98 % (1 g, 20 mmol)
was added. The reaction mixture was heated under reflux for
24 h and then cooled to room temperature. The product was
collected by filtration, washed with ethanol, dried, and re-
crystallized from water.
compound 9; it means that piperazine moiety potentiates
antitumor activity. Likewise, compounds 13 and 14 showed
high activity due to the presence of piperazine moiety.
However, the introduction of methoxy group at position
four of the 1-phenylpiperazine reduces the absorbance;
thus, compound 14 is more potent than compound 13. This
indicates that better activity could be attained by the
presence of a piperazine moiety substituted at position four
with phenyl group substituted with electron-donating
group, but not with electron-withdrawing or even unsub-
stituted group. It was shown that methoxy group at position
four of the Schiff base phenyl ring compound 18 displays
higher activity than nitro group at the same position as in
compound 19. Again, the presence of an electron-donating
group on an aromatic nucleus displays, in part, higher
antitumor activity.
1
Yield 84 %, mp [300 °C. H NMR (DMSO-d6): d 3.7
(s, 2H, NH2; D2O exchangeable), 6.3 (s, 1H, C6–H), 8.6 (s,
1H, CONH; D2O exchangeable), 10.2 (brs, 1H, NH; D2O
exchangeable), 11.0 (brs, 1H, NH; D2O exchangeable),
11.3 (brs, 1H, NH; D2O exchangeable). Anal. calcd. for
C8H7N5O4: C 40.51; H 2.97; N 29.53. Found: C 40.48; H
2.95; N 29.58.
Preparation of N-(aryl) (2,4,7-trioxo-1,2,3,4,7,8-
hexahydropyrido[2,3-d]pyrimidin-5-
yl)carbohydrazidocarbothioamides 4, 5
To a suspension of 3 (2.37 g, 10 mmol) in absolute ethanol
(50 ml), substituted isothiocyanate (10 mmol) was added.
The reaction mixture was heated under reflux for 24 h and
then cooled to room temperature. The product was col-
lected by filtration, washed with ethanol, dried, and
recrystallized from acetic acid.
Conclusion
A new series of pyrido[2,3-d]pyrimidines 6, 7; 10, 11; 13–16,
and 17–19 has been synthesized and evaluated for their
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Med Chem Res
N-(phenyl)
(2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido
(method 1), 60 % (method 2), mp [300 °C. IR (KBr, t,
cm-1): 1650 (C=O), 3210 (NH), 2600 (SH). 1H NMR
(DMSO-d6): d 6.3 (s, 1H, C6–H), 7.1 (t, J = 8.3 Hz, 1H,
Ar–H), 7.5 (t, J = 8.3 Hz, 2H, Ar–H), 7.8 (d, J = 8.3 Hz,
2H, Ar–H), 10.2 (brs, 1H, NH; D2O exchangeable), 10.6
(brs, 1H, NH; D2O exchangeable), 11.3 (brs, 1H, NH; D2O
exchangeable), 13.2 (s, 1H, SH). 13C NMR (DMSO-d6): d
98.8, 122.1, 129.1, 130.2, 132.0, 144.5, 151.1, 156.2,
162.1, 163.5, 170.4. Mass m/z (%): 355 (M??1, 5.28), 236
(100), 268 (39.09), 97 (69.19), 69 (70.33). Anal. calcd. for
C15H10N6O3S: C 50.84; H 2.84; N 23.72. Found: C 50.81;
H 2.80; N 23.76.
[2,3-d]pyrimidin-5-yl)carbohydrazidocarbothioamide
(4) Yield 80 %, mp [ 300 °C. IR (KBr, t, cm-1): 1548
(C=S), 1660, 1700 (C=O), 3160, 3200 (NH). 1H NMR
(DMSO-d6): d 6.4 (s, 1H, C6–H), 6.8 (t, J = 8.0 Hz, 1H,
Ar–H), 7.3 (t, J = 8.0 Hz, 2H, Ar–H), 7.6 (d, J = 8.0 Hz,
2H, Ar–H), 8.4 (brs, 1H, NH; D2O exchangeable), 9.5 (brs,
1H, NH; D2O exchangeable), 10.4 (brs, 1H, NH; D2O
exchangeable), 10.8 (brs, 1H, NH; D2O exchangeable),
11.4 (brs, 1H, NH; D2O exchangeable), 12.0 (brs, 1H, NH;
D2O exchangeable). 13C NMR (DMSO-d6): d 99.2, 112.7,
126.5, 128.7, 129.4, 139.0, 144.2, 150.6, 161.3, 162.6,
166.8, 182.4. Mass m/z (%): 372 (M?, 1.80), 77 (100), 206
(46.72), 118 (67.3), 91 (57.01). Anal. calcd. for
C15H12N6O4S: C 48.38; H 3.25; N 22.57. Found: C 48.30;
H 3.29; N 22.51.
5-[4-(4-Fluorophenyl)-5-sulfanyl-4H-[1,2,4]triazol-3-
yl]1H,3H,8H-pyrido[2,3-d]pyrimidine-2,4,7-trione
(7) Yield 70 % (method 1), 65 % (method 2),
mp [300 °C. IR (KBr, t, cm-1): 1660 (C=O), 3200 (NH),
N-(4-flourophenyl) (2,4,7-trioxo-1,2,3,4,7,8-hexahydropyr-
ido[2,3-d]pyrimidin-5-yl)carbohydrazidocarbothioamide
(5) Yield 75 %, mp [300 °C. IR (KBr, t, cm-1): 1542
1
2605 (SH). H NMR (DMSO-d6): d 6.7 (s, 1H, C6–H), 7.3
(d, J = 7.8 Hz, 2H, Ar–H), 7.6 (d, J = 7.8 Hz, 2H, Ar–H),
10.5 (brs, 1H, NH; D2O exchangeable), 10.9 (brs, 1H, NH;
D2O exchangeable), 11.4 (brs, 1H, NH; D2O exchange-
able), 13.1 (s, 1H, SH). 13C NMR (DMSO-d6): d 99.4,
120.5, 122.2, 126.8, 131.2, 144.1, 151.4, 157.1, 162.3,
163.6, 164.9, 170.6. Mass m/z (%): 372 (M?, 11.02), 227
(100), 200 (34.83), 168 (61.73), 115 (57.32). Anal. calcd.
for C15H9FN6O3S: C 48.38; H 2.44; N 22.57. Found: C
48.45; H 2.39; N 22.54.
1
(C=S), 1653, 1720 (C=O), 3170, 3202 (NH)). H NMR
(DMSO-d6): d 6.6 (s, 1H, C6–H), 7.2 (d, J = 7.5 Hz, 2H,
Ar–H), 7.7 (d, J = 7.5 Hz, 2H, Ar–H), 8.5 (brs, 1H, NH;
D2O exchangeable), 9.7 (brs, 1H, NH; D2O exchangeable),
10.5 (brs, 1H, NH; D2O exchangeable), 10.7 (brs, 1H, NH;
D2O exchangeable), 11.2 (brs, 1H, NH; D2O exchange-
able), 12.1 (brs, 1H, NH; D2O exchangeable). Mass m/
z (%): 390 (M?, 7.50), 127 (100), 368 (91.56), 200 (30.00),
168 (54.34). Anal. calcd. for C15H11FN6O4S: C 46.15; H
2.84; N 21.53. Found: C 46.22; H 2.83; N 21.57.
Preparation of 2-chloro-S-[3-(2,4,7-trioxo-1,2,3,4,7,8-
hexahydropyrido[2,3-d]pyrimidin-5-yl)-4-(aryl)-4H-
[1,2,4]triazol-4-yl]ethanethioates 8, 9
Preparation of 5-(4-aryl-5-sulfanyl-4H-[1,2,4]triazol-3-
yl)1H,3H,8H-pyrido[2,3-d]pyrimidine-2,4,7-triones 6, 7
Chloroacetyl chloride (1.13 g, 10 mmol) was added drop-
wise to a suspension of compounds 6 or 7 (10 mmol) in
toluene (50 ml). TEA (0.5 ml) was added to the reaction
mixture and heated under reflux for 6 h and then filtered
while hot. The precipitated solid was dried and recrystal-
lized from acetic acid.
Method 1 A solution of 4 or 5 (10 mmol) in an aqueous
potassium hydroxide solution (5 %, 50 ml) was heated
under reflux for 2 h and then concentrated. The precipitated
solid was dissolved in water and neutralized with concen-
trated solution of hydrochloric acid. The separated solid
was collected by filtration, dried, and recrystallized from
water.
2-Chloro-S-[3-(2,4,7-trioxo-1,2,3,4,7,8-hexahydropyri-
do[2,3-d]pyrimidin-5-yl)-4-(phenyl)-4H-[1,2,4]triazol-4-
yl]ethanethioate (8) Yield 65 %, mp [300 °C. IR (KBr,
t, cm-1): 1658 (C=O), 3016 (CH2), 3181 (NH). Mass m/
z (%): 430 (M?, 40.05), 86 (100), 348 (41.8), 277 (41.8),
58 (65.8). Anal. calcd. for C17H11ClN6O4S: C 47.39; H
2.57; N 19.51. Found: C 47.44; H 2.60; N 19.56.
Method 2 To a mixture of 3 (2.37 g, 10 mmol) and
potassium hydroxide (0.56 g, 10 mmol) in 95 % ethanol
(50 ml), substituted isothiocyanate (10 mmol) was added.
The reaction mixture was heated under reflux for 12 h and
then concentrated. The precipitated solid was dissolved in
water and neutralized with concentrated solution of
hydrochloric acid. The separated solid was collected by
filtration, dried, and recrystallized from water.
2-Chloro-S-[3-(2,4,7-trioxo-1,2,3,4,7,8-hexahydropyri-
do[2,3-d]pyrimidin-5-yl)-4-(4-fluorophenyl)-4H-
[1,2,4]triazol-4-yl]ethanethioate
(9) Yield
70 %,
mp [300 °C. IR (KBr, t, cm-1): 1662 (C=O), 3000 (CH2),
5-(4-Phenyl-5-sulfanyl-4H-[1,2,4]triazol-3-yl)1H,3H,8H-
pyrido[2,3-d]pyrimidine-2,4,7-trione (6) Yield 65 %
3176 (NH). 1H NMR (DMSO-d6): d 6.8 (s, 1H, C6–H), 7.2
123

Med Chem Res
1
(d, J = 7.6 Hz, 2H, Ar–H), 7.7 (d, J = 7.6 Hz, 2H, Ar–H),
10.2 (brs, 1H, NH; D2O exchangeable), 10.8 (brs, 1H, NH;
D2O exchangeable), 11.4 (brs, 1H, NH; D2O exchange-
able). Mass m/z (%): 448 (M?, 61.8), 109 (100), 312 (79.4),
176 (32.4), 55 (55.9). Anal. calcd. for C17H10ClFN6O4S: C
45.49; H 2.25; N 18.72. Found: C 45.54; H 2.32; N 18.76.
Yield 38 %, mp [300 °C. H NMR (DMSO-d6): d 4.3
(s, 2H, CH2), 6.7 (s, 1H, C6–H), 8.4 (brs, 1H, NH; D2O
exchangeable), 9.7 (brs, 1H, NH; D2O exchangeable), 10.2
(brs, 1H, NH; D2O exchangeable), 10.5 (brs, 1H, NH; D2O
exchangeable), 11.4 (brs, 1H, NH; D2O exchangeable).
Mass m/z (%): 313 (M?, 4.37), 219 (100), 178 (6.57), 135
(14.36), 92 (10.23). Anal. calcd. for C10H8ClN5O5: C
38.29; H 2.57; N 22.33. Found: C 38.33; H 2.50; N 22.39.
Preparation of S-[3-(2,4,7-trioxo-1,2,3,4,7,8-
hexahydropyrido[2,3-d]pyrimidin-5-yl)-4-(aryl)-4H-
[1,2,4]triazol-5-yl]-2-(4-phenylpiperazin-1-
yl)ethanethioates 10, 11
Preparation of 2,4,7-trioxo-N0-[(4-substituted piperazin-1-
yl)acetyl]-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-
5-carbohydrazides 13–16
A mixture of compounds 8 or 9 (4 mmol), 1-phenylpiper-
azine (1.2 g, 8 mmol), and potassium carbonate (0.54 g,
4 mmol) in DMF (10 ml) was stirred at room temperature
for 24 h. The reaction mixture was filtered. The residue
was washed with water, dried, and recrystallized from
acetic acid.
A mixture of compound 12 (1.22 g, 4 mmol), the appro-
priate 1-substituted piperazine (8 mmol), and potassium
carbonate (0.54 g, 4 mmol) in DMF (10 ml) was stirred at
room temperature for 24 h. The product was collected by
filtration, washed with water, dried, and recrystallized from
acetic acid.
S-[3-(2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyr-
imidin-5-yl)-4-(phenyl)-4H-[1,2,4]triazol-5-yl]-2-(4-phen-
2,4,7-Trioxo-N0-[(4-phenylpiperazin-1-yl)acetyl]-
1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbohy-
drazide (13) Yield 85 %, mp [300 °C. 1H NMR
(DMSO-d6): d 3.0–3.3 (m, 8H, piperazine), 3.7 (s, 1H,
CH2), 6.2 (s, 1H, C6–H), 6.8–7.4 (m, 5H, Ar–H), 8.1 (brs,
1H, CONH; D2O exchangeable), 9.2 (brs, 1H, NHCO; D2O
exchangeable), 10.3 (brs, 1H, NH; D2O exchangeable),
11.2 (brs, 1H, NH; D2O exchangeable), 11.3 (brs, 1H, NH;
D2O exchangeable). Anal. calcd. for C20H21N7O: C 54.67;
H 4.82; N 22.31. Found: C 54.59; H 4.75; N 22.26.
ylpiperazin-1-yl)ethanethioate
(10) Yield
55 %,
mp [300 °C. IR (KBr, t, cm-1): 1554 (C=N), 1631
(C=O), 2839, 2993 (CH2), 3422 (NH). Mass m/z (%): 556
(M?, 12.5), 178 (100), 100 (23), 89 (18.4), 63 (16.4). Anal.
calcd. for C27H24N8O4S: C 58.26; H 4.35; N 20.13. Found:
C 58.32; H 4.42; N 20.19.
S-[3-(2,4,7-trioxo-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyr-
imidin-5-yl)-4-(4-fluorophenyl)-4H-[1,2,4]triazol-5-yl]-2-
(4-phenylpiperazin-1-yl)ethanethioate (11) Yield 62 %,
mp [300 °C. IR (KBr, t, cm-1): 1550 (C=N), 1638
2,4,7-Trioxo-N0-[4-(4-methoxyphenylpiperazin-1-yl)ace-
tyl]-1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-car-
bohydrazide (14) Yield 75 %, mp [300 °C. 1H NMR
(DMSO-d6): d 3.3–3.7 (m, 8H, piperazine), 2.5 (s, 2H,
CH2), 3.7 (s, 3H, –OCH3), 6.4 (s, 1H, C6–H), 6.7 (d,
J = 8.2 Hz, 2H, Ar–H), 7.5 (d, J = 8.2 Hz, 2H, Ar–H),
8.5 (brs, 1H, CONH; D2O exchangeable), 9.1 (brs, 1H,
NHCO; D2O exchangeable), 10.2 (brs, 1H, NH; D2O
exchangeable), 11.1 (brs, 1H, NH; D2O exchangeable),
11.4 (brs, 1H, NH; D2O exchangeable). 13C NMR (DMSO-
d6): d 50.4, 53.1, 56.7, 59.1, 99.2, 112.9, 116.1, 116.4,
143.5, 147.4, 151.1, 153.7, 161.8, 162.9, 166.4, 172.2.
Anal. calcd. for C21H23N7O6: C 53.73; H 4.94; N 20.89.
Found: C 53.66; H 4.90; N 20.95.
1
(C=O), 2843, 2996 (CH2), 3425 (NH). H NMR (DMSO-
d6): d 2.8–3.0 (m, 8H, piperazine), 3.6 (s, 2H, CH2), 6.3 (s,
1H, C6–H), 6.8 (t, J = 8.5 Hz, 1H, Ar–H), 7.1 (t,
J = 8.5 Hz, 2H, Ar–H), 7.3 (d, J = 8.0 Hz, 2H, Ar–H),
7.5 (d, J = 8.5 Hz, 2H, Ar–H), 7.7 (d, J = 8.0 Hz, 2H,
Ar–H), 10.1 (brs, 1H, NH; D2O exchangeable), 10.6 (brs,
1H, NH; D2O exchangeable), 11.2 (brs, 1H, NH; D2O
exchangeable). Mass m/z (%): 574 (M?, 48.5), 120 (100),
416 (54.5), 163 (36.4), 77 (57.6). Anal. calcd. for
C27H23FN8O4S: C 56.44; H 4.03; N 19.50. Found: C 56.00;
H 4.08; N 19.55.
Preparation of N0-(chloroacetyl)-2,4,7-trioxo-1,2,3,4,7,8-
hexahydropyrido[2,3-d]pyrimidine-5-carbohydrazide
(12) Chloroacetyl chloride (1.13 g, 10 mmol) was added
dropwise to a suspension of compound 3 (2.37 g,
10 mmol) in ethanol (40 ml). The reaction mixture was
heated under reflux for 6 h and then filtered while hot. The
precipitated solid was dried and recrystallized from DMF.
2,4,7-Trioxo-N0-[4-(4-ethoxyphenylpiperazin-1-yl)acetyl]-
1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbohy-
drazide (15) Yield 72 %, mp [300 °C. IR (KBr, t,
cm-1): 1590, 1640 (C=O), 2820, 2962 (CH2), 3260, 3295
(NH). Mass m/z (%): 483 (M?, 15.86), 294 (13.0), 179
(11.36), 128 (8.69), 89 (5.68). Anal. calcd. for
123

Med Chem Res
C22H25N7O6: C 54.65; H 5.21; N 20.28. Found: C 54.68; H
5.28; N 20.21.
Ar–H), 7.7 (d, J = 8.5 Hz, 1H, Ar–H), 7.9 (d, J = 8.5 Hz,
1H, Ar–H), 8.1 (s, 1H, Ar–H), 8.4 (s, 1H, CH=N), 8.8 (brs,
1H, NH; D2O exchangeable), 9.8 (brs, 1H, NH; D2O
exchangeable), 10.4 (brs, 1H, NH; D2O exchangeable),
11.2 (brs, 1H, NH; D2O exchangeable). Mass m/z (%): 370
(M?, 1.80), 176 (100), 178 (12.91), 153 (3.02), 122 (4.15).
Anal. calcd. for C15H10N6O6: C 48.66; H 2.72; N 22.70.
Found: C 48.62; H 2.76; N 22.77.
2,4,7-Trioxo-N0-[(4-benzylpiperazin-1-yl)acetyl]-
1,2,3,4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-carbohy-
drazide (16) Yield 84 %, mp [300 °C. IR (KBr, t,
cm-1): 1580, 1620 (C=O), 2811, 2941 (CH2), 3264, 3296
(NH). Mass m/z (%): 453 (M?, 11.76), 55 (100), 275
(13.24), 178 (11.76), 78 (11.76). Anal. calcd. for
C21H23N7O5: C 55.62; H 5.11; N 21.62. Found: C 55.67; H
5.14; N 21.66.
Pharmacology
Bleomycin-dependant DNA damage assay
(Ayyad et al., 2011a, b)
Preparation of N0-arylidene-2,4,7-trioxo-1,2,3,
4,7,8-hexahydropyrido[2,3-d]pyrimidine-5-
carbohydrazides 17–19
Materials
(1) DNA, L-ascorbic acid, ethylenediaminetetraacetic
acid (EDTA), thiobarbituric acid (TBA), and bleo-
mycin sulfate were purchased all from Aldrich Co.,
USA.
To a suspension of compound 3 (2.37 g, 10 mmol) in
absolute ethanol (30 ml), the appropriate aromatic alde-
hyde (20 mmol) was added. The reaction mixture was
refluxed for 6 h. On cooling, the precipitated solid was
collected by filtration, washed with ethanol, dried, and
recrystallized from DMF.
(2) Magnesium chloride, ferric chloride, and hydrochlo-
ric acid are of high analytical grade, and are obtained
all from El-Nasr Co. for pharmaceutical chemicals,
Egypt.
N0-(4-chlorobenzylidene)-2,4,7-trioxo-1,2,3,4,7,8-hexahy-
dropyrido[2,3-d]pyrimidine-5-carbohydrazide (17) Yield
70 %, mp [300 °C. IR (KBr, t, cm-1): 1587 (C=N), 1677
(C=O, ring), 1735 (C=O), 3174, 3243 (NH). Mass m/z (%):
359 (M?, 5.2), 206 (100), 248 (19.6), 178 (15.3), 111
(11.3). Anal. calcd. for C15H10ClN5O4: C 50.08; H 2.80; N
19.47. Found: C 50.12; H 2.75; N 19.51.
Method The reaction mixture contained DNA (0.5
mg/ml), bleomycin sulfate (0.05 mg/ml), magnesium
chloride (5 mM), ferric chloride (50 lM), and samples to
be tested in a concentration of 0.1 mg/ml are dissolved in
dimethylsulfoxide. L-Ascorbic acid (0.1 mg/ml) was used
as a positive control. The mixture was incubated at 37 °C
for 1 h and the reaction was terminated by the addition of
0.05 ml EDTA (0.1 ml). The color was developed by
adding 0.5 ml TBA (1 % w/v) and 0.5 ml hydrochloric
acid (25 % v/v) followed by heating at 80 °C for 10 min.
After centrifugation, the extent of DNA damage was
measured by the increase in absorbance at 532 nm
(Table 1).
N0-(4-methoxybenzylidene)-2,4,7-trioxo-1,2,3,4,7,8-hexa-
hydropyrido[2,3-d]pyrimidine-5-carbohydrazide
(18) Yield 73 %, mp [300 °C. IR (KBr, t, cm-1): 1602
(C=N), 1670 (C=O, ring), 1716 (C=O), 3160, 3243 (NH).
1H NMR (DMSO-d6): d 4.1 (s, 3H, CH3), 6.7 (s, 1H, C6–
H), 7.4 (d, J = 8.2 Hz, 2H, Ar–H), 7.7 (d, J = 8.2 Hz, 2H,
Ar–H), 8.1 (s, 1H, CH=N), 8.6 (brs, 1H, NH; D2O
exchangeable), 9.7 (brs, 1H, NH; D2O exchangeable), 10.5
(brs, 1H, NH; D2O exchangeable), 11.3 (brs, 1H, NH; D2O
exchangeable). 13C NMR (DMSO-d6): d 56.1, 99.0, 113.4,
115.1, 126.4, 131.4, 143.8, 147.4, 150.9, 161.9, 162.7,
163.1, 169.2. Mass m/z (%): 355 (M?, 1.95), 161 (100),
257 (5.41), 178 (5.21), 98 (12.98). Anal. calcd. for
C16H13N5O5: C 54.09; H 3.69; N 19.71. Found: C 54.11; H
3.65; N 19.75.
Evaluation of antineoplastic activity against EAC
in mice (Ayyad et al., 2011a, b; Qureshi et al.,
2001; Bala et al., 2010)
Materials
(1) Ehrlich ascites cells The cells of Ehrlich ascites
tumor were obtained from the National Cancer
Institute (which is a certified institute by National
Medical Research Ethics Committee), Cairo, Egypt.
After harvesting and preparation of the cells, their
total number and viability were determined by
counting using Trypan blue. The desired concentra-
tion of tumor cells (2 9 106 cells per 0.2 ml) was
N0-(3-nitrobenzylidene)-2,4,7-trioxo-1,2,3,4,7,8-hexahy-
dropyrido[2,3-d]pyrimidine-5-carbohydrazide (19) Yield
68 %, mp [300 °C. IR (KBr, t, cm-1): 1590 (C=N), 1671
(C=O, ring), 1714 (C=O), 3162, 3241 (NH). 1H NMR
(DMSO-d6): d 6.9 (s, 1H, C6–H), 7.5 (t, J = 8.5 Hz, 1H,
123

Med Chem Res
Chan DCM, Fu H, Forsch RA, Queener SF, Rosowsky A (2005)
Design, synthesis and antifolate activity of new analogues of
piritrexim and other diaminopyrimidine dihydrofolate reductase
inhibitors with x-carboxyalkoxy or x-carboxy-1-alkynyl substi-
tution in the side chain. J Med Chem 48:4420–4431
Cordeu L, Cubedo E, Bandres E, Rebollo A, Saenz X, Chozas H,
Dominguez MV, Echeverria M, Mendivil B, Sanmartin C, Palop
JA, Font M, Foncillas JG (2007) Biological profile of new
apoptotic agents based on 2,4-pyrido[2,3-d]pyrimidine deriva-
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De P, Baltas M, Theys DL, Bruyere C, Kiss R, Belval FB, Saffon N
(2010) Synthesis and anticancer activity evaluation of 2(4-
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Eur J Med Chem 46:2031–2036
obtained by dilution with saline (0.9 % sodium
chloride solution). The viability of tumor cells
obtained and used in this experiment was always
higher than 90 %. Below this percentage, the cells
were discarded and the entire procedure was
repeated.
(2) 5-FU was obtained from Aldrich Co., USA.
(3) Mice Adult Swiss male albino mice (20–25 g) of both
sexes were used in this experiment. They were housed
in microlon boxes in a controlled environment
(temperature 25 2 °C and 12 h dark/light cycle)
with standard laboratory diet and after regimen.
Method The animals were divided into four groups. All
the animals in the treated groups (from groups 2 to 4) were
inoculated with 2 9 106 Ehrlich ascites cells/mouse on the
day 0. Treatment started 24 h after inoculation by intra-
peritoneal (IP) injection of the drug. The animals in group
3 were injected by the tested compound in a dose of
100 lg/mouse, which is nearly equivalent to 5 mg/kg of
body weight, while the standard group (group 4) had
received IP treatment with 5 mg/kg of body weight of
5-FU. The control group (group 2) was treated with the
same volume of 0.9 % sodium chloride solution. Treatment
was given for nine successive days (Tables 2, 3, 4).
Formagio ASN, Tonin LTD, Foglio MA, Madjarof C, Carvalho JE,
Costa WF, Cardoso FP, Sarragiotto MH (2008) Synthesis and
antitumoral activity of novel 3-(2-substituted-1,3,4-oxadiazol-5-
yl) and 3-(5-substituted-1,2,4-triazol-3-yl) b-carboline deriva-
tives. Bioorg Med Chem 16:9660–9667
Gangjee A, Adair O, Queener SF (2001) Synthesis of 2,4-diamino-6-
(thioarylmethyl)pyrido[2,3-d]pyrimidines as dihydrofolate reduc-
tase inhibitors. Bioorg Med Chem 9:2929–2935
Grande F, Aiello F, Grazia OD, Brizzi A, Garofalo A, Neamati N
(2007) Synthesis and antitumor activities of a series of novel
quinoxaline hydrazides. Bioorg Med Chem 15:288–294
Guo CC, Tong RB, Li KL (2004) Chloroalkyl piperazine and nitrogen
mustard porphyrins: synthesis and anticancer activity. Bioorg
Med Chem 12:2469–2475
Joska TM, Anderson AC (2006) Structure activity relationships of
Bacillus cereus and Bacillus anthracis dihydrofolate reductase:
toward the identification of new potent drug leads. Antimicrob
Agents Chemother 50:3435–3443
Acknowledgments We would like to thank Prof. Farid A. Badria,
Professor of Pharmacognosy, Faculty of Pharmacy, Mansoura Uni-
versity, for his cooperation in performing the biologic screening.
Kurumurthy C, Rao PS, Swamy BV, Kumar GS, Rao PS, Narsaiah B,
Velatooru LR, Pamanji R, Rao JV (2011) Synthesis of novel
alkyltriazole tagged pyrido[2,3-d]pyrimidine derivatives and
their anticancer activity. Eur J Med Chem 46:3462–3468
Mohamed NR, El-Saidi MMT, Ali YM, Elnagdi MH (2007) Utility of
6-amino-2-thiouracil as a precursor for the synthesis of bioactive
pyrimidine derivatives. Bioorg Med Chem 15:6227–6235
Qureshi S, Al-Shabana O, Al-Harbi M, Al-Bekairi A, Raza M (2001)
Boric acid enhances in vivo Ehrlich ascites carcinoma cell
proliferation in Swiss albino mice. Toxicology 165:1–11
Sanmartin C, Echeverria M, Mendivil B, Cordeu L, Cubedo E,
Foncillas JG, Font M, Palop JA (2005) Synthesis and biological
evaluation of new symmetrical derivatives as cytotoxic agent
and apoptosis inducers. Bioorg Med Chem 13:2031–2044
Shi F, Ding J, Zhang S, Hao WJ, Cheng C, Tu S (2011) Substrate
controlled chemoselective synthesis and potent cytotoxic activity
of novel 5,6,7-triarylpyrido[2,3-d]pyrimidin-4-one derivatives.
Bioorg Med Chem Lett 21:1554–1558
Tu S, Zhang J, Zhu X, Xu J, Zhang Y, Wang Q, Jia R, Jiang B, Zhang
J (2006) New potential inhibitors of cyclin dependent kinase 4:
design and synthesis of pyrido[2,3-d]pyrimidine derivatives
under microwave irradiation. Bioorg Med Chem Lett 16:
3578–3581
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piritrexim, a lipophilic inhibitor of human dihydrofolate reduc-
tase: synthesis, antitumor activity and molecular modeling
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