Synthesis and antitumor activity of novel pyrido[2,3-d][1,2,4]triazolo[4,3- a]pyrimidin-5-one derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.02.055

A series of new pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidines with different substituents at position 3 were synthesized. The effect of the newly synthesized compounds was tested in vitro on human breast adenocarcinoma cell line (MCF7). Some of the synthesized compounds exploited potent antitumor activity, especially the 3-amino derivative 12 which displayed the highest activity among the test compounds with IC₅₀ equal to 3.74 μg/mL.

Full text of DOI:10.1016/j.ejmech.2011.02.055

European Journal of Medicinal Chemistry 46 (2011) 2031e2036
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activity of novel pyrido[2,3-d][1,2,4]triazolo[4,3-a]
pyrimidin-5-one derivatives
*
Hala B. El-Nassan
Organic Chemistry Department, Faculty of Pharmacy, Cairo University, 33 Kasr El-Aini Street, Cairo 11562, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidines with different substituents at position
3 were synthesized. The effect of the newly synthesized compounds was tested in vitro on human breast
adenocarcinoma cell line (MCF7). Some of the synthesized compounds exploited potent antitumor
activity, especially the 3-amino derivative 12 which displayed the highest activity among the test
Received 2 September 2010
Received in revised form
21 February 2011
Accepted 22 February 2011
Available online 3 March 2011
compounds with IC50 equal to 3.74 mg/mL.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrido[2,3-d]pyrimidin-4-one
Pyrido[2,3-d][1,2,4]triazolo[4,3-a]
pyrimidin-5-one
Antitumor activity
MCF7
1. Introduction
2. Results and discussion
Cancer is the second leading cause of death in the world. In the
last few years, a large number of fused pyrimidine derivatives were
reported as anticancer agents used in clinics or in clinical trials
[1e7]. Pyrido[2,3-d]pyrimidines were reported to exhibit anti-
tumor activity which may be attributed to inhibition of cyclin
dependent kinase [1,2], check point kinase [3] or mammalian target
of rapamycin [4].
Another fused pyrimidine ring system was [1,2,4]triazolo[4,3-a]
pyrimidine and many derivatives of this class were reported to
exhibit antitumor activity [5e7].
The combination of these two classes to give the tricyclic
pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine ring system and
the subsequent influence on the biological activities are of
current interest [8e14]. None of these publication discussed the
effect of pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidines as anti-
tumor agents.
In this work, we aimed to synthesize new pyrido[2,3-d][1,2,4]
triazolo[4,3-a]pyrimidin-5-one derivatives bearing different
substituents at position 3 [H, alkyl, oxo, thioxo, alkylsulphanyl, aryl,
amino and chloromethyl groups] in order to, examine the effect of
substitution at position 3 on the antitumor activity.
2.1. Chemistry
The synthesis of the target compounds is outlined in Schemes
1e3.
The starting compound, 5-(4-chlorophenyl)-2,3-dihydro-7-
phenyl-2-thioxopyrido[2,3-d]pyrimidin-4(1H)-one (3) was prepared
via reacting 6-amino-2-thiouracil (1) and 3-(4-chlorophenyl)-1-
phenyl-2-propen-1-one (2) [15] in DMF according to the procedure
reported by Quiroga et al. [16].
Reacting compound 3 with hydrazine hydrate in ethanol afforded
2-hydrazinopyrido[2,3-d]pyrimidin-4(3H)-one 4. While conducting
the same reaction in DMF resulted in 5-oxo-1H-pyrido[2,3-d][1,2,4]
triazolo[4,3-a]pyrimidine 5.
The formation of compound 5 was confirmed by 1H NMR that
showed only one exchangeable singlet signal at d 13.6 ppm. Besides,
the mass spectrum of compound 5 showed the molecular ion peaks
at m/z 373 (M) and 375 (Mþ2) in the ratio of 3:1 [Cl pattern].
Besides, the same compound was obtained by reacting
compound 4 with triethyl orthoformate or by its heating under
reflux in DMF.
A possible mechanism for the formation of compound 5 in DMF
was outlined in Fig. 1.
On the other hand, reacting compound 4 with acetic anhydride
or propionic anhydride resulted in 3-methyl 6a and 3-ethyl 6b
* Tel.: þ20 2 23632245; fax: þ20 2 23635140.
E-mail address: hala_bakr@hotmail.com.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.055

2032
H.B. El-Nassan / European Journal of Medicinal Chemistry 46 (2011) 2031e2036
Cl
O
O
O
HN
+
NH
S
N
H
NH
2
Cl
N
N
H
S
2
1
3
NH NH /
2
NH NH / DMF
2
2
2
C H OH
2
5
Cl
Cl
O
DMF
O
NH
or
HC(OC H )
2
5 3
N
N
N
N
NHNH
2
N
H
N
N
4
5
Scheme. 1. Synthesis of the starting compound 3 and its reaction with hydrazine hydrate.
derivatives, respectively. The formation of compound 6a was
confirmed by 1H NMR that revealed the presence of a singlet signal
11.2 ppm in the spectrum of 7 and at
spectrum of 8.
d 8.2 and 13.6 ppm in the
at
d
3.0 ppm corresponding to methyl protons and an exchangeable
12.8 ppm corresponding to NH proton. Also, the
Alkylation of 3-thioxo derivative 8 with dimethyl sulfate, ethyl
iodide, benzyl chloride or ethyl chloroacetate gave the corre-
sponding 3-alkylsulphanyl derivatives 9aed. 1H NMR spectra of
compounds 9aed revealed the disappearance of the NH signals at
singlet signal at
d
mass spectrum of compound 6a showed the molecular ion peaks at
m/z 387 (M) and 389 (Mþ2) in the ratio of 3:1 [Cl pattern]. While,
the 1H NMR spectrum of compound 6b showed triplet and quartet
d
8.2 and 13.6 ppm and the appearance of only one exchangeable
signals at
d
1.0 and 2.2 ppm, respectively, corresponding to ethyl
singlet signal at 12.5e12.9 ppm.
d
protons as well as an exchangeable singlet signal at
corresponding to NH proton.
The reaction of compound 4 with ethyl chloroformate in dry
pyridine or carbon disulphide in ethanolic KOH solution resulted in
3-oxo and 3-thioxopyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidines 7
and 8, respectively. Both compounds were confirmed by 1H NMR
d
11.3 ppm
The 3-aryl derivatives 11ced could be obtained via two path-
ways; either by reacting compound 4 with benzoyl chloride or 4-
methylbenzoyl chloride in dry pyridine, or by oxidative cyclization
of the corresponding hydrazone 10a,b in bromine/acetic acid
mixture.
Reacting 2-hydrazino derivative
4 with ammonium iso-
spectra that showed two exchangeable singlet signals at
d 9.2 and
thiocyanate in acetic acid afforded the 3-amino derivative 12. The
Scheme. 2. Synthesis of 3-alkyl, 3-oxo, 3-thioxo and 3-alkylsulphanyl derivatives.

H.B. El-Nassan / European Journal of Medicinal Chemistry 46 (2011) 2031e2036
2033
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Scheme. 3. Synthesis of 3-aryl, 3-amino and 3-chloromethyl derivatives.
1H NMR spectrum of the latter compound showed an exchangeable
The IC50 of the synthesized compounds compared to the refer-
ence drug are shown in Table 1 and the results are represented
graphically in Fig. 2.
singlet signal at
d 6.9 ppm corresponding to NH2 protons as well as
NH signal at 13.6 ppm.
d
Finally, reacting compound 4 with chloroacetyl chloride in dry
From the results in Table 1, it was found that the best results were
obtained by compounds bearing thioxo group [compound 8], alkylthio
group [compounds 9a,b,d] or amino group [compound 12] at position
3. Regarding the alkylthio derivatives, better result was obtained by
methylsulphanyl derivative 9a than by ethylsulphanyl derivative 9b or
benzylsulphanyl derivative 9c. Compound 12 was the most potent
pyridine afforded the 3-chloromethyl derivative 13. Its 1H NMR
spectrum showed CH2 protons as a singlet signal at
NH proton as an exchangeable broad singlet signal at
d
4.2 ppm and
13.5 ppm.
d
2.2. In vitro anticancer screening
compound in this screening with IC50 equal to 3.74 mg/mL.
Derivatives substituted with aryl, ethyl or chloromethyl at
position 3 showed moderate cytotoxic activity with IC50 between
The newly synthesized compounds were evaluated for their in
vitro cytotoxic activity against human breast cancer cell line, MCF7.
Doxorubicin which is one of the most effective anticancer agents
was used as the reference drug in this study.
The relationship between surviving fraction and drug concen-
tration was plotted to obtain the survival curve of breast cancer cell
line (MCF7). The response parameter calculated was the IC50 value,
which corresponds to the concentration required for 50% inhibition
of cell viability.
9.07 and 14
On the other hand, derivatives substituted with H, CH3 or CO at
position 3 showed poor cytotoxic activity with IC50 > 20 g/mL.
It is worth noting that the starting compound 3 showed good
cytotoxic activity against MCF7 with IC50 equal to 5.11 g/mL.
mg/mL.
m
m
Further studies needed to be done to explore the mechanism of
action as well as the effect of substitution at other positions of the ring.
Cl
Cl
Cl
O
O
O
O
N
NH NH
2
NH
2
H
H
NH
N
NH
N
N
H
S
N
N
NHNH
N
2
N
N
N
H
3
4
Cl
- HN(CH )
3 2
O
N
N
N
H
N
N
5
Fig. 1. A possible mechanism for the formation of compound 5 in DMF.

2034
H.B. El-Nassan / European Journal of Medicinal Chemistry 46 (2011) 2031e2036
Table 1
purified and dried by standard techniques. Elemental microanal-
yses were performed at The Microanalytical Center, Cairo Univer-
sity, Cairo, Egypt, and were within ꢀ0.4%.
Results of in vitro cytotoxic activity of the synthesized
compounds on human breast adenocarcinoma cell line
(MCF7).
Compound no.
IC50 in mg/mL
4.1.1. 5-(4-Chlorophenyl)-2-hydrazino-7-phenylpyrido[2,3-d]
pyrimidin-4(3H)-one (4)
Doxorubicin
3
5
2.97
5.11
21.60
A
mixture of 2-thioxopyrido[2,3-d]pyrimidine 3 (1.46 g,
4 mmol) and hydrazine hydrate (99%, 3 mL, 60 mmol) in absolute
ethanol (20 mL) was heated under reflux for 15 h. The reaction
mixture was cooled and the solid formed was filtered, dried and
crystallized from DMF. Yield: 70%; mp: 277e278 ꢁC; IR (cmꢂ1):
6a
6b
7
>50
14.00
24.30
6.78
3.89
5.20
12.61
4.50
9.22
9.07
3.74
8
9a
9b
9c
9d
11a
11b
12
13
3400, 3302 (NH/NH2), 1685 (CO); 1H NMR (DMSO-d6)
d ppm 2.0
(br s, 2H, NH2, D2O exchangeable), 6.1 (br s, 1H, NH, D2O
exchangeable), 7.4e8.1 (m, 10H, AreH), 10.0 (br s, 1H, NH, D2O
exchangeable); MS m/z: 365 [(Mþ2)þ, 25.40%], 363 [Mþ, 100%];
Anal. Calcd for C19H14ClN5O: C, 62.73; H, 3.88; N, 19.25. Found: C,
62.95; H, 3.80; N, 19.22.
11.70
4.1.2. 6-(4-Chlorophenyl)-8-phenyl-1H-pyrido[2,3-d][1,2,4]triazolo
[4,3-a]pyrimidin-5-one (5)
3. Conclusion
4.1.2.1. Method A. A mixture of 2-thioxopyrido[2,3-d]pyrimidine 3
(0.73 g, 2 mmol) and hydrazine hydrate (99%, 0.5 mL, 10 mmol) in
DMF (7 mL) was heated under reflux for 15 h. The reaction mixture
was cooled and the solid formed was filtered, dried and crystallized
from DMF. Yield: 64%.
The objective of the present study was to synthesize new pyrido
[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones bearing different
substituents at position 3 and to examine the effect of substitution
at position 3 on the cytotoxic activity. Some of these new
compounds exhibited good antitumor activity against MCF7 when
compared to doxorubicin as a reference drug. The best results were
obtained by derivatives bearing thioxo, alkylthio or amino groups at
position 3. The 3-amino derivative 12 was the most potent
4.1.2.2. Method B. A mixture of 2-hydrazinopyrido[2,3-d]pyrimi-
dine 4 (0.37 g, 1 mmol) and DMF (5 mL) was heated under reflux for
3 h. The reaction mixture was cooled and the solid formed was
filtered, dried and crystallized from DMF. Yield: 74%.
compound in this screening with IC50 equal to 3.74 mg/mL.
4.1.2.3. Method C. A mixture of 2-hydrazinopyrido[2,3-d]pyrimi-
dine 4 (0.37 g, 1 mmol) and triethyl orthoformate (8 mL) was heated
under reflux for 3 h. The solid formed was filtered, dried and crys-
tallized from DMF. Yield: 88%. mp: > 330 ꢁC; IR (cmꢂ1): 3400 (NH),
4. Experimental part
4.1. General
1693 (CO); 1H NMR (DMSO-d6)
d
ppm 7.4e8.4 (m,10H, AreH), 9.4 (s,
1H, CH]N),13.6 (br s,1H, NH, D2O exchangeable); 13C NMR (DMSO-
Melting points were determined using a Griffin apparatus and
were uncorrected. IR spectra were recorded on Shimadzu IR 435
spectrophotometer and values were represented in cm1. 1H NMR
and 13C NMR were carried out on Varian Gemini 200 MHz spec-
trophotometer at The Microanalytical center, Cairo University,
Cairo, Egypt, using TMS as an internal standard and chemical shifts
d6)
d ppm 105.0, 117.9, 127.6, 128.8, 130.3, 130.8, 132.9, 136.4, 137.7,
150.3, 151.3, 152.8, 153.4, 154.3, 158.0, 159.4; MS m/z: 375 [(Mþ2)þ,
36.96%], 373 [Mþ, 100%]; Anal. Calcd for C20H12ClN5O: C, 64.26; H,
3.24; N, 18.74. Found: C, 64.49; H, 3.50; N, 18.40.
4.1.3. 6-(4-Chlorophenyl)-3-methyl-8-phenyl-1H-pyrido[2,3-d]
[1,2,4]triazolo[4,3-a]pyrimidin-5-one (6a)
were recorded in ppm on
d scale. The electron impact (EI) mass
spectra were recorded on Hewlett Packard 5988 spectrometer at
The Microanalytical center, Cairo University, Cairo, Egypt. Analytical
thin layer chromatography (TLC) on silica gel plates containing UV
indicator was employed routinely to follow the course of reactions
and to check the purity of products. All reagents and solvents were
A mixture of 2-hydrazinopyrido[2,3-d]pyrimidine 4 (0.37 g,
1 mmol) and acetic anhydride (4 mL) was heated under reflux for 5 h.
The reaction mixture was cooled and the solid formed was filtered,
dried and crystallized from DMF. Yield: 74%; mp: > 330 ꢁC; IR
(cmꢂ1): 3410 (NH), 1693 (CO); 1H NMR (DMSO-d6)
d ppm 3.0 (s, 3H,
50
45
40
35
30
25
20
15
10
5
CH3), 7.4e8.3 (m,10 H, AreH),12.8 (br s,1H, NH, D2O exchangeable);
MS m/z: 389 [(Mþ2)þ, 37.73%], 387 [Mþ, 100%]; Anal. Calcd for
C21H14ClN5O: C, 65.04; H, 3.64; N, 18.06. Found: C, 64.70; H, 4.10; N,
18.39.
4.1.4. 6-(4-Chlorophenyl)-3-ethyl-8-phenyl-1H-pyrido[2,3-d][1,2,4]
triazolo[4,3-a]pyrimidin-5-one (6b)
A mixture of 2-hydrazinopyrido[2,3-d]pyrimidine 4 (0.37 g,
1 mmol) and propionic anhydride (0.26 g, 2 mmol) in pyridine
(10 mL) was heated under reflux for 15 h. The reaction mixture was
cooled and the solid formed was filtered, washed with ethanol, dried
and crystallized from DMF. Yield: 57%; mp: > 330 ꢁC; IR (cmꢂ1): 3421
0
dox
3
5
6a 6b
7
8
9a 9b 9c 9d 11a 11b 12 13
(NH),1693 (CO); 1H NMR (DMSO-d6)
2.2 (q, 2H, CH2, J ¼ 7.5 Hz), 7.4e8.1 (m,10 H, AreH),11.3 (br s,1H, NH,
D2O exchangeable); Anal. Calcd for C22H16ClN5O: C, 65.76; H, 4.01; N,
17.43. Found: C, 65.71; H, 4.10; N, 17.77.
d
ppm 1.0 (t, 3H, CH3, J ¼ 7.5 Hz),
Compound no.
Fig. 2. IC50 in mg/mL of the synthesized compounds and doxorubicin against human
breast adenocarcinoma cell line (MCF7).

H.B. El-Nassan / European Journal of Medicinal Chemistry 46 (2011) 2031e2036
2035
4.1.5. 6-(4-Chlorophenyl)-8-phenyl-1H-pyrido[2,3-d] [1,2,4]
triazolo[4,3-a]pyrimidin-3,5(2H)-dione (7)
(NH), 2927, 2850 (CH aliphatic), 1701 (CO); 1H NMR (DMSO-d6)
ppm 2.0 (m, 3H, CH3), 3.8 (m, 2H, CH2), 4.0 (s, 2H, SCH2CO), 7.3e8.4
d
A mixture of 2-hydrazinopyrido[2,3-d]pyrimidine 4 (0.37 g,
1 mmol) and ethyl chloroformate (0.22 g, 2 mmol) in pyridine
(10 mL) was heated under reflux for 9 h. The reaction mixture was
cooled and the solid was filtered, washed with ethanol, dried and
crystallized from DMF. Yield: 78%; mp: > 330 ꢁC; IR (cmꢂ1): 3387,
(m, 10 H, AreH), 12.5 (br s, 1H, NH, D2O exchangeable); Anal. Calcd
for C24H18ClN5O3S: C, 58.60; H, 3.69; N, 14.24. Found: C, 58.88; H,
3.36; N, 14.43.
4.1.8. 2-Arylmethylenehydrazone-5-(4-chlorophenyl)-7-
phenylpyrido[2,3-d]pyrimidin-4(3H)-ones 10a,b
3340 (NH), 1690, 1654 (CO); 1H NMR (DMSO-d6)
d ppm 7.4e8.1 (m,
10H, AreH), 9.2 (br s, 1H, NH, D2O exchangeable), 11.2 (s, 1H, NH,
D2O exchangeable); Anal. Calcd for C20H12ClN5O2: C, 61.63; H, 3.10;
N, 17.97. Found: C, 61.98; H, 3.16; N, 17.77.
A mixture of 2-hydrazinopyrido[2,3-d]pyrimidine 4 (0.37 g,
mmol) and 4-chlorobenzaldehyde or 3-nitrobenzaldehyde
(2 mmol) in glacial acetic acid (15 mL) was heated under reflux for
4 h. The reaction mixture was cooled and the solid formed was
filtered, dried and crystallized from DMF.
1
4.1.6. 6-(4-Chlorophenyl)-5-oxo-8-phenyl-1H-pyrido[2,3-d] [1,2,4]
triazolo[4,3-a]pyrimidin-3(2H)-thione (8)
A mixture of 2-hydrazinopyrido[2,3-d]pyrimidine 4 (0.74 g,
2 mmol), KOH (0.23 g, 4 mmol) and carbon disulphide (4 mL) in
absolute ethanol (40 mL) was heated under reflux for 5 h. The
reaction mixture was evaporated to dryness and water (200 mL)
was added then, the alkaline solution was filtered. The filtrate was
acidified with conc. HCl (10 mL) and the separated solid was
filtered, dried and crystallized from DMF. Yield: 90%; mp: > 330 ꢁC;
4.1.8.1. 2-(4-Chlorophenyl)methylenehydrazone-5-(4-chlorophenyl)-
7-phenylpyrido[2,3-d]pyrimidin-4(3H)-one (10a). Yield: 91%; mp:
315e316 ꢁC; IR (cmꢂ1): 3500, 3300 (NH),1680 (CO); 1H NMR (DMSO-
d6)
d
ppm 7.4e8.4 (m, 14 H, AreH), 8.8 (s, 1H, CH¼N),11.2 (s, 1H, NH,
D2O exchangeable), 11.6 (s,1H, NH, D2O exchangeable); MS m/z: 487
[(Mþ2)þ, 30.21%], 485 [Mþ, 45.19%], 374 [MþeC6H4Cl, 100%]; Anal.
Calcd for C26H17Cl2N5O: C, 64.21; H, 3.52; N, 14.40. Found: C, 64.62;
H, 3.23; N, 14.52.
IR (cmꢂ1): 3450 (NH), 1705 (CO); 1H NMR (DMSO-d6)
d ppm 7.4e8.6
(m, 10H, AreH), 8.2 (br s, 1H, NH, D2O exchangeable), 13.6 (s, 1H,
NH, D2O exchangeable); MS m/z: 407 [(Mþ2)þ, 3.28%], 405 [Mþ,
7.61%], 190 [100%]; Anal. Calcd for C20H12ClN5OS: C, 59.19; H, 2.98;
N, 17.26. Found: C, 59.10; H, 2.90; N, 17.50.
4.1.8.2. 5-(4-Chlorophenyl)-2-(3-nitrophenyl)methylenehydrazone-
7-phenylpyrido[2,3-d]pyrimidin-4(3H)-one (10b). Yield: 92%; mp:
278e279 ꢁC; IR (cmꢂ1): 3400, 3383 (NH), 1693 (CO), 1539, 1350
(NO2); 1H NMR (DMSO-d6)
d ppm 7.4e8.5 (m, 14 H, AreH), 8.8 (s,
4.1.7. 3-Alkylsulphanyl-6-(4-chlorophenyl)-8-phenyl-1Hpyrido
[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones
1H, CH]N), 11.2 (s, 1H, NH, D2O exchangeable), 11.6 (s, 1H, NH, D2O
exchangeable); Anal. Calcd for C26H17ClN6O3: C, 62.85; H, 3.45; N,
16.91. Found: C, 63.21; H, 3.88; N, 16.46.
Compound 8 (0.40 g, 1 mmol) was dissolved in 10% ethanolic
KOH (2 g in 20 mL ethanol). The reaction mixture was cooled and
dimethyl sulfate, ethyl iodide, benzyl chloride or ethyl chlor-
oacetate (1 mmol) was added and the reaction was stirred at room
temperature for 2 h then left overnight. The reaction was diluted
with water (30 mL) and acidified with acetic acid (5 mL). The
separated solid was filtered, dried and crystallized from the
appropriate solvent.
4.1.9. 6-(4-Chlorophenyl)-3-(substituted phenyl)-8-phenyl-1H-
pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones 11aed
4.1.9.1. Method A (for the synthesis of compounds 11a,b). A mixture
of 2-hydrazinopyrido[2,3-d]pyrimidine 4 (0.37 g, 1 mmol) and
benzoyl chloride or 4-methylbenzoyl chloride (1.5 mmol) in dry
pyridine (8 mL) was heated under reflux for 20 h. The reaction
mixture was cooled and the solid formed was filtered, washed with
ethanol, dried and crystallized from DMF.
4.1.7.1. 6-(4-Chlorophenyl)-3-methylsulphanyl-8-phenyl-1H-pyrido
[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (9a). Crystallized from
ethanol. Yield: 51%; mp: 233e234 ꢁC; IR (cmꢂ1): 3387 (NH), 2927,
2850 (CH aliphatic),1705 (CO); 1H NMR (DMSO-d6)
d ppm 2.6 (s, 3H,
4.1.9.2. Method B (for the synthesis of compounds 11c,d). A mixture
of compounds 10a,b (1 mmol) and anhydrous sodium acetate
(0.21 g, 2.5 mmol) bromine (0.16 g, 1 mmol) and glacial acetic acid
(10 mL) was heated in a water bath at 80 ꢁC for 8 h. The reaction
mixture was poured onto water (50 mL) and the solid formed was
filtered, dried and crystallized from DMF.
SCH3), 7.2e8.2 (m, 10 H, AreH), 12.8 (br s, 1H, NH, D2O exchange-
able); Anal. Calcd for C21H14ClN5OS: C, 60.07; H, 3.36; N, 16.68.
Found: C, 60.28; H, 3.46; N, 16.90.
4.1.7.2. 6-(4-Chlorophenyl)-3-ethylsulphanyl-8-phenyl-1H-pyrido
[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (9b). Crystallized from
acetic acid. Yield: 94%; mp: 273e274 ꢁC; IR (cmꢂ1): 3394 (NH), 2927,
4.1.9.3. 6-(4-Chlorophenyl)-3,8-diphenyl-1H-pyrido[2,3-d][1,2,4]tri-
azolo[4,3-a]pyrimidin-5-one (11a). Yield: 52%; mp: 323e324 ꢁC; IR
2870 (CH aliphatic),1697 (CO); 1H NMR (DMSO-d6)
d ppm 1.4 (t, 3H,
(cmꢂ1): 3350 (NH), 1680 (CO); 1H NMR (DMSO-d6)
d ppm 7.3e8.3
CH3, J ¼ 7.2 Hz), 3.3 (q, 2H, CH2, J ¼ 7.2 Hz), 7.4e8.4 (m, 10 H, AreH),
12.5 (br s,1H, NH, D2O exchangeable); Anal. Calcd for C22H16ClN5OS:
C, 60.90; H, 3.72; N, 16.14. Found: C, 60.77; H, 3.48; N, 16.25.
(m, 15 H, AreH), 12.7 (br s, 1H, NH, D2O exchangeable); Anal. Calcd
for C26H16ClN5O: C, 69.41; H, 3.58; N,15.57. Found: C, 69.62; H, 3.48;
N, 15.90.
4.1.7.3. 3-Benzylsulphanyl-6-(4-chlorophenyl)-l-8-phenyl-1H-pyrido
[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (9c). Crystallized from
ethanol. Yield: 60%; mp: 198e199 ꢁC; IR (cmꢂ1): 3398 (NH), 2927,
4.1.9.4. 6-(4-Chlorophenyl)-3-(4-methylphenyl)-8-phenyl-1H-pyrido
[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (11b). Yield: 72%; mp:
342e343 ꢁC; IR (cmꢂ1): 3400 (NH), 1680 (CO); 1H NMR (DMSO-d6)
2854 (CH aliphatic),1701 (CO); 1H NMR (DMSO-d6)
d ppm 4.3 (s, 2H,
d
ppm 2.3 (s, 3H, CH3), 7.2e8.2 (m, 14 H, AreH), 12.8 (br s, 1H, NH,
SCH2), 7.2e8.2 (m, 15 H, AreH), 12.9 (br s, 1H, NH, D2O exchange-
able); Anal. Calcd for C27H18ClN5OS: C, 65.38; H, 3.66; N, 14.12.
Found: C, 65.70; H, 3.24; N, 14.39.
D2O exchangeable); MS m/z: 465 [(Mþ2)þ, 2.68%], 463 [Mþ, 6.11%],
450 [100%], 77 [69.64%]; Anal. Calcd for C27H18ClN5O: C, 69.90; H,
3.91; N, 15.10. Found: C, 70.28; H, 3.91; N, 15.42.
4.1.7.4. Ethyl [6-(4-chlorophenyl)-5-oxo-8-phenyl-1H-pyrido[2,3-d]
4.1.9.5. 3,6-Di(4-chlorophenyl)-8-phenyl-1H-pyrido[2,3-d][1,2,4]tri-
azolo[4,3-a]pyrimidin-5-one (11c). Yield: 66%; mp: > 330 ꢁC; IR
[1,2,4]triazolo[4,3-a]pyrimidin-3-ylsulphanyl]
acetate
(9d).
Crystallized from DMF. Yield: 83%; mp: 292e293 ꢁC; IR (cmꢂ1): 3398

2036
H.B. El-Nassan / European Journal of Medicinal Chemistry 46 (2011) 2031e2036
(cmꢂ1): 3350 (NH),1680 (CO); 1H NMR (DMSO-d6)
14 H, AreH), 12.7 (br s, 1H, NH, D2O exchangeable); Anal. Calcd for
C26H15Cl2N5O: C, 64.48; H, 3.12; N,14.46. Found: C, 64.50; H, 3.23; N,
14.67.
d
ppm 7.3e8.3 (m,
the cells to the wall of the plate. Test compounds were dissolved in
DMSO and diluted with saline to the appropriate volume. Different
concentrations of the test compound (0, 1, 2.5, 5 and 10 mg/mL)
were added to the cell monolayer. Triplicate wells were prepared
for each individual dose. Monolayer cells were incubated with the
test compound for 48 h at 37 ꢁC in atmosphere of 5% CO2. After 48 h,
cells were fixed with trichloroacetic acid, washed with water and
stained for 30 min with 0.4% (wt./vol) Sulforhodamine-B stain
dissolved with 1% acetic acid. Excess stain was removed by four
washes with 1% acetic acid and attached stain was recovered with
Tris EDTA buffer. Colour intensity was measured in ELISA reader.
The relation between surviving fraction and compound concen-
tration was plotted and IC50 [the concentration required for 50%
inhibition of cell viability] was calculated for each compound and
results are given in Table 1.
4.1.9.6. 6-(4-Chlorophenyl)-3-(3-nitrophenyl)-8-phenyl-1H-pyrido
[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-one (11d). Yield: 64%; mp:
> 330 ꢁC; IR (cmꢂ1): 3390 (NH), 1708 (CO), 1550, 1339 (NO2); 1H
NMR (DMSO-d6) d ppm 7.2e8.3 (m, 14 H, AreH), 12.6 (br s, 1H, NH,
D2O exchangeable); Anal. Calcd for C26H15ClN6O3: C, 63.10; H, 3.06;
N, 16.98. Found: C, 63.44; H, 3.22; N, 16.90.
4.1.10. 3-Amino-6-(4-chlorophenyl)-8-phenyl-1H-pyrido[2,3-d]
[1,2,4]triazolo[4,3-a]pyrimidin-5-one (12)
A mixture of 2-hydrazinopyrido[2,3-d]pyrimidine 4 (0.74 g,
2 mmol) and ammonium thiocyanate (2.38 g, 30 mmol) in glacial
acetic acid (20 mL) was heated under reflux for 10 h. The reaction
mixture was cooled, poured into water (50 mL) and the solid
formed was filtered, dried and crystallized from acetic acid. Yield:
77%; mp: > 330 ꢁC; IR (cmꢂ1): 3417, 3394 (NH/NH2), 1712 (CO); 1H
Acknowledgment
We are grateful to all members of the department of Cancer
Biology, National Cancer Institute, Cairo, Egypt, for carrying out the
cytotoxicity testing.
NMR (DMSO-d6) d ppm 6.9 (s, 2H, NH2, D2O exchangeable), 7.4e8.6
(m, 10 H, AreH), 13.6 (s, 1H, NH, D2O exchangeable); Anal. Calcd for
C20H13ClN6O: C, 61.78; H, 3.37; N, 21.61. Found: C, 61.70; H, 3.12;
N, 21.38.
References
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A mixture of 2-hydrazinopyrido[2,3-d]pyrimidine 4 (0.74 g,
2 mmol) and chloroacetyl chloride (0.23 g, 2 mmol) in pyridine
(15 mL) was heated under reflux for 15 h. The reaction mixture was
cooled, poured into water (50 mL) and the solid was filtered, dried
and crystallized from acetic acid. Yield: 72%; mp: > 330 ꢁC; IR
(cmꢂ1): 3390 (NH), 1690 (CO); 1H NMR (DMSO-d6)
d ppm 4.2 (s, 2H,
CH2Cl), 7.4e8.5 (m, 10H, AreH), 13.5 (br s, 1H, NH, D2O exchange-
able); Anal. Calcd for C21H13Cl2N5O: C, 59.73; H, 3.10; N, 16.79.
Found: C, 60.07; H, 3.15; N, 16.77.
4.2. Biological testing
4.2.1. Materials and methods
The human breast adenocarcinoma cell line (MCF7) was
obtained as a gift from NCI, MD, USA.
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All chemicals and solvents were purchased from SigmaeAldrich.
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4.2.1.1. Measurement of potential cytotoxicity. The cytotoxic activity
of the newly synthesized compounds was measured in vitro on
human breast adenocarcinoma cell line (MCF7) using Sulforhod-
amine-B stain (SRB) assay applying the method of Skehan et al. [17].
Cells were plated in 96-multiwell plate (104 cells/well) for 24 h
before treatment with the test compounds to allow attachment of