Synthesis and antitumor activity evaluation of some novel pyrazolotriazine derivatives

Article abstract of DOI:10.1080/00397911.2016.1262422

6-Aminopyrazolo[1,2-a][1,2,4]triazine-4,8-dione derivative 3 was obtained upon the reaction of the acid hydrazide derivative 2a with ethyl cyanoacetate. The reactions of 3 with several electrophiles such as aldehydes, isatin, acetic anhydride, phenyl isoc

Full text of DOI:10.1080/00397911.2016.1262422

Synthesis and antitumor activity evaluation of some novel pyrazolotriazine
derivatives
Saad R. M. Hassan1; Rasha S. Gouhar2; Magdy M. Hemdan1; Wael S. I. Abou-Elmagd1,
David S. A. Haneen1,; Kamal A. A. Kandeel1 , Ahmed S. A.Youssef1
1Chemistry Department, Faculty of Science, Ain Shams University, Abbassia, Cairo,
Egypt. , 2Therapeutic Chemistry Department, National Research Centre, Dokki, Cairo,
Egypt.
Address correspondence to David S. A. Haneen, E-mail: david.shoukry@yahoo.com.
Abstract
6-Aminopyrazolo[1,2-a][1,2,4]triazine-4,8-dione derivative 3 was obtained upon the
reaction of the acid hydrazide derivative 2a with ethyl cyanoacetate. The reactions of 3
with several electrophiles such as aldehydes, isatin, acetic anhydride, phenyl isocyanate,
benzoyl isothiocyanate and p-toluenesulphonyl chloride were studied. The structures of
1
the newly synthesized compounds were established on the basis of IR, H-NMR, mass
spectra, and elemental analyses. The antitumor activities of some selective compounds
were examined against two cell lines as liver carcinoma cell line (HEPG-2) and human
breast cancer cell line (MCF7).
GRAPHICAL ABSTRACT
KEYWORDS: pyrazolotriazine, pyrazoline-3,5-dione, antitumor activities, HEPG-2,
MCF7
1

INTRODUCTION
Among the wide variety of nitrogen heterocycles, 1,2,4-triazine and its fused derivatives
were explored for developing pharmaceutical and agrochemical molecules[1-2]
.
The pyrazolotriazine ring system is similar to adenine analogues antagonists in their
structure. Therefore, this ring has considerable biological and medicinal activities such as
antischistosomal, antitumor agents[3-6] and antimicrobial activity[7]. Also, the
Pyrazolotriazine derivatives were reported to have remarkable cytotoxic activity against
colon, breast and lung carcinoma cells[8]. Some derivatives showed selective cytotoxicity
in hypoxic and normoxic conditions [9]. The valuable biological and pharmaceutical
activities of these ring systems, prompted us to synthesis new derivatives of pyrazolo[1,2-
a][1,2,4] triazines for the evaluation of antitumor activities.
RESULTS AND DISCUSSION
The geometrical isomers of the acid hydrazide derivatives Z-(2a) & E-(2b) were
previously prepared by our research group upon hydrazinolysis of 4-benzylidene-2-
phenyl-1,3-oxazole-5(4H)-one 1 [10,11]. Treatment of the Z-(2a) with ethyl cyanoacetate in
refluxing ethanol gave 6-amino pyrazolo[1,2-a][1,2,4]triazines-4,8-diones derivative 3.
Similar treatment of the E-(2b) with ethyl cyanoacetate under the same conditions, gave
E-(2b) recovered unchanged. However, its treatment with ethyl cyanoacetate in the
presence of a catalytic amount of sodium ethoxide in refluxing ethanol affored the
pyrazoline-3, 5-dione derivative 4 (Scheme 1).
2

The difference in the chemical behavior of Z-(2a) and E-(2b) may be due to the steric
crowding between benzylidene and benzamido groups that forces the compound to
cyclize easily before or after the reaction with ethyl cyanoacetate to give compound 3.
The structures of compounds 3 and 4 were substantiated from their analytical and spectral
data. Their IR spectra exhibit bands corresponding to NH and C=O groups. The 1H-NMR
spectra are in accord with the proposed structures as they showed signals for protons of
NH, olefinic and aromatic groups as well as the signals of the coupling pattern of CH2-
CH moiety of compound 4. The higher δ value for the proton of NH pyrazolo suggests
the existence of compound 4 as its chelated form shown (Scheme 1). Further evidence
was gained from their mass spectra that showed the molecular ion peaks beside some of
abundant peaks (cf. experimental).
The Schiff bases were exhibited many biological activities such as antifungal,
antibacterial, antitumor, anti-inflammatory and antipyretic activities [12-15]. The presence
of free amino group in compound 3 initiated our interest to synthesis a new series of
Schiff bases in order to achieve heterocycles of anticipated biological activity.
Thus, the treatment of an ethanolic solution of compound 3 with different aromatic
aldehydes such as benzaldehyde, p-methoxybenzaldehyde and p-chlorobenzaldehyde in
the presence of a catalytic amount of acetic acid, afforded the expected Schiff bases 5a-c
beside small amounts of the unexpected product 6a-c, respectively (Scheme 2).
3

The structure of compounds 5a-c was established from their analytical and spectral data.
Thus, their IR spectra were devoid of any absorptions due to NH2 group. Further support
for the suggested structure was gained from their 1H-NMR and mass spectra (cf.
experimental). The structure of compounds 6a-c was identical in all respects (m.p., mixed
m.p., TLC) with authentic samples prepared by reacting the acid hydrazides 2a and/or 2b
with benzaldehyde, p-methoxybenzaldehyde and p-chlorobenzaldehyde, respectively.
The mechanistic pathway for the formation of 6a-c is depicted in (Scheme 3).
On the other hand, when compound 3 was treated with istain in refluxing ethanol and
a catalytic amount of acetic acid, it afforded the Schiff base derivative 7 (Scheme 2). The
structure of 7 was substantiated from its microanalytical and spectral data. The IR
spectrum of compound 7 showed the appearance of bands for NH group at 3200, 3169
cm-1. Its 1H-NMR spectrum showed the appearance of a broad singlet signal for NH
proton as well as signals for olefinic and aromatic protons. Further support for the
assigned structure of compound 7 was gained from its mass spectrum that showed its
correct molecular ion peak beside some of important peaks.
Refluxing of compound 3 in acetic anhydride afforded the diacetyl derivative 8a.
However, its heating on water bath with acetic anhydride yielded a mixture of diacetyl
and monoacetyl derivatives 8a & 8b (Scheme 4).
Their structures were elucidated from their microanalytical and spectral data. The IR, 1H-
NMR and mass spectra were in accord with the suggested structures (cf. experimental).
4

Treatment of compound 3 with phenyl isocyanate in dry benzene gave the urea derivative
9. However, its refluxing with benzoyl isothiocyanate in dry acetonitrile yielded the
thiourea derivative 10. The structures of compounds 9 & 10 were substantiated from their
analytical and spectral data. Their IR spectra show absorptions characteristic of NH and
C=O groups, as well as C=S group for compound 10. Their 1H-NMR spectra exhibit
signals for protons of NH, olefinic and aromatic groups. Further support for the assigned
structures of compounds 9 & 10 was found in their mass spectra. They revealed the
molecular ion peaks and other abundant peaks.
Heating of the thiourea derivative 10 with phenylhydrazine in ethanol/dioxane (1:1)
mixture in the presence of a catalytic amount of acetic acid afforded a mixture of two
compounds one of them was identical in all respects (m.p., mixed m.p., TLC) with
compound 3 and the other was proved to be the 1,2,4-triazole derivative 11 (Scheme 5).
The structure of compound 11 was evidenced by studying its spectral data that were in
accord with the proposed structure. A chemical proof for its structure was gained by
reaction of benzoyl isothiocyanate with phenylhydrazine under the same condition. The
product obtained was identical in all respects (m.p., mixed m.p., TLC) with 11. The
mechanistic pathway for the conversion of 10 to compounds 3 and 11 could be visualized
to proceed as shown in (Scheme 6).
Refluxing compound 3 with p-toluenesulphonyl chloride in dry benzene afforded p-
toluene sulfonamide derivative 12 (Scheme 7). The structure of 12 was established from
5

its analytical and spectroscopic data. The IR spectrum showed the appearance of an
absorption bands for NH, CH3 and S=O group. Inspection of the 1H-NMR spectrum of
compound 12 revealed the existence of a singlet signal for CH3 at 2.28 ppm, a broad
singlet signal corresponds to proton of NH group at 7.18 ppm, multiplet signals for
aromatic protons and singlet signals for olefinic protons. Furthermore, The EI-MS of
compound 12 showed the correct molecular ion peak beside some of abundant peaks.
Treatment of a solution of compound 3 in acetic acid with sodium nitrite followed by
aqueous solution of sodium azide and/or alkaline -naphthol gave the hydroxy derivative
13 (Scheme 7). This is due to the unstability of the formed intermediate diazonium salt
that underwent nucleophilic attack by H2O to give the hydroxy derivative 13.
The structure of compound 13 was devoid of any absorption corresponding to NH2
group; but shows bands for OH group. The 1H-NMR spectrum was in accord with the
proposed structure. The higher δ value for the signal of OH proton is in accord with its
existence as its chelated form has shown (Scheme 7). On the other hand, when the
compound 3 was treated with hydrazine hydrate in refluxing ethanol, it afforded the N-
substituted pyrazole-5-one derivative 14 (Scheme 7).
The structure of compound 14 was substantiated from its spectral and analytical data.
The IR spectrum exhibits bands corresponds to NH and C=O groups. Further evidence
was ascertained from its 1H-NMR spectrum that was consistent with the suggested
structure (cf. experimental). Furthermore, the structure of compound 14 was supported
6

from its mass spectrum. The mechanistic pathway for the formation of 14 is presented in
(Scheme 8).
BIOLOGICAL ACTIVITY
In Vitro Anticancer Screening (Antitumor Evaluation)
Compounds 2b, 3, 5a, 5b, 7, 8a, 10 and 12 were evaluated for their in vitro cytotoxic
activity against two cell lines as liver carcinoma cell line (HEPG-2), 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 applied method is similar to that reported by
Skehan, P. et al.[16] using Sulfo-Rhodamine-B stain (SRB) as shown in the experimental
part. The results of in vitro cytotoxic activity against a liver carcinoma cell line (HEPG2)
and a human breast carcinoma cell line (MCF7) were shown in tables 1 and 2,
respectively. The response parameter calculated was the IC50 value, which corresponds
to the concentration required for 50% inhibition of cell viability.
.
From tables 1 and 2, we found that the better results were obtained with the five
compounds (3, 7, 8a, 10 and 12) only those showed a cytotoxic activity (IC50) against a
liver carcinoma cell line (HEPG2) with a less than 50 µg/ml of these compounds. While
the compounds (2b, 5a and 5b) against a liver carcinoma cell line (HEPG2) and all of the
tested compounds against a human breast carcinoma cell line (MCF7) showed a cytotoxic
activity (IC50) with a more than 50 µg/ml. The IC50 values of the compounds (3, 7, 8a,
10 and 12) against a liver carcinoma cell line (HEPG2) compared to the reference drug
were shown in table 1.
7

We can conclude that, Compound 12 was the most potent due to it showed a higher
cytotoxic activity in this screening with IC50 equal to 6.8 µg/ml which can be attributed
to the presence of the sulfonamide group. While, Compounds 3, 8a and 10 showed a
moderate cytotoxic activity with IC50 equal to 18, 12 and 10 µg/ml which can be
attributed to the presence of the amino group, two acetyl groups and one lactam and two
thioamide groups, respectively. Compound 7 showed a poor cytotoxic activity with IC50
equal to 35.4 µg/ml.
EXPERIMENTAL
Melting points were uncorrected and were measured on a Gallen Kamp electric melting
point apparatus. The infrared spectra were recorded using potassium bromide disks on
FTIR Thermo Electron Nicolet 7600 (USA) infrared spectrometer at the Central
laboratory of Faculty of Science, Ain Shams University. The 1H-NMR spectra were run
at 300 MHz on a GEMINI 300 BB NMR spectrometer using tetramethyl silane (TMS) as
internal standard in deuterated dimethylsulphoxide (DMSO-d6) at the main defense
chemical laboratory. The mass spectra were recorded on a shimadzu GC-MS QP-
1000EX mass spectrometer operating at 70 ev at the Regional Center for Mycology &
Biotechnology of Al-Azhar University. The progress of all reactions was monitored by
the thin layer chromatography using Merck Kiesel gel 60 F254 aluminum backed plates.
General Procedure for the Reaction of the Acid Hydrazide 2a with Ethyl Cyanoacetate
To a solution of the acid hydrazide 2a (10 mmol) in ethanol (40 ml), ethyl cyanoacetate
(10 mmol) was added. The reaction mixture was refluxed for 6h., then left to cool at
8

room temperature. The precipitated solid obtained was filtered off and washed with small
amount of ethanol, recrystallized from ethanol to give compound 3.
6-Amino-3-Benzylidene-1-Phenyl-3h-Pyrazolo[1,2-a][1,2,4]Triazine-4,8-Dione (3)
Yellow crystals (34%), m.p. 188-189 oC, (ethanol); FTIR (KBr) cm1: 3334, 3272 (NH2),
3062, 3027 (aryl-H), 1704 (C=O amide), 1639 (C=N), 1609 (C=C), 766, 687 (δ5H). 1H-
NMR (DMSO-d6): δ 5.37 (br.s, 2H, NH2 exchangeable), 7.18 (s, 1H, =CHPh), 7.44 - 8.39
(m, 11H, ArH+ =CH pyrazolone). MS (70 ev) m/z (%): 331 (M.+ +1, 2), 330 (M.+, 8), 328
(12), 285 (22), 263 (43), 248 (25), 233 (33), 178 (25), 161 (42), 151 (47), 122 (29), 89
(30), 81 (59), 74 (94), 61 (39), 49 (23), 41 (100). Anal. Calcd. for C19H14N4O2 (330.34):
C, 69.08; H, 4.27; N, 16.96. Found: C, 69.23; H, 4.21; N, 17.09%.
BIOLOGICAL TESTING
Measurement of Potential Cytotoxicity by SRB Assay
Potential cytotoxicity of the compounds was tested using the method of Skehan et al.
(1990). Cells were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment
with the test compound to allow attachment of cell to the wall of the plate. Different
concentrations of the compound under test (0, 1, 2.5, 5, and 10 µg/ml) were added to the
cell monolayer in triplicate wells were prepared for each individual dose. Monolayer cells
were incubated with the compounds for 48 h at 37oC and in atmosphere of 5 % CO2.
After 48 h, cells were fixed, washed and stained with SRB stain. Excess stain was washed
with acetic acid and attached stain was recovered with Tris EDTA buffer. Color intensity
was measured in an ELISA reader. The relation between surviving fraction and drug
9

concentration is plotted to get the survival curve of each tumor cell line after the specified
compound and the IC50 was calculated.
REFERENCES
[1] Tomar, I.; Mishra, R. The molecule of diverse biological and medicinal importance.
Int. J. Pharm Sci. Res. 2011, 2, 758.
[2] Sztanke, K.; Pasternak, K.; Rajtar, B.; Sztanke, M.; Majek, M.; Polz-Dacewicz, M.
Identification of antibacterial and antiviral activities of novel fused 1,2,4-triazine ester.
Bioorg Med Chem Lett, 2007, 15, 5480.
[3] Rao, D. R.; Raychaudhuri, S. P.; Verma, V. S. Study on the Effect of Herbicides on
the Infectivity of Cucumber Mosaic Virus. Int. J. Tropical Plant Dis. 1994, 12, 177.
[4] Elfaham, H. A.; Abdel-Galil, F. M.; Ibraheim, Y. R.; Elnagdi, M. H. Activated nitriles
in heterocyclic synthesis: A novel synthesis of pyrazolo[1,5-a]pyrimidines and
pyrano[2,3-c]pyrazoles. J. Heterocycle. Chem. 1983, 20, 667.
[5] Hinshaw, B. C.; Lconoudakis, O.; Townsend, L. B. Abstracts 112d National Meeting
of the American Chemical Society, Washington, D. C., Sept. 1971, No MEDI-15.
[6] Ito, I. Japanese Patent 7030, 101, 1970. Chem. Abst. 1971, 74, 22827e.
[7] Bhavna, N. A.; Arun, R. P.; Hansa, P.; Vijayalakshmi, G. Synthesis of Some Novel
Pyrazolo [3, 4-e] [1, 2, 4] Triazines As Potential Antimicrobial Agents. Am. J. Pharm.
Health Res, 2014, 2(4), 2321.
[8] Cornnello, M.; Ciciani, G.; Mini, E.; Guerrini, G.; Caciagli, B.; Selleri, S.; Costanzo,
A.; Mazzei, T. Cytotoxic activity of 3-nitropyrazolo[5,1-c][1,2,4]benzotriazine
10

derivatives: a new series of anti-proliferative agents. Anticancer Drugs, 2005, 16 (6),
645.
[9] Ciciani, G.; Coronnello, M.; Guerrini, G.; Selleri, S.; Cantore, M.; Failli, P.; Mini, E.;
Costanzo, A. Synthesis of new pyrazolo[5,1-c][1,2,4] benzotriazines, pyrazolo[5,1-
c]pyrido[4,3-e][1,2,4] triazines and their open analogues as cytotoxic agents in normoxic
and hypoxic conditions. Bioorg. Med. Chem. 2008, 16, 9409.
[10] Youssef, A. S. A.; El-Mariah, F. A.; Abd-Elmottaleb, F. T.; Hashem, H. E. Utility of
4-Benzylidene-2-phenyl-5(4H)-oxazolone in Synthesis of Triazine, Oxadiazole and
Imidazole Derivatives of Anticipated Biological Activity. J. Heterocycle. Chem. 2015,
52, 1467.
[11] Youssef, A. S. A.; Kandeel, K. A.; Abou-Elmagd, W. S. I.; Haneen, D. S. A. Action
of Some Nitrogen and Carbon Nucleophile on 4-Arylidene-1,3-oxazolones. J.
Heterocycle. Chem. 2016, 53, 175.
[12] Shah, S.; Vyas, R.; Mehta, R. H. Synthesis, characterization and antibacterial
activities of some new Schiff base compounds. J Ind Chem Soc. 1992, 69 (9), 590.
[13] Pandeya, S. N.; Sriram, D.; Nath, G. Synthesis, antibacterial, antifungal and anti-
HIV activities of Schiff and Mannich bases derived from isatin derivatives and N-[4-(4'-
chlorophenyl)thiazol-2-yl] thiosemicarbazide. Eur J pharma Soc. 1999, 9 (1), 25.
[14] More, P. G.; Bhalvankar, R. B.; Paher, S. C. J. Synthesis and biological activities of
Schiff bases of aminothiazoles. Ind Chem Soc. 2001, 78, 474.
[15] Kuzmin, V. E.; Artemenko, A. G.; Lozytska, R. N.; Fedtchouk A. S.; Lozitsky V. P.;
Muratov E. N.; Mescheriakov A. K. Investigation of anticancer activity of macrocyclic
11

Schiff bases by means of 4D-QSAR based on simplex representation of molecular
structure. SAR & QSAR Environ Res. 2005, 16 (3), 219.
[16] Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren,
J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New colorimetric cytotoxicity assay for anti-
cancer drug screening. J. Nat. Cancer Inst., 1990, 82, 1107.
[17] Rajitha, G.; Saideepa, N.; Praneetha, P. Synthesis and evaluation of N-(α-benzamido
cinnamoyl) aryl hydrazone derivatives for anti-inflammatory and antioxidant activities.
Indian J. Chem. 2011, 50B, 729.
[18] Youssef, A. S. A.; Kandeel, K. A.; Abou-Elmagd, W. S. I.; Haneen, D. S. A.
Synthesis of Novel Heterocycles Derived from 4-Arylmethylene-2-phenyl-1,3-oxazole-
5(4H)-ones. J. Heterocycle. Chem. 2016, 53, 809.
[19] Yavari, I.; Hossaini, Z.; Sabbaghan, M. Synthesis of functionalized 1,2,4-triazole-3-
thiones from potassium isothiocyanate, acid chlorides and arylhydrazines. Iranian J. Org.
Chem. 2009, 1(3), 168.
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Table 1. Represents the results of in vitro cytotoxic activity against a liver carcinoma cell
line (HEPG2).
compound
number
2b
Concentration:µg/ml
IC50 value
0.000
5.000
12.500
25.000
50.000
1.000
0.712
0.666
0.646
0.619
----
18
1.000
0.666
0.600
0.380
0.344
3
1.000
1.000
1.000
0.835
0.770
0.645
0.822
0.787
0.514
0.699
0.680
0.555
0.709
0.703
0.426
----
5a
5b
7
----
35.4
1.000
1.000
0.877
0.755
0.475
0.404
0.270
0.381
0.283
0.373
12
10
8a
10
1.000
1.000
0.528
0.396
0.423
0.245
0.350
0.222
0.315
0.183
6.8
3.9
12
Doxorubicin
13

Table 2. Represents the results of in vitro cytotoxic activity against a human breast
carcinoma cell line (MCF7).
compound number
Concentration:µg/ml
IC50 value
0.000 5.000 12.500 25.000 50.000
1.000 0.882 0.812
1.000 0.988 0.943
0.708
0.939
0.718
0.939
----
----
2b
3
1.000 0.882 0.842
1.000 0.988 0.998
0.844
0.988
0.934
0.988
----
----
5a
5b
1.000 0.797 0.753
1.000 0.988 0.934
0.766
0.950
0.638
0.924
----
----
7
8a
1.000 0.783 0.726
1.000 0.857 0.773
1.000 0.691 0.424
0.645
0.726
0.372
0.597
0.685
0.296
----
----
2.9
10
12
Doxorubicin
14

Scheme 1: reaction of 2a, b with ethyl cyanoacetate
15

Scheme 2: Reactions of 3 with different aromatic aldehydes and istain
16

Scheme 3: Mechanistic pathway for the conversion of 3 to compounds 6a-c
17

Scheme 4: Reaction of 3 with acetic anhydride under different conditions
18

Scheme 5: Reactions of 3 with phenyl isocyanate and benzoyl isothiocyanate
19

Scheme 6: Mechanistic pathway for the conversion of 10 to compounds 3 and 11
20

Scheme 7: Reactions of 3 with p-toluenesulphonyl chloride, sodium azide and/or alkaline
-naphthol and hydrazine monohydrate.
21

Scheme 8: Mechanistic pathway for the conversion of 3 to compound 14
22