Synthesis and anticancer evaluation of some novel pyrimido[5,4-e][1,2,4]triazines and pyrazolo[3,4-d]pyrimidine using DMF-DMA as methylating and cyclizing agent

Background Described a series of main target compounds pyrimido[5,4-e][1,2,4]triazines is obtained via condensation of 6-hydrazinyluracil with different aromatic aldehydes to give the hydrazones followed by nitrosation with HNO2 then intramolecular cyclization. On the other hand, pyrazolopyrimidines can be obtained by the reaction of hydrazones with dimethylformamide-dimethylacetal (DMF-DMA), DMF-DMA in the presence of DMF or by refluxing the hydrazinyluracil with DMF-DMA in the presence of DMF directly. The newly synthesized compounds are evaluated in vitro for their anticancer activity against human lung carcinoma (A549). Results A newly substituted compounds of benzaldehyde-pyrimidin-4-yl)hydrazones (5a–f), pyrimido[5,4-e][1,2,4]triazines 6a–e, arylethylidenehydrazinylpyrimidine 7a,b and pyrazolopyrimidines 9,11 are screened for cytotoxic activity against human lung carcinoma (A549) cell line. They exhibited a good yield. Compound 6b shows the highest effect with IC50 value 3.6 μM, followed by compounds 9, 5a, 8, 5e, 6e, 5b, 5f, 7a, 5c, 6c, 7b, 6a, 11, 5d and 6d. Conclusion A simple and efficient route is used for the synthesis of pyrimido[5,4-e][1,2,4]triazines and pyrazolopyrimidines. The synthesized compounds are screened for antitumor activity.


Chemistry
In continuation to our research, the importance of fervenulin and its diverse pharmacological activity on the medical field, especially as antitumors, we became interested in the prospect of developing our strategies to synthesize new fervenulin analogues of pyrimidotriazine and pyrazolopyrimidine derivatives using 6-hydrazinyl-1-propyluracil (4) as a core for construction. This substrate is prepared via simple hydrolysis of 2,4,6-trichloropyrimidine [37] followed by N-1 selective alkylation using propyl iodide in DMSO in the presence of potassium carbonate as a basic medium [38] then hydrazinolysis of 6-chloro-1-propyluracil (3) with hydrazine hydrate [38][39][40]. Condensation of substrate 4 with 6-Azacytosine 6-azauracil Tirapazamine
In this regard, our strategy is directed towards the synthesis of new pyrimido [5,4-e] [1,2,4]triazine and pyrazolopyrimidine compounds because they have found wide applications in pharmaceutical fields. The structure of newly synthesized pyrimidotriazines and pyrazolopyrimidines are proven on the basis of their 1 H-NMR, mass spectral data, IR and elemental analysis. different aromatic aldehydes in ethanol at room temperature for 1 h leads to the formation of hydrazones 5a-f in a good yield (Scheme 1).
The IR spectra of hydrazones displayed the N-H stretching bands at 3271-3122 cm −1 . The stretching band of the two C=O groups (Amide I) is displayed within the range 1740-1625 cm −1 . Compound 5d showed O-H stretching bands at 3560 cm −1 while the nitro group in compound 5e shows strong asymmetric and symmetric NO 2 stretching bands at 1514 and 1337 cm −1 , respectively. The 1 H-NMR spectra supported the previous observation from the IR spectra, where N3-H and C6-NH is highly deshielded. They appeared around δ 10.75-10.05 ppm, while the α-CH of hydrazone appeared at the range δ 8.48-8.24 ppm. The C5-H was the most shielded as expected around δ 5.46-5.30 ppm. The downfield shift of the α-carbon of hydrazone appeared around δ 145 ppm in the 13 C-NMR spectra.
Pyrimidotriazines 6a-e is isolated by the nitrosation of hydrazone compounds 5a-e at C-5 with in situ prepared nitrous acid. The inseparable 5-nitroso-derivatives undergoes cyclization via the nucleophilic attack of the electron rich α-carbon of the hydrazones on the nitroso group to form hydroxylamine intermediates, which are converted into the target pyrimidotriazines 6a-e by protonation of the N-hydroxyl group followed by the elimination of H 3 O + (Scheme 2). The IR spectra of 6a-e displayed broad absorption bands of NH stretching in the region of 3180-3135 cm −1 . The two bands of C=O groups gave rise in the region of 1725-1670 cm −1 .
Moreover, the cyclization of the hydrazone series are confirmed in 1 H-NMR spectra through the disappearance of both the α-CH hydrazone at δ 8.48-8. 24  Another condensation reaction is obtained via condensation of 4 with different acetophenones by stirring at room temperature for 3-4 h (Scheme 3). The IR spectra of 7a,b displayed stretching bands at the range of 3188-3154 cm −1 due to N-H absorption and characteristic bands at the range 1715-1691 cm −1 due to absorption of C=O groups. The mass spectra of these compounds show the expected molecular ions, whereas their 1 H-NMR spectra exhibited two signals at δ 11.11-11.06 ppm and at δ 9.13-8.96 ppm ascribed for N3-H and C6-NH protons respectively. The singlet signals of the methyl group protons at the α-carbon appeared at δ 2.42-2.37 ppm while for the CH-5 position appeared at δ 5.48-5.39 ppm. 13 C-NMR confirmed the structure of 7a,b where the key signals at δ 78.8-79.8 ppm and δ 14.3-14.2 ppm are assigned to sp 2 carbon at position 5 and sp 3 carbon attached to the α-carbon respectively. The target compound 9 is prepared by refluxing of 7a with DMF-DMA for 12 h or DMF-DMA in presence of DMF as a solvent for 1 h (Scheme 3). DMF-DMA is a convenient electrophile to introduce one-carbon units. The reaction proceeds via nucleophilic attack of C-5 to electrophilic carbon center of acetal in DMF-DMA followed by intramolecular cyclization and elimination of dimethylamine. A subsequent methylation of NH-5 is observed which arises from O-CH 3 group of the acetal not N-CH 3 as illustrated in Scheme 4. The plausible mechanism is proved by isolation of the intermediate 8. This intermediate is easily identified in IR, Mass, 1 H-NMR and 13

Scheme 2
The plausible reaction mechanism formation of compounds 5a-f and 6a-e singlet signal at δ 8.08 ppm characteristic for CH-N proton, a singlet signal of α-C-CH 3 at δ 2.39 ppm and two characteristic N-CH 3 group at δ 3.19, 3.05 ppm of compound 8 and disappears in compound 9 due to the elimination of dimethylamine (Scheme 4).
Whereas, the alkylation on N-5 in compound 9 is proven without doubt by the disappearance of a singlet signal of NH-5 proton at δ 10.13 ppm and the appearance of the sp 3 singlet signal at δ 3.23 ppm characteristic of CH 3 appears in 1 H-NMR and a signal at δ 27.6 ppm in 13 C-NMR. In addition, the 1 H-NMR shows a singlet signal of CH-3 at δ 8.60 ppm and two doublet signals at δ 5.71-5.61 ppm corresponding to the two protons of methylene group which indicates that they are not magnetically equivalent. 13 C-NMR shows the appearance of signals at δ 134.2 and 101.7 ppm characteristic for C-3 and methylene carbon atom respectively.
Treatment of 4 with DMF-DMA in the presence of DMF by refluxing for 1 h yielded compound 11 (Scheme 3). IR spectra shows characteristic absorption band at 1751, 1698 cm −1 for C=O groups. 1 H-NMR spectrum displays three singlet signals at δ 8.41, 3.88 and 3.19 ppm for CH-3, N(2)-CH 3 and N(5)-CH 3 respectively. On the other hand, 13 C-NMR showed C-N(2) at δ 40.4 ppm and C-N(5) at δ 27.5 ppm which confirms the alkylation of N(5) with DMF-DMA. The plausible mechanism for this reaction is shown in (Scheme 5).

Cytotoxic activity
The in vitro growth inhibitory rates against human lung carcinoma (A549) cell line and effective antitumor doses (as measured by IC 50 ) of the synthesized compounds are investigated in comparison with the well-known anticancer standard drugs toxoflavin and 5-fluorouracil, using crystal violet colorimetric viability assay. Data generated are used to plot dose response curves and presented in Table 1 and Fig. 1. The results reveal that all the tested compounds show high variation in the inhibitory growth rates and activities to the tumor cell line in a concentration dependent manner as shown in (Table 1).
From the results in Fig. 1, it is clear that all the tested compounds are found to be very active at 500 μM against human lung carcinoma (A549) cell line after treatment for 72 h with inhibition ratio values between 60 and 97%. The difference between inhibitory activities of all compounds with different concentrations is statistically significant (p < 0.001).

Instruments
All melting points were determined with an electrothermal melting-temperature II apparatus and are uncorrected. Element analyses are performed at the regional

Scheme 5
The plausible reaction mechanism formation of compound 11 center for mycology and biotechnology at Al-Azhar University. The infrared (IR) spectra are recorded using potassium bromide disc technique on Nikolet IR 200 FT IR. Mass spectra are recorded on a DI-50 unit of Shimadzu GC/MS-QP 5050A at the regional center for mycology and biotechnology at Al-Azhar University. 1 H-NMR and 13 C-NMR spectra are determined on Bruker 400 MHz spectrometer using DMSO-d 6 as a solvent, applied nucleic acid research center, Zagazig University, Egypt. All reactions are monitored by TLC using precoated plastic sheets silica gel (Merck 60 F 254 ). Spots are visualized by irradiation with UV light (254 nm). The used solvent system is chloroform: methanol (9:1) and ethyl acetate: toluene (1:1). (2) was prepared according to the reported method [37]. 6-Chloro-1-propyluracil (3) was prepared according to the reported method [38].

Evaluation of the antitumor activity Mammalian cell lines
The cell line that used in this study was human lung carcinoma cell line (A549 cells) is obtained from tissue culture Unit, VACSERA, Cairo, Egypt.
The mammalian cells are propagated in Dulbecco's modified Eagle's [41] medium (DMEM) or RPMI-1640 depending on the type of cell line supplemented with 10% heat-inactivated fetal bovine serum, 1% l-glutamine, HEPES buffer and 50 µg/mL gentamycin. All cells are maintained at 37 °C in a humidified atmosphere with 5% CO 2 and are subcultured two times a week along experimentation.

i-Antitumor activity evaluation using viability assay
Antitumor activity assay is carried out according to the method described literature [42]. All the experiments concerning the cytotoxicity evaluation are performed and analyzed by tissue culture unit at the regional center for mycology and biotechnology RCMB, Al-Azhar University, Cairo, Egypt.

Procedure
The A549 tumor cells are seeded in 96-well plate in 100 µL of growth medium at a cell concentration of 1 × 10 4 cells/well. After 24 h of seeding, the monolayers are then washed with sterile phosphate buffered saline (0.01 M pH 7.2) and simultaneously the cells are treated with 100 µL from different dilutions of the test sample in fresh maintenance medium and incubated at 37 °C. Different two-fold dilutions of the tested compound (started from 500 to 0.25 µM) are added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette. The microtiter plates are incubated at 37 °C in a humidified incubator with 5% CO 2 for a period of 72 h. Untreated cells are served as controls. Three independent experiments are performed each containing six replicates for each concentration of the tested samples. The cytotoxic effects of the tested compounds are then measured using crystal violet staining viability assay. Briefly, after 72 h of treatment, the medium is removed, 100 μL of 0.5% of crystal violet in 50% methanol is added to each well and incubated for 20 min at room temperature and subsequently excess dye is washed out gently by distilled water. The plate is allowed to dry then the viable crystal violet-stained cells are lysed using 33% glacial acetic acid solution. Absorbance at 570 nm is then measured in each well using microplate reader (Sunrise, TECAN, Inc, USA). Toxoflavin and 5-fluorouracil are used as positive control. The absorbance is proportional to the number of surviving cells in the culture plate.