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A Novel Synthesis of Fused Uracils: Indenopyrimidopyridazines, Pyrimidopyridazines, and Pyrazolopyrimidines for Antimicrobial and Antitumor Evalution

A variety of different compounds of fused uracils were prepared simply by the heating of 6-hydrazinyl-1-methyl-, 6-hydrazinyl-1-propyl-, or 6-hydrazinyl-1,3-dipropyluracil under reflux with ninhydrin, isatin, benzylidene malononitrile, benzylylidene ethyl cyanoacetate, benzil, and phenacyl bromide derivatives. The newly synthesized compounds were completely screened for antimicrobial and antitumor activity.


Introduction
For the last several decades, fused pyrimidine derivatives have become a significant attraction in the field of medicinal chemistry research. This is attributed to the fact that pyrimidine is the basic unit of DNA and RNA structure. This fact explains the wide range of pharmacological activities of pyrimidine derivatives. Pyrazolo [3,4-d]pyrimidine derivatives are a class of fused pyrimidines possessing significant biological activities [1,2]. They act as purine analogs [3] and many of their derivatives act with antimicrobial [2,4,5], antiviral [1,6], antimetabolites [7], anticancer [8,9], anti-inflammatory [10][11][12], and xanthine oxidase inhibitor activities [13,14].
Furthermore, pyrimidopyridazine derivatives have a significant interest owing to the fact that they have a potent pharmacological effect as therapeutic agents [15][16][17]. They have monoamine oxidase (MAO) inhibitory effect and subsequent modification on the diazine ring results in different inhibitory activities [18]. MAO inhibitory drugs play an important role in clinical management of depression, as well as Alzheimer's disease [19].
On account of these facts, a new series of substituted pyrimidopyridazines and pyrazolopyrimidines have been synthesized starting from 6-hydrazinyluracil derivatives and their antimicrobial, as well as antitumor activity has been evaluated and reported. The mechanism formation of 4a-c is shown in Scheme 2. The mechanism formation of 4a-c is shown in Scheme 2. On the other hand, the reaction of 3b,c with even benzylidene malononitrile or benzylidene ethyl cyanoacetate derivative via Michael addition reaction by heating under reflux for 6-8 h in DMF in the presence of triethylamine as basic medium furnished the same products of pyrazolopyrimidines 6a-f, as shown in Scheme 3 by the elimination of malononitrile and ethyl cyanoacetate moieties, respectively, as shown in Scheme 4. Compounds 6a-f were confirmed on the basis of analytical and spectral data. The 1 H-NMR spectrum showed a characteristic singlet at δ 10.95, 11.19 ppm for NH(5) of compounds 6a and 6b, respectively, a singlet around δ 7.95-7.50 ppm for NH(1) and characteristic signals for the phenyl group around δ 7.73-6.72 ppm for compounds 6a-f. 13  On the other hand, the reaction of 3b,c with even benzylidene malononitrile or benzylidene ethyl cyanoacetate derivative via Michael addition reaction by heating under reflux for 6-8 h in dimethylformamide (DMF) in the presence of triethylamine as basic medium furnished the same products of pyrazolopyrimidines 6a-f, as shown in Scheme 3 by the elimination of malononitrile and ethyl cyanoacetate moieties, respectively, as shown in Scheme 4. Compounds 6a-f were confirmed on the basis of analytical and spectral data. The 1 H-NMR spectrum showed a characteristic singlet at δ 10.95, 11.19 ppm for NH(5) of compounds 6a and 6b, respectively, a singlet around δ 7.95-7.50 ppm for NH(1) and characteristic signals for the phenyl group around δ 7.73-6.72 ppm for compounds 6a-f. 13 C-NMR for compounds 6b and 6e showed 12 and 17 signals characteristic for carbon atoms respectively. The expected mechanism for the reaction of 6-hydrazinyl uracil with benzylidene malononitrile and/or benzylidene ethyl cyanoacetate (Scheme 4).   Heating of 3b,c under reflux conditions for 4-5 h with benzil in dimethylformamide in the presence of triethylamine furnished 7a,b in moderate yield, as shown in (Scheme 3). On the other hand, compound 7a was also obtained via heating of 3b with α-phenyl phenacyl bromide under reflux conditions for 5 h. Compounds 7a,b were confirmed on the basis of analytical and spectral data. 1 H-NMR spectra of 7a showed a singlet at δ 11.72 characteristic for NH (6) and signals of phenyl groups for 7a,b around δ 7.26-7.10 ppm. 13 C-NMR showed 16 signals characteristic for carbon atoms of compound 7a.
Finally, reaction of 3b with different phenacyl bromides such as phenacyl-, p-methoxyphenacyl-, and p-nitrophenacyl bromide by heating under reflux for 4-6 h in dimethylformamide in the presence of triethylamine afforded pyrimidopyridazines 8a-c in moderate yields (Scheme 3). Compounds 8a-c were identified on the basis of analytical and spectral data. The 1H-NMR spectra showed a singlet around δ 12.08-11.96 ppm characteristic for NH(6), a characteristic singlet aromatic proton at CH(4) around δ 8.69-8.43 ppm of pyridazine ring, and signals of phenyl groups around δ 8.69-7.00 ppm. 13 C-NMR showed 14 signals for compound 8b characteristic for carbon atoms.
The expected mechanism for the reaction of 6-hydrazinyl uracil with benzylidene malononitrile and/or benzylidene ethyl cyanoacetate (Scheme 4).

Antimicrobial Screening
As shown in Table 1, the newly-synthesized compounds tested displayed variable in vitro antibacterial and antifungal activities. From the screening results, it can be seen that compound 4b showed the highest activity against Gram-positive bacteria Bacillus subtilis compared with the standard drug, followed by compounds 4c, 5a, 6b, 6d, 4a, 5b, 6c and 6f, respectively. Similarly, compound 4b showed the highest activity against Gram-positive bacteria Streptococcus pneumonia in comparison to the standard drug, followed by compounds 5a, 4c, 6b, 6d, 6c, 4a, and 5b, respectively. On the other hand, compound 4b showed the highest activity against Gram-negative bacteria Escherichia coli compared with the standard drug, followed by compounds 4c, 6b, 5a, 6d, 4a, and 6c, respectively. However, the order of activity against Pseudomona aeruginosa was 4b, followed by compounds 5a, 4c, 6b, 6d, 6c, 4a, and 5b, respectively. Regarding the activity of the tested compounds against the tested filamentous fungus Aspergillus fumigatus, the order of activity being 4b, 4c, 6b, 5a, 6d, 4a, 6c, 6f, respectively. Compound 6f showed a weak antimicrobial effect on Gram-positive bacteria Bacillus subtilis as well as the tested filamentous fungus Aspergillus fumigatus. No antimicrobial activities were detected for compounds 7a and 7b. None of the tested compounds exert any activity against the pathogenic yeast species (Candida albicans) under these screening conditions. Scheme 4. The expected mechanism for the reaction of 6-hydrazinyluracil with malononitrile.
Heating of 3b,c under reflux conditions for 4-5 h with benzil in dimethylformamide in the presence of triethylamine furnished 7a,b in moderate yield, as shown in (Scheme 3). On the other hand, compound 7a was also obtained via heating of 3b with α-phenyl phenacyl bromide under reflux conditions for 5 h. Compounds 7a,b were confirmed on the basis of analytical and spectral data. 1 H-NMR spectra of 7a showed a singlet at δ 11.72 characteristic for NH (6) and signals of phenyl groups for 7a,b around δ 7.26-7.10 ppm. 13 C-NMR showed 16 signals characteristic for carbon atoms of compound 7a.
Finally, reaction of 3b with different phenacyl bromides such as phenacyl-, p-methoxyphenacyl-, and p-nitrophenacyl bromide by heating under reflux for 4-6 h in dimethylformamide in the presence of triethylamine afforded pyrimidopyridazines 8a-c in moderate yields (Scheme 3). Compounds 8a-c were identified on the basis of analytical and spectral data. The 1 H-NMR spectra showed a singlet around δ 12.08-11.96 ppm characteristic for NH(6), a characteristic singlet aromatic proton at CH(4) around δ 8.69-8.43 ppm of pyridazine ring, and signals of phenyl groups around δ 8.69-7.00 ppm. 13 C-NMR showed 14 signals for compound 8b characteristic for carbon atoms.
The expected mechanism for the reaction of 6-hydrazinyl uracil with benzylidene malononitrile and/or benzylidene ethyl cyanoacetate (Scheme 4).

Antimicrobial Screening
As shown in Table 1, the newly-synthesized compounds tested displayed variable in vitro antibacterial and antifungal activities. From the screening results, it can be seen that compound 4b showed the highest activity against Gram-positive bacteria Bacillus subtilis compared with the standard drug, followed by compounds 4c, 5a, 6b, 6d, 4a, 5b, 6c and 6f, respectively. Similarly, compound 4b showed the highest activity against Gram-positive bacteria Streptococcus pneumonia in comparison to the standard drug, followed by compounds 5a, 4c, 6b, 6d, 6c, 4a, and 5b, respectively. On the other hand, compound 4b showed the highest activity against Gram-negative bacteria Escherichia coli compared with the standard drug, followed by compounds 4c, 6b, 5a, 6d, 4a, and 6c, respectively. However, the order of activity against Pseudomona aeruginosa was 4b, followed by compounds 5a, 4c, 6b, 6d, 6c, 4a, and 5b, respectively. Regarding the activity of the tested compounds against the tested filamentous fungus Aspergillus fumigatus, the order of activity being 4b, 4c, 6b, 5a, 6d, 4a, 6c, 6f, respectively. Compound 6f showed a weak antimicrobial effect on Gram-positive bacteria Bacillus subtilis as well as the tested filamentous fungus Aspergillus fumigatus. No antimicrobial activities were detected for compounds 7a and 7b. None of the tested compounds exert any activity against the pathogenic yeast species (Candida albicans) under these screening conditions. The minimum inhibitory concentration of the six most active synthesized compounds were detected, as shown in Table 2. It was shown that 4b showed the highest potential where its minimum inhibitory concentration (MIC) was comparable with that of the standard compounds, whereas 4a showed the lowest potential and a very high MICs in comparison to the standard.

Anticancer Activity
The in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with the well-known anticancer standard drug 5-flourouracil under the same conditions using colorimetric viability assay. Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of cell population (IC 50 ) was determined. The results revealed that all the tested compounds showed inhibitory activity to the tumor cell lines in a concentration dependent manner. Cytotoxic activity was expressed as the mean IC 50 of three independent experiments. The results are represented in Table 3 and Figure 1a,b showed that compound 4a was the most active against the breast carcinoma cell line (MCF-7), compared with the reference drug with IC 50 values of 3.6 and 4.1 µg/mL, respectively. Interestingly, compounds 4a, 4c, and 8a exhibited potent antitumor activity against breast cancer, respectively, and were the most active among their analogues. Moreover, the other compounds were less active.

General
All melting points were determined with an Electrothermal Mel.-Temp. II (Registered trademark of Barnstead, Barnstead, NH, USA) apparatus and were uncorrected. Element analyses were performed at Regional Center for Mycology and Biotechnology at Al-Azhar University. The infrared (IR) spectra were recorded using a potassium bromide disc technique on a Nikolet IR 200 FT IR spectrometer (Thermo Electron Scientific Instruments LLC, Madison, WI, USA) and carried out in Taif University, Taif, KSA. Mass spectra were recorded on DI-50 unit of Shimadzu GC/MS-QP 5050A mass spectrometer (Shimadzu Corporation, Tokyo, Japan) at the Regional Center for Mycology and Biotechnology at Al-Azhar University. 1 H-NMR and 13 C-NMR spectra were recorded in DMSO-d6 as a solvent using a Varian Mercury spectrometer at 400 MHz and 125 MHz, respectively, Applied Nucleic

General
All melting points were determined with an Electrothermal Mel.-Temp. II (Registered trademark of Barnstead, Barnstead, NH, USA) apparatus and were uncorrected. Element analyses were performed at Regional Center for Mycology and Biotechnology at Al-Azhar University. The infrared (IR) spectra were recorded using a potassium bromide disc technique on a Nikolet IR 200 FT IR spectrometer (Thermo Electron Scientific Instruments LLC, Madison, WI, USA) and carried out in Taif University, Taif, KSA. Mass spectra were recorded on DI-50 unit of Shimadzu GC/MS-QP 5050A mass spectrometer (Shimadzu Corporation, Tokyo, Japan) at the Regional Center for Mycology and Biotechnology at Al-Azhar University. 1 H-NMR and 13 C-NMR spectra were recorded in DMSO-d 6 as a solvent using a Varian Mercury spectrometer at 400 MHz and 125 MHz, respectively, Applied Nucleic Acid Research Center, Zagazig University, Egypt. Chemical shifts (δ) are given in ppm and coupling constants (J) are given in Hz. All reactions were monitored by TLC using pre-coated plastic sheet silica gel (0.25 mm, 20 × 20 cm, 60F 254 , E. Merck KGaA, Konstanz, Germany) and spots were visualized by irradiation with UV light (254 nm). The used solvent system was chloroform:methanol (9:1) and ethyl acetate:toluene (1:1).
The newly-synthesized target compounds were tested in vitro against different types of bacteria, Streptococcus pneumoniae and Bacillus subtilis as examples of Gram-positive bacteria, and Pseudomonas aeruginosa and Escherichia coli as examples of Gram-negative bacteria. Fungi, as well as bacteria, were used for testing the antifungal activity of the synthesized compounds. Aspergillus fumigates and Candida albicans were used as example of fungi and yeast, respectively. The stock solution of concentrations (1 mg/mL) of the synthesized compounds were used. The plates were incubated at 37 • C for 24 h for bacteria and yeast, and for 48-72 h for fungi. Tetracycline was used as the standard antibacterial drug while amphotericin B was used as the standard antifungal drug. The diameters of the inhibition zones (mm) were measured and used as criterion for the antimicrobial activity.

Determination of the Minimum Inhibitory Concentration (MIC)
Serial dilutions of the promising compounds were subjected to MIC determination. The different concentrations of each compound were tested with the modified agar diffusion cylinder method as was described before.

Evaluation of the Antitumor Activity Using Viability Assay
All human anticancer cell lines were obtained from the American Type Culture Collection. The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were maintained at 37 • C in a humidified atmosphere with 5% CO 2 and were subcultured two to three times a week. For antitumor assays, the tumor cell lines were suspended in medium at concentrations of 5 × 10 4 cell/well in Corning ® 96-well tissue culture plates, then incubated for 24 h. The tested compounds were then added into 96-well plates (three replicates) to achieve eight concentrations for each compound. Six vehicle controls with media or 0.5% DMSO were run for each 96-well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by staining the cells with crystal violet [37,38], followed by cell lysing using 33% glacial acetic acid and read the absorbance at 590 nm using microplate reader (Sunrise, TECAN, Inc., Morrisville, NC, USA) after well mixing. The percentage of viability was calculated as [1−(ODt/ODc)] ×100%, where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to obtain the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots [37].

Conclusions
The newly synthesized compounds of indeno [2,1-c]pyrimido [5,4-e]pyridazines, oxoindolinylidene hydrazinyl pyrimidines, pyrazolo [3,4-d]pyrimidines and pyrimido [4,5-c]pyridazines were prepared by a simple method. The novel compounds were screened for both antimicrobial and anticancer activities. Compound 4b showed a very high MICs in comparison to the standard drug tetracycline. Compounds 4a, 4c and 8a exhibited potent antitumor activity against breast cancer in comparison to the standard drug 5-flourouracil.