One pot synthesis, antimicrobial and antioxidant activities of fused uracils: pyrimidodiazepines, lumazines, triazolouracil and xanthines

Background Uracil derivatives have a great attraction because they play an important role in pharmacological activities. Pyrimidodiazepines, lumazines, triazolopyrimidines and xanthines have significant wide spectrum activities including anticancer, antiviral as well as antimicrobial activities. Results A newly synthesized compounds pyrimido[4,5-b][1, 4]diazepines 5a–e, 6a–d, lumazines 7a–d, triazolo[4,5-d]pyrimidine 8 and xanthines 9, 10 was prepared in a good yields. The antimicrobial and antioxidant activities of compounds 5a, 5b, 6a, 6d and 8 exhibited a wide range activity against the pathogenic tested microbes (Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Candida albicans, and Saccharomyces cerevisiae). Compound 8 showed activity against the fungus Aspergillus niger. The highest antioxidant activity was noticed for compound 5a. Conclusions A series of novel pyrimido[4,5-b][1, 4]diazepines 5a–e, 6a–d, lumazines 7a–d, triazolo[4,5-d]pyrimidine 8 and xanthines 9, 10 was prepared from 5,6-diamino-1-(2-chlorobenzyl)uracil 3 in good yields. Compounds 5a–e, 6a–d were prepared by sequential manipulation of 3 with α,β-unsaturated ketones. Lumazines 7a–d were obtained from 3 by treatment with phenacyl bromides in the presence of TEA. Compound 8 was prepared by treatment of 3 with HNO2, while xanthines 9, 10 were obtained from 3 by consecutive acetylation then intramolecular cyclodehydration or heating with malononitrile under solvent-free condition. The antimicrobial and antioxidant activity of this series was evaluated in vitro and they showed either weak or moderate activities.Graphical abstract Several pyrimido[4,5-b][1,4]diazepines, lumazines, triazolo-, and imidazolopyrimidines were synthesized from the starting compound 4,5-diaminouracils. The newly synthesized compounds were screened for both antimicrobial and antioxidant activities.

To solve these problems, researchers are required to modify the structure of uracil and subsequently these problems can be overcome by innovation of new derivatives with beneficial pharmacological and pharmacokinetic effects. These new fused uracil derivatives as antibacterial agents can be obtained via replacement at N-1, N-3, C-5 and C-6 positions with different substituents on uracil ring. Seven-member heterocyclic compounds containing nitrogen atom, such as 1,4-diazepine derivatives, are considered as an important drug discovery because they have a wide range of antimicrobial activities [29].
The purpose of this study is to evaluate the in vitro effect of antimicrobial fused uracil derivatives, pyrimidodiazepines, lumazines, triazolouracil and xanthines. Simultaneously, a MIC-kinetic curve for the inhibition activity of the new molecules was also obtained. The structure of newly synthesized uracil-based derivatives was proven on the basis of their 1 H-NMR, mass spectral data, IR and elemental analysis.

Chemistry
To our endeavor toward developing new uracil-based architectures of potential pharmacological significance, 5,6-diamino-1-(2-chlorobenzyl)uracil 3 [30] was chosen as scaffold for annulations of the target congeners. This substrate was prepared from 1-(2-chlorobenzyl) urea by consecutive cyclization with ethylcyanoacetate in the presence of sodium ethoxide [31][32][33], nitrosation with in situ prepared HNO 2 [30,34] then reduction with (NH 4 ) 2 S [30] (Scheme 1). Series 5a-e was prepared in moderate yield (49-66%) by refluxing compound 3 with different arylidene ethylcyanoacetates in DMF containing TEA for 6-7 h. All derivatives were recrystallized from DMF/EtOH. The reaction proceeded through Michael addition reaction via the formation of non-isolated Michael adduct intermediate that undergo cyclocondensation accompanied by elimination of EtOH followed by oxidation affording the corresponding 1-(2-chlorobenzyl)-8-hydroxy-6-(aryl)-2,4-dioxo-2,3,4,5-tetrahydro-1Hpyrimido [4,5-b][1, 4]diazepine-7-carbonitrile. The IR spectra of these diazepines displayed the C≡N stretching band at 2222-2217 cm −1 confirming cyclization, the stretching band of the two C=O groups (Amide I) was red-shifted within the range 1690-1610 cm −1 . Derivatives 5d, e displayed two separate bands for the two C=O groups. The imide linkages in this series displayed ketoiminol tautomerism, since they showed O-H stretching bands 3634-3617 cm −1 and additional O-H stretching bands in compound 5d at 3495 cm −1 and N-H stretching bands 3164-3141 cm −1 . The nitro group in compound 5e showed strong asymmetric and symmetric NO 2 stretching bands at 1518 and 1350 cm −1 , respectively. The intrinsic significance of the IR spectra is that they exclude the possibility of the cyclization pass way that lead to compounds 4a-e due to absence of any blue-shifted C=O stretching bands.
The 1 H-NMR spectra supported the previous observation from the IR spectra, where compounds 4a-e are excluded, as the ethyl fingerprint signals were not observed. The singlet of the NCH 2 protons (δ 5.25-5.23 ppm) were the most shielded as expected, while the C8-OH and N3-H were highly deshielded. They appeared around δ 14.0 and 11.4 ppm, due to flanking of the N3-H between the two C=O groups and strong magnet anisotropic effect of the imine linkage on C8-OH group. Thus, the N5-H signal is most likely to be overlapped with the signals of the aromatic protons. The downfield shift of the C=O groups in the 13 C-NMR spectra, for instance 5b, is typical for imides as sequel of bond order reduction by keto-iminol tautomerism or overlap of the nitrogen's lone-pair of electrons with the π-cloud of the C=O group.
Refluxing of 3 with different arylidenemalononitriles in refluxing DMF containing TEA afforded the corresponding 8-Amino-1-aryl-6-(4-chlorophenyl)-2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrimido [4,5-b][1, 4]diazepine-7-carbonitriles (6a-d) in 53-69% yields after recrystallization from DMF/ EtOH (Scheme 1). The reaction proceeded exactly as for compounds 5a-e; Michael addition then cycloaddition on one nitrile group as unique possible lane. The IR spectra were in accordance with the proposed structures and the common bands with compounds 5a-e were within similar frequencies ranges. The most interesting conclusion from comparing the 1 H-NMR spectra of these derivatives with compounds 5a-e is the absence of the signal at δ 14.36-13.98 ppm in compounds 6a-d. This confirms without doubt that this signal is attributed to the C8-OH group in compounds 5a-e, the group that does not exist in compounds 6a-d. The signals at δ 7.77-7.54 ppm are believed to be for the C8-NH 2 protons. A reasonable mechanism for this reaction is shown in (Scheme 2).
Pteridine is a basic component of folic acid, bacteria use it as starting material for its own multi stage tetrahydrofolic acid`s (FH 4 ) biosynthesis and, consequently the production of nucleic acid bases necessary for its replication. Sulphonamides (sulpha drugs) are common inhibitors of FH 4 biosynthesis and act as bacteriostatic. Therefore, substrate 3 was treated with different phenacyl bromides in refluxing DMF containing TEA to afford lumazines 7a-d in good yields as potential folate antagonists (Scheme 3).
Formation of lumazines 7a-d, presumably proceeded via S N 2 alkylation of C5-NH 2 followed by aromatization through synchronous dehydration and oxidation steps (Scheme 4).
The IR spectra of this series showed the N-H stretching bands within the range 3174-3100 cm −1 . The two C=O groups gave rise to two bands ≈1725 and ≈1680 cm −1 . Pteridine 7d displayed the two characteristic bands of the NO 2 group at 1515, 1368 cm −1 .
The 1 H-NMR spectra of compounds 7a, b and d showed characteristic singlet for the N-H protons at δ 12.15-12.00 ppm and a singlet at δ 9.32-9.14 ppm for H-6. Compound 7b showed a signal at δ 3.82 ppm for the methyl group, besides the CH 2 signal at δ 5.44 ppm. The shift of the CH 3 signal was observed at δ 42.2 ppm in the 13 C-NMR spectrum. Triazolopyrimidine 8 was prepared in good yield by cyclocondensation of substrate 3 with in situ prepared HNO 2 at ambient temperature. The triazole's N-H signal was abnormally observed highly deshielded at δ 15.76 ppm, beside the pyrimidine N3-H at δ 11.61 ppm.
The shift of the CH 2 carbon was observed normally at δ ≈44.30 ppm in the 13 C-NMR spectrum.
Xanthine 9 was prepared in 72% yield by refluxing of substrate 3 with Ac 2 O in AcOH. The 1 H-NMR spectrum showed characteristic two broad singlets for the 2N-H protons at δ 13.19 and 11.15 ppm. The CH 3 signal appeared upfiled at δ 2.31 ppm and its carbon appeared at δ 14.20 ppm in the 13 C-NMR spectrum. Surrogate 10 was prepared in 77% yield from compound 3 by heating TEA 5a -e Scheme 2 Plausible mechanism for the formation of compounds 5a-e and 6a-d with CH 2 (CN) 2 under solvent-free condition. The IR spectrum displayed the C≡N stretching band at proper frequency 2200 cm −1 , while the 1 H-NMR disclosed two signals at δ 5.08 ppm for the NCH 2 protons and at δ 4.10 ppm for the protons in the CH 2 CN group. This series displayed, in their EI-MS spectra, molecular ions peaks corresponding to the mass of each formula and their elemental analyses agreed as well.

Biological activity Antimicrobial activity
Antimicrobial activity assay results (Table 1) revealed that compound 6b exhibited low to moderate activity only against Pseudomonas aeruginosa. Compound 7a exhibited low to moderate activity only against Saccharomyces cerevisiae. Some other compounds (5a, 5b, 6a, 6d and 8) exhibited activities against wide range of pathogenic tested microbes. The minimal inhibitory concentrations (MIC) of these compounds had been measured (Table 2). MIC is the lowest concentration of substance that inhibits the growth of microorganism.
Compound 5a exhibited low activity against Staphylococcus aureus; low to moderate activity against P. aeruginosa, Bacillus subtilis and S. cerevisiae, but showed moderate to strong activities against Candida albicans (Fig. 1).
Compound 5b exhibited low to moderate activity against S. aureus, B. subtilis and C. albicans, but showed moderate to strong activities against P. aeruginosa and S. cerevisiae (Fig. 2).
Compound 6a exhibited moderate activity against S. aureus, B. subtilis and C. albicans, but showed low activity against P. aeruginosa, and showed no activity against S. cerevisiae and Aspergillus niger (Fig. 3).
Compound 6d exhibited moderate to strong activity against all test microbes except for the fungus A. niger (Fig. 4). Compound 8 was the only compound that exhibited activity against the fungus A. niger. Also, it exhibited moderate activity against S. aureus; strong activity against S. cerevisiae, and moderate to strong activity against P. aeroginosa but showed no activity against B. subtilis and C. albicans (Fig. 5). Plausible mechanism for the formation of lumazines 7a-d.

Antioxidant activity
The percentages of antioxidant activity (AA%) of compounds (5a-e, 6a-d, 7a-c and 8-10) have been measured (Table 3) and the results revealed that the compound 5a showed the highest activity (39.9%) followed by the compound 8. The lowest antioxidant activity recorded for the compound 6c is 1.9. Two compounds 7a and 7b showed no antioxidant activity.

Experimental section
Materials and instruments All melting points were determined by an Electrothermal Mel.-Temp. II 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 potassium bromide disc technique on Nikolet IR 200 FT IR. Mass spectra were recorded on DI-50 unit of Shimadzu GC/MS-QP 5050A at the Regional Center for Mycology and Biotechnology at Al-Azhar University. The proton nuclear magnetic resonance ( 1 H-NMR) spectra were recorded on Bruker 400 MHz Spectrometer and 13 C-NMR spectra were run at 125 MHz in dimethylsulfoxide (DMSO-d6) and TMS as an internal standard, Applied Nucleic Acid Research Center, Zagazig University, Egypt. All new compounds gave corresponding elemental analyses (C, H, N, typically ±0.3%). All reactions were monitored by TLC using precoated plastic sheets silica gel (Merck 60 F 254 ) 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).

Biological activity assay Antimicrobial activity assay
The antimicrobial activity was measured using two different agar diffusion methods; paper-disk and agar-well diffusion methods. Samples were dissolved in DMSO. Aliquots of 20 µl (conc. 50 mg/ml) were soaked on filter paper disks (5 mm diameter, Wattman no. 1) and left to dry under aseptic conditions for 1 h. Paper-disk diffusion assay [35] with some modifications has been followed to measure the antimicrobial activity. Twenty milliliters of medium seeded with test organisms were poured into 9 cm sterile Petri dishes. After solidification, the paper disks were placed on the inoculated agar plates and allowed to diffuse the loaded substances into refrigerator at 4 °C for 2 h to allow the diffusion of substances. The plates were incubated for 24 h at 35 °C. Both bacteria and yeasts were grown on nutrient agar medium (g/l): Beef extract, 3; peptone, 10; and agar, 20. The pH was adjusted to 7.2. Fungal strain was grown on potato dextrose agar medium (g/l): Potato extract, 4; Dextrose, 20; Agar No. 1 15 (pH 6). The diameter of inhibition zone was measured. In the agar-well diffusion method [36], cups (5 mm in diameter), were cut using a sterile cork borer and the agar discs were removed. Cups were filled with 20 μl of samples. Benzylpenicillin and Nystatin were used as antibacterial and antifungal control, respectively. After incubation, the diameter of inhibition zones was measured against a wide range of test microorganisms comprising: Gram positive bacteria; (B. subtilis ATCC6633 and S. aureus ATCC6538-P), Gram negative bacteria (P. aeruginosa ATCC 27853), yeasts (C. albicans ATCC 10231 and S. cerevisiae ATCC 9080) and the fungus A. niger NRRL A-326. Minimal inhibition concentrations (MIC) of the active compounds have been determined using disk diffusion method according to methods described in [37,38]. Tenth fold dilutions of starting concentration had been done to make different concentrations.

Antioxidant activity assay
The percentage of antioxidant activity (AA%) was measured using DPPH free radical assay as described by [39]. The samples were reacted with DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) in DMSO solution. The reaction mixture consisted of 50 µl (conc. 2.5 mg/ml) of each sample, 3 ml of 0.5 mM DPPH/DMSO solution. The reduction of DPPH by antioxidant compounds changes the color from deep violet into light yellow. The absorbance was read at 517 nm after 60 min of reaction using a UV-Vis spectrophotometer (Shimadzu). The mixture of DMSO (3 ml) and sample (50 µl) serve as blank. The control is 3 ml of prepared DPPH solution (0.5 mM). The scavenging activity percentage (AA%) was calculated according to Ref. [40].