Synthesis and Biological Evaluation of New Chromenes and Chromeno[2,3-d] pyrimidines

A simple and efficient approach has been developed to synthesise novel and functionalised 5H-chromeno[2,3-d] pyrimidines derivatives (4a–h). This approach entails treating 2-amino-3-cyano-4H-chromenes (3a–h) with formamidine acetate under microwave irradiations and solvent-free conditions. All structures of new compounds obtained in this study were characterised by IR, MS, 1H and 13C NMR analysis. Additionally, the synthesised compounds were investigated for their antibacterial and antioxidant potential. Compounds 3b, 3c, 3e, 4c and 4e showed significant activities.


Introduction
The fusion of chromene fragments with different heterocycle scaffolds gives rise to a new class of hybrid heterocycles: 5H-chromeno [2,3-d] pyrimidine. These motifs are wellestablished in the literature as important biologically effective heterocyclic compounds. 1,2,3,4,5 These compounds are the subject of many research studies due to their large potential for pharmacological activities. These activities include antitumoral, 6 antibacterial, 7 anti-fungicidal, analgesic, 8 anti-allergic, 9 cardiotonic, hepatoprotective, 10 antihypertensive, 11 activity.
In continuation of our efforts toward constructing heterocyclic compounds, 12,13,14,15 the present work reports an efficient process for the synthesis of 5H-chromeno [2,3-d] pyrimidines derivatives. The key step in our strategy is based on the condensation of 2-amino-3-cyano-4H-chromenes with formamidine acetate. The structures of these new compounds were characterised by spectroscopic analysis and evaluated for their antioxidant and antibacterial activities.

Results and Discussion
The synthesis of the new 5H-chromeno[2,3-d] pyrimidine derivatives was obtained through a two-step method, as shown in Scheme 1. The first step was based on the synthesis of 2-amino-3-cyano-4H-chromenes (3a-h). This step was followed by cyclisation and condensation to the 5H-chromeno [2,3-d] pyrimidines in the second step.

Synthesis of 2-amino-3-cyano-4H-chromenes (3a-h)
The general synthesis of 2-amino-3-cyano-4H-chromenes includes the reaction of arylidene malononitriles and β-dicarbonyl compounds in the presence of piperidine, 16 triethylamine, 17 Ca(OH) 2 18 or TFE. 19 Most of these methods involve the use of volatile solvents, expensive reagents and require longer reaction times. We found that these reactions can be carried out without a base and a catalyst. Consequently, 2-amino-3-cyano-4H-chromenes (3a-h) were obtained by condensation of stoichiometric amounts of substituted resorcinol, malononitrile and aromatic aldehydes under microwave for 5 min (Scheme 2).
The results obtained for the preparation of compound 3a-h, with excellent yield (82-97%), are reported in Table 1. The use of microwave allowed us to reduce the reaction time from 24 h to 5 min. NMR spectra of 3a-h showed characteristic signals for 4H-chromenes: singlets at δ H 4.49-4.67 ppm in the 1 HNMR and at δ C 54.87-56.97 ppm in the 13 CNMR. The IR spectra showed a CN stretch at n 2197-2223 cm −1 , NH 2 stretch at n 3420-3362 cm −1 , CO stretch at n 1670-1675 cm −1 and OH at n 3420-3446 cm −1 . For all the compounds, the mass spectra gave additional evidence for the proposed structures.
The proposed mechanism for the formation of 2-amino-3cyano-4H-chromenes (3a-h) is described in Scheme 3. First, we have the formation of alkene I by the Knoevenagel condensation of aromatic aldehyde and malononitrile. A
The results obtained for the synthesis of compound 4a-h with good yield (85-95%) are summarised in Table 2.
The mechanism proposed for forming 4-amino-5Hchromeno [2,3-d] pyrimidines (4a-h) was described in Scheme 5. The reaction begins with the formation of the intermediate I.
The latter undergoes a nucleophilic addition of the NH 2 group Scheme 3 Proposed mechanism for the formation of 2-amino-3-cyano-4H-chromenes derivatives (3a-h)  The formation of III is followed by intramolecular cyclisation of the NH 2 group with the CN group to give intermediate IV.
After rearrangement and a step of aromatisation, 4-amino-5Hchromeno[2,3-d] pyrimidine was obtained. The antibacterial activity was evaluated using the broth microdilution method to estimate the Minimal Inhibitory Concentration (MIC) according to the Clinical and Laboratory Standards Institute (CLSI) recommendations (CLSI, 2012). A series of two-fold dilutions from 1024 to 2 µg ml −1 were prepared in a 96 well sterile microplate. These dilutions were inoculated with 100 µl of a solution containing 10 6 colony forming units (CFU).

Determination of antibacterial and antioxidant activity -The disc diffusion method
Gentamicin and Ciprofloxacin were used as positive controls. The microplate was incubated at 37°C for 24 h. The MIC was defined as the low concentration in which there was no turbidity.

Scavenging activity
In our study, the scavenging activity of the compounds was carried out by the technique of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging. The effect of each compound on DPPH was measured by the procedure described by Da Silva Pinto et al. 26 A volume of 5 µl of various concentrations of each compound was added to 1950 µl of the methanol solution of DPPH. The negative control was prepared by mixing 50 µl of methanol with 1950 µl of the methanol solution of DPPH. The absorbance was measured at 517 nm against a blank of each concentration. These measurements were done after incubation in the dark for 30 min, using a spectrophotometer. The positive control was a solution of the synthetic antioxidant butylated hydroxyanisole (BHA) with concentrations ranging from 0.02 to 0.2 mg ml -1 . The absorbance was measured in the same conditions as the samples. The results were expressed taking into account the average of three measurements obtained for each sample, and the percentage of DPPH reduction was calculated using the formula: where DPPH (%): Reduction percentage of DPPH. A Control : Absorbance of the negative control tube. A Sample : Absorbance of the sample.
The IC 50 value is the concentration that was reduced to 50% of the radical DPPH. It was determined graphically for each compound from the curve of the percentage reduction depending on the concentration.

Ferric reducing antioxidant power assay (FRAP)
The reducing power was determined by the measurement of absorbance at 700 nm. 27 A volume of 0.1 ml of our compounds in different concentrations (initial) was mixed with 0.25 ml of sodium phosphate buffer (0.2 M; pH = 6.6) and 0.25 ml of potassium ferricyanide 1% (w/v), and the mixture was incubated at 50°C for 20 min. After 0.25 ml of trichloroacetic acid (TCA), 10% (w/v) were added, the mixture was centrifuged at 650 g for 10 min the supernatant (0.5 ml) was mixed with 0.5 ml distilled water and 0.1 ml ferric chloride solution (FeCl 3 ) 0.1% (w/v). 28 The IC 50 value (mg compound/ ml) is the effective concentration at which the absorbance was 0.5 for reducing power and was obtained by interpolation from linear regression analysis. For the same conditions, BHA and ascorbic acid were used as a positive control for comparison.

Total antioxidant capacity (TAC)
The total antioxidant capacity of our compounds was evaluated with the phosphor-molybdenum technique. 29 An aliquot of 0.2 ml of different concentrations of our compounds was combined with 2 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were incubated in a thermal block at 95°C for 90 min. The absorbance of each solution was measured at 695 nm  Chem., 2021, 75, 150-155 https://journals.co.za/content/journal/chem/ against a blank. The antioxidant capacity was expressed in milligram equivalent of ascorbic acid per gram of compound (mg EAA g −1 C). The range of the calibration curve of the ascorbic acid was 0.10 to 0.80 mg ml −1 ( Table 3).
The MIC results of the synthesis products against references bacteria are shown in Table 4.
The higher activity against gram-negative bacteria was obtained toward Pseudomonas aeruginosa. The product 4e showed a MIC equal to 16 µg ml −1 . The products 3e, 3b and 3c showed MICs equal to 64 µg ml −1 . Compound 3d showed a MIC equal to 256 µg ml −1 . However, other gram-negative bacteria exhibited resistance to products tested. For grampositive bacteria, low MICs were obtained by the products 3b and 3c and 4e against the Bacillus cereus strain; the MICs range was 32 to 128 µg ml −1 . The compound 3e showed a MIC equal to 128 µg ml −1 against Bacillus cereus and Staphylococcus aureus.
The products 3b and 3c have Cl or Br halogen groups respectively at the 4-position, while the 3e has two Cl located in the 2-and 4-positions on the aromatic (phenyl) ring. The products with the electron-withdrawing groups in position 4 showed antibacterial activity with respect to gram-positive and -negative bacteria. These results are in accordance with the work of Nirav et al., 30 which focused on the synthesis and evaluation of the antimicrobial activity of a new series of 4H-chromenes against Staphylococcus aureus (S. aureus), and Bacillus subtilis (B. subtilis). The best result has been observed with chromene, which has a chlorine group. In the study of Sudhan and Mansoor, 31 the synthesis and evaluation of the antimicrobial activity of a new series of 7,8-dihydro-2-(2-oxo-2H-chromen-3yl)-5-aryl-cyclopenta[b]pyranopyrimidine-4,6-5H-dione were investigated; the results suggest that aromatic substituents strongly influence antibacterial and antifungal activities.
In the chromeno[2,3-d] pyrimidines, the products 4e, 4c, 4d and 4b showed interesting activities against Bacillus cereus (B. cereus) and Pseudomonas aeruginosa (P. aeruginosa). The best results were obtained with compound 4e, where the structure of the chromeno pyrimidine is composed of two fused heterocyclic units, namely, the substituted 4H-chromene and the pyrimidine. The pyrimidine has an amine in the 4-position of the ring. Chlorine is well known to have good bacterial activities, as shown in the case of 4e.
These results agree with the work of Agrod, 32 who found that the chlorine group linked to chromenopyrimidine heterocycles has good antibacterial activities compared to their analogues. The modification of the 4H-chromene structure through the chromenopyrimidine products allowed the improvement of the antibacterial activity.
In the study of Sankappa et al., 33  In the antioxidant study, two products (3e and 3d) of the 4H-chromenes family showed good DPPH scavenging activity. The products 3b and 3c have Cl or Br respectively at the 4-position, while the 3e has two Cl located in the 2-and 4-positions of the aromatic (phenyl) ring. The grafting of electron-withdrawing halide substituents in position 4 improve the DPPH trapping activity. In iron reduction and antioxidant capacity, the situation is reversed; products 3b and 3c showed the most interesting reduction.
Concerning chromenopyrimidines, the 4c product showed the most interesting DPPH and iron reduction trapping activity. The grafting of electron-withdrawing halide substituents on the phenyl nucleus seems to improve this activity. The most important antioxidant capacity was obtained with 4e. It seems that the grafting of the electron-donating groups improves this activity.
Finally, we can say that the importance of this research work lies in the possibility that an in-depth investigation of the structure-activity relationship, toxicity and biological effects of these compounds may be useful for designing more potent oxidising agents at higher concentrations for therapeutic purposes.

Experimental
The melting points were measured using a Bank Kofler HEIZBANK apparatus standard WME 50-260°C and were uncorrected. IR spectra were obtained with solids with a Fourier transform Perkin Elmer Spectrum One with ATR accessory. Only significant absorptions are listed in the results. The 1 H NMR spectra were recorded at 400 MHz on a Brüker AC 400   Chem., 2021, 75, 150-155 https://journals.co.za/content/journal/chem/ spectrometer, and 13 C NMR spectra were recorded on the same spectrometers at 100.6 MHz. Samples were dissolved in DMSO-d 6 , values for δ are in parts per million relative to tetramethyl silane (TMS) as an internal standard. Mass spectra were recorded on a QTOF Micro (Waters). Microwave irradiation experiments use a microwave reactor (Biotage). The multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet).

General procedure 1 for the synthesis of 2-amino-3-cyano-4H-chromenes (3a-h)
A mixture of the aromatic aldehyde (2a-e) (10 mmol), substituted resorcinol (1a-b) (10 mmol), malononitrile (10 mmol), H 2 O (2 ml) and carbonate of ammonium (0.5 g) was irradiated in a single-mode microwave at 300 W for 5 min under 12 bar pressure. The completion of the reaction was determined by thin-layer chromatography (TLC). After the completion of the reaction, the residue that formed was diluted with 30 ml of CH 2 Cl 2 . The organic layer obtained was washed with water (3 × 20 ml), then with a solution of saturated NaCl (10 ml), dried over MgSO 4 , filtered and evaporated under vacuum. The compounds 3a-h were obtained as white solids.

General procedure 2 for the synthesis of-amino-5H-chromeno[2,3-d] pyrimidines (4a-h)
A mixture of 3a-h (5 mmol) and formamidine acetate (15 mmol, 1,56 g) was irradiated in a single-mode microwave at 300 W for 15 min and under 12 bar pressure. TLC determined the completion of the reaction. After the completion of the reaction, the residue that formed was diluted with 30 ml of CH 2 Cl 2 . The organic layer obtained was washed with water (3 × 20 ml), then with a solution of saturated NaCl (10 ml), dried over MgSO 4 , filtered and evaporated under vacuum to afford desired compounds 4a-h.

Conclusions
In conclusion, we have successfully developed a novel and efficient approach for synthesising new chromenes and 5H-chromeno [2,3-d] pyrimidines derivatives under solventfree conditions and microwave irradiations with high yields. The originality of our synthetic strategy is based on the use of formamidine acetate as cyclisation agents. This easy to execute methodology with rapid access and good yields open a new route for synthesising various substituted nitrogen heterocycles of biological and pharmaceutical. Evaluating the antioxidant and antibacterial activity of the various compounds tested against three bacterial strains showed good activity.