Synthesis and Biological Evaluation of Newly Synthesized Flavones

Introduction

Flavonoids constitute a diverse group comprising over 4000 polyphenolic compounds inherent in plant-derived foods. The primary basis for the systematic classification of these compounds is the saturation level and aperture of the principal pyran ring, which leads to the formation of flavones, flavanols, flavonols, isoflavones, and flavanonols. They share a fundamental phenyl-benzopyrone structure (C₆-C₃-C₆)^1,2. Flavones hold a distinctive position within the field of natural and synthetic organic chemistry due to their consequential biological actions, encompassing antioxidant^3-7, anxiolytic⁸, antitumor^9-11, anti-inflammatory^12-14, bactericidal¹⁵, antiulcer, and thrombosis¹⁶ effects.

There are several approaches for preparing chalcone, which is a crucial precursor for the synthesis of flavones. Chalcones are usually produced by the Claisen-Schmidt condensation reaction which involves substituted benzaldehyde and acetophenone followed by a strong base ( NaOH, KOH, KOtBu and MgO). Moreover, chalcones can be synthesized through advanced methods such as ultrasonic vibration and microwave irradiation procedures^17-19.

Chalcones, represent major alternate metabolites in the flavonoid (Fig.1) class, abundantly present in edible plants. Their radical quenching property, owing to phenolic groups, has spurred research into chalcone-rich plant extracts for therapeutically beneficial compounds. Historically, chalcones like chalconarngenin, phloretin, and phloridzin have been associated with diverse therapeutic applications^14-19.

Figure 1: Proposed structure of flavones. Click here to View Figure

Studies reveal that both natural and synthetic flavonoid and their analogs display a broad spectrum of pharmaceutical actions, including antioxidant, antitubercular, antimicrobial, anti-inflammatory, anti-plasmodial, antiviral, antitumor, antileishmanial, modulation of P-glycoprotein-mediated multi-drug resistance, and immune supportive potential^20,21. Interestingly, substances that possess a chalcone-based structure or template also have a significant therapeutic impact on the nervous, cerebrovascular, and cardiovascular systems. In addition to their anti-spasmodic, sedative, anti-thrombic, tranquilizing, analgesic, vasodilatory, anesthetic, anti-coagulating, estrogenic,anti-convulsant, and diuretic qualities.

Furthermore, flavonoid are regarded as vital pharmacophores in numerous bioactive natural commodities, showcasing varied biological actions. Typical examples of naturally existing bioactive chalcones include xanthohumol and cardamonin, with antimutagenic, vasorelaxant, anti-inflammatory, and broad-spectrum tumor chemo-preventive properties^22,23. In the current investigation, chalcones are used as the primary precursor for the creation of active flavonoids and their biological evaluation.

Materials and Methods

In the present investigation, we utilized A.R. grade chemicals and reagents (2-Hydroxy acetophenone, 2-chlorobenzaldehyde, and DMSO) obtained from E. Merck. The development of reactions was monitored using thin-layer chromatographic precoated plates (E. Merck). The FT-IR spectra of the compound were recorded from the NICOLET 6700, Thermo Scientific spectrophotometer. The ¹HNMR signals were captured at optimal temperature via a 400 MHz liquid-state NMR Spectro-meter in DMSO-d6 (Brüker Biospin), with tetramethyl silane serving as the internal standard.

Antibacterial activity has been determined via the Kuber agar well diffusion technique²⁶. The concentration of the compound for testing is 50 mg/ml, and the test organisms used were E. coli and S. aureus, 8 mm diameter well was made in an agar plate in which the 50 µl compound was loaded, and incubated at 37℃ for 24 hours, next day the plates were observed for the presence of the zone of inhibition.

Extensive mechanism for the synthesis of substituted chalcone

The chalcone of every target has been created by utilizing a standard Claisen-Schmidt condensation^24,25 reaction to react equimolar amounts of substituted acetophenone with substituted benzaldehyde. This reaction creates an α, β-unsaturated ketone bridge connecting ring A and ring B of the chalcone basic structure. In the present investigation, the synthesis procedure has been completed in two steps as follows-

Step one

To a mixture comprising 2-hydroxyacetophenone (1 equivalent) and a substituted aromatic aldehyde (2-chlorobenzaldehyde), 10 ml of ethanol was included dropwise with stirring. The mixture was sustained in an ice bath for 15–20 minutes, and freshly prepared 10 ml of 60% NaOH was added dropwise to the above mixture with persistent stirring. The reaction solution was standing for five hours, and the reaction development was scrutinized by thin-layer chromatography. Subsequently, it was acidified with diluted hydrochloric acid, and the precipitate was filtered and washed with cold water, dried, and recrystallized by ethanol.

Yield: 78%

Color: Pale yellow

MP   : 144ºC

Molecular Mass: 258.90 g/mol

Figure 2: Synthesis of Chalcones ((E)-3-(2 chlorophenyl)-1 (2 hydroxyphenyl) prop-2en-1 one) (Compound-1) Click here to View Figure

Step two

To equimolar quantities of the compound obtained from the preceding step, DMSO and a catalytic amount of solid iodine were added dropwise. The mixture of chemical reactions were refluxed for about 2 hours and monitored by TLC. It was then poured into crushed ice. The resultant mixture was recrystallized using diethyl ether. The ethereal layer was extracted and dried under a vacuum. The synthetic pathway is presented in fig. 3.

Yield: 76%

Color: Silvery white

MP   : 146ºC

Molecular Mass: 256.69 g/mol

Figure 3: Synthesis of Flavone derivatives (2 (2-chlorophenyl)-4 H chromen 4-one.) (Compound-2). Click here to View Figure

Results and Discussion

The two flavonoids (Compound-1 and Comound-2) were prepared by the Claisen-Schmidt condensation procedure. Physical analysis suggested that the newly prepared compounds are solids with different colors (Pale yellow and silvery white), and the resultant yield is adequate. Both compounds are soluble in the general organic solvent viz; DMF, DMSO, hexene, chloroform, and benzene, etc.

FT-IR Interpretation of synthesized compounds

Chalcone (Compound-1)

FT-IR spectrum of Chalcone (Compound-1) (fig. 4) is not overly elaborate having characteristic bends. The broad bands at the higher frequency site 3523 cm^-1 is revealed the υ(O-H) stretching vibration of the -OH functional group^27,28. The stretching frequency υ(C-H) is usually displayed as a weak intensity band with a range of 3035-3050 cm^-1 and it appears at 3041 cm^-1 as a weak band¹. The well-defined bands at 1649 cm^-1 is the individual properties of the carbonyl Compound its due to υ(C=O) group. The sharp υ(C=C) band at 1554 cm^-1 is owing to the phenyl ring vibration^29,30. The medium band at the lower site 767 cm^-1 is due to the υ(C-Cl) vibration of the chlorine and bond in between the phenyl ring³¹.

Figure 4: FT-IR spectra of the (E)-3-(2 chlorophenyl)-1(2-hydroxyphenyl) prop-2en-1 one (Compound-1). Click here to View Figure

Flavone (Compound-2)

FT-IR spectra of Flavone compound-2 (Fig. 5) showed useful information about the newly prepared compound. Aromatic compounds usually displayed several weak singles in the region 3110-3045 cm^-1 due to the υ(C-H) stretching^32,33. In the IR spectrum, the bend appears at 3041 cm^-1 as a weak signal is due to the aromatic C-H group³⁴. The broad band’s at the higher frequency site (3523 cm^-1) disappears in the compound-2 is confirmed the oxidation of chalcone compound-1 into flavone compound-2. The well-defined bands at 1639 cm^-1 is the individual properties of the carbonyl Compound its due to υ(C=O) group. The medium band at 1571 cm^-1 are due to the C-C skeletal mode of vibration of the aromatic ring^35-36. The sharp band at 1386 cm^-1 is due to the phenyl ring υ(C=C) vibration^29-30. The medium band at the lower frequency site 746 cm^-1 is due to the chlorophenyl, υ(C-Cl) vibration³¹.

Figure 5: FT-IR spectrum of the 2 (2-chlorophenyl)-4 H chromen 4 –one (Compound-2). Click here to View Figure

¹HNMR and ¹³CNMR interpretation of newly synthesized compounds

Chalcone (Compound-1)

In the ¹HNMR band, a significant chemical shift value appears as a singlet at 8.69 ppm, assigned to the O-H proton, exhibiting high de-shielding due to intra-molecular hydrogen bonding with the carbonyl oxygen atom. A doublet was displayed at 7.89 ppm and 7.67 with huge coupling constant J = 15.5 and 15.5 Hz, respectively³⁷, which are characteristics of trans olefinic protons (α and β). Other signals and the total proton count align precisely with the identified compound’s structure. Despite the synthesized compound having 15 carbons, the ¹³C NMR spectrum displays thirteen signals, ascribed to the symmetric character of the 4-chlorophenyl ring³⁸.

Figure 6: 1HNMR spectrum of the (E)-3-(2 chlorophenyl)-1(2-hydroxyphenyl)  prop-2en-1 one (Compound-1). Click here to View Figure

Figure 7: 13C NMR spectrum of the (E)- 3 – (2 chlorophenyl)-1 (2 hydroxyphenyl) prop-2en-1 one (Compound-1). Click here to View Figure

Flavone (Compound-2)

In the ¹H NMR signal, the bend at 8.22–7.55 ppm (doublet) and 7.64–7.43 ppm (double doublet) is due to the ring hydrogen (d, 2H, Ar and dd, 2H, Ar) respectively. The doublet bends at 7.92–7.60 ppm and a double doublet at 7.50–7.47 ppm is due to the chlorophenyl ring (dd, 2H, Ar–Cl and d, 2H, Ar–Cl) respectively. The strong bend at 6.99 ppm is due to the (s, 1H, =CH-) proton present in this compound³⁹. All the aromatic protons are observed in predictable regions. In the ¹³C NMR signals the aromatic (=CH-) carbon appeared at 108.03 ppm and C-O carbon appeared at 155.85 and 157.45. The bend at 177.52 is due to the (C=O) carbon. Newly synthesized compound having 15 carbons, the ¹³C NMR spectrum displays thirteen signals, ascribed to the symmetric character of the 4-chlorophenyl ring³⁸. The ¹³C NMR signals offer direct evidence about the carbon skeleton of the newly synthesized compound.

Figure 8: 1HNMR spectrum of the 2 (2-chlorophenyl)-4 H chromen 4 –one (Compound-2). Click here to View Figure

Figure 9: 13C NMR spectrum of the 2 (2-chlorophenyl)-4 H chromen 4 –one (Compound-2). Click here to View Figure

Biological Evaluation

The antibacterial action of the two newly produced compounds, (compound-1 and compound-2), were tested against G^– microbe (E. coli) and G⁺ microbe (S. aureus) by the agar well diffusion procedure. The results revealed that the synthesized compounds exhibited superior antimicrobial activity against G⁺ microbe S. aureus, with zones of inhibition measuring 28 mm and 25 mm for compound-1 and compound-2, respectively. Conversely, in the case of G^– microbe E. coli, the antibacterial activity was observed to be 22 mm and 19 mm for compound-1 and compound-2, respectively (Fig.10).     

Figure 10: Antibacterial activity (zone of inhibition in mm) of Compound-1 and Compound-2. Click here to View Figure

Conclusion

Synthesis of flavonoids was effectively completed through a condensation reaction and the purity of compounds were determined using melting point and TLC measurements. In order to synthesized compound further confirmed by the FT-IR, ¹HNMR, and ¹³C NMR spectral studies.                 Antibacterial activity of the synthesized, Compound-1 and Compound-2, demonstrated notable activity against G⁺ microbe (S. aureus) and G^– microbe (E. coli). Specifically, superior antibacterial efficacy was observed against S. aureus, emphasizing the potential significance of these synthesized flavonoids in combating bacterial infections. The comprehensive synthesis and characterization, coupled with promising antibacterial properties, underscore the utility of the developed compounds for further exploration in pharmaceutical and medicinal applications.

Acknowledgment

We would like to thank IIT Indore for the spectral analysis and the head of the microbiology department at Govt. Holkar Science College, Indore, for their assistance with the biological studies.

Conflict of Interest

According to the authors, there are no conflicts of interest associated with this work’s publishing.

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