1 Introduction

Chalcone 1 (1,3-diphenyl-2-propene-1-one) is the simplest molecule whose basic structural motif can be seen in a vast array of natural products and in many synthesized 1,3-diaryl-α,β-unsaturated carbonyl compounds (Fig. 1). These molecules are collectively known as chalcones or chalconoids. The name chalcone is derived from the Greek word for copper ‘χαλκοσ’ or “chalkos” due to the reddish-brown colours that many early chalcones possessed. A recent CAS SciFinder search of the word “chalcones” for example recovered over 32,800 references. Chalcones have been shown to have a wide range of promising biological activities and are effective against various diseases; examples of such molecules and their antimicrobial targets are shown in Fig. 2 [1,2,3,4]. Anti-inflammatory, anticancer, antidiabetic, anti-tumor, anti-fungal and antibacterial properties, for example, have been reported for chalones and for many of their derivatives [1, 3, 5,6,7,8]. The presence of the ketoethylenic group as the pharmacophore in these and other molecules have been postulated to account for the various biological and antimicrobial properties [5, 9,10,11].

Fig. 1
figure 1

The four possible stereoisomers of chalcone 1

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Fig. 2
figure 2

Schematic overview of some important chalcone derivative

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Chalcones are also considered to belong to the class of, and are precursors to, the flavonoids which are naturally found widely in fruits, teas and soy-based foods [2, 12]. Pérez-González et al. [13] employed an extensive CADMA protocol to evaluate a total of 568 chalcone derivatives for their potential as antioxidant neuroprotectors. Russell and Gines [14] recently presented a timely review of the chemotherapeutic potential of chalcones with a particular emphasis for their antiproliferative potential against breast cancer.

Lastly, chalcones are not only important in natural product and medicinal and biological chemistry, but are also useful for their structural physicochemical properties. Consequently, many synthetic routes have been designed to synthesize novel chalcone derivatives [12]. Among these routes, the Claisen–Schmidt condensation reaction is commonly and easily employed. In principle, chalcone 1 could have four different stereoisomers which can be due to both the cis or trans orientation of the hydrogen atoms on the alkene double bond giving E or Z geometrical isomers; and also from the two possible s-cis or s-trans conformers due to the orientation of the carbonyl double bond relative to the alkene double bond. These are shown as structures 1a–d in Fig. 1.

The s-cis-E-isomer 1a is the more stable form since there is less steric crowding between the two bulky groups around the double bond [4]. Density functional theory (DFT) computational studies have also shown this to be the case [15,16,17]. Several early and more recent studies have employed DFT studies with chalcones in order to evaluate the potential influences of substituents on both the A and the B aryl groups. Hicks et al. [18] correlated DFT-computed electron affinities with experimentally derived reduction potentials of a series of mono-, di- tri- and tetra-substituted chalcones. Kozlowski et al. [19] studied the DFT-computed conformational and electronic data of 11 differently-substituted hydroxylated chalcones to correlate their effect(s) with experimentally-derived antioxidant properties as determined by the 2,2-diphenyl-1-pycril-hydrazyl (DPPH) free-radical scavenging assay. A search of the recent literature has identified several other DFT studies with chalcones. Mittal et al. [20] reported on the DFT conformational and electronic study of a retrochalcone isolated from  licorice root, namely echinatin, and its ionic form in both the gas phase and aqueous solution. Omar et al. [21] conducted a DFT computational study on seven mono-substituted A-ring derivatives at the 2-, 3- and 4-positions, using both the B3LYP/6-31G** and TD-B3LYP/6-31++G** for potential optoelectronic application. Wong et al. [22] conducted a comprehensive B3LYP/6-31++G** DFT structure–property relationship study of three newly synthesized B-ring 2-chloro-4-fluoro chalcone derivatives for their linear and non-linear optical properties. Another DFT study recently conducted by Ahmad et al. [23] evaluated the photophysical properties of two A-ring 2,4-dichlorinated chalcones and their 2-methoxy-3-cyanopyridine derivatives formed with malononitrile.

It has been noted that relatively fewer chalcone derivatives bearing electron-withdrawing groups have been synthesized and their physicochemical properties reported. The objective of the study presented herein therefore was to synthesize and compare the structural properties of B-ring o-, m-, p-hydroxy and their corresponding mono- o-, m-and p-nitro derivatives, and to conduct a preliminary study of their antibacterial and antioxidant properties. As well, to gain insight into these experimental properties, DFT including TD-DFT calculations were undertaken to obtain the optimized structure and visualize the frontier molecular orbitals (FMOs) and other quantum chemical properties.

2 Experimental

2.1 Materials

All newly-synthesized products were purified by crystallization from MeOH or EtOH to give brown crystals. 1H NMR spectra were recorded on a Bruker Ascend™ 400 Spectrometer (400 MHz for 1H) in CDCl3 or with DMSO-d6 as the internal standard. The 13C NMR spectra were recorded on a Bruker Ascend™ 600 Spectrometer. IR spectra were measured using a PerkinElmer Frontier FTIR/NIR Spectrometer. Acetophenone (Daejung Chemicals & Metals), 2-nitrobenzaldehyde (Alfa Aesar), 2-hydroxybenzaldehyde (Sigma Aldrich), 3- and 4-hydroxybenzaldehydes and 3- and 4-nitrobenzaldehydes (Alfa Aesar), sodium hydroxide (Alfa Aesar), methanol (Alfa Aesar) were purchased from local suppliers and were used directly without further purification.

2.2 Synthesis of Chalcone Derivatives

In a typical synthesis, a solution of acetophenone (5.0 mmol, 1.0 equiv.) in MeOH (15.0 mL) was added benzaldehyde (5.0 mmol, 1.0 equiv.). Aqueous 40% NaOH was added dropwise to the reaction mixture, with stirring. The solution was stirred at room temperature for a further 1–3 h to allow for the complete precipitation of product. The product formation was confirmed by TLC using a 3:1 n-hexane:ethyl acetate eluent mixture. Hydrochloric acid (1.0 M) was added to neutralize the excess NaOH. Finally, the reaction mixture was filtered and the precipitated product was purified by crystallization from MeOH or EtOH to afford brown solid crystals.

2.2.1 (E)-3-(2-hydroxyphenyl)-1-phenylprop-2-en-1-one, 2a [24,25,26,27,28]

Yield: 82%; White color (m.p. 153–154 °C, lit [24] 152–153 °C); 1H NMR (600 MHz, DMSO-d6): δ 10.3 (s, 1H), 8.10–8.05 (m, 3H), 7.86 (m, 2H), 7.64 (d, J = 6.6 Hz, 1H), 7.57 (d, J = 7.8 Hz, 2H), 7.28 (t, 1H), 6.95 (d, J = 7.8 Hz, 1H), 6.88 (t, 1H) ppm; 13C NMR (150 MHz, DMSO-d6): δ 189.6, 157.3, 139.6, 138.0, 133.0, 132.1, 128.8, 128.7, 128.4, 121.4, 121.0, 119.5, 116.3 ppm; IR (KBr): 3435 (br.), 3061 (w), 2749 (w), 1683 (s), 1603 (m), 1550 (m), 766 (m) cm−1.

2.2.2 (E)-3-(3-hydroxyphenyl)-1-phenylprop-2-en-1-one, 3a [29,30,31,32,33]

Yield: 80%; Brown color (m.p. 159–160.8 °C, lit [30] 150–153 °C); 1H NMR (400 MHz, CDCl3): δ 8.00 (d, J = 7.2 Hz, 2H), 7.74 (d, J = 16.0 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 7.51 (m, 3H), 7.29–7.21 (m, overlapped with H2O peak, 2H), 7.12 (s, 1H), 6.89 (d, J = 8.0, 1H) ppm; 13C NMR (150 MHz, CDCl3): δ 190.9, 156.3, 144.9, 138.2, 136.6, 133.1, 130.3, 128.8, 128.7, 122.6, 121.3, 117.9, 115.1 ppm; IR (KBr): 3318 (br.), 1650 (s), 1602 (st), 1568 (st), 1487 (m), 1265 (m), 984 (m) cm−1.

2.2.3 (E)-3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one, 4a [29, 30, 34, 35]

Yield: 74%; Yellow color (m.p. 184–184.5 °C, lit [35] 183–184 °C); 1H NMR (400 MHz, CDCl3): δ 8.01 (m, 2H), 7.77 (d, J = 15.6 Hz, 1H), 7.59–7.49 (m, 5H), 7.39 (d, J = 15.6 Hz, 1H), 6.88 (m, 2H) ppm; 13C NMR (150 MHz, CDCl3): δ 191.3, 158.4, 145.2, 138.6, 132.9, 132.6, 130.7, 128.8, 127.7, 119.9, 116.2 ppm; IR (KBr): 3195 (br.), 1648 (s), 1599 (s), 1557 (s), 1511 (sh), 1349 (m), 1217 (sh), 1167 (s) cm−1.

2.2.4 (E)-3-(2-nitrophenyl)-1-phenylprop-2-en-1-one, 5a [36,37,38,39]

Yield: 72%; Ash grey (m.p. 141 °C, lit [37] 139–140 °C); 1H NMR (400 MHz, CDCl3): δ 8.13 (d, J = 15.6 Hz, 1H), 8.08 (dd, J = 1.2 Hz and 0.8 Hz, 1H), 8.03–8.00 (m, 2H), 7.75–7.70 (dd, J = 1.6 Hz and 1.6 Hz, 1H), 7.70–7.68 (m, 1H), 7.62–7.58 (m, 2H), 7.57–7.49 (m, 2H), 7.31 (d, J = 15.6 Hz, 1H) ppm; 13C NMR (150 MHz, CDCl3): δ 190.7, 148.7, 140.3, 137.5, 133.7, 133.28, 131.5, 130.5, 129.4, 128.9, 128.9, 127.5, 125.1 ppm; IR (KBr): 3052 (w), 1670 (s), 1606 (sh), 1570 (s), 1514 (s), 869 (m) cm−1.

2.2.5 (E)-3-(3-nitrophenyl)-1-phenylprop-2-en-1-one, 6a [37, 40]

Yield: 80%; Colorless solid (m.p. 140.1–141 °C, lit [37] 143–144 °C); 1H NMR (400 MHz, CDCl3): δ 8.51 (t, 1H), 8.27–8.24 (m, 1H), 8.06–8.03 (m, 2H), 7.92–7.91 (m, 1H), 7.83 (d, J = 15.6 Hz, 1H), 7.67 (s, 1H), 7.64–7.60 (m, 2H), 7.55–7.51 (m, 2H) ppm; 13C NMR (150 MHz, CDCl3): δ 189.8, 148.9, 141.8, 137.7, 136.8, 134.5, 133.5, 130.2, 128.9, 128.7, 124.8, 124.8, 122.5 ppm; IR (KBr): 3069 (m), 1663 (s), 1611 (sh), 1528 (s), 1223 (s), 995 (m) cm−1.

2.2.6 (E)-3-(4-nitrophenyl)-1-phenylprop-2-en-1-one, 7a [34, 37, 38, 40]

Yield: 82%; Brown color (m.p. 161–162 °C, lit [40] 160–163 °C); 1H NMR (400 MHz, CDCl3): δ 8.28 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 7.6 Hz, 2H), 7.79 (t, 3H), 7.62 (d, J = 5.6 Hz, 2H), 7.53 (t, 2H) ppm; 13C NMR (150 MHz, CDCl3): δ 189.8, 148.7, 141.6, 141.2, 137.7, 133.5, 129.1, 128.9, 128.7, 125.9, 124.4 ppm; IR (KBr): 3077 (w), 1660 (s), 1609 (sh), 1515 (s), 1338 (s), 983 (s) cm−1.

2.3 Results and Discussion

The preparation of the six targeted compounds 2–7 using base-catalyzed Claisen–Schmidt condensation reactions of acetophenone and the corresponding substituted benzaldehydes, are shown in Scheme 1. The starting materials were commercially obtained and were used directly without further purification. The reactions were conducted with stirring at room temperature in methanol solvent. The target product(s) precipitated within few hours, and after reaction completions were ascertained by TLC monitoring, were purified by crystallization and were characterized by 1H NMR and other spectroscopic methods. Although some of those compounds were reported elsewhere and cited, important additional properties including, for example, the absorption behavior and theoretical studies have not previously been reported.

Scheme 1
scheme 1

Synthesis of acetophenone-based chalcone derivatives 2a–7a. (Note: for simplicity only the s-cis-E stereoisomers are shown here)

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2.3.1 Absorption Spectra

All of the synthesized compounds are coloured and their absorption spectra are shown in Fig. 3. Since the polarity of a solvent affects the intensity and positions of the absorptions in the spectra of solutes [41] the spectra of 2 to 7 were recorded in both polar CH2Cl2 and polar aprotic DMSO solvents, at 4.0 × 10–5 M concentrations. As it can be seen from Fig. 3, the absorption maxima (in CH2Cl2) of 2–7 are in the range of 297 nm (\(\varepsilon\) = 1.7 × 104 dm3 mol−1 cm−1) to 335 nm (\(\varepsilon\) = 2.1 × 104 dm3 mol−1 cm−1). In DMSO, however, the absorption maxima were in the range of 300 nm (\(\varepsilon\) = 2.3 × 104 dm3 mol−1 cm−1) to 418 nm (\(\varepsilon\) = 1.3 × 104 dm3 mol−1 cm−1). A strong charge-transfer interaction [42] between the solvent and compounds can account for the latter data. Hence, 2 revealed two significant absorption peaks at 326 nm (\(\varepsilon\) = 1.1 × 104 dm3 mol−1 cm−1) and 418 nm (\(\varepsilon\) = 1.3 × 104 dm3 mol−1 cm−1) in DMSO.

Fig. 3
figure 3

Absorption spectra of compounds 3a–7a: A In CH2Cl2 (E-2a not shown); and B In DMSO solvent, concentration is 4.0 × 10–5 mol/L)

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2.3.2 Antibacterial Activities

Four pathogenic bacteria, Bacillus cereus ATCC 10876, Staphylococcus aureus ATCC 9144, E. coli ATCC 35150, Salmonella typhi ATCC19430 were used to check the antibacterial activities of compounds 2a–7a. The antibacterial susceptibility tests were performed using the modified agar diffusion method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [43, 44]. Plates were incubated for 18–24 h at 37 ± 2 °C and resulting zones of inhibition were measured (mm). Inoculums of the microorganisms were prepared to give a final concentration of 5.0 × 105 CFU/mL in Mueller Hinton (MH) Broth. Bacterial suspensions (50 μL) were added to each plate of MH Agar media. Solutions (1 mg/mL) of the samples were prepared in aqueous 10% DMSO and 200 μL aliquots of each sample to be tested were added to each well of the test plates of the organisms. Ciprofloxacin (1.0 mg) was used as positive control and 10% DMSO as negative control. The antibacterial effectiveness of the compounds with the panel of both gram-positive and gram-negative pathogenic bacteria are recorded in Table 1. The obtained effective results indicated that the compound exhibited a varying degree of antibacterial activity against tested microorganisms (Fig. 4). However, we did not obtain any measurable activities apart from 2a (Table 1), possibly due the inability of the compounds to diffuse in the medium [7].

Table 1 Antibacterial activity against bacterial species
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Fig. 4
figure 4

Antibacterial activities of representative samples 2a, 3a and 5a (effective results were found only for 2a)

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2.3.3 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Assessments of Compounds 2a–7a

The broth micro-dilution method was used to determine the MIC and MBC values according to the CLSI guidelines [44]. Five solutions of concentrations 1.5, 1.0, 0.5, 0.15 and 0.10 mg/mL of samples of each of the compounds 2a–7a were prepared in aqueous 10% DMSO. Inoculums of the microorganisms were prepared to give a final concentration of 5.0 × 105 CFU/mL in each well. Samples (100 μL) were added to each well of the microplate. Bacterial suspensions (50 μL) were added to each well except for the negative controls. Ciprofloxacin (1.0 mg/mL) was used as the positive control. Microbial growth was assessed measuring the absorbance at 690 nm. For the MBC evaluations the inoculated plates were incubated at 37 ± 2 °C for 18–24 h. However, 0.06–1.0 µg/mL concentrations of ciprofloxacin are required to inhibit the growth of the Gram positive and Gram negative bacteria tested. In comparison, 500 µg/mL of the chalcone compounds tested were required for bacterial growth inhibition. Also, 5 µg of ciprofloxacin is used to determine the bactericidal effect of the antibiotic. Whereas, the potency of the tested sample was found 1 µg/mg. The results from the microdilution assay for each sample tested and derived from the four test organisms are reported in Fig. S13 (SI). At the highest concentrations of samples i.e. 1.5, 1.0 and 0.50 mg/mL, the growth of E. coli culture were significatively inhibited. The same results were obtained against Bacillus cereus, Staphylococcus aureus and Salmonella typhi for the same sample concentrations.

The experiments conducted showed that the bacterial growth-inhibiting concentration of the 2a sample was equal to 0.50 mg/mL. The identified MIC values are reported in Table 2. Therefore, the obtained results showed that the lowest MIC value was (0.50 mg/mL) on E. coli and for other three organisms.

Table 2 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values; (− −) no growth, (−) growth inhibition, (+, ++) growth for 2a
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2.3.4 Antioxidant Activities of Compounds 2a–7a

The antioxidant activities of 2a–7a were measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals scavenging method, according to Braca et al. [45], with some modifications. From each of the stock solutions (2000 ppm) of compounds 2a–7a, sample aliquots (2.5 mL) were mixed with 2.5 mL of 0.0040% DPPH solution. After incubating the mixtures for 30 min at room temperature and in the dark, their absorption spectra were measured. The percentages of DPPH inhibition were calculated using the following formula:

$$\% \ \text{of DPPH inhibition} = \left(\frac{{{\text{A}}}_{{\text{c}}}-{{\text{A}}}_{{\text{s}}}}{{{\text{A}}}_{{\text{c}}}}\right)\times 100$$

where, Ac and As represent the absorbance of the control and sample respectively.

The DPPH scavenging capacity expressed as a percentage of scavenging by 1000 ppm of each sample is shown in Table 3. Only compound 4a showed a modest DPPH scavenging ability at 22% whereas all of the others showed less than 10% scavenging ability. The synthesized chalcone derivatives therefore did not exhibit any noteworthy measurable antioxidant activity.

Table 3 Antioxidant properties of compounds by DPPH method
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3 Density Functional Theory (DFT) Computational Studies

Since the existence of four stereoisomers shown as 1a–d can be surmised for chalcone 1 itself (Fig. 1), DFT calculations for these four stereoisomers in the gas phase were conducted and are shown in Table 4. Similarly, but in addition, for each of the other B-ring ortho- derivatives 2a–d and 3a–d; and meta- substituted and 5a–d and 6a–d, respectively, there are also two regioisomers as well, for each. We have labelled these correspondingly as 2a′–d′ and 3a′–d′; and 5a′–d′ and 6a′–d′ respectively and conducted gas phase calculations with these as well, and the results are summarized in Table 4 also. All single-point DFT calculations were performed using the long-range corrected hybrid functional B3LYP in conjunction with the 6-311+G(d,p) basis set. All calculations were conducted in the gas phase using Gaussian 16, Revision C.01 [46]. The molecular structures of all the structures were drawn using the GaussView 6.0.16 program. A vibrational analysis was carried out for each optimized molecular structure to ensure they were in a vibrational energy minimum and had no imaginary frequencies. The individual geometry-optimized structures are shown in Figs. 5 and 6 and their energies are summarized in Table 4. The energies of the less-energetically favored configuration for each compound are presented as ΔE values relative to the most-energetically favored configuration for that compound. The DFT calculations clearly confirm that that for the mono hydroxy-group B-ring derivatives the (E)-geometrical isomers are relatively more stable than their (Z)-geometrical isomers, as are their corresponding s-cis conformers. The order of stability is s-cis-(E) > s-trans-(E) > s-cis-(Z) > s-trans-(Z). In the case of the corresponding nitro group-containing derivative 5a′, the order is different: s-trans-(E) > s-cis-(Z) > s-trans-(Z) > s-cis-(Z) which can be ascribed to the additional intramolecular electrostatic interactions that can form with the larger polar nitro group.

Table 4 Single-point DFT (gas phase calculations) of the stereoisomers of chalcones 1–7 using the long-range corrected hybrid functional B3LYP in conjunction with the 6-311++G(d,p) basis set
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Fig. 5
figure 5

The gas phase optimized molecular structures of the various stereoisomers of chalcone hydroxy derivatives 2–4 and regioisomers 2′–3′. Color code: carbon = grey; oxygen atom = red; hydrogen atom = white

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Fig. 6
figure 6

The gas phase optimized molecular structures of the various stereoisomers of chalcone nitro derivatives 5–7 and regioisomers 5′–6′. Color code: carbon = grey; oxygen atom = red; hydrogen atom = white; nitrogen atom = blue

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Figure 7 shows that the relative magnitudes of the energy differences of each group of stereoisomers with respect to the more stable regioisomers vary. The ortho-nitro regioisomers (i.e., 2′ vs 6′ positions on the B-ring) show both the largest and smallest contrasts. The Gibbs free energy (G) values can also reveal useful information of the drug-like properties of a molecule[47] i.e. for a series of derivatives the more effective drug molecule typically has a higher negative value of G. For the three hydroxy-group compounds 2–4 a correlation can be seen for the 4 > 3 > 2 DPPH scavenging abilities noted in Table 3 and the optimized Gibbs free energy values which are in the same order. For the three nitro-group compounds 5–7 a correlation is not as obvious since there were no significant differences observed in their scavenging abilities. The energies of the frontier molecular orbitals (FMOs) [48, 49], of the E isomers of compounds 2a–7a, which are the optimized most energy-favoured structural isomers of 2–7 respectively, were also computed and are shown in Table 4. These FMOs are for the highest occupied molecular orbitals (HOMOs) and for the lowest unoccupied molecular orbitals (LUMOs). From Koopmans’ theorem [49], the difference between the HOMO and LUMO energy values (or H–L energy gap) of a species is related to its polarizability. Molecules with higher polarizability and lower H–L energy gaps will show more biological activity properties [50]. Conversely, the larger the energy gap, the lower the reactivity of the molecule. Several other useful quantum chemical properties e.g., the electronegativity (χ), chemical potential (µ), hardness (η), softness (S), and electrophilic index (ω) values were calculated using the common equations (see details in Table S1). The H–L energy gap data suggests that the order of stability for the hydroxy-substituted B ring compounds (Figs. 8, 9) is in the approximate order of 4 > 3 > 2 which is consistent with the DPPH-scavenging abilities of these compounds.

Fig. 7
figure 7

Relative energy differences with respect to the most stable regio and stereoisomers from the data from Table 4

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Fig. 8
figure 8

HOMOs and LUMOs structures of optimized (B3LYP, 6-311++G(d,p) structures of 2a–3a and their respective regioisomers 2a′–3a′ and 4a. All energy units are in eV

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Fig. 9
figure 9

HOMOs and LUMOs structures of optimized (B3LYP, 6-311++G(d,p) structures of 5a–6a and their respective regioisomers 5a′–6a′ and 7a. All energy units are in eV

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For the nitro-group analogues, the H–L energy gap data order is similar, for the para > meta > ortho isomers, although the nitro compounds differ in magnitude to their hydroxy counterparts, in their ionization potential, electro affinity, chemical potential and electrophilicity index values (Fig. 10).

Fig. 10
figure 10

Molecular electrostatic potential (MEP) maps of 2–7

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4 Conclusions

The limited study presented herein provides some insights into the various geometrical and regioisomeric structural aspects of the corresponding hydroxy- and nitro-B ring substituted chalones 2–7 using DFT computations. The antioxidant DPPH-scavenging study showed that for 2–4 the correction was better with regard to their Gibbs free energies. For the antimicrobial study activity was only observed with compound 2. Our study will be on-going with other ring-B substituted derivatives of chalcone which will guided by the insights gained from the present study reported herein.