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. 2012 Jun 1;17(6):6684–6696. doi: 10.3390/molecules17066684

Synthesis and Antibacterial Activity of Some Heterocyclic Chalcone Analogues Alone and in Combination with Antibiotics

Thanh-Dao Tran 1,*, Thi-Thao-Nhu Nguyen 1, Tuong-Ha Do 2, Thi-Ngoc-Phuong Huynh 1, Cat-Dong Tran 3, Khac-Minh Thai 1,*
PMCID: PMC6268422  PMID: 22728362

Abstract

A series of simple heterocyclic chalcone analogues have been synthesized by Claisen Schmidt condensation reactions between substituted benzaldehydes and heteroaryl methyl ketones and evaluated for their antibacterial activity. The structures of the synthesized chalcones were established by IR and 1H-NMR analysis. The biological data shows that compounds p5, f6 and t5 had strong activities against both susceptible and resistant Staphylococcus aureus strains, but not activity against a vancomycin and methicillin resistant Staphylococcus aureus isolated from a human sample. The structure and activity relationships confirmed that compounds f5, f6 and t5 are potential candidates for future drug discovery and development.

Keywords: chalcone, heterocyclic chalcone analogues, synthesis, antibacterial, synergitic activity, antibiotics combination, MRSA

1. Introduction

In the past, many decades since penicillin was discovered and introduced as a powerful antibacterial agent, antibiotics have become critical in the fight against infectious diseases caused by bacteria and other microbes. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains [1,2,3]. At present, the appearance of more and more pathogenetic bacterial species resistant to conventional antibiotics has resulted in either high expenses or failure in the treatment of infectious diseases. An alarming increase in resistance of bacteria that cause community acquired infections has also been documented, especially in Staphylococci and Pneumococci, which are prevalent causes of disease and mortality. In addition, the risk of opportunistic fungal infections increases rapidly accompanied with AIDS disease, and as an obviously consequence invasive infections represent a major cause of mortality for these patients [4]. With the emergence of new microbial strains resistant to many conventional available antibiotics there is growing interest in the discovery of new antibacterial agents [1,2]. According to the known structure and activity relationships, it is considered that certain small heterocyclic molecules act as highly functionalized scaffolds and are known pharmacophores of a number of biologically active and medicinally useful molecules [5,6].

Chalcones (1,3-diphenyl-propene-1-one) belonging to the flavonoid family, are natural and synthetic products that have been reviewed for their wide range of biological activities as antibacterial [7,8], anti-tumor [9,10,11], anti-inflammatory [12,13,14] and antioxidant agents [15,16,17,18], etc. Although studies on the bioavailability of heterocyclic chalcones from natural sources are limited, they have been reported as having a wide range of biological activities, especially antibacterial [19,20,21,22,23,24,25,26,27,28], and antifungal activities [29]. In an effort to diversify the biological activities of conventional chalcones, a series of heterocyclic chalcone analogues in which an electron rich nitrogen or oxygen as well as thiophene heterocycle replaces the benzene ring were synthesized. Herein, we report the synthesis of some novel heterocyclic chalcone analogues (Figure 1) using a conventional base catalyzed Claisen Schmidt condensation reaction and their possible antibacterial activity alone and in combination with antibiotics.

Figure 1.

Figure 1

Structure of heterocyclic chalcones obtained from the Claisen Schmidt condensation between heteroaryl methyl ketones and substituted benzaldehydes.

2. Results and Discussion

2.1. Chemistry

A number of known and novel heterocyclic chalcone analogues were prepared via Claisen Schmidt condensation reactions [14,30] between appropriate benzaldehydes and heteroaryl methyl ketones like pyridine-2-yl methyl ketone, thiophene-2-yl methyl ketone and furan-2-yl methyl ketone (Scheme 1). The purity of the products and progress of the reaction was checked by TLC on silica gel plates. The condensation step was carried out over 6 h to 36 h. After recrystallization from methanol all corresponding chalcones were obtained in 43–63% yields.

Scheme 1.

Scheme 1

Claisen-Schmidt condensation.

The structures of the synthesized compounds were confirmed by IR, 1H-NMR and mass spectrometry measurements. The 1H-NMR spectrum of the synthesized chalcones displayed two doublets at δ 7.2–8.4 ppm with a characteristic coupling constant (J) of 15–16 Hz, which confirms the formation of chalcones (possessing a α,β-unsaturated ketone). This higher coupling constant value indicates all synthetic compounds were geometrically pure and were exclusively trans (E) isomers. This condensation produced three series of heterocyclic chalcones, which differ in the A ring like pyridine-2-yl-chalcones (p1-7), furan-2-yl-chalcones (f1-7) and thiophene-2-yl-chalcone (t1-7), as listed in Table 1.

2.2. Antimicrobial Activity

The antimicrobial assays were carried out by the dilution method using the following bacterial strains: methicillin-sensitive Staphylococcus aureus ATCC 29213 (SA or MSSA), methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA) and a vancomycin and methicillin resistant Staphylococcus aureus isolated from a human sample. Table 2 summarizes the results obtained for the MICs of the heterocyclic chalcones against the three bacterial strains. Of the 21 heterocyclic chalcones, five compounds had strong activity against the tested S. aureus, including two compounds pertaining to the pyridine-2-yl-chalcone group (p5 and p6), two compounds pertaining to the furan-2-yl-chalcone group (f5, f6) and one compound pertaining to the thiophene-2-yl- chalcone group (t5).

Results of determining the antibacterial coordination ability of the test substances and antibiotics are shown in Table 3. Interestingly, while the five abovementioned chalcones (p5, p6, f5, f6, t5) showed moderate antibacterial effects, they might increase dramatically the activity of some combined antibiotics. The chalcones in these combinations also exhibited an antibacterial effect better than that seen when they were used alone (Table 4). In the checkerboard technique, results of quantitatively determining antibacterial coordination ability proved that combinations between antibiotics and chalcone derivatives f5 and p6 had synergistic effects against MRSA ATCC 43300. Such combinations are mixtures of compound f5 and oxacillin; compound p6 and vancomycin. However, no synergistic effect of the combinations of the synthesized heterocylic chalcones and test antibiotics could be found on the local hospital isolated MRSA.

Table 1.

The structures of the 21 chalcones with their substitution patterns.

graphic file with name molecules-17-06684-i001.jpg

Compounds A ring (R) Substitution in B ring Formula Isolated yield (%)
C2 C3 C4 C5
p1 graphic file with name molecules-17-06684-i002.jpg H NO2 H H C14H10N2O3 45
p2 H H N(CH3)2 H C16H16N2O 62
p3 H H OCH3 OCH3 C16H15NO3 58
p4 H OCH3 OCH3 OCH3 C17H17NO4 51
p5 OH H H H C14H11NO2 51
p6 H OH H H C14H11NO2 50
p7 H H OH H C14H11NO2 58
f1 graphic file with name molecules-17-06684-i003.jpg H NO2 H H C13H9NO4 43
f2 H H N(CH3)2 H C15H15O2 61
f3 H H OCH3 OCH3 C15H14O4 55
f4 H OCH3 OCH3 OCH3 C16H16O3 51
f5 OH H H H C13H10O5 54
f6 H OH H H C13H10O3 58
f7 H H OH H C13H10O3 62
t1 graphic file with name molecules-17-06684-i004.jpg H NO2 H H C13H9NO3S 46
t2 H H N(CH3)2 H C15H15NOS 63
t3 H H OCH3 OCH3 C15H14O3S 50
t4 H OCH3 OCH3 OCH3 C16H16O4S 53
t5 OH H H H C13H10O2S 55
t6 H OH H H C13H10O2S 52
t7 H H OH H C13H10O2S 58

Table 2.

Anti-Staphylococcus aureus activity of the synthetic compounds (minimum inhibitory concentration in µg/mL).

Chalcones a\Bacteria Pyridin-2yl-chalcones Furan-2yl-chalcones Thiophen-2yl-chalcones
p5 p6 p6 f5 f6 f7 t5 t6
MSSA ATCC 25923 128 64 512 64 128 256 32 256
MRSA ATCC 44330 128 128 512 64 128 256 64 128
MRSA-I (isolated) b - 64 - 32 512 - 256 -

a: the remaining compounds (p1-4; f1-4; t1-4 and t7) were inactive at a concentration of 512 µg/mL; b: isolated MRSA (MICs: oxacillin 256; vancomycin 64 µg/mL); (-): inactive at a concentration of 512 µg/mL; ND: not determined.

Table 3.

Antibacterial coordination ability results.

Antibiotics p6 f5 f6 t5
SA MRSA Isolated MRSA SA MRSA Isolated MRSA SA MRSA Isolated MRSA SA MRSA Isolated MRSA
Vancomycin - + - - + - - + - + + -
Doxycycline - - - - - - - - - - - -
Ciprofloxacin + - - - - - + - - - - -
Gentamicin - - - - - - - - - - - -
Chloramphenicol - - - - - - - - - - - -
Oxacicllin - - - - + - - + - - + -
Erythromycin - - - - - - - - - + - -

(SA): MSSA ATCC 25923; (MRSA): MRSA ATCC 43300; (isolated MRSA): MRSA with MICs of oxacicllin 256; 64 µg/mL and vancomycin 64 µg/mL.

Table 4.

Anti-SA and anti-MRSA activity of chalcones in combination with antibiotics.

Mix a MSSA ATCC 25923 MRSA ATCC 43300
MICs (µg/mL) FICI Interpretation Increasing rate (fold) b MICs (µg/mL) FICI Interpretation Increasing rate (fold) b
Alone Mix a Alone Mix a
p6 64 32 0.75 Additive ND ND ND ND ND
Cipro 0.5 0.125
p6 ND ND ND ND ND 128 32 0.31 Synergistic 4
Vanco 1 0.0625 16
f5 ND ND ND ND ND 64 16 0.31 Synergistic 4
Oxa 2 0.125 16
f6 128 64 0.75 Additive 2 ND ND ND ND ND
Cipro 0.5 0.125 4
f6 ND ND ND ND ND 128 64 0.625 Additive 2
Vanco 1 0.125 8
f6 ND ND ND ND ND 128 64 0.56 Additive 2
Oxa 2 0.125 16
t5 32 32 1.06 Indifferent 1 ND ND ND ND ND
Ery 0.5 0.03 1.67
t5 32 16 1 Additive 2 ND ND ND ND ND
Cipro 0.5 0.25 2
t5 ND ND ND ND ND 64 32 1.0 Additive 2
Vanco 1 0.5 2
t5 ND ND ND ND ND 64 32 0.56 Additive 2
Oxa 2 0.125 16

(a) Mix = Combination; (b) Rate in increasing antibacterial activity of antibiotic/chalcone in combination compared to that of in alone (fold); ND = not dertemined. Vanco: vancomycin; Cipro: ciprofloxacin; Ery: erythromycin; Oxa: oxacillin.

Some synthesized heterocyclic chalcones exhibit strong antibacterial activities and may act by damaging the cell wall of SA, which is clearly similar to the observed mechanism of a well-known cell membrane permeabilizer, polymyxin B [27]. However, the molecular mechanism of action is not yet clearly understood. Some preliminary remarks on structure-activity relationship can be drawn from the bioactivity results as follows:

  • (i) A free hydroxyl group in position 2/3 in the B ring (phenyl moiety) appears to be very important for anti-Staphylococcus aureus activity (p5, p6, f5, f6, t5). In case of substitution of a hydroxyl group in position 4 (p7, f7, t7), chalcones were inactive on the tested Staphylococcus aureus strains.

  • (ii) Substitution in the B ring with a nitro group in position 3 (as in p1, f1, t1) or with two/three methoxyl groups in positions 3/4/5 (as in p3, p4, f3, f4, t3 and t4) might also be responsible for the decrease in the anti-Staphylococcus aureus activity;

  • (iii) The chalcones possessing two/three methoxy groups in B ring were inactive, regardless of their A ring structures. This means that methoxy groups seem to abolish the hydrophilic property of the phenol hydroxy moiety which can affect penetration of antibiotics through bacterial cell walls.

  • (iv) The chalcones possessing a hydroxyl group in B ring (p6, f6 and t5) demonstrated strongly positive interactions with antibiotic such as ciprofloxacin on methicillin-sensitive-Staphylococcus aureus. These chalcones increased significantly the activity of combined antibiotics like vancomycin and oxacillin up to sixteen-fold on methicillin -resistant-Staphylococcus aureus. These combinations could lead to develop new treatments for MRSA infectious diseases;

  • (v) The analysis of structural influence of A ring on anti-Staphylococcus aureus activity suggested that a furan-2-yl moiety may be more important than the thiophene-2-yl or pyridine-2yl- moieties.

3. Experimental

3.1. General

Melting points (mp) were taken in open capillary tubes using a Gallenkamp apparatus and are uncorrected. The IR spectra were recorded on a Shimadzu FTIR 8201 PC spectrophotometer. 1H-NMR spectra were determined on a Bruker Ultrashield 500 spectrometer using tetramethylsilane (TMS) as an internal reference. All the starting materials are commercially available.

3.2. General Procedure for the Synthesis of Chalcones [14,30]

A solution of heteroaryl methyl ketone (5 mM) and aromatic aldehyde (5 mM) in methanol (15 mL) was cooled to 5–10 °C in an ice bath. The cooled solution was treated with a small portion of pulverized KOH (10 mM). The reaction mixture was magnetically stirred for 60 min and then left overnight or longer, as monitored by thin layer chromatography using n-hexane-acetone (5:1) as developing solvent. The resulting dark solution was diluted with ice water and carefully acidified using dilute hydrochloric acid. The chalcones, which separated as a yellow solid, were collected by filtration after washing with water and further purified by crystallization from methanol. When needed, the crude product was further purified by silica gel column chromatography.

Characterization data of the synthesized heterocyclic chalcone analogues:

1-(Pyridine-2-yl)-3-(3-nitrophenyl)-2-propene-1-one (p1). Mp: 180 °C; IR (νcm−1, KBr): 1674 (υC=O) and 1616 (υC=C). 1H-NMR (DMSO-d6, δ ppm): 8.83 (d, J = 4.5 Hz, 1H, H6′); 8.63 (s, 1H, H2); 8.40 (d, J = 16 Hz, 1H, Hβ); 8.31 (d, J = 7.5 Hz, 1H, H4); 8.29 (d, J = 2 Hz, 1H, H3′); 8.14 (d, J = 8 Hz, 1H, H6); 8.10 (m, 1H, H4′); 7.98 (d, J = 16 Hz, 1H, Hα); 7.77 (t, 1H, H5); 7.74 (m, 1H, H5′). MS (ESI, positive scan): m/z 277.23 [M+Na]+.

1-(Pyridine-2-yl)-3-(4-dimethylaminophenyl)-2-propene-1-one (p2). Mp: 138 °C; IR: (νcm−1, KBr): 1656 (υC=O), 1604 (υC=C). 1H-NMR (DMSO-d6, δ ppm): 8.78 (d, J = 4.5 Hz, 1H, H6); 8.08 (d, J = 8 Hz, 1H, H3′); 8.02 (d, 1H, H4′); 8.01 (d, J = 16 Hz, 1H, Hβ); 7.79 (d, J = 16 Hz, 1H, Hα); 7.67-7.66 (m, 1H, H5’); 7.65 (d, J = 9 Hz, 2H, H2 and H6); 6.77 (d, J = 9 Hz, 2H, H3 and H4); 3.02 (s, 6H, 2 × CH3). MS (ESI, positive scan): m/z 255.35 [M+H]+.

1-(Pyridine-2-yl)-3-(3,4-dimethoxyphenyl)-2-propene-1-one (p3). Mp: 105 °C; IR (νcm−1, KBr): 1666 (υC=O); 1596 (υC=C); 1271 (υC-O-). 1H-NMR (DMSO-d6, δ ppm): 8.80 (m, 1H, H6′); 8.13 (d, J = 16 Hz; 1H, Hβ); 8.09 (d, J = 7.5 Hz, 1H, H3′); 8.04 (m, 1H, H4′); 7.82 (d, J = 16 Hz, 1H, Hα); 7.68 (m, 1H, H5′); 7.40 (d, J = 8 Hz, 1H, H2); 7.37 (d, J = 1.5 Hz, 1H, H6); 7.04 (d, J = 8 Hz, 1H, H5); 3.83 (s, 6H, 2 × -OCH3). MS (ESI, positive scan): m/z 292.34 [M+Na]+.

1-(Pyridine-2-yl)-3-(3,4,5-trimethoxyphenyl)-2-propene-1-one (p4). Mp: 160 °C; IR (νcm−1, KBr): 1668 (υC=O); 1610 (υC=C); 1124 (υC-O-). 1H-NMR (CDCl3, δ ppm): 8.76 (m, 1H, H6′); 8.20 (s, 1H, H3′); 8.17 (d, J = 16 Hz, 1H, Hβ); 7.89 (t, 1H, H4′); 7.87 (d, J = 16 Hz, 1H, Hα); 7.51 (m, 1H, H5′); 6.96 (s, 2H, H2 and H6); 3.94 (s, 6H, 2 × -OCH3); 3.91 (s, 3H, -OCH3). MS (ESI, positive scan): m/z 322.14 [M+H]+.

1-(Pyridine-2-yl)-3-(2-hydroxyphenyl)-2-propene-1-one (p5). Mp: 140 °C; IR (νcm−1, KBr): 1660 (υC=O); 1591 (υC=C); 3375 (υO-H). 1H-NMR (DMSO-d6, δppm): 10.32 (s, 1H, -OH); 8.79 (m, 1H, H6′); 8.30 (d, J = 16 Hz, 1H, Hβ); 8.10 (d, J = 1 Hz, 6.5 Hz, 1H, H3′); 8.08 (m, 1H, H4′); 8.04 (m, 1H, H5′); 7.70 (d, J = 1 Hz, 7.5 Hz, 1H, H3); 7.67 (d, J = 16 Hz, 1H, Hα); 7.29 (m, 1H, H4); 6.96 (d, J = 1 Hz, 8 Hz, 1H, H6); 6.90 (t, 1H, H5). MS (ESI, positive scan): m/z 248.07 [M+Na]+.

1-(Pyridine-2-yl)-3-(3-hydroxyphenyl)-2-propen-1-one (p6). Mp: 131 °C; IR (νcm−1, KBr): 1660 (υC=O); 1566 (υC=C); 3359 (υO-H). 1H-NMR (DMSO-d6, δ ppm): 9.70 (s, 1H, OH); 8.80 (d, J = 4.5 Hz, 1H, H6′); 8.19 (d, J = 16.5 Hz, 1H, Hβ); 8.10 (d, J = 8 Hz, 1H, H3′); 8.05 (t, 1H, H4′); 7.75 (d, J = 16 Hz, 1H, Hα); 7.69 (m, 1H, H5′); 7.27 (t, 1H, H5); 7.23 (d, J = 8 Hz, 1H, H6); 7.18 (s, 1H, H2); 6.88 (d, J = 1 Hz, 7.5 Hz, 1H, H4). MS (ESI, positive scan): m/z 248.07 [M+Na]+.

1-(pyridine-2-yl)-3-(4-hydroxyphenyl)-2-propene-1-one (p7). Mp: 137 °C; IR (νcm−1, KBr): 1664 (υC=O); 1579 (υC=C); 3100 (υO-H). 1H-NMR (DMSO-d6, δ ppm): 10.12 (s, 1H, -OH); 8.77 (d, J = 5.5 Hz, 1H, H6′); 8.08 (d, J = 16 Hz, 1H, Hβ); 8.03 (m, 2H, H3′, H4′); 7.77 (d, J = 16 Hz, 1H, Hα); 7.71 (m, 1H, H5′); 7.69 (d, 2H, H2, H6); 6.84 (d, 2H, H3, H5). MS (ESI, positive scan): m/z 248.07 [M+Na]+.

1-(Furan-2-yl)-3-(3-nitrophenyl)-2-propene-1-one (f1). Mp: 185 °C; IR (νcm−1, KBr): 1656 (υC=O); 1604 (υC=C). 1H-NMR (DMSO-d6, δ ppm): 8.73 (s, 1H, H5’); 8.31 (d, J = 8 Hz, 1H, H4); 8.27 (d, J = 2 Hz, 8 Hz, 1H, H6); 8.10 (t, 1H, H2); 7.94 (d, J = 3.5 Hz, 1H, H3′); 7.91 (d, J = 16 Hz, 1H, Hβ); 7.85 (d, J = 16 Hz, 1H, Hα); 7.76 (t, 1H, H5); 6.82 (m, 1H, H4′). MS (ESI, positive scan): m/z 266.2 [M+Na]+.

1-(Furan-2-yl)-3-(4-dimethylaminophenyl)-2-propene-1-one (f2). Mp: 104 °C; IR (νcm−1, KBr): 1643 (υC=O); 1612 (υC=C). 1H-NMR (DMSO-d6, δ ppm): 8.00 (d, J = 1 Hz, 1H, H5’); 7.67 (d, J = 16 Hz, 1H, Hβ); 7.66 (d, J = 9 Hz, 3H, H2, H6 and H3′); 7.40 (d, J = 16 Hz, 1H, Hα); 6.76 (m, 3H, H3, H5 and H4′); 3.00 (s, 6H, 2 × -CH3). MS (ESI, positive scan): m/z 242.2 [M+H]+.

1-(Furan-2-yl)-3-(3,4-dimethoxyphenyl)-2-propene-1-one (f3). Mp: 105 °C; IR (νcm−1, KBr): 1651 (υC=O); 1595 (υC=C); 1263(υC-O-). 1H-NMR (DMSO-d6, δ ppm): 8.04 (d, J = 1 Hz, 1H, H5′); 7.77 (d, J = 3 Hz; 1H, H3′); 7.70 (d, J = 16 Hz, 1H, Hβ); 7.56 (d, J = 16 Hz, 1H, Hα); 7.47 (s, 1H, H2); 7.37 (m, 1H, H6); 7.02 (d, J = 8.5 Hz, 1H, H5); 6.78 (m, 1H, H4′); 3.83 (s, 6H, 2 × -OCH3). MS (ESI, positive scan): m/z 281.4 [M+Na]+.

1-(Furan-2-yl)-3-(3,4,5-trimethoxyphenyl)-2-propene-1-one (f4). Mp: 158 °C; IR (νcm−1, KBr): 1654 (υC=O); 1604 (υC=C); 1122 (υC-O-). 1H-NMR (CDCl3, δ ppm): 7.80 (d, J = 16 Hz, 1H, Hβ); 7.66 (d, J = 1 Hz, 1H, H5′); 7.34 (d, J = 16 Hz, 1H, Hα); 7.34 (d, J =3.5 Hz, 1H, H3′); 6.88 (s, 2H, H2 and H6); 6.61 (m, 1H, H4’); 3.93 (s, 6H, 2 × -OCH3); 3.90 (s, 3H, -OCH3). MS (ESI, positive scan): m/z 311.06 [M+Na]+.

1-(Furan-2-yl)-3-(2-hydroxyphenyl)-2-propene-1-one (f5). Mp: 138 °C; IR (νcm−1, KBr): 1643 (υC=O), 1577 (υC=C); 3124 (υO-H). 1H-NMR (DMSO-d6, δ ppm): 10.24 (s, 1H, -OH); 8.03 (d, J = 16 Hz, 1H, Hβ); 8.03 (d, J = 1 Hz, 7.5 Hz, 1H, H5′); 7.78 (d, J = 1.5 Hz, 6 Hz, 1H, H6′); 7.67 (d, J = 3.5 Hz, 1H, H3′); 7.64 (d, J = 16 Hz, 1H, Hα); 7.27 (m, 1H, H4); 6.93 (d, J = 0.5 Hz, 8Hz, 1H, H3); 6.87 (t, 1H, H4′); 6.76 (m, 1H, H5). MS (ESI, positive scan): m/z 248.07 [M+Na]+.

1-(Furan-2-yl)-3-(3-hydroxyphenyl)-2-propene-1-one (f6). Mp: 141 °C; IR (νcm−1, KBr): 1643 (υC=O); 1577 (υC=C); 3309 (υO-H). 1H-NMR (DMSO-d6, δ ppm): 9.61 (s, 1H, -OH); 8.05 (d, 1H, H5′); 7.79 (d, 1H, H3′); 7.65 (d, J = 16 Hz, 1H, Hβ); 7.58 (d, J = 16 Hz, 1H, Hα); 7.25 (t, 1H, H4′); 7.25 (d, 1H, H6); 7.19 (s, 1H, H2); 6.87 (m, 1H, H5); 6.78 (m, 1H, H4). MS (ESI, positive scan): m/z 248.07 [M+Na]+.

1-(Furan-2-yl)-3-(4-hydroxyphenyl)-2-propene-1-one (f7). Mp: 142 °C; IR (νcm−1, KBr): 1647 (υC=O); 1610 (υC=C); 3200 (υO-H). 1H-NMR (DMSO-d6, δ ppm): 10.09 (s, 1H, -OH); 8.02 (s, 1H, H5′); 7.72 (s, 1H, H3′); 7.71(d, J = 7.5 Hz, 2H, H2 and H6); 7.66 (d, J = 17 Hz, 1H, Hβ); 7.48 (d, J = 16.5 Hz, 1H, Hα); 6.83 (m, 2H, H3 and H5); 6.76 (m, 1H, H4′). MS (ESI, positive scan): m/z 248.06 [M+Na]+.

1-(Thiophene-2-yl)-3-(3-nitrophenyl)-2-propene-1-one (t1). Mp: 159 °C; IR (νcm−1, KBr): 1651 (υC=O); 1600 (υC=C). 1H-NMR (DMSO-d6, δ ppm): 8.77 (d, J = 1.5 Hz, 1H, H5′); 8.43 (d, J = 5 Hz, 1H, H3′); 8.33 (d, J = 8 Hz, 1H, H4); 8.27 (d, J = 2 Hz, 8 Hz, 1H, H6); 8.09 (d, J = 16 Hz, 1H, Hβ); 8.10 (d, J = 1 Hz, 1H, H2); 7.85 (d, J = 15.5 Hz, 1H, Hα); 7.77 (t, 1H, H5); 7.54 (m, 1H, H4′). MS (ESI, positive scan): m/z 282.02 [M+Na]+.

1-(Thiophene-2-yl)-3-(4-dimethylaminophenyl)-2-propene-1-one (t2). Mp: 131 °C; IR (νcm−1, KBr): 1633 (υC=O); 1612 (υC=C). 1H-NMR (CDCl3, δ ppm): 7.83 (d, J = 15.5 Hz, 1H, Hβ); 7.83 (d, J = 9 Hz, 1H, H5′); 7.62 (d, J = 6 Hz, 1H, H3′); 7.55 (d, J = 9 Hz, 2H, H2 and H6); 7.23 (d, J = 15.5 Hz, 1H, Hα); 7.16 (t, 1H, H4′); 6.70 (d, J = 8.5 Hz, 2H, H3 and H5); 3.04 (s, 6H, 2 × -CH3). MS (ESI, positive scan): m/z 258.08 [M+H]+.

1-(Thiophene-2-yl)-3-(3,4-dimethoxyphenyl)-2-propene-1-one (t3). Mp: 99 °C; IR (νcm−1, KBr): 1639 (υC=O); 1573 (υC=C); 1265 (υC-O-). 1H-NMR (DMSO-d6, δ ppm): 8.32 (m, 1H, H5′); 8.03 (m, 1H, H3′); 7.75 (d, J = 15.5 Hz, 1H, Hβ); 7.68 (d, J = 15.5 Hz, 1H, Hα); 7.52 (s, 1H, H2); 7.40 (m, 1H, H6); 7.31 (m, 1H, H4’); 7.02 (d, J = 8.5 Hz, 1H, H5); 3.83 (s, 6H, 2 × -OCH3). MS (ESI, positive scan): m/z 297.02 [M+Na]+.

1-(Thiophene-2-yl)-3-(3,4,5-trimethoxyphenyl)-2-propene-1-one (t4). Mp: 156 °C; IR (νcm−1, KBr): 1645 (υC=O); 1596 (υC=C); 1124 (υC-O-). 1H-NMR (CDCl3, δ ppm): 7.88 (m, 1H, H5’); 7.77 (d, J = 15.5 Hz, 1H, Hβ); 7.69 (d, J = 1 Hz, 5 Hz, 1H, H3′); 7.30 (d, J = 15.5 Hz, 1H, Hα); 7.19 (t, 1H, H4′); 6.87 (s, 2H, H2 and H6); 3.93 (s, 6H, 2 × -OCH3); 3.91 (s, 3H, -OCH3). MS (ESI, positive scan): m/z 327.04 [M+Na]+.

1-(thiophene-2-yl)-3-(2-hydroxyphenyl)-2-propene-1-one (t5). Mp: 158 °C; IR (νcm−1, KBr): 1637 (υC=O); 1560 (υC=C); 3332 (υO-H). 1H-NMR (DMSO-d6, δ ppm): 10.30 (s, 1H, -OH); 8.20 (d, J = 1 Hz, 3 Hz, 1H, H5′); 8.04 (d, J = 16 Hz, 1H, Hβ); 8.03 (d, 1H, H6); 7.86 (d, J = 1.5 Hz, 8 Hz, 1H, H3); 7.77 (d, J = 15.5 Hz, 1H, Hα); 7.26-7.30 (m, 2H, H4, H5); 6.94 (d, J = 1 Hz, 4Hz, H3); 6.88 (t, 1H, H4′). MS (ESI, positive scan): m/z 253. [M+Na]+.

1-(Thiophene-2-yl)-3-(3-hydroxyphenyl)-2-propene-1-one (t6). Mp: 130 °C; IR (νcm−1, KBr): 1643 (υC=O); 1577 (υC=C); 3334 (υO-H). 1H-NMR (DMSO-d6, δppm): 9.62 (s, 1H, -OH); 8.31 (d, J = 4 Hz, 1H, H5′); 8.05 (d, J = 5 Hz, 1H, H3′); 7.77 (d, J = 15.5 Hz, 1H, Hβ); 7.63 (d, J = 16 Hz, 1H, Hα); 7.31 (t, 1H, H4′); 7.30 (d, 1H, H6); 7.27 (t, 1H, H5); 7.23 (s, 1H, H2); 6.87 (m, 1H, H4). MS (ESI, positive scan): m/z 253. [M+Na]+.

1-(Thiophene-2-yl)-3-(4-hydroxyphenyl)-2-propene-1-one (t7). Mp: 151 °C; IR (νcm−1, KBr): 1643 (υC=O); 1610 (υC=C); 3172 (υO-H). 1H-NMR (DMSO-d6, δ ppm): 10.09 (s, 1H, -OH); 8.25 (m, 1H, H5’); 8.01 (m, 1H, H3′); 7.73 (m, 2H, H2 and H6); 7.65 (s, 2H, Hβ and Hα); 6.83 (m, 2H, H3 and H5). MS (ESI, positive scan): m/z 253. [M+Na]+.

3.3. Antibacterial Activity

3.3.1. Antibiotics and Antibacterial Agents

Standard powder forms of vancomycin and ciprofloxacin (Sigma Chemical Co., Ltd.), chloramphenicol, doxycycline and gentamicin (Shanghai Fine Chemicals Co., Ltd.), were stored at 2 to 8 °C until use. Antibiotics discs like gentamycin (10 µg), cloramphenicol (30 µg), vancomycin (30 µg), clindamycin (2 µg), doxycycline (30 µg), ciprofloxacin (5 µg) and erythromycin (15 µg) were supplied by Nam Khoa Co., Ltd., Vietnam.

Three standard strains of bacteria: Staphylococcus aureus ATCC 25923 and methicillin-resistant Staphylococcus aureus ATCC 43300 and isolated MDR MRSA preserved and activated atour Department of Microbiology were used in the study. The bacterial culture media were purchased from Merck. Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB) were used for bacteria culture and Mueller-Hinton Agar (MHA) was used for testing antibacterial activity.

3.3.2. Antibacterial Susceptibility Testing

The disc diffusion method was carried out for the antibacterial tests [31]. Briefly a suspension of bacterial strains (100 µL) containing 106 cfu/mL of bacteria was spread on Mueller Hinton agar (MHA) medium. The disc (6 mm in diameter), impregnated with 10 µL of the test compound at the concentration of 102.4 mg/mL in DMSO were placed on the inoculated agar. Negative control was prepared using the same solvent (DMSO), which was employed to dissolve the test compounds. Antibiotics (2–30 µg/disc, 6 mm in diameter) were used as references. The inoculated plates were incubated at 35–37 °C for 24 h. The diameter of inhibitory zones (mm) was measured (Table 2).

3.2.3. Qualitative Determination of the Interaction between the Chalcones and Antibiotics

A routine Kirby-Bauer antibiotics susceptibility test [32,33] was performed to determine the inhibitory zone of selected compounds. Briefly, sterile MHA medium is melted and poured into Petri dishes to get an agar layer about 3–4 millimeters thick. A sterile cotton stick is dipped into the prepared bacterial suspension (prepared to a final concentration of approximately 108 CFU/mL), then pressed on the tube wall to dry, then the bacteria are spread on the agar surface evenly. Opened boxes are put in the incubator in 3–5 min to drain. Wells (6 mm in diameter) were punched in the agar. The distances from the hole (synthesized chalcone analogues) and disc (antibiotics) were measured so that margins of two antibiotic inhibitory zones would meet or slightly overlap. The chalcone samples prepared in solutions at rational concentration (1,024 µg/mL) in DMSO were filled into the wells. After allowing the test compounds to diffuse into agar (15 min at 37 °C) the plates were further incubated at 37 °C for a period of 18 h (for MSSA) or 24 h (for MRSA). A positive interaction between a specific antibiotic and a test substance was reported when the inhibitory zone of each compound extended towards the other (Table 3).

3.2.4. Measurement of MIC Values

The MICs of antibiotics and selected active chalcones were determined by the microdilution method as described by The National Committee for Clinical Laboratoty Standard (NCCLS) [34,35]. Each test compound was run in duplicate. The test plates were incubated at 35–37 °C for 24 h. The MIC was taken as the minimum concentration of the dilutions that inhibited the growth of the test microorganism. The concentration of the solvents used in the following assays was maintained at less than 2% so that no inhibition of organisms or interference occurred.

3.2.5. Evaluation of Combined Activity

Antibacterial interactions were determined using the checkerboard assay as previously described [36]. The range of tested compounds concentration used in the checkerboard analysis was such that the dilution range encompassed the MIC for each drug used in the analysis. The first compound of the combination was serially diluted along the ordinate, while the second compound was diluted along the abscissa. Broth micro-dilution plates were inoculated with each S. aureus strain to yield ~5 × 105 CFU/mL in a 100 μL final volume, and incubated for 18 h–24 h at 37 °C. Synergy has been defined as requiring a fourfold reduction in the MIC of both antibiotics in combination, compared with each used alone, measuring the fractional inhibitory concentration index (FICI). The FICI was calculated for each combination (A and B as tested compounds) using the following formula [36]:

graphic file with name molecules-17-06684-i005.jpg

The FICI was interpreted as follows: synergistic, FICI ≤ 0.5; additive, 0.5 < FICI ≤ 1; indifferent, 1 < FICI ≤ 2; antagonistic, FICI > 2.

4. Conclusions

The study has proved that the synthesized heterocyclic chalcone analogues have some anti-Staphyloccus aureus effects. The results, based on the potentially active chalcone skeleton, have pointed out the importance of the positions of the phenolic hydroxy groups in the B ring for obtaining antibacterial activity. In addition, the furan-2-yl moiety is more active than either the thiophene-2-yl or pyridine-2-yl one in anti-Staphylococcus aureus activity. Compounds p5, f6 and t5 exhibited potent inhibitory actvity against methicillin-resistant Staphylococus aureus in combination with vancomycin and oxacillin. They may provide a template to design new combinations of antibiotics and non-antibiotics to treatment of Staphylococus aureus infections.

Acknowledgements

This work was supported by the Vietnam’s National Foundation for Science and Technology Development - NAFOSTED (Grant # 104.01.105.09 to Thanh-Dao Tran). The authors are grateful to the Center for Education and Research of Natural Products, University of Medicine and Pharmacy at Ho Chi Minh City and MSc Quang-Nam Nguyen for kindly providing IR, MS measurements.

Conflict of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of the compounds p1-7, f1-7 and t1-7 are available from the authors.

References and notes

  • 1.Neu H.C. The crisis in antibiotic resistance. Science. 1992;257:1064–1073. doi: 10.1126/science.257.5073.1064. [DOI] [PubMed] [Google Scholar]
  • 2.Michel M., Gutmann L. Methicillin-resistant Staphyllococus aureus and vancomycin-resistan enterococci: therapeut realities and possibilities. Lancet. 1997;349:1901–1906. doi: 10.1016/S0140-6736(96)11192-2. [DOI] [PubMed] [Google Scholar]
  • 3.Normark B.H., Normark S. Evolution and spread of antibiotic resistance. J. Intern. Med. 2002;252:91–106. doi: 10.1046/j.1365-2796.2002.01026.x. [DOI] [PubMed] [Google Scholar]
  • 4.Shekarchi M., Pirali-Hamedani M., Navidpour L., Adib N., Shafiee A. Synthesis, Antibacterial and Antifungal Activities of 3-Aryl-5-(pyridin-3-yl)-4,5-dihydropyrazole-1-carbothioamide Derivatives. J. Iran. Chem. Soc. 2008;5:150–158. doi: 10.1007/BF03245828. [DOI] [Google Scholar]
  • 5.Silverman R.B. Organic Chemistry of Drug Design and Drug Action. Academic Press; San Diego, CA, USA: 1992. [Google Scholar]
  • 6.Thompson L.A., Ellman J.A. Synthesis and applications of small molecule libraries. Chem. Rev. 1996;96:555–600. doi: 10.1021/cr9402081. [DOI] [PubMed] [Google Scholar]
  • 7.Alcaraz L.E., Blanco S.E., Puig O.N., Tomas F., Ferretti F.H. Antibacterial activity of Flavonoids against methicillin- resistant Staphylococcus aureus strains. J. Theor. Biol. 2000;205:231–240. doi: 10.1006/jtbi.2000.2062. [DOI] [PubMed] [Google Scholar]
  • 8.Baviskar B.A., Baviskar B., Shiradkar M.R., Deokate U.A., Khadabadi S.S. Antimicrobial activity of some novel. Benzimidazolyl chalcones. E-J. Chem. 2009;6:196–200. doi: 10.1155/2009/746292. [DOI] [Google Scholar]
  • 9.Echeverria C., Santibañez J.F., Donoso-Tauda O., Escobar C.A., Ramirez-Tagle R. Structural antitumoral activity relationships of synthetic chalcones. Int. J. Mol. Sci. 2009;10:221–231. doi: 10.3390/ijms10010221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Modzelewska A., Pettit C., Achanta G., Davidson N.E., Huang P., Khan S.R. Anticancer activities of novel chalcone and bis-chalcone derivatives. Bioorg. Med. Chem. 2006;14:3491–3495. doi: 10.1016/j.bmc.2006.01.003. [DOI] [PubMed] [Google Scholar]
  • 11.Vogel S., Heilmann J. Synthesis, cytotoxicity, and antioxidative activity of minor prenylated chalcones from Humulus lupulus. J. Nat. Prod. 2008;71:1237–1241. doi: 10.1021/np800188b. [DOI] [PubMed] [Google Scholar]
  • 12.Babasaheb P.B., Sachin A.P., Rajesh N.G. Synthesis and biological evaluation of nitrogen-containing chalcones as possible anti-inflammatory and antioxidant agents. Bioorg. Med. Chem. Lett. 2010;20:730–733. doi: 10.1016/j.bmcl.2009.11.068. [DOI] [PubMed] [Google Scholar]
  • 13.Vogel S., Barbic M., Jürgenliemk G., Heilmann J. Synthesis, cytotoxicity, anti-oxidative and anti-inflammatory activity of chalcones and influence of A-ring modifications on the pharmacological effect. Eur. J. Med. Chem. 2010;45:2206–2213. doi: 10.1016/j.ejmech.2010.01.060. [DOI] [PubMed] [Google Scholar]
  • 14.Tran T.-D., Park H., Kim H.P., Ecker G.F., Thai K.-M. Inhibitory activity of prostaglandin E2 production by the synthetic 2'-hydroxychalcone analogues: Synthesis and SAR study. Bioorg. Med. Chem. Lett. 2009;19:1650–1653. doi: 10.1016/j.bmcl.2009.02.001. [DOI] [PubMed] [Google Scholar]
  • 15.Beom-Tae K., Kwang-Joong O., Jae-Chul C., Ki-Jun H. Synthesis of dihydroxylated chalcone derivatives with diverse substitution patterns and their radical scavenging ability toward DPPH free radicals. Bull. Korean Chem. Soc. 2008;29:1125–1130. doi: 10.5012/bkcs.2008.29.6.1125. [DOI] [Google Scholar]
  • 16.Doan T.N., Tran T.-D. Synthesis, antioxidant and antimicrobial activities of a novel series of chalcones, pyrazolic chalcones, and allylic chalcones. Pharmacol. Pharm. 2011;2:282–288. [Google Scholar]
  • 17.Sivakumar P.M., Prabhakar P.K., Doble M. Synthesis, antioxidant evaluation, and quantitative structure-activity relationship studies of chalcones. Med. Chem. Res. 2011;20:482–492. doi: 10.1007/s00044-010-9342-1. [DOI] [Google Scholar]
  • 18.Vogel S., Ohmayer S., Brunner G., Heilmann J. Natural and non-natural prenylated chalcones: synthesis, cytotoxicity and anti-oxidative activity. Bioorg. Med. Chem. 2008;16:4286–4293. doi: 10.1016/j.bmc.2008.02.079. [DOI] [PubMed] [Google Scholar]
  • 19.Munawar M.A., Azad M., Siddiqui H.L. Synthesis and antimicrobial studies of some quinolinylpyrimidine derivatives. J. Chin. Soc. 2008;55:394–400. [Google Scholar]
  • 20.Azad M., Munawar M.A., Siddiqui H.L. Antimicrobial activity and synthesis of quinoline-base chalcones. J. Appl. Sci. 2007;7:2485–2489. doi: 10.3923/jas.2007.2485.2489. [DOI] [Google Scholar]
  • 21.Kalirajan R., Sivakumar S.U., Jubie S., Gowramma B., Suresh B. Synthesis and biological evaluation of some heterocyclic derivatives of chalcones. Int. J. ChemTech Res. 2009;1:27–34. [Google Scholar]
  • 22.Talia J.M., Debattista N.B., Pappano N.B. New antimicrobial combinations: Substituted chalcones- oxacillin against methicillin resistant Staphylococcus aureus. Braz. J Microbiol. 2011;42:470–475. doi: 10.1590/S1517-83822011000200010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Eumkeb G., Siriwong S., Phitaktim S., Rojtinnakorn N., Sakdarat S. Synergistic activity and mode of action of flavonoids isolated from smaller galangal and amoxicillin combinations against amoxicillin-resistant Escherichia coli. J. Appl. Microbiol. 2012;112:55–64. doi: 10.1111/j.1365-2672.2011.05190.x. [DOI] [PubMed] [Google Scholar]
  • 24.Sato Y., Shibata H., Arakaki N., Higuti T. 6,7-dihydroxyflavone dramatically intensifies the susceptibility of methicillin-resistant or -sensitive Staphylococcus aureus to beta-lactams. Antimicrob. Agents Chemother. 2004;48:1357–1360. doi: 10.1128/AAC.48.4.1357-1360.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Liu M.H., Otsuka N., Noyori K., Shiota S., Ogawa W., Kuroda T., Hatano T., Tsuchiya T. Synergistic effect of kaempferol glycosides purified from Laurus nobilis and fluoroquinolones on methicillin-resistant Staphylococcus aureus. Biol. Pharm. Bull. 2009;32:489–492. doi: 10.1248/bpb.32.489. [DOI] [PubMed] [Google Scholar]
  • 26.Tiwari B., Pratapwar A.S., Tapas A.R., Butle S.R., Vatkar B.S. Synthesis and antimicrobial activity of some chalcone derivatives. Int. J. ChemTech Res. 2010;2:499–503. [Google Scholar]
  • 27.Sivakumar P.M., Priya S., Doble M. Synthesis, biological evaluation, mechanism of action and quantitative structure-activity relationship studies of chalcones as antibacterial agents. Chem. Biol. Drug Des. 2009;73:403–415. doi: 10.1111/j.1747-0285.2009.00793.x. [DOI] [PubMed] [Google Scholar]
  • 28.Zheng C.J., Jiang S.M., Chen Z.H., Ye B.J., Piao H.R. Synthesis and anti-bacterial activity of some heterocyclic chalcone derivatives bearing thiofuran, furan, and quinoline moieties. Arch. Pharm. Chem. Life Sci. 2011;344:689–695. doi: 10.1002/ardp.201100005. [DOI] [PubMed] [Google Scholar]
  • 29.Lopez S.N., Castelli M.V., Zacchino S.A., Domnguez J.N., Lobo G., Charris-Charris J., Cortes J.C.G., Ribas J.C., Devia C., Rodrguez A.M., et al. In Vitro Antifungal evaluation and structure-activity relationships of a new series of chalcone derivatives and synthetic analogues, with inhibitory properties against polymers of the fungal cell wall. Bioorg. Med. Chem. 2001;9:1999–2013. doi: 10.1016/S0968-0896(01)00116-X. [DOI] [PubMed] [Google Scholar]
  • 30.Funiss B.S., Hannford A.J., Smith P.W.G., Tatchell A.R. Vogel’s Textbook of Practical Organic Chemistry. 5th. Prentice Hall; Upper Saddle River: NJ, USA: 2004. pp. 1032–1035. [Google Scholar]
  • 31.National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A2. 3rd. Vol. 22. NCCLS; Wayne, PA, USA: 2002. pp. 1–30. [Google Scholar]
  • 32.Bauer A.W., Kirby W.M.M., Sherris J.C., Turck M. Antibiotic Susceptibility Testing by a Standardized Single Disk Method. Am. J. Clin. Pathol. 1966;36:493–496. [PubMed] [Google Scholar]
  • 33.Jorgensen J.H., Turnidge J.D. In: Manual of Clinical Microbiology. 9th. Murray P.R., Baron E.J., Jorgensen J.H., Landry M.L., Pfaller M.A., editors. ASM Press; Washington, DC, USA: 2007. pp. 1152–1172. [Google Scholar]
  • 34.National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved standard M7-A6. 6th. NCCLS; Wayne, PA, USA: 2003. [Google Scholar]
  • 35.National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing. Informational supplement M100-S13. 12th. NCCLS; Wayne, PA, USA: 2003. [Google Scholar]
  • 36.Bajaksouzian S., Visalli M.A., Jacobs M.R., Appelbaum P.C. Activities of levofloxacin, ofloxacin, and ciprofloxacin, alone and in combination with amikacin, against acinetobacters as determined by checkerboard and time-kill studies. Antimicrob. Agents Chemother. 1997;41:1073–1076. doi: 10.1128/aac.41.5.1073. [DOI] [PMC free article] [PubMed] [Google Scholar]

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