Synthesis and antimicrobial activity of novel chalcone derivatives

Article abstract of DOI:10.1007/s11164-013-1076-5

A series of novel 3-[N, N-bis(2-hydroxyethyl)-amino]-chalcone derivatives 3a-3j were synthesized by the aldol condensation of [N, N-bis(2-hydroethyl)-3- amino]-acetophenone 2 with aromatic aldehydes. Their structures were further confirmed by ESI-HRMS, ¹H NMR, IR and elemental analysis. X-ray analysis reveals crystal 3b is a monoclinic system with P2₁/n space group. The antimicrobial activities of the newly synthesized chalcones in vitro were evaluated and the results indicated that most compounds presented moderate to good antimicrobial activities, especially the antifungal capability. Compounds 3a, 3d, 3f and 3g revealed obvious potency against Candida albicans with MIC values of 32 μg/mL, which were better compared with others.

Full text of DOI:10.1007/s11164-013-1076-5

Res Chem Intermed
DOI 10.1007/s11164-013-1076-5
Synthesis and antimicrobial activity of novel chalcone
derivatives
•
Xianwen Fang Bingqin Yang Zhao Cheng
•
•
•
Pengfei Zhang Meipan Yang
Received: 12 October 2012 / Accepted: 23 January 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract A series of novel 3-[N, N-bis(2-hydroxyethyl)-amino]-chalcone deriv-
atives 3a–3j were synthesized by the aldol condensation of [N, N-bis(2-hydroethyl)-
3-amino]-acetophenone 2 with aromatic aldehydes. Their structures were further
1
confirmed by ESI-HRMS, H NMR, IR and elemental analysis. X-ray analysis
reveals crystal 3b is a monoclinic system with P21/n space group. The antimicrobial
activities of the newly synthesized chalcones in vitro were evaluated and the results
indicated that most compounds presented moderate to good antimicrobial activities,
especially the antifungal capability. Compounds 3a, 3d, 3f and 3g revealed obvious
potency against Candida albicans with MIC values of 32 lg/mL, which were better
compared with others.
Keywords Chalcone ꢀ Synthesis ꢀ Crystal structure ꢀ Broth tube dilution method ꢀ
Antimicrobial activity
Introduction
Chalcones are the main substructures of some natural compounds and important
precursors for the biosynthesis of flavonoids. To date, chalcones have been
identified in a variety of plant species such as fruits, vegetables, spices, tea and soy-
Electronic supplementary material The online version of this article (doi:
10.1007/s11164-013-1076-5) contains supplementary material, which is available to authorized users.
X. Fang (&) ꢀ B. Yang ꢀ Z. Cheng ꢀ P. Zhang ꢀ M. Yang
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education,
College of Chemistry & Materials Science, Northwest University, Xi’an 710069, China
e-mail: fxw325@126.com
B. Yang
e-mail: yangbq@nwu.edu.cn
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X. Fang et al.
Fig. 1 The basic framework of
chalcone
O
2'
2
3
3'
4'
B
A
R
6
6'
4
R'
5
5'
based foodstuffs [1]. The naturally occurring and synthesized chalcone compounds,
with a common 1,3-diphenyl-2-propen-1-one framework (Fig. 1), have been
reported to possess various pharmacological activities, such as antioxidation [2]
anticancer [3], antileishmanial [4], cytotoxicity [5, 6], antituberculosis[7] and
antimalarial [8]. Recent reports indicated the importance of chalcones as antiin-
flammatory and antifungal agents involved in the inhibition of cell migration and
the prevention of TNF-a and lipopolysaccharide (LPS) induced neutrophil
adhension [9]. Sivakumar et al. [10] found that a coating of chalcone on the
polymeric surfaces could reduce the bacterial adhesion due to its bactericidal (cell
membrane disruption) [11] activities.
Infectious diseases, such as Candida albicans infection, is the major cause of
infectious death among patients with chemotherapy-induced myelosuppression
neutropenia [12], which is particularly dangerous in people with compromised
immune systems such as cancer patients (undergoing immunosuppressive therapy)
and AIDS patients. Moreover, increasing numbers of multidrug-resistant microbial
pathogens such as methicillin-resistant staphylococcus aureus (MRSA) and
vancomycin intermediate-resistant staphylococcus aureus (V-ISA) have made the
treatment of infectious diseases a serious and challenging therapeutic problem in
recent decades [13]. As pathogenic bacteria and fungi continuously evolve
mechanisms of resistance to currently used antibiotics, the discovery and design
of novel and potent antibacterial and antifungal drugs are badly needed to overcome
microbial resistance and develop effective therapies [14].
With the attempt to search for better and more potential biological activity, we
focus on the chalcone derivatives containing different substitutions on one or both
of the phenyl rings, which may show potential antifungal and antibacterial
properties. Here, we report the kind of newly designed chalcone analogues with
N-alkyl moiety and different substitutions in a chalcone framework, and the
potential antifungal and antibacterial activities of them are also described along with
some of their chemical properties.
Experimental
Chemistry and apparatus
Melting points were taken on a XT-4 micro-melting point apparatus and are
uncorrected. FT-IR spectra were recorded in KBr disks on an EQUINOX-55 FTIR
spectrometer. 1H NMR spectra were run on an INOVA-400 NMR spectrometer with
TMS as internal standard. Elemental analyses were carried out on a Vario EL-III
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Synthesis and antimicrobial activity
CHNOS analyzer. Electron Spray Ionization-Mass Spectrometry (ESI–MS) anal-
yses were carried out in positive ion modes using a Thermo Finnigan LCQ
Advantage MAX LC/MS/MS. Column flash chromatography was carried out on
Merck silica gel (250-400 mesh ASTM). The processes of the reactions were
monitored by analytical thin-layer chromatography (TLC) using Merck silica gel
plates 60 GF-254. Solvents and chemicals used for synthesis were commercially
available and were used without further purification unless otherwise noted.
Synthesis
3-Aminoacetophenone (1)
Iron powder (3.9 g, 60 mL) was activated by refluxing it with the mixture of 10 mL
distilled water, ethanol (40 mL), glacial acetic acid (1 mL) and NH4Cl (0.2 g,
4 mmol) for 0.5 h under vigorous stirring at 80 °C, then ethanol (10 mL) and
3-nitroacetophenone (1.65 g, 10 mmol) were added to the mixture within 10 min.
The mixture was then heated slowly under vigorous stirring for 2 h. The hot
reaction mixture then filtered and the residue was washed with ethanol 3 times.
After evaporation of alcohol, the residue was diluted with water and extracted with
V (CHCl3) : V (CH3OH) = 2:1 for 3 times, the extraction was dried over anhydrous
Na2SO4 and the solvent was evaporated at reduced pressure. The crude was purified
on a flash column [V (CH3CO2Et) : V (petroleum ether) = 2:1 as eluent] to give
3-aminoacetophenone 1. Yield 84 % of white solid. m.p. 96–97 °C; 1H NMR
(400 MHz, CDCl3) d: 7.32 (dd, J = 7.6, 0.8 Hz, 1H, H-5), 7.24 (t, J = 7.8 Hz, 2H,
H-1 and H-4), 6.89–6.84 (m, 1H, H-3), 3.90 (s, 2H, –NH2), 2.55 (s, 3H, –CH3);
IR(KBr) m: 3,467, 3,369, 3,222, 1,668, 1,628, 1,597, 1,490, 1,457, 1,356, 1,324,
1,236, 777, 683 cm-1; Anal. Calc. for C8H9NO: C, 71.09; H, 6.7; N, 10.36; Found:
C, 70.81; H, 6.73; N, 10.72.
(E)-[N,N-bis(2-hydroethyl)-3-amino]-acetophenone (2)
A mixture of 3-aminoacetophenone (6.4 g, 47.5 mmol), 2-chlorothanol (16 mL,
240 mmol) and CaCO3 (6.5 g, 65 mmol) in water (60 mL) was heated under reflux
with vigorous stirring for 7 h. After hot filtering, the unreacted CaCO3 was washed
with few portions of hot water, then the filtrate was extracted with CH2Cl2
(40 ml 9 3) dried over anhydrous MgSO4 and concentrated under reduced pressure
to afford a yellow oil. The residue was further purified by flash column
chromatography [V (CH3CO2Et) : V (petroleum ether) = 2:1 as eluent] to give
the required diol 2. Yield: 68 % of faint yellow liquid. 1H NMR (400 MHz, CDCl3)
d: 7.20–7.24 (m, 3H, Ar–H), 6.85–6.87 (m, 1H, Ar–H), 4.74 (s, 2H, –OH), 3.77 (s,
4H, –OCH2–), 3.53–3.54 (d, J = 4.8 Hz, 4H, –NCH2–), 2.49 (s, 3H, –CH3). IR
(KBr) m: 3,378, 2,933, 2,880, 1,673, 1,597, 1,494, 1,444, 1,357, 1,267, 1,178, 1,010,
779, 688 cm-1. HRMS calcd for C12H17NO2: 246.1105 (M?Na)?; Found:
246.1101; Anal. Calc. for C12H17NO2: C, 64.55; H, 7.67; N, 6.27; Found: C,
64.61; H, 7.68; N, 6.26.
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X. Fang et al.
General procedure for the preparation of compounds 3a-3i
To the solution of [N, N-bis(2-hydroethyl)-3-amino]-acetophenone (3 mmol) and
substituted aromatic aldehydes (3 mmol) in ethanol (9 mL) a solution of 2.5 M
sodium hydroxide of (1 mL) was added slowly within 10 min in an ice bath. After
stirring for 4–6 h at room temperature, the formed precipitate was left. The mixture
was extracted with CH2Cl2 (10 mL 9 3) dried over MgSO4. After the solvent was
evaporated under reduced pressure, the crude product was obtained, the crude
chalcones was further purified by flash column chromatography SiO2. The yield,
melting point and spectral date of each compound had been collected as below.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-phenyl-prop-2-en-1-one (3a)
1
Yield: 87 % of light yellow solid. m.p. 94–96 °C; H NMR (400 MHz, CDCl3)
d: 7.74 (dd, J = 15.7, 2.2 Hz, 1H, H-17), 7.64–7.58 (m, 2H, H-16 and Ar–H),
7.48–7.44 (m, 1H, Ar–H), 7.42–7.38 (m, 3H, Ar–H), 7.31–7.26 (m, 3H, Ar–H),
6.93–6.91 (m, 1H, Ar–H), 4.17 (s, 1H, –OH), 4.02 (s, 1H, –OH), 3.87–3.82 (m, 4H,
–OCH2–), 3.63–3.58 (m, 4H, –NCH2–). IR (KBr) m: 3,239, 2,958, 2,926, 1,665,
1,590, 1,489, 1,443, 1,353, 1,320, 1,272, 1,066, 998, 764, 688 cm-1. HRMS calcd
for C19H21NO3: 334.1414 (M?Na)?; Found: 334.1407. Anal. Calc. for C19H21NO3:
C, 73.29; H, 6.80; N, 4.50; Found: C, 73.36; H, 6.78; N, 4.51.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(4-chlorophenyl)prop-2-en-1-
1
one (3b) Yield: 83.5 % of orange red powder solid. m.p. 115–117 °C; H NMR
(400 MHz, CDCl3) d: 7.69 (d, J = 15.7 Hz, 1H, H-17), 7.55 (d, J = 8.4 Hz, 2H,
H-16 and Ar–H), 7.44 (s, 1H, Ar–H), 7.40–7.36 (m, 2H, Ar–H), 7.34–7.27 (m, 3H,
Ar–H), 6.94–6.90 (m, 1H, Ar–H), 3.87 (t, J = 4.7 Hz, 4H, –OCH2–), 3.68 (s, 2H, –
OH), 3.63 (t, J = 4.7 Hz, 4H, –NCH2–). IR(KBr) m: 3,417, 2,923, 1,655, 1,587,
1,488, 1,446, 1,391, 1,228, 1,072, 815, 770, 727, 680 cm-1. HRMS calcd for
C19H20ClNO3: 346.1204 (M?H)?; Found: 346.1201. Anal. Calc. for C19H20ClNO3:
C, 65.99; H, 5.83; N, 4.05; Found: C, 66.06; H, 5.81; N, 4.04.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(2-chlorophenyl)prop-2-en-1-
one (3c) Yield: Yield: 91 % of deep yellow solid. m.p. 88–90 °C; 1H NMR
(400 MHz, CDCl3) d: 8.13 (d, J = 15.8 Hz, 1H, H-17), 7.73 (dd, J = 6.8, 2.5 Hz,
1H, H-16) 7.43 (d, J = 15.7, 9.0 Hz, 2H, Ar–H), 7.35–7.28 (m, 5H, Ar–H), 6.92 (d,
J = 7.7 Hz, 1H, Ar–H), 3.88 (t, J = 4.8 Hz, 4H, –OCH2–), 3.83 (s, 2H, –OH), 3.64
(t, J = 4.7 Hz, 4H, –NCH2–). IR(KBr) m: 3,415, 3,268, 2,924, 2,867, 1,658, 1,592,
1,496, 1,462, 1,434, 1,391, 1,266, 1,183, 1,082, 749, 684 cm-1. HRMS calcd for
C19H20ClNO3: 346.1204 (M?H)?; Found: 346.1197. Anal. Calc. for C19H20ClNO3:
C, 65.99; H, 5.83; N 4.05; Found: C, 65.92; H, 5.85; N, 4.06.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(2,4-dichlorophenyl)prop-2-
1
en-1-one (3d) Yield: 82 % of yellow powder solid. m.p. 102–103 °C; H NMR
(400 MHz, CDCl3) d: 8.03 (d, J = 15.8 Hz, 1H, H-17), 7.65 (d, J = 8.5 Hz, 1H,
H-16), 7.45 (d, J = 1.9 Hz, 1H, Ar–H), 7.38 (d, J = 15.8 Hz, 1H, Ar–H), 7.33–7.26
(m, 4H, Ar–H), 6.95–6.89 (m, 1H, Ar–H), 3.93 (s, 2H, -OH), 3.86 (t, J = 4.7 Hz,
4H, –OCH2–), 3.62 (t, J = 4.6 Hz, 4H, –NCH2–). IR(KBr) m: 3,251, 2,918, 2,861,
1,660, 1,587, 1,492, 1,465, 1,351, 1,226, 1,081, 1,002, 818, 773, 679 cm-1. HRMS
calcd for C19H19Cl2NO3: 380.0815 (M?H)?; Found: 380.0810. Anal. Calc. for
C19H19Cl2NO3: C, 60.01; H, 5.04; N, 3.68; Found: C, 60.13; H, 5.03; N, 3.66.
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Synthesis and antimicrobial activity
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(4-bromophenyl)prop-2-en-1-
1
one (3e) Yield: 84 % of orange red lumpish solid. m.p. 121–122 °C; H NMR
(400 MHz, CDCl3) d: 7.66 (d, J = 15.7 Hz, 1H, H-17), 7.53 (d, J = 8.5 Hz, 2H,
H-16 and Ar–H), 7.47 (d, J = 8.5 Hz, 2H, Ar–H), 7.42 (d, J = 15.7 Hz, 1H, Ar–H),
7.33–7.26 (m, 3H, Ar–H), 6.90 (dd, J = 7.9, 1.4 Hz, 1H, Ar–H), 4.03 (s, 2H, –OH),
3.85 (s, 4H, –OCH2–), 3.61 (t, J = 4.8 Hz, 4H, –NCH2–). IR(KBr) m: 3,376, 2,922,
1,656, 1,587, 1,486, 1,444, 1,318, 1,231, 1,060, 1,000, 854, 769, 725, 681 cm-1
.
HRMS calcd for C19H20BrNO3: 390.0699 (M?H)?; Found: 390.0690. Anal. Calc.
for C19H20BrNO3: C 58.47; H, 5.17; N, 3.59; Found C, 58.59; H, 5.15; N, 3.58.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(2-fluorophenyl)prop-2-en-1-
one (3f) Yield: 91 % of Orange red powder solid. m.p. 80–82 °C; 1H NMR
(400 MHz, CDCl3) d: 7.82 (d, J = 15.9 Hz, 1H, H-17), 7.60 (m, 1H, H-16), 7.53 (d,
J = 15.9 Hz, 1H, Ar–H), 7.39–7.32 (m, 1H, Ar–H), 7.28 (dd, J = 12.7, 4.8 Hz, 3H,
Ar–H), 7.17 (t, J = 7.5 Hz, 1H, Ar–H), 7.10 (dd, J = 10.4, 8.7 Hz, 1H, Ar–H), 6.89
(dd, J = 7.4, 2.2 Hz, 1H, Ar–H), 4.46 (s, 2H, –OH), 3.82 (d, J = 4.2 Hz, 4H, –
OCH2–), 3.59 (t, J = 4.8 Hz, 4H, –NCH2–). IR(KBr) m: 3,486, 3,376, 2,930, 2,875,
1,652, 1,583, 1,488, 1,450, 1,353, 1,221, 1,067, 1,027, 855, 752 cm-1. HRMS calcd
for C19H20FNO3: 330.1500 (M?H)?; Found: 330.1502. Anal. Calc. for
C19H20FNO3: C, 69.29; H, 6.12; N, 4.25; Found: C, 69.36; H, 6.10; N, 4.24.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(4-dimethylaminophe-
1
nyl)prop-2-en-1-one (3g) Yield: 76 % of kermesinus solid. m.p. 106–108 °C; H
NMR (400 MHz, CDCl3) d: 7.73 (d, J = 15.5 Hz, 1H H-17), 7.52 (d, J = 8.3 Hz,
2H, H-16 and Ar–H), 7.25 (5H, t, J = 10.8 Hz, Ar–H), 6.87 (dd, J = 4.3, 2.2 Hz,
1H, Ar–H), 6.68 (d, J = 8.3 Hz, 2H, Ar–H), 3.93 (s, 2H, OH), 3.86 (t, J = 4.4 Hz,
4H, –OCH2–), 3.61 (t, J = 4.2 Hz, 4H,–NCH2–), 3.03{s, 6H,–N(CH3)2}. IR(KBr)
m: 3,421, 2,882, 1,638, 1,606, 1,564, 1,523, 1,439, 1,367, 1,225, 1,166, 1,066, 809,
777, 734, 678 cm-1. HRMS calcd for C21H26N2O3: 377.1836 (M?Na)?; Found:
377.1835. Anal. Calc. for C21H26N2O3: C, 71.16; H, 7.39; N, 7.90; Found: C, 71.23;
H, 7.37; N, 7.88.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(4-fluorophenyl)prop-2-en-1-
1
one (3h) Yield: 89 % of light yellow powder solid. m.p. 103–105 °C; H NMR
(400 MHz, CDCl3) d: 7.70 (d, J = 15.7 Hz, 1H, H-17), 7.60 (dd, J = 7.4, 5.7 Hz,
2H, H-16 and Ar–H), 7.40–7.24 (m, 4H, ArH), 7.09 (t, J = 8.1 Hz, 2H, Ar–H), 6.90
(d, J = 7.6 Hz, 1H, H-2), 4.20 (s, 2H, –OH), 3.84 (t, J = 4.5 Hz, 4H, –OCH2–),
3.6–3.55 (m, 4H, –NCH2–). IR(KBr) m: 3,247, 2,927, 2,871, 1,661, 1,589, 1,502,
1,440, 1,354, 1,223, 1,184, 1,067, 1,002, 830, 783, 699 cm-1. HRMS calcd for
C19H20FNO3: 330.1500 (M?H)?; Found: 330.1503. Anal. Calc. for C19H20FNO3: C
69.29, H 6.12, N 4.25; Found: C 69.42, H 6.10, N 4.24.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(4-nitrophenyl)prop-2-en-1-
1
one (3i) Yield: 85 % of kermesinus solid. m.p. 164–166 °C; H NMR (400 MHz,
CDCl3) d: 7.87 (d, J = 8.4 Hz, 2H, H-17 and H-16), 7.77 (d, J = 15.4 Hz, 1H, Ar–
H), 7.59 (dd, J = 25.3, 8.6 Hz, 3H, Ar–H), 7.30 (d, J = 15.5 Hz, 1H, Ar–H), 6.70
(d, J = 8.8 Hz, 2H, Ar–H), 3.86-3.78 (m, 4H, –OCH2–), 3.7-3.63 (m, 4H, –NCH2–).
IR(KBr) m: 3,424, 2,925, 1,655, 1,590, 1,513, 1,497, 1,448, 1,387, 1,232, 1,103,
1,052, 840, 777, 747 cm-1. HRMS calcd for C19H20N2O5: 379.1264 (M?Na)?;
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X. Fang et al.
Found: 379.1262. Anal. Calc. for C19H20N2O5: C 64.04, H 5.66, N 7.86; Found: C
64.10, H 5.65, N 7.84.
(E)-30-[N,N-Bis(2-hydroxyethyl)-amino-phenyl]-1-(2-sulfophenyl)prop-2-en-1-one
(3j) To the solution of [N, N-bis(2-hydroethyl)-3-amino]-acetophenone (3 mmol) and
2-formylbenzenesulfonic acid sodium salt (3 mmol) in ethanol (9 mL) a solution of
2.5 M sodium hydroxide of (1 mL) was added slowly within 10 min in an ice bath.
After stirring for 4–6 h at room temperature, the reactant was poured into water, then
2 M HCl was added to precipitate the product. The crude solid was recrystallized
from water, giving a gray solid. Yield: 87 %. m.p. 243–245 °C; 1H NMR (400 MHz,
DMSO-d6) d: 8.77 (d, J = 14.7 Hz, 1H, H-17), 7.98–7.70 (m, 3H, Ar–H), 7.47 (d,
J = 39.0 Hz, 5H, Ar–H), 6.01 (s, 4H, –OH, and Ar–H), 3.64 (s, 4H, –OCH2–), 3.54
(s, 4H, –NCH2–). IR(KBr) m: 3,364, 3,037, 2,949, 1,658, 1,582, 1,466, 1,380, 1,237,
1,169, 1,079, 1,018, 862, 758, 710, 615 cm-1. HRMS calcd for C19H21NSO6:
390.1005(M–H)-; Found: 390.1022. Anal. Calcd for C19H21NSO6: C, 55.20; H, 4.88;
N, 3.41; Found: C, 55.34; H, 4.87; N, 3.41.
Pharmacology
The following micro-organisms were used for detecting antimicrobial activity:
Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Candida
albicans ATCC 10231, Pseudomonas aeruginosa ATCC 27853, Serratia typhi
ATCC 14028 and Escherichia coli ATCC 25922. All were obtained from the
Institute of Microbiology of Shaanxi province.
The preliminary screening for antibacterial as well as antifungal activity of the
ten tested compounds in DMSO (25 mg/mL) was evaluated using plate-hole
diffusion assay [15, 16]. A twofold serial dilution technique [17] was used to
determine the minimum inhibitory concentration (MIC) values of the screened
compounds. The MIC value was considered as quantitative method and was used for
evaluation of the antimicrobial activity of the synthesized compounds against the
given microbes.
Preliminary screening for antimicrobial activity
Suspensions of the above-mentioned micro-organisms were prepared by inoculating
fresh stock cultures into separate broth tubes, each containing (8 mL) of Muller–
Hinton broth for colonies of bacterial strains and fungal strains. The inoculated
tubes were incubated at 37 and 28 °C for 24 h, for bacterial and fungal strains,
respectively. Then, 250 lL of bacterial culture (*106–108 bacteria every mL) was
added aseptically to 20 mL of solid culture (a mixture of Tryptone, Sabouraud
dextrose agar and Yeast Extract) at 45 °C, mixed well, and poured into a sterile
Petri dish (90 mm). A sterile cork-borer of 6 mm diameter was used to make 2 wells
on each of the Petri dish. One was filled with 10 lL of the screened compounds. In
order to check the effect of the solvent, a control test was also performed containing
only DMSO. Then the cultures were incubated at 37 and 28 °C for 24 h for the
bacterial and fungal strains. After incubation, the zone of inhibition on the plates
was recorded. The compounds with the diameter of the growth-inhibition zones
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Synthesis and antimicrobial activity
greater than 6 mm were considered positive, and can be screened for further
determination of the minimum inhibitory concentration (MIC) values. The test was
repeated three times for each compound. All the new compounds (3a–3j) were
subjected to preliminary biological screening.
Determination of the minimum inhibitory concentration (MIC)
A broth tube dilution method [17, 18] was followed to determined the MIC values
for all the screened compounds against the micro-organisms, and for comparison
ciprofloxacin was used as a reference. For sample preparation, each of the test
compounds and reference were dissolved in DMSO at a concentration of 1,280 lg/
mL, and further dilutions of the compounds and reference in culture medium MHB
(Mu¨ller–Hinton Broth) were prepared at the required quantities of 128, 64, etc.,
down to 0.125 lg/mL. Bacterial strains and fungal strains were maintained on MHA
(Mu¨ller–Hinton Agar) medium at 37 and 28 °C for 24 h, respectively. The
microbial inocula were prepared by suspension in 10 mL of culture medium for
colonies from culture on MHA. The cell density of each inoculum was adjusted in
culture medium of a 0.5 McFarland standard. The final amount applied was 105
CFU/mL for bacteria and fungi. The microbial inocula were added to the twofold
diluted samples. MIC values were read after incubation at 37 and 28 °C for bacterial
and fungal strains, respectively. After 24 h, the last tube with no growth of micro-
organisms was recorded to represent the MIC value expressed in mg/mL. All
experiments were carried out three times.
Results and discussion
Synthesis
The N-alkoxylated chalcones were synthesized by the base-catalyzed Claisen–
Schmidt condensation [19] of 3-[N,N-bis(2-hydroxyethyl)-amino]-acetophone with
appropriate aromatic aldehydes; the synthesis route is illustrated in Scheme 1.
Intermediate 3-[N,N-bis(2-hydroxyethyl)-amino]-acetophone 2 was synthesized
through the hydroxyethylation of 3-aminoacetophenone 1 with 2-chlorothanol
[20]. While 3-amino-aceophenone 1 was prepared from 3-nitroacetophenone by
reduction with Fe/NH4Cl-CH3COOH [21]. Ten substitued chalcone derivatives
were synthesized, and their structures were confirmed by IR, 1H NMR, mass
spectrometry techniques, and elemental analyses. Crystal of chalcone 3b was also
obtained through the solvents evaporation method, and its X-ray single crystal
diffraction data was collected. All the spectral and crystal data are given as
supporting information.
Crystal and molecular structure
Chalcones are cross-conjugated molecules, and the carbonyl group in these systems
breaks the conjugation system into two independent parts to have a 2D b character
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X. Fang et al.
O
O
O
HO
HO
H2N
N
O2N
b
a
1
2
R1
OHC
R3
O
R1
HO
HO
N
R2
c
R3
R2
3c: R1 = R2 = H; R3 = Cl
3a-j
3a: R1 = R2 = R3 = H
3d: R1 = R2 = Cl; R3 = H
3g: R1 = R3 = H; R2 =F
3b: R1 = R3 = H; R2 = Cl
3e: R1 = R3 = H; R2 = Br 3f: R1 = F; R2 = R3 = H
3h: R1 = R3 = H; R2 =N(CH3)2
3i: R1 = R3 = H; R2 = NO2 3j : R2 = R3 = H; R1 =SO3H
Scheme 1 Synthetic scheme for the synthesis of compounds 1, 2, 3a–3j. Reagents and conditions: a Fe,
NH4Cl, CH3COOH, ethanol, reflux, 2 h; b CaCO3, 2-chlorothanol, H2O, 120 °C, 7 h; c 2.5 M NaOH,
ethanol, r.t., 3–6 h
Fig. 2 A molecular drawing of the asymmetric unit of the compound 3b
[22]. In the molecular structure of chalcone 3b, a Cl atom and a N,N-bis(2-
hydroxyethyl)-amino group existed in the para position of the B ring and the meta-
position of the other, respectively (Fig. 2), and an electron acceptor carbonyl (C=O)
group at the middle form a donor-p-accept-p-withdrawing (D-p-A-p-W) system,
where charge transfer takes place from the donor end to the withdrawing end. The
electron transfer mode may help to align the molecule in a parallel head-to-tail
alignment in the crystal packing.
X-ray analysis reveals crystal 3b is a monoclinic system with an P21/n space
group. The bond lengths and angles are generally normal in compound 3b, which is
in good agreement with the literature report [23]. The two benzene rings [C3, C2,
C4, C5, C6, C1] and [C10, C11, C12, C13, C14, C15] are fairly planar, respectively,
and the dihedral angles between the aromatic rings are 27.94°. In the crystal lattice,
there are several potentially weak hydrogen bond inter- and intra-molecular
ꢀꢀꢀ
ꢀꢀꢀ
˚
interactions [O2–H2 O3, 2.763 A, 175°, symmetry code i: (x, y, z); O3–H3 O2,
123

Synthesis and antimicrobial activity
ꢀꢀꢀ
˚
˚
2.720 A, 165°, symmetry code: (x, y, z); C11–H11 O3, 3.292 A, 136°, symmetry
code: (x, y, z)]. The crystal packing is further stabilized by these hydrogen bonds.
Biological evaluation
Ten of newly synthesized chalcones were evaluated for their antibacterial activity
in vitro against Gram-positive bacteria: S. aureus and B. subtilis; Gram-negative
bacteria: P. aeruginosa, E. coli and S. typhi; and anti-fungal activity against
C. albicans. The results of preliminary biological screening of all the newly
synthesized chalcones against the above strains are presented in Table 1. As we can
see, most chalcones exhibited moderate antibacterial activity for Gram-positive
bacteria (S. aureus and B. subtilis) and Gram-negative strains (P. aeruginosa,
S. typhi and E. coli) except 3b, 3c, 3h and 3i. Compounds 3d and 3e did not show
any activity at all to B. subtilis. Also, 3e showed no potency against E. coli and
P. aeruginosa. Interestingly, unexpected results were obtained that all the chalcones
revealed moderate to good activity against fungus (C. albicans). Thus, we screened
all the other compounds except 3b, 3c, 3h, 3j for further determining of MIC values.
The minimum inhibitory concentration [24] (MIC) values of the screened
compounds are outlined in Table 2. Compound 3f with 2-F in the B ring exhibited a
high potency against C. albicans and S. typhi, with MIC values of 32 and 64 lg/mL,
respectively. This compound is also effective in inhibiting the growth of the rest of
microorganism. Compounds 3 g and 3j possessing 4-F and 2-SO3H in the B ring
revealed good antifungal activity against C. albicans with MIC value of 32 lg/mL,
while compounds 3e and 3d have a lower degree of potency against all the tested
microbes. Compound 3a with no substituent in the B ring showed good antifungal
activity, and moderate to obvious antibacterial activity. Compounds 3a, 3g and 3j
Table 1 Antimicrobial activitya of the newly synthesized chalconesa (Cchalcones = 25 mg/mL)
Compounds
Fungi
Gram-positive bacteria
Gram-negative bacteria
C. albicans
B. subtilis
S. aureus
P. aeruginosa
S. typhi
E. coli
3a
3b
3c
3d
3e
3f
???
?
??
-
??
-
??
-
??
-
??
-
?
??
-
-
-
-
-
???
?
??
??
??
??
-
??
-
??
??
??
??
-
-
-
-
???
???
?
??
??
-
??
??
-
??
??
-
3g
3h
3i
?
-
-
-
-
-
3j
???
??
??
??
? ?
??
Values were the averages of duplicate experiments
a
Activity was determined from plate-hole diffusion assays with values to indicate the diameters of zones
of growth inhibition around the holes. The diameter of zones of inhibition
??? [10 mm, ?? 8-10 mm, ? 6-8 mm, - \6 mm
123

X. Fang et al.
Table 2 MIC values of antimicrobial activity of the screened compounds
Compounds
MIC(ug mL-1
)
Fungi
Gram-positive bacteria
Gram- negative bacteria
C. albicans
S. aureus
B. subtilis
P. aeruginosa
S. typhi
E. coli
3a
32
64
128
Nta
128
[128
Nt
64
128
Nt
3d
128
128
32
128
128
128
128
128
0.25
128
128
64
3e
Nt
Nt
3f
128
128
128
0.125
128
128
128
2.00
128
3g
32
128
128
16
[128
[128
0.125
3j
32
Ciprofloxacinb
2.00
Minimum inhibitory concentration of the screened compounds were determined using broth tube tech-
nique, values were the average ones of duplicate experiments
a
Nt not tested
b
Ciprofloxacin was used as a control
revealed potent inhibitory effects against C. albicans, which were as active as the
standard drug (ciprofloxacin) but less active than it. Combining the results of the
preliminary biological screening test, it seemed that with substituent as fluorine or
no substituent in the B ring, the newly synthesized chalcones showed potent
inhibitory effects against both all the tested microbes, especially the fungi. While
substituent such as with the chloride, nitro, and dimethylamino groups or bromine in
the B ring, the new chalcones revealed less or even no inhibitory effects. And as we
have seen, there is no significant difference of antimicrobial activity observed when
the electron-withdrawing groups took the place of electron-donating ones on the
phenyl ring, and those are consistent with the related report [25]. Thus, it was
predicted that the electronic effect of the substituent on the phenyl rings may not be
the key factor influencing antimicrobial activity as much as we assumed.
Conclusion
In conclusion, a series of novel 30-[N, N-Bis(2-hydroxyethyl)-amino]-chalcone
derivatives were synthesized, characterized and evaluated for their antimicrobial
activity. The results of preliminary screening of 10 compounds showed that only 6
of them revealed antimicrobial activities, and of those some presented moderate to
good potency against the selected microorganisms, especially against C. albicans.
Compounds 3a, 3d, 3f and 3g revealed obvious potency against C. albicans with
MIC value of 32 lg/mL, and this reveals that these compounds may be of potential
value in using as antifungal agents. The results may provide us a new prospect of the
structure modifications of chalcones derivatives for further biological investigations.
On the other hand, more chalcone derivatives of this kind with different
substitutions need to be designed and synthesized for further investigation of the
analysis of structure–activity relationships (SAR).
123

Synthesis and antimicrobial activity
X-ray crystallography data
All measurements of compound 3b was made on a Bruker Smart APEX II CCD
diffractometer with graphite monochromated Mo–Ka radiation. The data was
collected at a temperature of 296(2) K using the x-2h scan technique. Crystal data
˚
and structural refinement parameters, and selected bond angles (A) and bond lengths
(°) of 3b are summarized in Table S1 and Table S2, respectively (see supporting
information). CIF files of compound 3b have been deposited with the Cambridge
Structural Database (CCDC No. 886491). Copies of the data can be obtained free of
charge via www.ccdc.cam.ac.uk/conts/retrieving.html or from the CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK; Tel: (?44) 1223-336-408; fax: (?44) 1223-336-
033. E-mail:deposit@ccdc.cam.ac.uk.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No.
21172178). The authors are grateful to Prof. KeWu Yang for his useful comments on biological activity
test.
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