In vitro and in vivo synergistic interaction of substituted chalcone derivatives with norfloxacin against methicillin resistant Staphylococcus aureus

Article abstract of DOI:10.1039/c4ra10842f

Thirty chalcone derivatives were synthesized via a base catalyzed Claisen Schmidt condensation and evaluated for their anti-methicillin-resistant Staphylococcus aureus (MRSA) activity alone and in combination with norfloxacin. Among these, 5 derivatives namely trans-3-(1H-indol-3-yl)-1-(4′-benzyloxyphenyl)-2-propen-1-one (2), 1-(4″-biphenyl)-3-(3′4′-dihydroxyphenyl)-2-propen-1-one (11), 1-(4″-hydroxy-3″-methylphenyl)3-(4′-hydroxyphenyl)-2-propen-1-one (14), 3-(4′-chlorophenyl)-1-(4″-hydroxyphenyl)2-propen-1-one (17), and LTG-oxime (27) showed significant antibacterial activity with MIC 12.5-50 g mL^-1 respectively. In combination studies, derivatives 2 and 14 significantly reduced the MIC of norfloxacin by up to 16 fold (FICI < 0.5), while derivatives 11, 17 and 27 reduced it by up to eight fold (FICI ≤ 0.5). Flow cytometry analysis results clearly indicated that derivatives 2 and 14 significantly promote the accumulation and inhibition of the Et-Br efflux, which was further validated through spectrofluorimeter using clinical isolate MRSA-ST2071. In systemically infected Swiss albino mice model, both the compounds significantly (P < 0.001, P < 0.01) lowered the systemic bacterial load in blood, liver, kidney, lung and spleen tissues. This study supports the promising use of chalcones in the development of economical antibacterial combinations.

Full text of DOI:10.1039/c4ra10842f

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In vitro and in vivo synergistic interaction of
substituted chalcone derivatives with norfloxacin
against methicillin resistant Staphylococcus aureus
Cite this: RSC Adv., 2015, 5, 5830
a
b
b
b
*
Rashmi Gaur,† Vivek Kumar Gupta,† Anirban Pal, Mahendra Padurang Darokar,
Rajendra Singh Bhakuni and Brijesh Kumarc
*
a
Thirty chalcone derivatives were synthesized via a base catalyzed Claisen Schmidt condensation and
evaluated for their anti-methicillin-resistant Staphylococcus aureus (MRSA) activity alone and in
combination with norfloxacin. Among these,
5
derivatives namely trans-3-(1H-indol-3-yl)-1-(40-
benzyloxyphenyl)-2-propen-1-one (2), 1-(400-biphenyl)-3-(3040-dihydroxyphenyl)-2-propen-1-one (11), 1-
(400-hydroxy-300-methylphenyl)3-(40-hydroxyphenyl)-2-propen-1-one (14), 3-(40-chlorophenyl)-1-(400-
hydroxyphenyl)2-propen-1-one (17), and LTG-oxime (27) showed significant antibacterial activity with
MIC 12.5–50 mg mLꢀ1 respectively. In combination studies, derivatives 2 and 14 significantly reduced the
MIC of norfloxacin by up to 16 fold (FICI < 0.5), while derivatives 11, 17 and 27 reduced it by up to eight
fold (FICI # 0.5). Flow cytometry analysis results clearly indicated that derivatives 2 and 14 significantly
promote the accumulation and inhibition of the Et-Br efflux, which was further validated through
spectrofluorimeter using clinical isolate MRSA-ST2071. In systemically infected Swiss albino mice model,
both the compounds significantly (P < 0.001, P < 0.01) lowered the systemic bacterial load in blood, liver,
kidney, lung and spleen tissues. This study supports the promising use of chalcones in the development
of economical antibacterial combinations.
Received 20th September 2014
Accepted 5th December 2014
DOI: 10.1039/c4ra10842f
www.rsc.org/advances
clinicians to manage these infections.4,5 Therefore, efforts are
being directed towards the design, synthesis and evaluation of
Introduction
the substituted chemical therapeutics as an alternative to the
existing antimicrobial therapy.
Methicillin-resistant Staphylococcus aureus (MRSA) are increas-
ingly encountered and difficult to treat with available antibi-
otics.1 Most multidrug-resistant S. aureus strains are
nosocomially acquired (HA-MRSA) and cause an array of site
specic infections in hospitalized patients, including blood-
stream infections, pneumonia, surgical site infections, and
urinary tract infections.2 However, in the past few years, they
also increase in community as community-acquired MRSA (CA-
MRSA) which are more virulent than HA-MRSA.3 Most of the
MRSA strains are now becoming resistant to most of the clini-
cally used antibiotics like penicillins and other b-lactam anti-
microbial drugs, macrolide and quinolones. Due to the
emergence of glycopeptides resistance strains like vancomycin
hetero-intermediate (hVISA), vancomycin-intermediate (VISA),
and vancomycin-resistant (VRSA) S. aureus; it is difficult for
Chalcone is an open-chain avonoid with a,b-unsaturated
carbonyl group and is one of the important compound groups
of avonoid derived from nature which are considered as
precursors for the preparation of various avonoids and exhibit
interesting pharmacological activities.6 Flavonoids represent an
outstanding class of naturally occurring compounds with a 1,3-
diarylpropane skeleton, which may assume different cyclic or
alicyclic arrangements, according to varying levels of oxidation.
Thousands of chalcone derivatives have been synthesized
chemically, till date. Chalcones have received a great deal of
attention due to their relatively simple structures and wide
variety of pharmacological activities. Both natural and synthetic
chalcones exhibit a wide variety of activities like antimicro-
bial,6,7 antimalarial8 anti-inammatory,9 anti-cancer10 antioxi-
dant11 and many more. These activities are largely being
attributed due to the unsaturated ketone moiety.12–14 Recently,
many reports have conrmed the anti-MRSA activity of such
avonoid derivatives alone and in combination with some
antibiotics by increasing the antibacterial efficacy of antibiotics
used together or to restore the effect of separate invalid anti-
biotics.15–17 In the present study 30 chalcone analogues with
different substituent were synthesized and evaluated for their
aMedicinal Chemistry Division, CSIR-Central Institute of Medicinal and Aromatic
Plants, Lucknow-226015, India. E-mail: bhakunirs2000@yahoo.com; rs.bhakuni@
cimap.res.in; Fax: +91 522 2342666; Tel: +91 522 2359628
bMolecular Bioprospection Department, CSIR-Central Institute of Medicinal and
Aromatic Plants, Lucknow-226015, India. E-mail: mpdarokar@yahoo.com; mp.
darokar@cimap.res.in; Fax: +91 522 2342666; Tel: +91 522 2718532
cSophisticated Analytical Instrumentation Facility, CSIR-Central Drug Research
Institute, Lucknow 226001, India
† Both authors contributed equally to the paper.
5830 | RSC Adv., 2015, 5, 5830–5845
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antibacterial and synergistic interaction with noroxacin (a,b,c ¼ interchangeable); ESI-MS, MeOH (Positive): m/z 280
against clinical isolates of S. aureus (MRSA) through in vitro and [M + H]+, 302 [M + Na]+, (Negative): 278 [M ꢀ H]ꢀ, HRMS (ESI)
in vivo assays.
calcd for C18H14FNO [M + H]+ 280.1132, found 280.1129.
trans-3-(1H-Indol-3-yl)-1-(40-benzyloxyphenyl)-2-propen-1-one (2).
Yellow solid; 60%, yield; mp 74–75 ꢁC; IR nmax (KBr): 3448, 1562,
1120, 735 (NH), 1654 (chalcone C]O), 1523, 1495, 1437
(aromatics) cmꢀ1; 1H NMR (300 MHz, acetone-d6): d 5.29 (2H, s,
Materials and methods
Chemistry
General methods. Melting points were determined on a H2-700), 7.21–7.24 (2H, m, H-50, H-60), 7.26–7.29 (1H, m, H-400),
Toshniwal melting point apparatus and are uncorrected. IR 7.33–7.36 (3H, m, H-1000, H-1100, H-1200), 7.46–7.50 (2H, m, H-40, H-
spectra were recorded on a Perkin-Elmer 1719 FT-IR spectro- 500), 7.51–7.55 (2H, m, H-900, H-1300), 7.68 (1H, d, J ¼ 15.6 Hz, H-2),
photometer. NMR spectra were obtained in acetone-d6, DMSO-d6 7.70 (1H, s, H-200), 7.72 (1H, d, J ¼ 7.8, 2.1 Hz, H-70), 8.01 (1H, d, J
and pyridine-d5 on a Bruker Avance, 300 MHz and 400 MHz ¼ 2.7 Hz, H-20), 8.06–8.10 (1H, m, H-600), 8.10 (1H, d, J ¼ 15.6 Hz,
instrument using TMS as internal standard. The chemical shi H-3), 10.92 (1H, brs, NH); 13C NMR (75 MHz, acetone-d6): d 70.1
values are reported in ppm and coupling constants in Hz. ESI-MS (C-700), 112.7 (C-40), 114.0 (C-10), 114.2 (C-200), 116.8 (C-2), 119.3
spectra were recorded on a Perkin-Elmer Turbo Mass/Shimadzu (C-400), 120.8 (C-600), 121.1 (C-70), 121.6 (C-60), 123.2 (C-50), 126.0
LC-MS. TLC analyses were carried out on precoated silica gel 60 (C-30), 128.0 (C-900, C-1300), 128.2 (C-500), 128.8 (C-1000, C-1200), 130.0
F254 plates (Merck) using solvent system, CHCl3 : MeOH (9 : 1). (C-1100), 132.3 (C-20), 137.7 (C-80), 138.3 (C-100), 139.6 (C-3), 141.1
The compounds were visualized by either exposure of TLC plates (C-800), 159.3 (C-300), 189.0 (C-1); ESI-MS, MeOH (Positive): m/z 354
to I2 vapors or by spraying with vanillin–sulfuric acid reagent, [M + H]+, (Negative): 352 [M ꢀ H]ꢀ, HRMS (ESI) calcd for
followed by heating at 110 C for 15 minutes. Silica-gel, 60–120 C24H20NO2 [M + H]+ 354.1488, found 354.1488.
ꢁ
mesh (spectrochem) was used in the column chromatography for
trans-3-(1H-Indol-3-yl)-1-(benzofuran)-2-propen-1-one (3). Dark
the purication of metabolites. The compounds were identied brown solid; 40%, yield; mp 59–60 ꢁC; IR nmax (KBr): 3395, 1151,
by their spectral IR, ID (1H, 13C, DEPT) and 2D (COSY, HSQC, 1156, 736 (NH), 1654 (chalcone C]O), 1509, 1483, 1427
1
HMBC) NMR and ESIMS analysis. HPLC analysis was performed (aromatics) cmꢀ1; H NMR (300 MHz, acetone-d6): d 7.09–7.13
using a Shimadzu LC-10AD Liquid Chromatography equipped (2H, m, H-50, H-60), 7.35 (1H, d, J ¼ 15.6 Hz, H-2), 7.47–7.51 (3H,
with two LC-10A pumps controlled by a CBM-10 interface m, H-40, H-500, H-600), 7.53 (1H, d, J ¼ 15.6 Hz, H-3), 7.61 (1H, d, J
module, SPD-M10A VP diode array detector, and a SIL-10ADVP ¼ 2.7 Hz, H-20), 7.92 (1H, dd, J ¼ 8.4, 2.1 Hz, H-70), 8.01 (1H, dd, J
auto injector. Data were collected and analyzed using a class ¼ 6.9, 2.7 Hz, H-400), 8.12 (1H, dd, J ¼ 7.2, 2.4 Hz, H-800), 8.62 (1H,
LC-10 Work Station. The samples were analyzed by using reverse s, H-200), 13.05 (1H, brs, NH); 13C NMR (75 MHz, acetone-d6): d
phase chromatography on waters Spherisorb ODS2 (250 ꢂ 4.6 112.9 (C-40), 113.3 (C-10), 120.6a (C-2), 121.8a (C-70), 123.4 (C-60),
mm i.d.,10 mm) column using binary gradient elution with 124.7 (C-50), 125.7b (C-30, C-200), 125.9b (C-700), 126.6 (C-400), 127.9
acetonitrile and water containing 0.1% TFA mobile phase (C-500), 128.7 (C-300), 129.0 (C-600), 131.7 (C-800) 133.1 (C-20), 136.6c
(30 : 70) at a ow rate of 0.6 mL minꢀ1, a column temperature of (C-80), 138.4c (C-100), 142.7 (C-3), 198.9 (C-1), (a,b,c ¼ inter-
25 ꢁC and UV detection at l 254 nm.
General synthesis of chalcone derivatives
Synthesis of indolyl chalcones, 1–4. To a solution of indol-3- H]+ 288.1019, found 288.1019.
changeable); ESI-MS, MeOH (Positive): m/z 288 [M + H]+,
(Negative): 286 [M ꢀ H]ꢀ, HRMS (ESI) calcd for C19H14NO2 [M +
carboxaldehyde A (1 mmol) and appropriate acetophenone B
trans-3-(1H-Indol-3-yl)-1-(20-chlorophenyl)-2-propen-1-one (4).
(1 mmol) in anhydrous methanol (20 mL), sodium hydroxide Yellow shiny crystals, 85% yield, mp 208–210 ꢁC ESI-MS, MeOH
was added; 10% for compounds 1, 2, 30% for 3 and 40% for 4 (Positive): m/z 282 [M + H]+, (Negative): 281 [M ꢀ H]+, molecular
and stirred the reaction mixture at RT for 2–8 h. The contents of formula C17H12NO.18a
reaction mixture were poured into ice-cold water and neutral-
trans-3-(1H-Indol-3-yl)-1-(40-hydroxyphenyl)-2-propen-1-one (5).
ized with dilute hydrochloric acid. The solid so obtained was To a solution of A (indol-3-carboxaldehyde) (1 mmol) and p-
ltered, column chromatographed and recrystallized from hydroxyacetophenone B (1 mmol) piperidine (10 mL) was added
ethanol to afford pure 1–4.
and reuxed the reaction mixture for 2 h. The contents of
trans-3-(1H-Indol-3-yl)-1-(40-ouro-30-methylphenyl)-2-propen-1- reaction mixture were poured into ice-cold water. The solid so
one (1). Orange powder; 20% yield; mp 59–60 ꢁC; IR nmax (KBr): obtained was column chromatographed in (CHCl3 : MeOH),
3422, 1548, 1154, 737 (NH), 1653 (chalcone C]O), 1520, 1491, ltered, dried and recrystallized from ethanol to afford pure 5.
1
1440 (aromatics) cmꢀ1; H NMR (300 MHz, acetone-d6): d 2.35 Yellow crystals; 45% yield; mp 181–182 ꢁC; ESI-MS, MeOH
(3H, s, CH3), 7.19 (1H, d, J ¼ 9.3 Hz, H-500), 7.25–7.29 (2H, m, H- (Positive): m/z 264 [M + H]+, (Negative): 262 [M ꢀ H]+, molecular
50, H-60), 7.54 (1H, dd, J ¼ 8.1, 1.2 Hz, H-40), 7.70 (1H, d, J ¼ 15.6 formula C17H13NO2.18b
Hz, H-2), 7.98 (1H, d, J ¼ 2.7 Hz, H-200), 8.04 (1H, dd, J ¼ 8.7, 2.7
Synthesis of chalcones (6–24). To a solution of respective ace-
Hz, H-600), 8.08 (1H, brs, H-20), 8.11 (1H, d, J ¼ 15.6 Hz, H-3), 8.10 tophenone B (3-acetylindole for 6–11) (1 mmol) and appropriate
(1H, d, J ¼ 8.1, 1.2 Hz, H-70), 10.90 (1H, brs, NH); 13C NMR (75 aldehyde A (1 mmol) in methanol (20 mL) was added SOCl2
MHz, acetone-d6): d 14.3 (CH3), 113.0 (C-40), 114.3 (C-10), 115.4a (2 mL) and stirred the reaction mixture for 2 h. The contents of
(C-500), 115.7a (C-2), 121.1 (C-70), 121.9 (C-60), 123.6 (C-50), 126.3 reaction mixture were poured into ice-cold water. The solid so
(C-30), 128.9 (C-600), 132.6b (C-200), 132.7b (C-20), 136.1 (C-100), obtained was column chromatographed in (CHCl3 : MeOH),
138.6 (C-80), 139.3 (C-3), 162.7c (C-300), 166.0c (C-400), 188.3 (C-1) ltered, dried and recrystallized from ethanol to afford pure 6–24.
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113.0 (C-400), 118.5 (C-100), 122.6 (C-700), 122.7 (C-600), 123.9 (C-500),
126.3 (C-2), 126.8 (C-300), 127.1 (C-50), 127.3 (C-60), 130.4 (C-40),
131.5 (C-30), 134.6 (C-10), 135.6 (C-200), 137.5 (C-3), 137.7 (C-800),
138.2 (C-20), 184.5 (C-1); ESI-MS, MeOH (Positive): m/z 262 [M +
H]+, 284 [M + Na]+, (Negative): 260 [M ꢀ H]ꢀ, HRMS (ESI) calcd
for C18H16NO [M + H]+ 262.1226, found 262.1224.
trans-1-Indolyl-3-(20,30,40-trimethoxyphenyl)-2-propen-1-one (6).
Creamish powder; 70% yield; mp 163–164 ꢁC; IR nmax (KBr):
3431 1586, 1201, 754 (NH), 1640 (chalcone C]O), 1525, 1493,
1
1414 (aromatics) cmꢀ1; H NMR (300 MHz, DMSO-d6): d 3.67
(3H, s, OCH3), 3.76 (6H, s, 2 ꢂ OCH3), 6.71 (1H, d, J ¼ 9.0 Hz, H-
50), 7.10–7.14 (2H, m, H-500, H-600), 7.40 (1H, d, J ¼ 6.6 Hz, H-400),
7.60 (1H, d, J ¼ 15.6 Hz, H-2), 7.66 (1H, d, J ¼ 9.0 Hz, H-60), 7.74
(1H, d, J ¼ 15.56 Hz, H-3), 8.25 (1H, d, J ¼ 6.6 Hz, H-700), 8.57
(1H, brs, H-200), 11.79 (1H, brs, NH); 13C NMR (75 MHz, DMSO-
d6): d 56.8, 61.3, 62.3 (3 ꢂ OCH3), 109.2 (C-50), 113.0 (C-400), 118.6
(C-100), 122.6 (C-600, C-700), 122.4 (C-10), 123.4 (C-60), 123.9 (C-500),
124.0 (C-2), 126.8 (C-300), 134.6 (C-3), 135.1 (C-200), 137.7 (C-800),
142.7 (C-30), 153.5 (C-20), 155.8 (C-40), 184.7 (C-1); ESI-MS, MeOH
(Positive): m/z 338 [M + H]+, (Negative): 336 [M ꢀ H]+, HRMS
(ESI) calcd for C20H20NO4 [M + H]+ 338.1386, found 338.1386.
trans-3-(30-Ethoxy-40-acetylphenyl)-1-indolyl-2-propen-1-one (7).
trans-1-Indolyl-3-(thiophenyl)-2-propen-1-one (10). Creamish
max
ꢁ
white powder; 70% yield; mp 181–182 C; IR n
(KBr): 3448
1578, 1199, 754 (NH), 1632 (chalcone C]O), 1523, 1493, 1438,
1
1315 (aromatics) cmꢀ1; H NMR (300 MHz, DMSO-d6): d 6.03
(1H, d, J ¼ 4.2 Hz, H-20), 6.08–6.12 (2H, m, H-500, H-600), 6.33–6.35
(1H, m, H-400), 6.36 (1H, d, J ¼ 15.3 Hz, H-2), 6.45 (1H, brs, H-40),
6.55 (1H, d, J ¼ 4.2 Hz, H-30), 6.66 (1H, d, J ¼ 15.3 Hz, H-3), 7.50
(1H, d, J ¼ 7.5 Hz, H-700), 7.52 (1H, brs, H-200), 10.95 (1H, brs,
NH); 13C NMR (75 MHz, DMSO-d6): d 113.0 (C-400), 118.3 (C-100),
122.6 (C-7000), 122.7 (C-600), 124.0 (C-500), 124.0 (C-2), 126.7 (C-300),
129.3 (C-2 ), 129.7 (C-30), 132.0 (C-40), 133.3 (C-3), 135.4 (C-200),
137.7 (C-800), 141.0 (C-10), 184.0 (C-1); ESI-MS, MeOH (Positive):
m/z 254 [M + H]+, 276 [M + Na]+, (Negative): 252 [M ꢀ H]ꢀ, HRMS
(ESI) calcd for C15H12NOS [M + H]+ 254.0634, found 254.0635.
1-400-(Biphenyl)-3-3040-(dihydroxyphenyl)-2-propen-1-one (11).
35% yield; mp 198–200 ꢁC; IR nmax (KBr): 3448 (OH), 1654
max
ꢁ
Light yellow crystals; 70% yield; mp 154–155 C; IR n
(KBr):
1741 (ester CO), 1660 (chalcone C]O), 1603, 1478, 1356, 1260,
1061, 967, 875, 706 (NH) cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d
1.31 (3H, t, J ¼ 6.9 Hz, OCH2CH3), 2.25 (3H, s, COOCH3), 4.15
(2H, q, J ¼ 6.9 Hz, OCH2–), 7.16 (1H, d, J ¼ 7.5 Hz, H-50), 7.40
(1H, d, J ¼ 6.6 Hz, H-60), 7.21–7.24 (2H, m, H-500, H-600), 7.58 (1H,
s, H-20), 7.50 (1H, dd, J ¼ 8.4, 1.5 Hz, H-400), 8.72 (1H, d, J ¼ 2.7
Hz, H-200), 7.60 (1H, d, J ¼ 15.0 Hz, H-2), 7.79 (1H, d, J ¼ 15.6 Hz,
H-3), 8.34 (1H, dd, J ¼ 8.1, 1.8 Hz, H-700), 12.11 (1H, brs, NH); 13C
NMR (75 MHz, DMSO-d6): d 15.4 (OCH2CH3), 21.2 (COOCH3),
65.0 (OCH2–), 113.0 (C-400), 113.9 (C-20), 122.3a (C-50), 118.6 (C-
100), 139.9 (C-2), 122.6a (C-600), 123.9 (C-700), 124.0 (C-500), 126.7 (C-
300), 135.0 (C-10, C-40), 135.6 (C-200), 137.7 (C-800), 141.8 (C-3), 151.8
(C-30), 169.3 (COOCH3), 184.4 (C-1); ESI-MS, MeOH (Positive): m/
z 350 [M + H]+, HRMS (ESI) calcd for C21H20NO4 [M + H]+
350.1386, found 350.1386.
(chalcone CO), 1561, 1459, 1401, 1035 (aromatics) cmꢀ1 1H
;
NMR (300 MHz, DMSO-d6):d 6.70 (1H, d, J ¼ 8.1Hz, H-50), 7.06
(1H, d, J ¼ 8.1 Hz, H-60), 7.16 (1H, s, H-20), 7.26-7.29 (3H, m, H-
900, H-1000, H-1100), 7.56–7.58 (2H, m, H-2, H-3), 7.58 (2H, d, J ¼
7.8Hz, H-800, H-1200), 7.67 (2H, d, J ¼ 8.1 Hz, H-300, H-500), 8.03
(2H, d, J ¼ 8.1, H-200, H-600); 13C NMR, DEPT (75 MHz, DMSO-d6):
d 116.3 (C-20), 116.6 (C-50), 119.2 (C-2), 123.2 (C-60), 127.1 (C-10),
127.7 (C-800, C-1200), 127.8 (C-300, C-500), 129.1 (C-1000), 129.4 (C-900,
C-1100), 129.9 (C-200, C-600), 137.6 (C-100), 139.8 (C-700), 145.0 (C-400),
145.8 (C-3), 146.5 (C-30), 149.7 (C-40), 189.3 (C-1); ESI-MS, MeOH
(Positive): m/z 317 [M + H]+, HRMS (ESI) calcd for C21H17O3 [M +
H]+ 317.1172, found 317.1172.
trans-1-Indolyl-3-(30,50-dimethoxy-40-hydroxyphenyl)-2-propen-
max
ꢁ
1-one (8). Creamy crystals; 70% yield; mp 210–211 C; IR n
trans-1-(400-Biphenyl)3-(30-ethoxy-40-hydroxyphenyl)-2-propen-1-
(KBr): 3445, 1580, 1191, 740 (NH), 3445 (OH), 1640 (chalcone
max
1
C]O), 1522, 1491, 1404 (aromatics) cmꢀ1; H NMR (300 MHz,
ꢁ
one (12). 55% yield; mp 116–118 C; IR n
(KBr): 3444 (OH),
1652 (chalcone CO), 1563, 1459, 1401, 1034 (aromatics) cmꢀ1
;
DMSO-d6): d 1H NMR (300 MHz, DMSO-d6): d 3.51 (6H, brs, 2 ꢂ
OCH3), 6.49 (2H, br s, H-20), 6.60–6.63 (2H, m, H-500, H-600), 6.90
(1H, dd, J ¼ 6.9, 1.5 Hz, H-400), 6.99 (1H, d, J ¼ 15.3Hz, H-2), 7.04
(1H, d, J ¼ 15.3 Hz, H-3), 7.70 (1H, dd, J ¼ 8.1, 1.8 Hz, H-700), 8.06
(1H, d, J ¼ 2.7 Hz, H-200), 11.43 (1H, brs, NH); 13C NMR (75 MHz,
DMSO-d6): d 57.0 (2 ꢂ OCH3), 107.0 (C-20, C-60), 113.0 (C-400),
118.5 (C-100), 122.4 (C-2), 122.6 (C-700), 122.8 (C-600), 124.0 (C-500),
126.4 (C-10), 126.6 (C-300), 135.2 (C-200),137.6 (C-800), 138.6 (C-40),
141.8 (C-3), 148.9 (C-30, C-50), 185.1 (C-1); ESI-MS (Positive): m/z
324 [M + H]+, (Negative): 322 [M ꢀ H]ꢀ, HRMS (ESI) calcd for
1H NMR (300 MHz, DMSO-d6): d 1.33 (3H, t, J ¼ 6.6 Hz,
OCH2CH3), 4.10 (2H, q, J ¼ 6.0 Hz, OCH2–), 6.83 (1H, d, J ¼ 8.1
Hz, H-50), 7.25 (1H, d, J ¼ 7.8 Hz, H-60), 7.44–7.47 (3H, m, H-20,
H-900, H-1100), 7.73 (1H, d, J ¼ 16.0 Hz, H-2), 7.69 (1H, d, J ¼ 16.0
Hz, H-3), 7.69–7.72 (2H, m, H-800, H-1200), 7.80 (2H, d, J ¼ 6.9 Hz,
H-300, H-500), 8.19 (2H, d, J ¼ 7.2 Hz, H-200, H-600); 13C NMR, DEPT
(75 MHz, DMSO-d6): d 15.5 (OCH2CH3), 64.9 (OCH2–), 113.8 (C-
20), 116.5 (C-50), 119.4 (C-2), 125.0 (C-60), 127.1 (C-10), 127.7 (C-300,
C-500), 127.8 (C-800, C-1200), 129.2 (C-1000), 129.9 (C-900, C-1100),
130.0 (C-200, C-600), 137.6 (C-100), 139.8 (C-40), 145.0 (C-700), 145.8
(C-3), 148.0 (C-400), 150.8 (C-30), 189.3 (C-1); ESI-MS, MeOH
(Positive): m/z 345 [M + H]+, HRMS (ESI) calcd for C23H21O3 [M +
H]+ 345.1485, found 345.1485.
C
19H18NO4 [M + H]+ 324.1230, found 324.1229.
trans-1-Indolyl-3-(20-methylphenyl)-2-propen-1-one (9). Obtained
max
ꢁ
as brown solid; 80% yield; mp 140–142 C; IR n
(KBr): 3422,
1562, 1156, 748 (NH), 1639 (chalcone C]O), 1520, 1442, 1492
1
(aromatics) cmꢀ1; H NMR (300 MHz, DMSO-d6): d 2.34 (3H, s,
1-(300-Chloro-400-methoxyphenyl)-40-hydroxyphenyl-2-propenen-
max
ꢁ
CH3), 7.13–7.18 (5H, m, H-30, H-40, H-50, H-500, H-600), 7.40 (1H,
dd, J ¼ 8.1 Hz, 2.1 Hz, H-400), 7.63 (1H, d, J ¼ 15.6 Hz, H-2), 7.82
(1H, d, J ¼ 15.6 Hz, H-3), 7.87 (1H, dd, J ¼ 7.5, 2.4 Hz, H-60), 8.25
(1H, dd, J ¼ 6.6, 2.1Hz, H-700), 8.63 (1H, d, J ¼ 3.0 Hz, 1H, H-200),
12.05 (1H, brs, NH); 13C NMR (75 MHz, DMSO-d6): d 20.2 (CH3),
1one (13). Orange powder; 55% yield; mp 135–138 C; IR n
(KBr): 3407 (OH), 1658 (chalcone CO), 1560, 1458, 1400, 1035
1
(aromatics) cmꢀ1; H NMR (400 MHz, DMSO-d6):d 3.93 (3H, s,
OCH3), 6.84 (2H, d, J ¼ 8.14 Hz, H-30, H-50), 7.24 (1H, d, J ¼ 8.40
Hz, H-500), 7.68 (1H, d, J ¼ 17.36 Hz, H-3), 7.71 (1H, d, J ¼ 17.92
Hz H-2), 7.71–7.74 (2H, m, H-20, H-60), 8.11 (1H, d, J ¼ 8.56 Hz,
5832 | RSC Adv., 2015, 5, 5830–5845
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H-600), 8.16 (1H, s, H-200); 13C NMR, DEPT (100 MHz, DMSO-d6): d
56.9 (OCH3), 112.8 (C-500), 116.3 (C-30, C-50), 118.3 (C-2), 122.0 (C-
300), 126.2 (C-100), 129.8 (C-600), 130.4 (C-200), 131.5 (C-20, C-60),
131.7 (C-10), 144.9 (C-3), 158.5 (C-40), 160.5 (C-400), 186.9 (C-1);
ESI-MS, MeOH (Positive): m/z 289 [M + H]+, HRMS (ESI) calcd
for C16H14ClO3 [M + H]+ 289.0625, found 289.0625.
1-(400-Hydroxy-300-methylphenyl)3-(40-hydroxyphenyl)-2-propen-
1-one (14). Yellow shiny crystals, 88% yield, mp 198–200 ꢁC ESI-
MS, MeOH (Positive): m/z 255 [M + H]+, (Negative): 253 [M ꢀ H]+,
molecular formula C16H14O3.18c
trans-3-(Benzofuranyl)-1-(40-hydroxyphenyl)-2-propen-1-one (15).
Purple shiny crystals, 80% yield, mp 185–187 ꢁC; IR nmax (neat):
1659 (chalcone), 1592, 1510, 1452, 1031, 984, (aromatics), 750
(furan moiety) cmꢀ1; 1H NMR (300 MHz, acetone-d6): d 6.98 (2H,
dd, J ¼ 8.7, 1.2 Hz, H-30, H-50), 7.29 (1H, dd, J ¼ 7.8, 0.9 Hz, H-500),
7.46 (1H, dd, J ¼ 8.1, 1.2 Hz, H-600), 7.49 (1H, d, J ¼ 8.1 Hz, H-700),
7.61 (1H, d, J ¼ 15.8 Hz, H-2), 7.73 (3H, dd, J ¼ 8.7, 2.1 Hz, H-20,
H-60, H-4 00), 7.70 (1H, d, J ¼ 15.6 Hz, H-3), 7.87 (1H, brs, H-200);
13C, DEPT-NMR (75 MHz, acetone-d6): d 112.5 (C-200), 113.4 (C-
700), 115.5 (C-30, C-50), 119.6 (C-400), 123.7 (C-500), 124.3 (C-600),
127.9 (C-10), 127.9 (C-300), 128.5 (C-2), 131.0 (C-20, C-60), 143.9 (C-
3), 154.5 (C-800), 156.0 (C-100), 161.8 (C-40), 179.0 (C-1); ESI-MS
(Positive): m/z 265 [M + H]+, (Negative): 263 [M ꢀ H]ꢀ, HRMS
(ESI) calcd for C17H13O3 [M + H]+ 265.0864, found 265.0859.
3-(20-Chlorophenyl)-1-(400-hydroxyphenyl)-2-propen-1-one (16).
Orange powder, 65% yield, mp 192–193 ꢁC; ESI-MS, MeOH
(Positive): m/z 259 [M + H]+, (Negative): 257 [M ꢀ H]+, molecular
formula C15H11O2Cl.18d
3-(20,40-Dimethoxyphenyl-30-hydroxy)-1-(400-hydroxyphenyl)-2-
ꢁ
propen-1-one (22). Orange crystals; yield: 85%; mp 240–242 C;
ESI-MS, MeOH (Positive): m/z 285 [M + H]+, (Negative): 283 [M ꢀ
H]+, ESI-MS, MeOH (Positive): m/z 301 [M + H]+, molecular
formula C17H16O5.22
3-(30,40-Dimethoxy-40-hydroxyphenyl)-1-(300-hydroxyphenyl)-2-
propen-1-one (23). Orange crystals, 90% yield; mp 160–162 ꢁC; IR
nmax (KBr): 3418 (OH), 1659 (chalcone CO), 1551, 1462, 1388,
1
1030 (aromatics) cmꢀ1; H NMR (300 MHz, DMSO-d6): d 3.81
(2 ꢂ OCH3), 6.88–6.92 (2H, m, H-20, H-60), 6.94 (1H, d, J ¼ 8.4 Hz,
H-400), 7.28 (1H, d, J ¼ 8.1 Hz, H-500), 7.73 (1H, d, J ¼ 15.6 Hz H-2),
7.74 (2H, s, H-200), 7.61 (1H, d, J ¼ 15.3 Hz, H-3), 8.04 (1H, d, J ¼
8.1 Hz, H-600); 13C NMR, DEPT (75 MHz (DMSO-d6): d 56.3, 56.5
(2 ꢂ OCH3), 111.4 (C-20, C-60), 112.4 (C-200), 116.2 (C-400), 120.5
(C-2), 124.3 (C, C-300, C-600), 128.5 (C-40), 130.2 (C-100), 131.9
(C-500), 144.1 (C-3), 149.8 (C-30, C-50), 188.2 (C-1); ESI-MS, MeOH
(Positive): m/z 301 [M + H]+, 323 [M + Na]+, HRMS (ESI) calcd for
C
17H17O5 [M + H]+ 301.1070, found 301.1070.
3-(30-Ethyloxy-40-hydroxyphenyl)-1-(400-hydroxyphenyl)-2-propen-
1-one (24). Orangish yellow; yield: 91%; mp 250–252 ꢁC; ESI-MS,
MeOH (Positive): m/z 285 [M + H]+, (Negative): 283 [M ꢀ H]+,
molecular formula C17H16O4.23
Isoliquiritigenin (25). To a mixture of 4-hydroxybenzaldehyde
(1.12 g, 9.1 mmol), 2,5-dihydroxyacetophenone (1.36 g, 8.94
mmol), and ethanol (1.12 mL) was added potassium hydroxide
solution (8 mL, 60% w/w). The mixture was heated at 100 ꢁC for
2 h. The reaction mixture poured over ice cold water (30 mL).
The mixture was acidied to pH 5 with conc. HCl (13 mL). The
solid was ltered, washed with water (50 mL) and dried at 80 ꢁC.
The crude product was column chromatographed over silica gel
using ethyl acetate–hexane (40 : 60) as eluent to yield the
compound 25 (1.1 g, 48%) as a yellow solid.34
3-(40-Chlorophenyl)-1-(400-hydroxyphenyl)-2-propen-1-one (17).
Orange crystals, 72% yield, mp 196–198 ꢁC; ESI-MS, MeOH
(Positive): m/z 259 [M + H]+, (Negative): 257 [M ꢀ H]+, molecular
formula C15H11O2Cl.19
1-(500-Chlorothiophen-2-yl)-3-(40-hydroxyphenyl)-2-propen-1-one
(18). Yellow powder; yield: 71%; mp 210–212 ꢁC; ESI-MS, MeOH
(Positive): m/z 265 [M + H]+, (Negative): 263 [M ꢀ H]+, molecular
formula C13H9 ClO2S.20a
Liquiritigenin (26). A mixture of 25 (2.5 g, 9.76 mmol), meth-
anol (125 mL) and conc. HCl (10 mL) was reuxed for 48 h. The
reaction mixture was concentrated under vacuum and diluted
with water (100 mL). The product was ltered, washed with
water and dried. The compound was obtained as a pale yellow
1-(Furanyl)-3-(40-hydroxyphenyl)-2-propen-1-one (19). Light
yellow solid, yield: 75% mp 160–162 ꢁC IR nmax (neat): 1657
(chalcone), 1594, 1511, 1463, 1029, 985 (aromatics), 793 (furan
moiety) cmꢀ1; 1H NMR (300 MHz, acetone-d6): d 6.69 (1H, d, J ¼
3.6, H-400), 7.02 (2H, d, J ¼ 8.7 Hz, H-30, H-50), 7.52 (1H, d, J ¼ 3.0,
H-300),7.51 (1H, d, J ¼ 15.6, H-2), 7.72 (2H, d, J ¼ 3.6 Hz, H-200),
7.74 (2H, d, J ¼ 8.7, H-20, H-60), 7.76 (1H, d, J ¼ 15.6 Hz, H-3);
13C, DEPT-NMR (75 MHz, acetone-d6): d 112.7 (C-300), 115.5 (C-
30, C-50), 117.7 (C-200), 119.7 (C-2), 128.0 (C-10), 130.8 (C-20, C-60),
143.2 (C-3), 147.3 (C-400), 154.3 (C-100), 161.6 (C-40), 177.3 (C-1);
ESI-MS (Positive): m/z 215 [M + H]+, 213 [M ꢀ H]ꢀ, HRMS
(ESI) calcd for C13H11O3 [M + H]+ 215.0702, found 215.0702.
3-(20,30,40-Trimethoxyphenyl)-1-(400-hydroxyphenyl)-2-propen-1-
one (20). Yellow powder; yield: 81%; mp 192–194 ꢁC; ESI-MS,
MeOH (Positive): m/z 315 [M + H]+, (Negative): 313 [M ꢀ H]+,
molecular formula C18H18O5.20b
solid (2.33 g, 92%). Mp: 235–237 C.34
ꢁ
LTG-oxime (27). It was prepared by treating (0.256 g, 0.001
mol) 26 with hydroxylamine hydrochloride (0.069 g, 0.01 mol) in
ethanol and sodium acetate trihydrate (0.136 g, 0.001 mol) in
water. The solution was heated on a water bath for 4 h with
constant stirring. On cooling and water addition, the cream
compound precipitated out. It was ltered, washed with water,
and dried. The product was crystallized from ethanol to yield
max
ꢁ
0.235 g (91.7%), mp 125–126 C; IR n
(KBr): 3398 (phenolic
OH) 3600–2700, 1615, 1467, 1375, 931, 824 (CN-OH), 1517, 1243,
(aromatics) cmꢀ1; 1H NMR (300 MHz, CD3OD): d 2.62 (1H, dd, J
¼ 17.1, 12.0 Hz, H-3a), 3.35 (1H, dd, J ¼ 17.1, 3.0 Hz, H-3b), 4.91
(1H, dd, J ¼ 12.0, 2.7 Hz, H-2), 6.31 (1H, d, J ¼ 2.4 Hz, H-8), 6.41
(1H, dd, J ¼ 8.4, 2.1 Hz, H-6), 6.79 (2H, d, J ¼ 8.4 Hz, H-30, H-50),
7.26 (1H, d, J ¼ 8.4 Hz, H-20, H-60), 7.66 (1H, d, J ¼ 8.7 Hz, H-5);
13C and DEPT NMR (75 MHz, MeOD): d 31.1 (CH2, C-3), 78.4
(CH, C-2), 104.3 (CH, C-8), 110.9 (CH, C-6), 111.7 (C, C-4a), 116.2
(CH, C-30, C-50), 126.0 (CH, C-5), 128.7 (CH, C-20, C-60), 132.4 (C,
C-10), 150.8 (C]O, C-4), 158.4 (C, C-40), 159.2 (C, C-8a) 161.1 (C,
3-(20,40-Dimethoxyphenyl)-1-(400-hydroxyphenyl)-2-propen-1-one
(21). Dark pink; yield: 85%; mp 215–217 ꢁC; ESI-MS, MeOH
(Positive): m/z 285 [M + H]+, (Negative): 283 [M ꢀ H]+, molecular
formula C17H16O4.21
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C-7); ESI-MS, MeOH (Positive): m/z 272 [M + H]+, 294 [M + Na]+, 131.9 (C-20, C-60), 133.4 (C-600), 133.8 (C-2a), 134.1 (C-2b), 145.0
(Negative): m/z 270 [M ꢀ H]ꢀ, HRMS (ESI) calcd for C15H14O4N (C-3), 161.3 (C-40), 165.6 (C-400), 166.4 (C-200), 192.7 (C-1); ESI-MS,
[M + H]+ 272.0921, found 272.0922.38
MeOH (Positive): m/z 337 [M + H]+, HRMS (ESI) calcd for
LTG-oxime-tribenzoate (28). LTG-oxime 27 (0.198 g, 0.70
mmol) and benzoyl chloride (0.5 mL) were stirred in pyridine (2
mL) at room temperature. Aer completion, the reaction
mixture was work-up as usual which afforded a residue. The
residue upon crystallization in ethyl acetate–hexane furnished
dark orange crystals, 0.372 g (96%); mp 90–91 ꢁC. IR nmax (KBr):
3393 (phenolic OH), 1738 (ester CO), 1518, 1234, 1023
(aromatics) cmꢀ1; 1H NMR (300 MHz, CDCl3): d 3.03 (1H, dd, J ¼
13.5, 13.5 Hz, Ha-3), 3.74 (1H, d, J ¼ 15.9 Hz, Hb-3), 5.25 (1H, d, J
¼ 12.6 Hz, H-2), 6.92–6.95 (2H, m, H-6, H-8), 7.31 (2H, d, J ¼ 8.1
Hz, H-30, H-50), 7.50–7.54 (9H, m, H-4a, H-4b, H-4c, H-5a, H-5b,
H-5c, H-6a, H-6b, H-6c), 8.18–8.23 (6H, m, H-3a, H-3b, H-3c, H-
7a, H-7b, H-7c), 7.63 (1H, d, J ¼ 8.1 Hz, H-20, H-60), 8.31 (1H, d, J
¼ 9.0 Hz, H-5); 13C and DEPT NMR (75 MHz, CDCl3): d 32.9 (C-
3), 77.4 (C-2), 111.8 (C-8), 116.4 (C-6), 114.6 (C-4a), 122.6 (C-30, C-
50), 127.3 (C-5), 128.0 (C-20, C-60), 129.0 (C-4a/C-4b/C-4c, C-6a/C-
6b/C-6c), 129.6 (C-10), 130.1 (C-3a/C-3b/C-3c, C-7a/C-7b/C-7c),
134.2, 133.9 (C-5a/C-5b/C-5c), 136.9 (C-2a/C-2b/C-2c), 151.6 (C-
4), 155.0 (C-40), 156.7 (C-7), 158.9 (C-8a), 164.0/165.0/165.6 (C-
1a/C-1b/C-1c); ESI-MS, MeOH (Positive): m/z 704 [M + H]+, 726
[M + Na]+, HRMS (ESI) calcd for C46H26O7N [M + H]+ 704.1709,
found 704.1673.
C
21H21O4 [M + H]+ 337.1435, found 337.1435.
Bacterial strains and antimicrobial agents
The reference strain of S. aureus MTCC-96 (SA-96) was procured
from Microbial Type Culture Collection, CSIR-Institute of
Microbial Technology, Chandigarh, India. Clinical isolates of S.
aureus (MRSA) were obtained from the Clinical Microbiology
Laboratory of Sanjay Gandhi Post Graduate Institute of Medical
Sciences, Lucknow, India, which are being maintained in their
repository. The numbers mentioned alongside the strains
represent the repository accession number. Clinical isolates
were characterized and maintained as reported earlier24 Nor-
oxacin (Sigma-Aldrich, St. Louis, MO, USA) was used as posi-
tive control.
Anti-staphylococcal activity
Determination of minimum inhibitory concentration (MIC).
The antibacterial activity of substituted chalcone derivatives
was determined by the broth microdilution assay using 96 ‘U’-
bottom micro-titre plates as per CLSI guidelines (Wayne, 2006).
Derivatives were serially diluted twofold (in the range 100–0.195
mg mLꢀ1) in Mueller Hinton Broth (MHB). The broth was
inoculated with 10.0 mL of diluted 24 h-grown culture of the test
organism with a titre equivalent to 0.5 McFarland standards.
The inoculated plates were then incubated at 37 ꢁC for 16–24 h
and the growth was recorded spectrophotometrically at 600 nm
using a SpectraMax 190 microplate reader (Molecular Devices,
Sunnyvale, CA, USA). The minimum inhibitory concentration
(MIC) value was determined from the turbidimetric data as the
lowest concentration of derivatives showing growth inhibition
equal to or greater than 80% as compared to the control.
Experimental observations were performed in triplicate to rule
out any error during the procedure. An antibiotic noroxacin
were used as the positive control.
Synthesis of 29–30. 2,4-Dihydroxyavanone (1 eq.) and allyl
bromide (3 eq.) was reuxed in acetone in the presence of
K2CO3 for 2 h. Two spots developed on TLC which were sepa-
rated by column chromatography in CHCl3 : MeOH (Scheme 2).
40,200,400-Triallyloxyisoliquiritigenin (29). 45% yield; yellow,
viscous compound; IR nmax (KBr): 1654 (chalcone CO), 1551,
1450, 1411, 1022 (aromatics) cmꢀ1; 1H NMR (300 MHz, DMSO-
d6):d 4.61 (2H, d, J ¼ 5.08 Hz, H2-1b), 4.65 (2H, d, J ¼ 5.16 Hz, H2-
1c), 4.69 (2H, d, J ¼ 4.64 Hz, H2-1a), 5.24–5.27 (3H, m, Ha-3a,
Ha-3b, Ha-3c), 5.39–5.42 (3H, m, Hb-3a, Hb-3b, Hb-3c), 6.03–
6.07 (3H, m, H-2a, H-2b, H-2c), 6.65 (1H, d, J ¼ 8.64 Hz, H-500),
6.99 (2H, d, J ¼ 8.48 Hz, H-30, H-50), 6.70 (1H, s, H-300), 7.52 (1H,
d, J ¼ 15.60 Hz H-2), 7.45 (1H, d, J ¼ 15.60 Hz, H-3), 7.58 (2H, d, J
¼ 8.60 Hz, H-20, H-60), 7.63 (1H, d, J ¼ 8.56 Hz, H-600); 13C NMR,
DEPT (75 MHz, DMSO-d6): d 69.0 (C-1c), 68.7 (C-1b), 69.3 (C-1a),
100.7 (C-300), 115.6 (C-30, C-50), 117.9 (C-3b), 118.1 (C-3a), 118.3
(C-3c), 122.4 (C-100), 125.3 (C-2), 127.9 (C-10), 130.5 (C-20, C-60),
132.4 (C-600), 133.5 (C-2b), 133.6 (C-2c), 133.7 (C-2a), 141.5 (C-3),
159.3 (C-200), 160.4 (C-40), 163.0 (C-400), 107.3 (C-500), 189.8 (C-1);
ESI-MS, MeOH (Positive): m/z 377 [M + H]+, HRMS (ESI) calcd
for C24H25O4 [M + H]+ 377.1743, found 377.1743.
Interaction study of substituted chalcones derivatives with
noroxacin
The interactions of substituted chalcones derivatives with nor-
oxacin against clinical isolates (MRSA) were assessed through
checkerboard method.25 The synergy between substituted
chalcones derivatives and noroxacin was evaluated as a frac-
tional inhibitory concentration index (FICI). The FIC was
calculated as the MIC of an antibiotic or chalcone derivatives in
combination, divided by the MIC of the antibiotic or chalcone
derivatives alone. The FIC was then summed to derive the FIC
index, which indicated the interaction when the index values
were the following: FICI # 0.5 ¼ synergy, FICI > 4.0 ¼ antago-
nism, and FICI > 0.5–4 ¼ no interaction.26
40,400-Diallyloxy,200-hydroxyisoliquiritigenin (30). 35% yield;
yellow, viscous compound; IR nmax (KBr): 3448 (OH), 1657
(chalcone CO), 1546, 1454, 1408, 1015 (aromatics) cmꢀ1 1H
;
NMR (300 MHz, DMSO-d6):d 4.64–4.67 (4H, m, H2-1a, H2-1b),
5.29 (2H, d, J ¼ 9.9 Hz, Ha-3a, Ha-3b), 5.41 (2H, d, J ¼ 17.1 Hz,
Hb-3a, Hb-3b), 6.08–6.11 (2H, m, H-2a, H-2b), 6.58 (1H, d, J ¼
9.0 Hz, H-500), 7.04 (2H, d, J ¼ 8.4 Hz, H-30, H-50), 6.51 (1H, d, J ¼
1.8 Hz, H-300), 7.83–7.87 (4H, m, H-2, H-3, H-20, H-60), 8.26 (1H, d,
J ¼ 9.0 Hz, H-600); 13C NMR, DEPT (75 MHz, DMSO-d6): d 69.2 (C-
1a), 69.5 (C-1b), 102.6 (C-300), 108.5 (C-500), 114.8 (C-100), 116.0 (C-
30, C-50), 118.6 (C-3a), 118.8 (C-3b), 119.4 (C-2), 128.2 (C-10),
À
Á
MIC of drug 1 in combination
FIC drug 1 ¼
MIC of drug 1 on its own
5834 | RSC Adv., 2015, 5, 5830–5845
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aureus (SA-MTCC96). Chalcone derivatives at graded doses of
100, 50, 25 and 12.5 mg kgꢀ1 body weight comprised the
treatment groups. The vehicle control group was administered
with an equivalent volume of 0.1% cremophor (Fluka, USA). The
treatment commenced 2 h aer the infusion of infection and
continued till day 7 post-infection once daily. Blood was
collected from the retro-orbital plexus 24 h aer the last dose for
bacterial load which was estimated through plate counting on
brain heart infusion agar. All the animals were then sacriced
for the collection of lung, liver, kidney and spleen tissues. The
tissue homogenates were prepared in 2 mL of chilled, normal
saline solution with a glass tissue homogenizer under aseptic
conditions. Homogenates were suitably diluted and plated on
agar plates to enumerate the bacterial load per gram of
tissue.24,29 Bacterial elimination was assessed by comparing the
reduction of bacterial load of each organ in the infected groups
and vehicle control.
MIC of drug 2 in combination
MIC of drug 2 on its own
FIC ðdrug 2Þ ¼
FIC Index (FICI) ¼ FIC (drug 1) + FIC (drug 2)
Bacterial-killing assay
The in vitro bactericidal activity of selective chalcones deriva-
tives in combination with noroxacin against clinical isolates of
S. aureus (MRSA-ST 2071) was studied at different MIC combi-
nation in accordance to the method described by McKay et al.
2009.27 Each analysis was done in triplicate with a control
without test sample. Time kill curves were derived by plotting
log10 CFU mLꢀ1 against time (h).
Ethidium bromide accumulation assay (ow cytometric
analysis)
Statistical analysis
One-way analysis of variance was used to analyse the mean
values obtained for the treatment and vehicle groups. Tukey's
test was used to compare the treatment and vehicle groups and
statistical signicance was set at P # 0.001, P < 0.01.
Ethidium bromide accumulation assay was done as the
methods describe previously.28 Bacterial strainꢁ(MRSA-ST2071)
was cultured in 10 mL of MHB medium at 37 C and 200 rpm
until an OD600 of 0.6. Aliquots of 1.0 mL were centrifuged at
16 000g for 5 minutes, the supernatant discarded and the pellet
washed twice with PBS. The absorbance of bacterial suspension
Ethical clearance
was adjusted to 0.3 using Phosphate Buffer Saline (PBS) without The study was approved by the Institutional Bio-safety
glucose. Et-Br was added at a nal concentration of 1 mg mLꢀ1 Committee and Institutional Animal Ethics Committee under
and reserpine was added to a nal concentration of 20 mg mLꢀ1
.
the Committee for the Supervision and Experimentation on
Following incubation at 25 ꢁC for 60 minutes, aliquots of 0.5 mL Animals, Ministry of Environment, Government of India.
were taken for uorescence measurement as Et-Br was excited
at 488 nm and the uorescence detected through 585 nm lter
(FL-2 channel) using the ow cytometer FACS Calibur™ (BD
Results
Synthesis of substituted chalcones
Biosciences, San Jose, CA, USA). Data was collected for at least
10 000 events per sample.
Analogues based on the a,b-unsaturated ketone core can be
prepared by Claisen–Schmidt condensations between corre-
sponding aldehydes and ketones. Indolyl chalcones 1–4 were
prepared by the reaction of indol-3-carboxaldehyde with
appropriate acetophenone in the presence of NaOH at RT
(Scheme 1).30–32 Further, the reaction of 3-acetylindole with
appropriate aldehyde in the presence of SOCl2 resulted in the
formation of indolyl chalcones 6–10 (ref. 33). Claisen–Schmidt
condensation reactions were carried out between indole-3-
carboxaldehyde and p-hydroxyacetophenone catalyzed by
piperidine, which efficiently provided indole-chalcone 5. As
piperidine acts as a catalyst, we initially carried out the aldol
condensations in the presence of catalytic amounts of piperi-
dine. However, we generally observed higher yields when
Ethidium bromide efflux assay
The spectrouorometric determination of ethidium bromide
(Et-Br) efflux was performed as described previously.28 Bacterial
(MRSA-ST2071) culture was grown to reach optical density (OD)
of 0.6 at 600 nm. The cells were collected by centrifugation and
washed with PBS. The suspension (0.3 OD) was exposed to 5 mg
mLꢀ1 Et-Br for 60 min at 25 ꢁC in the presence of chalcone
derivatives at 10 mmg mLꢀ1. The cells were harvested by
centrifugation and re-suspended in fresh buffer. Loss of uo-
rescence was recorded for 30 min at 1 min intervals at an
excitation and emission wavelength of 530 nm and 585 nm
respectively using spectrouorimeter (FLUO star omega; BMG
Labtech, Ortenberg, Germany).
In vivo efficacy of substituted chalcone derivatives in
systemically infected Swiss mice model
The therapeutic efficacies of identied chalcone derivatives
were evaluated through intraperitoneal (i.p.) route. Five groups,
each with six Swiss mice (5–6 weeks old weighing 18 to 22 g),
Scheme 1 General routes used for the chalcone synthesis. Reagents
and conditions: (a) NaOH, MeOH, RT; (b) SOCl2, MeOH, RT; (c)
were infected by intravenous injection of 0.2 mL (106 CFU) of S. piperidine, reflux for 5.
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Table 1 Antibacterial activity of substituted chalcone derivatives against clinical isolates of S. aureus (MRSA)
Minimum inhibitory concentration (mg mLꢀ1
)
MRSA ST- MRSA ST- MRSA ST- MRSA B- MRSA P- MRSA ST- MRSA ST- MRSA B- MRSA ST- MRSA P-
Structure of test compound SA-96 1745
2071
>100
2438
>100
10732
>100
8029
>100
5457
>100
10342
>100
10760
>100
3151
>100
6642
>100
>100 >100
12.5 50
50
12.5
50
12.5
50
50
25
25
50
>100 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100 >100
>100
100
>100
>100
>100
>100
>100
100
>100
>100
>100
50
100
50
50
50
50
50
50
50
>100 >100
>100 >100
100 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
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Table 1 (Contd.)
Minimum inhibitory concentration (mg mLꢀ1
)
MRSA ST- MRSA ST- MRSA ST- MRSA B- MRSA P- MRSA ST- MRSA ST- MRSA B- MRSA ST- MRSA P-
Structure of test compound SA-96 1745
2071
>100
2438
>100
10732
>100
8029
>100
5457
>100
10342
>100
10760
>100
3151
>100
6642
>100
100 >100
>100 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50
50
50
25
25
25
50
25
25
50
25
>100 >100
100 >100
12.5 50
>100
>100
50
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
12.5
>100
>100
25
>100
>100
25
>100
>100
12.5
12.5
12.5
12.5
>100 >100
>100
>100
>100
100
>100
>100
>100
100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
100 >100
50
50
50
25
50
25
50
50
50
50
50
>100 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
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Table 1 (Contd.)
Minimum inhibitory concentration (mg mLꢀ1
)
MRSA ST- MRSA ST- MRSA ST- MRSA B- MRSA P- MRSA ST- MRSA ST- MRSA B- MRSA ST- MRSA P-
Structure of test compound SA-96 1745
2071
>100
2438
>100
10732
>100
8029
>100
5457
>100
10342
>100
10760
>100
3151
>100
6642
>100
>100 >100
100 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100 >100
>100 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100 >100
50
50
100
100
100
100
25
50
50
25
50
50
50
50
50
50
50
100
100
100
100
100
50
25
100
50
100
50
50
25
50
50
50
25
50
50
100
50
100
50
100
50
100
25
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Table 1 (Contd.)
Minimum inhibitory concentration (mg mLꢀ1
)
MRSA ST- MRSA ST- MRSA ST- MRSA B- MRSA P- MRSA ST- MRSA ST- MRSA B- MRSA ST- MRSA P-
Structure of test compound SA-96 1745
2071
>100
2438
>100
10732
>100
8029
>100
5457
>100
10342
>100
10760
>100
3151
>100
6642
>100
>100 >100
100 >100
100 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
piperidine was used in excess. In almost all instances, the benzyloxyphenyl)-2-propen-1-one (2), 1-(400-biphenyl)-3-(3040-
product was precipitated from the solution and was thus easily dihydroxyphenyl)-2-propen-1-one (11), 1-(400-hydroxy-300-methyl-
puried from excess catalyst (or starting material) by simple phenyl)-3-(40-hydroxyphenyl)-2-propen-1-one (14), 3-(40-chlor-
rinsing. Compounds 14–24 were synthesized by reported ophenyl)-1-(400-hydroxyphenyl)-2-propen-1-one (17), LTG-oxime
procedure and their structures were elucidated on the basis of (27) exhibits signicant antibacterial activity against all clinical
spectral data reported in the literature.18,20,34 Natural avanone isolates of S. aureus (MRSA) as well as reference strain SA-
liquiritigenin was synthesized by reported procedure by cycli- MTCC96 with MIC value ranging from 12.5–50 mg mLꢀ1
zation of trihydroxy chalcone isoliquritigenin34 and its anti- (Table 1). Noroxacin as positive control, exhibit MIC in the
bacterial activity reported by us is comparable with that range 125–500 mg mLꢀ1 against all the tested clinical isolates of
reported in literature.35–37 Oxime derivative of avanone was MRSA.
synthesized by reported procedure earlier described by us.38
Oxime derivative of avanone 26 was further benzoylated in the
presence of benzoyl chloride in pyridine. Allyl ether derivatives
In vitro combination study
of isoliquiritigenin resulted from ring opening of natural The combination of most active substituted chalcone deriva-
avanone liquiritigenin in the presence of allyl bromide and tives (2, 11, 14, 17 and 27) and noroxacin were evaluated
through checker board assay. The result showed synergistic
interaction of chalcone derivatives with 2–16 fold reduction in
MIC of noroxacin (FICI ¼ 0.312 to 0.50) against all the clinical
isolates of methicillin resistance S. aureus (Table 2).
The combination of noroxacin with compound 2 showed
synergistic interaction (2–16 fold reduction in MIC of nor-
K2CO3 in acetone in reux condition.
All new structures of compounds were satisfactorily
1
conrmed by IR, H NMR and EI-MS data. IR spectra showed
absorptions at 1650–1720 cmꢀ1 (C]O), and sharp bands at
3200 cmꢀ1 (NH). In the 1H NMR spectra chalcones showed
peaks in the region of d 2.54–2.78 (CH3), 2.88–2.98, 7.14–7.26
(CO–CH) and 7.53–7.58 (Ar–CH). The Ca–Cb double bond in the oxacin) against ve clinical isolates (MRSA-ST1745, ST2071,
enone moiety of chalcones can potentially adopt either a Z or an P8029, ST5457 and ST10760) with FICI ranging from 0.312 to
E conguration. The 1H NMR spectrum of each compounds
0.50.
Combination of noroxacin with compound 11 showed
synergistic interaction (2–8 fold reduction in MIC of nor-
oxacin) against four clinical isolates (MRSA-ST1745, ST2071,
P8029, and ST10760) with FICI ranging from 0.375 to 0.50.
Combination of noroxacin with compound 14 exhibits
strongest synergistic interaction (4–16 fold reduction in MIC of
noroxacin) against seven clinical isolates (MRSA-ST1745,
ST2071, ST2438, P8029, ST5457, ST10342 and ST3151) with
exhibited CH]CH protons around 7.14–7.63 ppm, with J > 15,
would suggest that the compounds were produced with an (E)
conguration. A library of compounds (chalcones, avanones
and their derivatives) were synthesized in our lab but the most
active ones are being reported here.
Anti-staphylococcal activity
Minimum Inhibitory Concentration (MIC). The results of FICI ranging from 0.312 to 0.50.
MIC assay showed that, among 30 substituted chalcone deriv-
Combination of noroxacin with compound 17 showed
atives only ve derivatives namely trans-3-(1H-indol-3-yl)-1-(40- synergistic interaction (2–8 fold reduction in MIC of
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noroxacin) against three clinical isolates (MRSA-ST1745,
ST2071, and ST10760) with FICI ¼ 0.375.
Similarly combination of compound 27 with noroxacin,
reveal synergistic interaction (2–8 fold reduction in MIC of
noroxacin) against four clinical isolates (MRSA-ST1745,
ST2071, ST2438, and ST10760) with FICI ranging from 0.375
to 0.50 (Table 2). Since clinical isolate MRSA-ST2071 also
exhibited higher level of resistance towards the maximum
number of antibiotics (data not shown).
Combination effect of chalcone derivatives with noroxacin
on bacterial killing kinetics
Times kill kinetics study of substituted chalcone derivatives
(2, 11, 14, 17 and 27) with noroxacin alone as well as in
combination were evaluated at two different MIC concentra-
tions. At 1/2MIC and MIC concentration derivatives 2 and 14
diminish the viability of bacterial cells up to 2 and 3 folds, while
derivatives 11, 17 and 27 reduce up to 1.5 folds respectively aer
24 h of incubation.
Combination of noroxacin with derivative 2 (1/2MIC Comp
2 + 1/2MIC Nor, MIC Comp 2 + MIC Nor), diminish in viability
of bacterial cells up to 5 and 10 folds at 1/2MIC and MIC
respectively aer 24
h incubation. The combination of
compound 14 with noroxacin at 1/2MIC concentration
(1/2MIC Comp 14 + ½MIC Nor) diminish the viability of
bacterial cells up to 6 folds aer 24 h of incubation, while at
MIC concentrations (MIC Comp 14 + MIC Nor) not a single
colony was observed aer 24 h of incubation indicating
bactericidal effect of combination.
Combination of noroxacin with derivatives 11, 17, and 27 at
different MIC concentrations, diminish the viability of bacterial
cells only up to 3 folds (Fig. 1). Since maximum synergy (up to 16
folds) of derivatives 2 and 14 was observed against clinical
isolate MRSA-ST2071, it was selected for further experiments.
Ethidium bromide accumulation and efflux assay
Taking advantage of Et-Br being a substrate for many MDR
efflux pump, the ability of both compound 2 and 14 to accu-
mulate and inhibit efflux of Et-Br in bacterial cells as Et-Br
bound to intracellular nucleic acid were measured via ow
cytometer and spectrouorimeter. Et-Br accumulation assay
using ow cytometer clearly indicate that both substituted
chalcone derivative 2 and 14 blocks the MDR efflux pump as
shown by the increase in Et-Br accumulation as increase in Et-Br
uorescence in the bacterial cells. A signicant shi (1.739 for
compound 2 and 2.374 fold for compound 14) in the intensity of
red uorescence was observed inside the bacterial cells as
compare to untreated control (Fig. 2). Similarly, spectrouo-
rimeter analysis revealed that rapid decrease in Et-Br efflux over
the time period of 30 min. As shown in Fig. 1 only control cells
without compounds (2 and 14) extruded Et-Br, resulting
signicant decrease in uorescence over the time period 30
min. In presence of both compound 2 and 14, loss of ores-
cence was signicantly reduced, reecting strong interference
with Et-Br efflux by both compounds (Fig. 3).
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Fig. 1 Time kill kinetics study of chalcone derivatives 2 (a) 11 (b), 14 (c), 17 (d) and 27 (e) alone as well as in combination with Norfloxacin at
different MIC concentrations against clinical isolates MRSA ST-2071. The data are expressed as mean ꢃ SEs.
Discussion
Currently, most of the pathogenic bacteria have been emerged
as multidrug resistant (MDR) strains as they were resistant
towards numerous classes of antibiotics through involvement
of efflux pump.39 Efflux is a process by which antimicrobial
compounds are removed from cell by membrane based
proteins, resulting in sub-lethal antimicrobial concentration at
the active site.35–37 S. aureus have an array of antiporter type
efflux proteins, energized by proton motive force or ATP
hydrolysis that excludes
a wide range of antimicrobial
compounds,40 which result in resistance to broad spectrum of
compounds including uoroquinolones, biocides (acriavine
and cetrimide) and dye such as ethidium bromide.41,42 There are
many natural compounds as reserpine,43 avonolignan and
avone compounds,44,45 N-caffeoylphenalkylamide derivatives,46
piperine and piperine analogues,47 have been reported as drug
resistance reversal agents. Some synthetic inhibitors such as
indoles,48 phenothiazines and thioxanthenes,49,50 quinolines
and quinolones51 and tricyclic compounds52,53 have also been
reported as drug resistance reversal agents. Many more chal-
cones have been reported for its antibacterial and resistance
modifying activity,15,54,55 which encourage us to design and
synthesizes some substituted chalcone derivatives. Keeping the
above, thirty chalcone derivatives were synthesized and inves-
tigated for their antibacterial and drug resistance reversal
potential against clinical isolates of methicillin resistance
S. aureus (MRSA).
Fig. 2 Ethidium bromide (Et-Br) efflux inhibition assay in presence of
chalcone derivatives 2 and 14 in clinical isolates MRSA-ST2071.
Reserpine used as positive control.
In vivo anti-staphylococcal activity of identied substituted
chalcone derivatives
The staphylococcal loads on various tissues (liver, spleen, kidney
and lung) and blood upon treatment with both substituted chal-
cone derivatives (2 and 14) at various doses ranging from 12.5 to
100 mg kgꢀ1 body weight are shown in Fig. 4 and 5 respectively. In
animals treated with compound 2, a signicant reduction of
staphylococcal load was observed (P < 0.01, P < 0.001) in blood and
different tissues such as liver, spleen, kidney and lung. Similarly,
signicant reduction of staphylococcal load was also observed
(P < 0.001) in blood and different tissue when animal treated with
compound 14 in a dose-dependent manner as compared to
untreated control.
On the basis of MIC data, among thirty derivatives, only ve
derivatives namely trans-3-(1H-indol-3-yl)-1-(40-benzyloxyphenyl)-
2-propen-1-one (2), 1-(40-biphenyl)-3-(3040-dihydroxyphenyl)-2-
propen-1-one (11), 1-(400-hydroxy-300-methylphenyl)-3-(40-hydroxy-
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Fig. 3 Ethidium bromide (Et-Br) accumulation by chalcone derivatives 2 and 14 treated cells of MRSA-ST2071. Cells treated with chalcone
derivatives 2 and 14 were examine immediately (a), after 15 min (b), and after 30 min (c) exposure to Et-Br. Reserpine used as positive control.
phenyl)-2-propen-1-one (14), 3-(40-chlorophenyl)-1-(400-hydroxy- 14 had most synergistic effect against MRSA in which the rates in
phenyl)-2-propen-1-one (17), LTG-oxime (27) showed signicant increasing susceptibility of bacteria with noroxacin were up to
antibacterial activity against all clinical isolates of MRSA. The sixteen fold, respectively.
results of quantitatively determining antibacterial coordination
Times kill kinetics study revealed that combination of
ability proved that combinations between noroxacin and chal- compound 2 with noroxacin diminish the viability of bacterial
cone derivatives (2, 11, 14, 17 and 27) were predominantly cells up to 5 and 10 folds at 1/2MIC and MIC respectively aer
synergistic as 2–16 fold reduction in MICs of noroxacin was 24 h incubation. The combination of 14 with noroxacin at
observed. The combination of noroxacin with derivatives 2 and 1/2MIC diminish the viability of bacterial cells up to 6 folds,
Fig. 4 Efficacy of chalcone derivative (2) at various doses in terms of reduction of bacterial burden (S. aureus MTCC-96) in multiple organs and
blood. The infection was induced through the intravenous injection of S. aureus cells (0.5Mc Farland) in a volume of 0.2 mL. The data are
expressed as mean ꢃ SEs.
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Fig. 5 Efficacy of chalcone derivative 14 at various doses in terms of reduction of bacterial burden (S. aureus MTCC-96) in multiple organs and
blood. The infection was induced through the intravenous injection of S. aureus cells (0.5Mc Farland) in a volume of 0.2 mL. The data are
expressed as mean ꢃ SEs.
while aer 24 h, at MIC concentrations not a single colony was showed better activity than its ring cyclized derivatives (ava-
observed indicating bactericidal combinations. Bactericidal nones, 26–28).
activity dened as decrease in 3 log 10 CFU mLꢀ1 correspond-
ing to 99.9% killing.46
Since both chalcone derivatives 2 and 14 were found to act
synergistically with noroxacin, as they were evaluated for their
From the results of bioactivities, no clear cut structure– efflux pump modulation potentials. Results of an Et-Br accu-
activity relationship can be drawn due to diversity of chalcone mulation assay using ow cytometer clearly indicate that both
derivatives but presence of a free hydroxyl group in the aromatic chalcone derivative 2 and 14 modulate the bacterial efflux pump
ring appears to be very important for anti-MRSA activity alone or as shown by the increase in Et-Br uorescence in the cells which
in combination with antibiotics; methylation to hydroxyl group might be due to accumulation of Et-Br inside the cells. Similar
might also be responsible for the abolishment in the anti MRSA observations were reported by Kalle and Rizvi, 2011,28,54 wherein
activity. It was found that ring opened chalcone derivatives celecoxib was found to inhibit bacterial MDR efflux pump.
Scheme 2 Reagents and conditions: (a) 60%, aq. KOH, 100 ꢁC, 2 h, 48%, (b) concd HCl, MeOH, reflux, 48 h, 92%, (c) NH2OH$HCl, CH3COONa,
EtOH, reflux; (d) pyridine, benzoyl chloride acetone, (e) allyl bromide, K2CO3, reflux.
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Similarly, spectrouorimeter assay revealed the decrease in Et- this work. Financial support from the National Medicinal Plant
Br efflux over the time period of 30 min as compared to Board (NMPB), New Delhi, is duly acknowledged.
untreated bacterial cells indicates towards a strong interference
of substituted chalcone derivatives in modulation of efflux
pump. Although there are many reports on in vitro antibacterial
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Abbreviations
MIC
Minimum inhibitory concentrations
DEPT Distortionless enhancement by polarization transfer
ESI-
MS
Electrospray ionization mass spectrometry
COSY Correlation spectroscopy
HSQC Heteronuclear single quantum correlation
HMBC Heteronuclear multiple-bond correlation spectroscopy
CFU
CLSI
FICI
MIC
Colony-forming unit
Clinical and Laboratory Standards Institute
Fractional Inhibitory Concentration Index
Minimum Inhibitory Concentration
MTCC Microbial Type Culture Collection
MFS Major facilitator superfamily
MRSA Methicillin-resistant Staphylococcus aureus
NOR Noroxacin
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Acknowledgements
The authors are grateful to the, Director, CIMAP, AcSIR-CIMAP
and SAIF-CDRI, Lucknow for providing necessary facilities for
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