7-Hydroxy-(E)-3-phenylmethylene-chroman-4-one analogues as efflux pump inhibitors against Mycobacterium smegmatis mc2 155

Article abstract of DOI:10.1016/j.ejmech.2013.06.024

Efflux pump (EP) induces resistance in mycobacteria and hence could be explored as a new target for the discovery of anti-TB agents. In search for efflux pump inhibitors from natural products, bonducellin, a homoisoflavonoid was isolated from Caesalpinia

Full text of DOI:10.1016/j.ejmech.2013.06.024

European Journal of Medicinal Chemistry 66 (2013) 499e507
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
7-Hydroxy-(E)-3-phenylmethylene-chroman-4-one analogues as
efflux pump inhibitors against Mycobacterium smegmatis mc2 155
,
,
,
*
Somendu K. Roya 1, Neela Kumari b 1, Shiv Gupta a, Sonika Pahwa b, Hemraj Nandanwar b
**
,
Sanjay M. Jachak a
,
a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Mohali 160062, Punjab,
India
b Bioactive Screening Laboratory, CSIR-Institute of Microbial Technology, Sector-39A, Chandigarh 160036, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Efflux pump (EP) induces resistance in mycobacteria and hence could be explored as a new target for the
discovery of anti-TB agents. In search for efflux pump inhibitors from natural products, bonducellin, a
homoisoflavonoid was isolated from Caesalpinia digyna roots and evaluated for modulation and EP
inhibitory activity. Bonducellin showed modulation in the MIC of EtBr by eight fold at a concentration of
62.5 mg/L and also showed significant EP inhibitory activity. A synthetic scheme was designed to prepare
analogues of 7-hydroxy-(E)-3-phenylmethylene-chroman-4-one by modification at the phenylme
thylene-ring and the synthesized compounds were evaluated in accumulation and efflux assays. Ana-
logues 1, 7e11, 13e15, 17 and 19 were found to be good modulators and decreased the MIC of EtBr by ꢀ4
fold at sub-inhibitory concentration. The compounds 8, 13 and 17 were the most potent inhibitors of
ethidium bromide efflux in Mycobacterium smegmatis mc2 155.
Received 23 October 2012
Received in revised form
21 March 2013
Accepted 3 June 2013
Available online 19 June 2013
Keywords:
Caesalpinia digyna
Homoisoflavonoid
Bonducellin
Ó 2013 Elsevier Masson SAS. All rights reserved.
Efflux pump
1. Introduction
putative efflux pumps but with the exception of LfrA, it is not clear
whether other genes contribute to the intrinsic drug resistance to
There is an emergence of multi-drug resistance (MDR) and
extensively drug-resistance (XDR) to the first line and second line
anti-TB drugs because of mutation of the target protein, enzymatic
inactivation of the antibiotic or inhibition of accumulation of an-
tibiotics by over expression of Efflux Pump (EP) within the cell of
bacteria. Hence, understanding drug resistance in Mycobacterium
tuberculosis could serve as one of the measures for the disease
control strategy. Numerous putative efflux pumps have been re-
ported in M. tuberculosis [1]. The active multidrug efflux pumps
(EPs) have been described as one of the mechanisms responsible for
resistance in Mycobacteria [2]. Drug efflux pumps have been
identified in several mycobacteria including Mycobacterium smeg-
matis. M smegmatis LfrA, a major facilitator superfamily transporter,
was the first multidrug efflux pump reported for mycobacteria. The
M smegmatis genome is reported to contain several genes encoding
this bacterium. However, Li X-Z et al showed that the homologues
of M. tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA) and
Rv3065 (mmr and emrE) were detectable in the M. smegmatis mc2
155, by reverse transcription-PCR technique [3]. It is also reported
that deletion of lfrA (strain XZL1675) and mmr homologue rendered
the strains more susceptible to several antimicrobials, such as
ethidium bromide (EtBr), acriflavine and fluoroquinolones (Two to
eight fold decrease in MICs) [3,4]. In recent years, it is observed that
efflux pumps have important functions in other cellular functions,
such as physiological homeostasis, resistance to stress conditions,
lipid transport, and virulence in M. tuberculosis [5]. Bioinformatics
as well as direct and indirect evidence have established efflux as a
mechanism of drug resistance in M. tuberculosis [6]. M. smegmatis
mc2 155 is being used in recent years as the model system for
expressing putative EP genes and to study the efflux mechanism,
because it is rapidly growing strain and non-pathogenic to humans
[4,7]. In search for bacterial efflux pump inhibitors (EPIs), natural
products have been explored in the past and provided several small
molecule natural product compounds as potential EPIs [8].
* Corresponding author. Tel.: þ91 172 6665338; fax: þ91 172 2690585.
** Corresponding author. Tel.: þ91 172 2214683; fax: þ91 172 2214692.
Considering the ethnomedicinal use of Caesalpinia digyna for the
treatment of TB [9], homoisoflavonoids were isolated from C. digyna
[10] and evaluated for modulation and efflux pump inhibitory (EPI)
E-mail
addresses:
raj_nandanwar@yahoo.com,
hemraj@imtech.res.in
(H. Nandanwar), sanjayjachak@niper.ac.in, sjachak11@gmail.com (S.M. Jachak).
1
Authors have equally contributed.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.024

500
S.K. Roy et al. / European Journal of Medicinal Chemistry 66 (2013) 499e507
2.2. Biology
2.2.1. Antimicrobial activity and modulating activity
Isolated and synthetic homoisoflavonoids were evaluated for
MIC and modulation of EtBr’s MIC in M. smegmatis mc2 155. For a
test compound to qualify as EPI, it should not possess an intrinsic
antibacterial activity. Therefore, the MIC for all the tested com-
pounds was determined to exclude intrinsic antimicrobial activity
for screening modulation effect in MIC of EtBr. Bonducellin (13) and
compound 15 showed the best modulating activity at 62.5 mg/L
and reduced MIC of EtBr by 8-fold against M. smegmatis. The
compounds 1 and 17 also modulated EtBr’s MIC by 8-fold but at
125 mg/L concentration (Table 1). Modulating activity of the iso-
lated and 19 synthetic homoisoflavonoids was further evaluated
with clinically used antibiotics (isoniazid and ofloxacin) using the
same protocol but none of these compounds modulated the MIC of
isoniazid and ofloxacin. The compounds that showed at least 4 fold
or more reduction in MIC of EtBr with modulation factor (MF) ꢀ4 (1,
7e11, 13e15, 17 and 19) were further studied for the accumulation
and efflux effect. This is based on the literature information that the
compounds with MF less than 4, generally do not show significant
accumulation and EPI activity in M. smegmatis mc2 155 [17,18].
Fig. 1. Structure of isolated homoisoflavonoids studied for modulation effect.
activity in M. smegmatis mc2 155. Standardized homoisoflavonoid-
enriched fraction [11] prepared from methanol extract of C. digyna
roots and four isolated homoisoflavonoids viz. bonducellin, 8-
methoxybonducellin, intricatinol, eucomin (Fig. 1) were studied
for modulation activity. The homoisoflavonoid enriched fraction
and bonducellin showed significant modulation of MIC of ethidium
bromide (EtBr) against M. smegmatis mc2 155. Further bonducellin
with
a basic structural skeleton, 7-hydroxy-(E)-3-phenylmet
hylene-chroman-4-one, exhibited significant accumulation and
efflux effects. Based on these observations, a synthetic scheme was
designed and analogues of 7-hydroxy-(E)-3-phenylmethylene-
chroman-4-one were synthesized by modification in phenyl
methylene-ring using various aldehydes [12e14]. The synthesized
analogues were evaluated for MIC and modulation of EtBr, isoniazid
(INH) and ofloxacin against M. smegmatis mc2 155. Those com-
pounds showing modulation were further tested for accumulation
and EPI activity. The dose dependent effect was determined for the
most active compounds.
2.2.2. Accumulation assay and efflux pump inhibitory assay
The accumulation and efflux assay were validated using stan-
dards verapamil, carbonyl cyanide m-chlorophenyl hydrazone
(CCCP) and reserpine (Fig. 2). Compounds (1, 7e11, 13e15, 17)
which modulated the MIC of EtBr by ꢀ4 fold, were subjected to
accumulation and efflux assays (Fig. 3). Among all tested com-
pounds, 1, 7e11, bonducellin (13) and 17 were found to be a better
modulators than the standard verapamil against M. smegmatis mc2
155 (Fig. 3). Compounds 14 and 15 have shown comparable activity
as that of verapamil but greater than CCCP.
2. Results and discussion
2.1. Chemistry
2.2.3. Dose dependent activity
Dose dependent effect of 1, 8e11, 13, and 17 were studied in
accumulation and efflux assays at a concentration of 1/2, 1/4 and 1/
8 of their MIC. The higher concentration of compounds exhibited
stronger accumulation and slower efflux of EtBr in M. smegmatis
mc2 155 cells (Fig. 4). The results revealed that the tested com-
pounds enhanced the accumulation and inhibited efflux of EtBr in a
dose-dependent manner during the assay period. All the above
mentioned compounds exhibited better EPI activity than the
standard, verapamil at all the tested concentrations 1/2, 1/4 and 1/8
of their MIC value. These results suggest that bonducellin (13) and
the synthetic homoisoflavonoid analogues (1, 8e11, and 17) are
potential EtBr efflux inhibitors in M. smegmatis mc2 155.
Bonducellin, 8-methoxybonducellin, intricatinol, and eucomin
were isolated from the methanolic extract of C. digyna as reported
previously by us [10]. The methoxy group at the para position in
phenylmethylene-ring of bonducellin was replaced by several other
functional groups as shown in Scheme 1 by condensation of 7-
hydroxy-chroman-4-one (II) with aromatic aldehydes to give the
compound 1e19 [15]. 7-Hydroxy-chroman-4-one (II) was synthe-
sized by acylation of resorcinol with 3-chloropropionic acid in the
presence of trifluoromethanesulphonic acid to yield an interme-
diate I which was then cyclized using sodium hydroxide (Scheme
1). The E-configuration of the synthesized homoisoflavonoids (1e
19) was confirmed by a characteristic signal (a singlet) of vinylic
proton of the trans (E) double bond at C3eC9, appearing downfield
at dH 7.58e7.79 ppm as compared to dH 6.78e7.07 ppm for the Z-
isomer [14,16].
It is reported that the presence of an intrinsic EP system in
M. smegmatis is responsible for the development of intrinsic
resistance to the EtBr and other antibiotics [19]. Rapid occurrence of
HO
O
HO
OH Cl
Step-2
2.0 M NaOH
2 h
Step-1
HO
OH
+
O
CF3SO3H(3 equiv)
75-80 oC, 1 h
Cl
OH
O
O
R
C
I
II
Comp No.
Comp No.
R
R
11
o-OCH3
12 m-OCH3
1
-H
Step-3
HO
O
R
HO
O
B
2
o-Br
m-Br
p-Br
o-Cl
m-Cl
p-Cl
o-F
Acetic acid,
dry HCl gas
rt, 24 h
3
13
14
15
16
17
18
19
p-OCH3
m-OH
p-OH
o-CH2CHCH2
m-CH2CHCH2
p-CH2CHCH2
p-OCF3
A
+
OHC
4
5
O
6
O
II
7
1-19
8
9
m-F
p-F
10
Scheme 1. Method used for the synthesis of (E)-3-benzylidene-7-hydroxychroman-4-one analogous.

S.K. Roy et al. / European Journal of Medicinal Chemistry 66 (2013) 499e507
501
Table 1
dependent efflux of ethidium bromide and berberine from
S. aureus [20].
MIC and modulation factors of methanol extract, homoisoflavonoid rich fraction,
bonducellin from C. digyna and compounds 1e19 for M. smegmatis mc2 155.
Compound/Extract
MIC
Value
(mg/L)
Concentration as
modulator (mg/L)
Modulation
factor (MF)
FICIa
2.2.4. Structure activity relationship (SAR)
In this study we observed that four isolated and nineteen syn-
thetic homoisoflavonoids showed weak amtimycobacterial activity
at ꢀ125 mg/L. The isolated homoisoflavonoids except bonducellin
(13) showed very weak modulating activity (MF ꢁ 2) at sub-
inhibitory concentration of 62.5 mg/mL (Table 1). Compounds 1,
7e11, 13e15, 17 and 19 showed a modulation effect on EtBr’s
antimycobacterial activity when added at sub-inhibitory concen-
trations. The identified modulators 1, 7e11, 13e15, 17 and 19 were
further tested for the accumulation and efflux assay. The results
revealed that the compounds 1, 8e11, 13 and 17 enhanced the
accumulation and inhibited the efflux of EtBr in the M. smegmatis
cells more potently than verapamil (Fig. 4). Compounds 8 and 17
found to be the best EPIs since they showed much better accu-
mulation as well as efflux inhibition of EtBr in M. smegmatis cells at
31.25 mg/L as compared to verapamil at 150 mg/L concentration.
Based on above observations we propose the SAR as below:
(i) The 7-Hydroxy-(E)-3-phenylmethylene-chroman-4-one is
required to be present in all the tested compounds for modulation
effect (Table 1 and Fig. 5). (ii) The hydroxy group at C-8 and or C-5
or a methoxy group at C-8 reduces the modulation effect. (iii) The
compounds with substituents such as fluoro, methoxyl, allyloxy on
the phenylmethylene-ring (8e10, 13, 17) enhanced accumulation
and efflux pump inhibitory activity as compared to 7-Hydroxy-(E)-
3-phenylmethylene-chroman-4-one. (iv) The fluoro substituent at
ortho, meta and para (8, 9, 10); methoxyl substituent (13) at para
and allyloxy substituent (17) at meta position on the phenly-
methylene ring potentiated the accumulation and EPI activity. (v)
Whereas, a chloro- and trifluromethane substituents (7, 19) at para;
methoxyl substituent (11) at ortho and hydroxyl substituent (14, 15)
at meta and para positions on the phenlymethylene ring led to
decrease in the accumulation and EPI activity (Fig. 5).
Methanol extract
HRF
E-Eucomine
250
500
500
125
250
250
62.5
62.5
31.2
62.5
62.5
125
62.5
62.5
125
125
62.5
125
125
62.5
125
125
125
125
125
125
62.5
125
125
62.5
125
125
40
2
4
4
2
8
4
2
2
8
4
2
2
2
2
2
4
4
4
4
4
2
4
16
8
2
8
4
2
4
4
2
4
2
1
0.75
0.6
Bonducellinb
125
0.62
0.50
1
E-8-methoxybonducellin
Intricatinol
1
125
125
500
1
0.37
0.37
1
0.75
0.75
1
2
3
4
5
6
7
8
9
10
11
12
14
15
125
500
500
125
250
250
125
250
250
250
500
500
250
1
0.75
0.75
0.75
0.75
0.75
0.75
0.50
0.56
0.37
0.75
0.62
0.50
0.75
0.75
0.40
0.63
0.52
1.14
16
17
500
250
18
19
250
250
256
300
Reserpine
Verapamil
40
80
16
CCCP
25
MIC of EtBr ¼ 16 mg/L.
a
FICI (fractional inhibitory concentration index) ꢁ0.5 synergism; ꢀ4.0 antago-
nism; 4.0 > FICI > 0.5 no interaction.
b
Bonducellin ¼ 13 (Synthetic).
3. Conclusion
bacterial resistance against antibiotics can be achieved in several
ways: (i) mutation of target proteins, (ii) active efflux systems and
reduced cell-wall permeability, and (iii) enzymatic inactivation of
the antibiotic. The characterization of mycobacterial EPs is a rela-
tively new field compared to the characterization of EPs from other
Gram-positive and Gram-negative bacteria. Also due to the high
incidence levels of MDR-TB cases, there is an urgent need to identify
and characterize new anti-TB compounds or enhancer/EPIs for
existing TB compounds. The efflux of an antibiotic in bacteria may
be inhibited by plant-derived compounds which can be evidenced
by an example of 50-methoxyhydnocarpin (50-MHC). 50-MHC did
not possess any antibacterial activity but inhibited the MDR-
In summary, four isolated and nineteen synthetic homoiso-
flavonoids were screened for their antimycobacterial activity and
modulating activities on the MIC of EtBr. An EtBr accumulation and
efflux assay were used to determine their potential to inhibit EtBr
efflux. The compounds 1, 8e11, 13 and 17 showed potent accumula-
tion and efflux inhibition of ethidium bromide in M. smegmatis mc2
155, among the tested nineteen homoisoflavonoids as compared to
verapamil. We successfully demonstrated that particularly com-
pounds 8 and 17 achieved considerable results as modulator and
decreased the EtBr efflux in M. smegmatis mc2 155 at levels either
comparable to or lower than the known EPIs. Homoisoflavones have
Fig. 2. Effect of verapamil (150 mg/L), CCCP (12.5 mg/L) and reserpine (128 mg/L), on EtBr accumulation (A) and efflux assay (B) in M. smegmatis mc2 155 cells.

502
S.K. Roy et al. / European Journal of Medicinal Chemistry 66 (2013) 499e507
Fig. 3. Effect of test compounds (1, 7e11, 13e15, 17 and 19) at half concentration of MIC; on EtBr accumulation (A) and efflux assay (B) in M. smegmatis mc2 155 cells.
not been described as bacterial efflux inhibitors before and thus
represent a promising class of compounds to be studied further.
presence of trifluoromethanesulphonic acid (3 equiv.), stirred at
75e80 ꢂC. After 1 h the solution was cooled to room temperature
and poured into CHCl3 (200 mL). This solution was slowly poured
into water (200 mL), and the phases were separated. The aqueous
phase was partitioned with 2 ꢃ 200 mL of CHCl3. The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4 and concentrated under vacuum to give semisolid com-
pound I (7.24 g).
Step B. Cyclization of I: Compound I (7.24 g) was added to 400 mL
of 2 M NaOH at 5 ꢂC and stirred for 20 min. The solution was
allowed to reach room temperature for 2 h and again cooled to 5 ꢂC.
The pH of the solution was adjusted to 2 with 6 M H2SO4. The acidic
mixture was extracted with 3 ꢃ 200 mL of EtOAc, washed with
brine, dried over anhydrous Na2SO4 and concentrated under vac-
uum to give crude solid. Crude solid was subjected to CC over silica
gel (#100e200) eluting with petroleum ether and EtOAc (75:25) to
give pure II (3.50 g).
4. Experimental
4.1. Materials and equipments
All Chemicals were purchased from Sigma Aldrich. Uncorrected
melting points were determined on a digital melting point appa-
ratus of PERFIT, India. 1H and 13C NMR spectra were recorded in
deuterated solvents on Bruker 400 Ultra ShieldÔ NMR spectro-
photometer with TMS as an internal standard. The Mass spectra
were recorded on MaXisÔ UHR-TOF and Thermo Finnigan LCQ
Mass spectrometer. IR spectra were recorded on Nicolet FT-IR
(Impact 410, Japan) spectrophotometer.
4.2. General procedure for synthesis of 7-Hydroxy-(E)-3-
phenylmethylene-chroman-4-one analogues (1e19)
Step C. Synthesis of homoisoflavonoids by acidic condensation:
Compound II (100 mg, 0.61 mM) was added in a solution of
Step A. Acylation of resorcinol: Resorcinol (9.00 g, 82 mM) was
substituted benzaldehyde (0.62 mM) in acetic acid (20
mL) and dry
acylated by the equi-molar amount of 3-chloropropionic acid in the
HCl gas was passed through this mixture for 5 min. The reaction

S.K. Roy et al. / European Journal of Medicinal Chemistry 66 (2013) 499e507
503
Fig. 4. Dose dependent Effect of Comp (1, 8e11, 13 and 17) on the accumulation (A) and Efflux (B) of EtBr from M. smegmatis mc2 155 cells.
mixture was allowed to stand at room temperature for 24 h. The
obtained solid mixture washed with water to remove acids and
concentrated on a rotary evaporator. Afterwards the mixture was
purified by CC on Silica gel (#230e400) eluting with petroleum
ether and EtOAc (85:15) to give pure compounds (1e19).
1H, H-30), 6.42 (dd, J ¼ 2.4, 8.7 Hz, 1H, H-50), 7.63 (d, J ¼ 8.7 Hz, 1H,
H-60), 12.53 (s, 1H, 20-OH). 13C NMR (100 MHz, CDCl3):
103.6, 108.2, 113.7, 132.1, 163.1, 165.2, 200.6 [12].
d 38.5, 40.3,
4.2.2. 7-Hydroxy-chroman-4-one (II)
Yield: 3.50 g, 72.8%; mp: 150e152 ꢂC (lit 145 ꢂC). 1H NMR
4.2.1. 20,40-Dihydroxy-3-chloro-propiophenone (I)
(400 MHz, CD3OD):
d
2.70 (t, J ¼ 5.1 Hz, 2H, H2-3), 4.46 (t,
Yield: 7.24 g, 44.25%. 1H NMR (400 MHz, CDCl3):
d
3.40 (t,
J ¼ 5.1 Hz, 2H, H2-2), 6.31 (s, 1H, H-8), 6.47 (d, J ¼ 8.6 Hz, 1H, H-6),
J ¼ 6.7 Hz, 2H, H2-2), 3.90 (t, J ¼ 6.7 Hz, 2H, H2-1), 6.39 (d, J ¼ 2.4 Hz,
7.70 (d, J ¼ 8.6 Hz, 1H, H-5). 13C NMR (100 MHz, CD3OD):
d 36.8,

504
S.K. Roy et al. / European Journal of Medicinal Chemistry 66 (2013) 499e507
Fig. 4. (continued).
67.0, 102.2, 110.2, 113.9, 128.6, 164.4, 165.1, 191.9. MS (APCI) m/z:
165 [M þ H]þ [12].
129.4, 129.7, 131.1, 134.3, 136.4, 163.6, 165.5, 181.5. IR (KBr, cmꢄ1
)
3333, 1657, 1612. HRMS (ESI) m/z: Calcd for C16H12O3Na [M þ Na]þ
275.0684; found 275.0680.
4.2.3. 7-Hydroxy-(E)-3-phenylmethylene-chroman-4-one (1)
Yield: 40.10 mg, 26.1%; mp: 95e96 ꢂC. 1H NMR (400 MHz,
4.2.4. 7-Hydroxy-(E)-3-[(2-bromophenyl)methylene]chroman-4-
one (2)
CD3OD):
d
5.32 (s, 2H, H2-2), 6.32 (s, 1H, H-8), 6.54 (d, J ¼ 8.7 Hz, 1H,
H-6), 7.36 (d, J ¼ 7.1 Hz, 2H, H-20 þ H-60), 7.43e7.50 (m, 3H, H-
Yield: 170.21 mg, 84.6%; mp: 209e210 ꢂC. 1H NMR (400 MHz,
30 þ H-40 þ H-50), 7.77 (s, 1H, H-9), 7.82 (d, J ¼ 8.7 Hz, 1H, H-5). 13C
DMSO-d6):
d
5.20 (s, 2H, H2-2), 6.33 (s,1H, H-8), 6.58 (d, J ¼ 8.1 Hz,1H,
NMR (100 MHz, CD3OD):
d
67.4, 102.3, 111.0, 114.3, 128.4, 129.2,
H-6), 7.31 (d, J ¼ 7.3 Hz, 1H, H-60), 7.40 (t, J ¼ 7.3 Hz, 1H, H-50), 7.50 (t,
Fig. 5. Structure activity relationship of homoisoflavonoids.

S.K. Roy et al. / European Journal of Medicinal Chemistry 66 (2013) 499e507
505
J ¼ 7.3Hz,1H, H-40), 7.65 (s,1H, H-9), 7.78 (d, J ¼ 8.1 Hz,1H, H-5)7.79 (t,
1H, H-6), 7.32e7.39 (m, 3H, H-30 þ H-50 þ H-60), 7.53 (m, 1H, H-40),
7.67 (s, 1H, H-9), 7.78 (d, J ¼ 7.2 Hz, 1H, H-5). 13C NMR (100 MHz,
J ¼ 7.3 Hz, 1H, H-30).13C NMR (100 MHz, DMSO-d6):
d 67.5,102.9,111.9,
114.5, 124.6, 128.4, 130.0, 131.4, 131.7, 132.9, 133.4, 134.3, 134.4, 163.3,
165.5, 179.9. IR (KBr, cmꢄ1) 3482, 1655, 1624, 1078. HRMS (ESI) m/z:
Calcd for C16H11BrO3Na [M þ Na]þ 352.9789; found 352.9792.
DMSO-d6): d 67.9, 103.0, 111.9, 114.5, 116.2, 122.1, 125.2, 128.0, 130.0,
131.6, 132.3, 133.5, 160.5, 163.3, 165.5, 179.9. IR (KBr, cmꢄ1) 3390,
1664, 1602, 1250. HRMS (ESI) m/z: Calcd for C16H11FO3Na [M þ Na]þ
293.0589; found 293.0591.
4.2.5. 7-Hydroxy-(E)-3-[(3-bromophenyl)methylene]chroman-4-
one (3)
4.2.11. 7-Hydroxy-(E)-3-[(3-fluorophenyl)methylene]chroman-4-
one (9)
Yield: 182.72 mg, 90.8%; mp: 229e230 ꢂC. 1H NMR (400 MHz,
DMSO-d6):
d
5.33 (s, 2H, H2-2), 6.34 (s, 1H, H-8), 6.58 (d, J ¼ 8.5 Hz,
Yield: 137.03 mg, 83.3%; mp: 145e146 ꢂC. 1H NMR (400 MHz,
1H, H-6), 7.40e7.46 (m, 3H, H-20 þ H-40 þ H-60), 7.66 (s, 2H, H-
DMSO-d6):
d
5.35 (s, 2H, H2-2), 6.34 (s,1H, H-8), 6.59 (d, J ¼ 8.1 Hz,1H,
50 þ H-9), 7.76 (d, J ¼ 8.5 Hz, 1H, H-5). 13C NMR (100 MHz, DMSO-
H-6), 7.26e7.32 (m, 3H, H-20 þ H-40 þ H-60), 7.53 (m,1H, H-50), 7.67 (s,
d6):
d 67.7, 102.9, 111.9, 114.5, 122.5, 129.3, 130.0, 131.2, 132.5, 132.7,
1H, H-9), 7.78 (d, J ¼ 7.3 Hz, 1H, H-5). 13C NMR (100 MHz, DMSO-d6):
132.9, 134.2, 136.9, 163.2, 165.4, 179.8. IR (KBr, cmꢄ1) 3394, 1665,
1623, 1073. HRMS (ESI) m/z: Calcd for C16H11BrO3Na [M þ Na]þ
352.9789; found 352.9793.
d 67.7, 102.9, 111.8, 114.5, 116.6, 117.1, 126.6, 130.0, 131.1, 132.5, 134.4,
136.7, 162.5, 163.2, 165.4, 179.8. IR (KBr, cmꢄ1) 3405, 1689, 1610,1244.
HRMS (ESI) m/z: Calcd for C16H11FO3Na [M þ Na]þ 293.0589; found
293.0588.
4.2.6. 7-Hydroxy-(E)-3-[(4-bromophenyl)methylene]chroman-4-
one (4)
4.2.12. 7-Hydroxy-(E)-3-[(4-fluorophenyl)methylene]chroman-4-
one (10)
Yield: 193.02 mg, 95.9%; mp: 202e203 ꢂC. 1H NMR (400 MHz,
DMSO-d6):
d
5.32 (s, 2H, H2-2), 6.33 (s, 1H, H-8) 6.56 (d, J ¼ 8.5 Hz,
Yield: 149.05 mg, 90.6%; mp: 195e197 ꢂC. 1H NMR (400 MHz,
1H, H-6), 7.38 (d, J ¼ 8.4 Hz, 2H, H-30 þ H-50), 7.63 (s, 1H, H-9), 7.68
DMSO-d6):
d
5.33 (s, 2H, H2-2), 6.33 (d, J ¼ 2.1 Hz, 1H, H-8) 6.57 (dd,
(d, J ¼ 8.4 Hz, 2H, H-20 þ H-60) 7.75 (d, J ¼ 8.5 Hz, 1H, H-5). 13C NMR
J ¼ 2.1, 8.5 Hz, 1H, H-6), 7.32 (t, J ¼ 8.8 Hz, 2H, H-30 þ H-50), 7.51 (m,
(100 MHz, DMSO-d6):
d 67.7, 102.9, 111.8, 114.5, 123.4, 130.0, 132.1,
J ¼ 8.8 Hz, 2H, H-20 þ H-60) 7.67 (s, 1H, H-9), 7.77 (d, J ¼ 8.5 Hz, 1H,
132.2, 132.6, 133.7, 134.6, 163.1, 165.4, 179.9. IR (KBr, cmꢄ1) 3453,
H-5). 13C NMR (100 MHz, DMSO-d6):
d 67.7, 102.9, 111.8, 114.6, 116.3,
1666, 1623, 1067. HRMS (ESI) m/z: Calcd for
C
16H11BrO3Na
130.0, 131.0, 131.3, 133.0, 134.7, 162.9, 163.0, 165.3, 179.9. IR (KBr,
[M þ Na]þ 352.9789; found 352.9791.
cmꢄ1
) 3238, 1675, 1612, 1243. HRMS (ESI) m/z: Calcd for
C
16H11FO3Na [M þ Na]þ 293.0589; found 293.0590.
4.2.7. 7-Hydroxy-(E)-3-[(2-chlorophenyl)methylene]chroman-4-
one (5)
4.2.13. 7-Hydroxy-(E)-3-[(2-methoxyphenyl)methylene]chroman-
4-one (11)
Yield: 160.95 mg, 92.3%; mp: 225e226 ꢂC.; 1H NMR (400 MHz,
DMSO-d6):
d
5.21 (d, J ¼ 1.2 Hz, 2H, H2-2), 6.33 (d, J ¼ 2.2 Hz, 1H, H-
Yield: 101.66 mg, 59.1%; mp: 166e168 ꢂC. 1H NMR (400 MHz,
8), 6.57 (dd, J ¼ 2.2, 8.7 Hz, 1H, H-6), 7.31 (dd, J ¼ 1.9, 7.2 Hz, 1H, H-
60), 7.45 (m, 2H, H-40 þ H-50), 7.59 (dd, J ¼ 1.9, 7.2 Hz, 1H, H-30), 7.70
(s, 1H, H-9), 7.76 (d, J ¼ 8.72 Hz, 1H, H-5). 13C NMR (100 MHz,
DMSO-d6): d
3.85 (s, 3H, 20-OCH3), 5.21 (s, 2H, H2-2), 6.33 (d,
J ¼ 2.1 Hz, 1H, H-8), 6.57 (dd, J ¼ 2.1, 8.7 Hz, 1H, H-6), 7.04 (t,
J ¼ 7.2 Hz, 1H, H-50), 7.12 (d, J ¼ 8.4 Hz, 1H, H-30), 7.16 (d,
J ¼ 8.2 Hz, 1H, H-60), 7.44 (t, J ¼ 7.6 Hz, 1H, H-40), 7.75 (s, 1H, H-9),
DMSO-d6): d 67.7, 102.9, 111.9, 114.5, 127.8, 130.0, 130.2, 131.4, 131.5,
132.1, 132.5, 133.3, 134.1, 163.3, 165.5, 179.8. IR (KBr, cmꢄ1) 3516,
7.77 (d, J ¼ 8.7 Hz, 1H, H-5). 13C NMR (100 MHz, DMSO-d6)
d 56.0,
1658, 1600, 1075. HRMS (ESI) m/z: Calcd for
C
16H11ClO3Na
68.1, 102.9, 111.7, 111.8, 114.7, 120.7, 122.9, 129.9, 130.8, 131.1, 131.8,
131.9, 158.2, 163.2, 165.2, 180.0. IR (KBr, cmꢄ1) 3414, 1613. HRMS
(ESI) m/z: Calcd for C17H14O4Na [M þ Na]þ 305.0789; found
305.0790.
[M þ Na]þ 309.0294; found 309.0291.
4.2.8. 7-Hydroxy-(E)-3-[(3-chlorophenyl)methylene]chroman-4-
one (6)
Yield: 163.4 mg, 93.7%; mp: 232e233 ꢂC. 1H NMR (400 MHz,
4.2.14. 7-Hydroxy-(E)-3-[(3-methoxyphenyl)methylene]chroman-
4-one (12)
DMSO-d6):
d
5.31 (s, 2H, H2-2), 6.32 (s,1H, H-8), 6.56 (d, J ¼ 8.5 Hz,1H,
H-6), 7.35 (s,1H, H-50), 7.50 (m, 3H, H-20 þ H-40 þ H-60), 7.63 (s,1H, H-
Yield: 144.84 mg, 84.2%; mp: 215e216 ꢂC. 1H NMR (400 MHz,
9), 7.74 (d, J ¼ 8.5 Hz, 1H, H-5). 13C NMR (100 MHz, DMSO-d6):
d
67.2,
DMSO-d6): d
3.80 (s, 3H, 30-OCH3), 5.35 (s, 2H, H2-2), 6.33 (s, 1H, H-
102.4, 111.4, 114.0, 128.5, 129.1, 129.5, 130.5, 132.2, 133.4, 133.7, 136.1,
162.6, 164.9, 179.3. IR (KBr, cmꢄ1) 3374, 1660, 1620, 1069. HRMS (ESI)
m/z: Calcd for C16H11ClO3Na [M þ Na]þ 309.0294; found 309.0289.
8), 6.56 (d, J ¼ 8.5 Hz, 1H, H-6), 6.97e6.99 (m, 2H, H-20 þ H-40), 7.02
(d, J ¼ 8.2 Hz, 1H, H-60), 7.40 (t, J ¼ 7.3 Hz,1H, H-50), 7.66 (s, 1H, H-9),
7.76 (d, J ¼ 8.5 Hz, 1H, H-5). 13C NMR (100 MHz, DMSO-d6):
d 55.2,
67.4, 102.4, 111.3, 114.1, 115.2, 115.3, 122.2, 129.5, 129.8, 131.1, 135.2,
135.3, 159.3, 162.6, 164.8, 179.5. IR (KBr, cmꢄ1) 3390, 1645, 1614.
HRMS (ESI) m/z: Calcd for C17H14O4Na [M þ Na]þ 305.0789; found
305.0790.
4.2.9. 7-Hydroxy-(E)-3-[(4-chlorophenyl)methylene]chroman-4-
one (7)
Yield: 168.99 mg, 96.9%; mp: 201e203 ꢂC (lit 196e198 ꢂC) [14].
1H NMR (400 MHz, DMSO-d6):
d 5.33 (s, 2H, H2-2), 6.34 (d,
J ¼ 1.7 Hz, 1H, H-8), 6.56 (dd, J ¼ 1.7, 8.72 Hz, 1H, H-6), 7.45 (d,
J ¼ 8.4 Hz, 2H, H-30 þ H-50), 7.54 (d, J ¼ 2.1, 8.4 Hz, 2H, H-20 þ H-60),
7.65 (s, 1H, H-9), 7.76 (d, J ¼ 8.5 Hz, 1H, H-5). 13C NMR (100 MHz,
4.2.15. 7-Hydroxy-(E)-3-[(4-methoxyphenyl)methylene]chroman-
4-one (13)
Yield: 66.40 mg, 38.6%; mp: 210e211 ꢂC (lit 206e208 ꢂC) [12]. 1H
DMSO-d6):
d
67.7, 102.9, 111.8, 114.5, 129.2, 130.0, 132.0, 132.3, 133.3,
NMR (400 MHz, DMSO-d6): d
3.81 (s, 3H, 40-OCH3), 5.35 (d,
134.5, 134.6, 163.1, 165.3, 179.9. IR (KBr, cmꢄ1) 3437, 1671, 1601,
J ¼ 1.6 Hz, 2H, H2-2), 6.31 (d, J ¼ 2.2 Hz, 1H, H-8), 6.54 (dd, J ¼ 2.2,
8.7 Hz, 1H, H-6), 7.03 (d, J ¼ 8.7 Hz, 2H, H-30 þ H-50), 7.39 (d,
J ¼ 8.7 Hz, 2H, H-20 þ H-60), 7.63 (s, 1H, H-9), 7.73 (d, J ¼ 8.7 Hz, 1H,
1060. APCI (ESI) m/z: 288.16 [M þ H]þ.
4.2.10. 7-Hydroxy-(E)-3-[(2-fluorophenyl)methylene]chroman-4-
one (8)
H-5). 13C NMR (100 MHz, DMSO-d6):
d 55.8, 68.0, 102.9, 111.6, 114.7,
114.8, 127.0, 129.3, 129.9, 132.6, 135.7, 160.7, 162.9, 165.1, 180.0. IR
(KBr, cmꢄ1) 3394, 1603. MS (APCI) m/z: Calcd for C17H14O4 [M þ H]þ
283.10; found 283.18.
Yield: 132.09 mg, 80.2%; mp: 132e133 ꢂC. 1H NMR (400 MHz,
DMSO-d6):
d
5.24 (s, 2H, H2-2), 6.35 (s, 1H, H-8), 6.59 (d, J ¼ 8.1 Hz,

506
S.K. Roy et al. / European Journal of Medicinal Chemistry 66 (2013) 499e507
4.2.16. 7-Hydroxy-(E)-3-[(3-hydroxyphenyl)methylene]chroman-
4-one (14)
20 þ H-60), 7.79 (s, 1H, H-9), 7.84 (d, J ¼ 8.8 Hz, 1H, H-5). 13C NMR
(100 MHz, CD3OD):
d 67.2, 102.3, 111.2, 114.3, 125.2, 125.3, 129.5,
Yield: 81.58 mg, 49.9%; mp: >270 ꢂC. 1H NMR (400 MHz, DMSO-
130.1, 133.3, 134.1, 138.1, 138.2, 163.7, 165.6, 180.8. IR (KBr, cmꢄ1
)
d6):
d
5.32 (s, 2H, H2-2), 6.32 (s, 1H, H-8), 6.55 (d, J ¼ 8.0 Hz, 1H, H-6),
3445, 1660, 1624. HRMS (ESI) m/z: Calcd for
C17H11F3O3Na
6.84e6.78 (m, 3H, H-60 þ H-40 þ H-20), 7.29e7.27 (m, 1H, H-50), 7.58
[M þ Na]þ 343.0558; found 343.0550.
(s, 1H, H-9), 7.74 (d, J ¼ 8.0 Hz, 1H, H-5). 13C NMR (100 MHz, DMSO-
d6):
d 67.9, 102.9, 111.7, 114.7, 117.0, 117.0, 121.4, 129.9, 130.3, 131.3,
4.3. Biology
135.7, 136.0, 157.9, 163.1, 165.2, 180.0. IR (KBr, cmꢄ1) 3446,1654,1629.
HRMS (ESI) m/z: Calcd for C16H12O4Na [M þ Na]þ 291.0633; found
291.0620, Calcd for C16H13O4 [M þ H]þ 269.0814; found 269.0805.
4.3.1. Bacterial strain growing conditions
M. smegmatis mc2 155 (ATCC 700084) was provided by Dr. S.
Majumdar, IMTECH, Chandigarh, India. Mycobacterial strain was
grown in 10% oleic acidealbumineglucose complex (OADC) sup-
plemented Middlebrook 7H11 agar medium.
4.2.17. 7-Hydroxy-(E)-3-[(4-hydroxyphenyl)methylene]chroman-
4-one (15)
Yield: 52.31 mg, 32.0%; mp: 252e253 ꢂC (lit 248e249 ꢂC)
[14,21]. 1H NMR (400 MHz, CD3OD):
d 5.23 (s, 2H, H2-2), 6.20 (s, 1H,
4.3.2. MIC determination
H-8), 6.42 (d, J ¼ 8.5 Hz, 1H, H-6), 6.78 (d, J ¼ 7.2, 2H, H-30 þ H-50),
This assay comprised MIC determination of standards and test
compounds as per the reported method [22]. All the compounds are
freely soluble in DMSO. Briefly, the compounds was first dissolved in
DMSO and then diluted in 7H9/OADC-supplemented medium across
a 96 well Microtiter plate in twofold serial dilution. Bacterial inocula
equivalent to the 0.5 McFarland standards were prepared in normal
saline and diluted to give the final density of 0.5 ꢃ 105 cfu/mL. The
7.13 (d, J ¼ 7.2 Hz, 2H, H-20 þ H-60), 7.60 (s, 1H, H-9), 7.69 (d,
J ¼ 8.5 Hz, 1H, H-5). 13C NMR (100 MHz, CD3OD):
d 67.6, 102.4, 110.8,
114.4, 115.4, 125.7, 128.1, 129.3, 132.1, 136.8, 159.1, 163.4, 165.2, 181.7.
IR (KBr, cmꢄ1) 3451, 1612. MS (APCI) m/z: Calcd for C16H12O4
[M þ H]þ 269.07; found 269.10.
4.2.18. 7-Hydroxy-(E)-3-[(2-allyloxyphenyl)methylene]chroman-4-
one (16)
inoculum (100 mL) was added to all the wells and the microtiter plate
was incubated at 37 ꢂC for 72 h. The MIC was recorded as the lowest
concentration at which no bacterial growth was observed. This was
Yield: 64.37 mg, 33.2%; mp: 185e186 ꢂC. 1H NMR (400 MHz,
DMSO-d6):
d
4.63 (d, J ¼ 1.9 Hz, 2H, H2-100), 5.19 (s, 2H, H2-2), 5.25 (d,
facilitated by the addition of 20 mL of MTT at the concentration of
J ¼ 10.5 Hz,1H, H-3b00), 5.37 (d, J ¼ 17.3 Hz,1H, H-3a00), 6.04 (m,1H, H-
200), 6.31 (s, 1H, H-8), 6.55 (d, J ¼ 8.6 Hz, 1H, H-6), 7.02 (t, J ¼ 7.5 Hz,
1H, H-50), 7.10 (d, J ¼ 8.3 Hz, 1H, H-30), 7.15 (d, J ¼ 7.4 Hz, 1H, H-60),
7.41 (t, J ¼ 7.7 Hz,1H, H-40), 7.74 (d, J ¼ 8.6 Hz,1H, H-5), 7.77 (s,1H, H-
10 mg/mL in MeOH to each well and incubated at 37 ꢂC for 20 min
where bacterial growth was indicated by purple coloration adhered
to cells. Appropriate DMSO, cell and sterile saline controls were
carried out in the same set of experiment.
9). 13C NMR (100 MHz, DMSO-d6):
d 68.1, 69.0, 102.9, 111.8, 112.9,
114.7, 118.1, 120.9, 123.2, 130.0, 131.0, 131.1, 131.8, 131.9, 133.8, 157.1,
163.2, 165.2, 180.4. IR (KBr, cmꢄ1) 3442, 1654, 1616. HRMS (ESI) m/z:
Calcd for C19H16O4Na [M þ Na]þ 331.0946; found 331.0950.
4.3.3. Modulation assay
Test compounds were further screened for their synergistic ef-
fects with EtBr prior to efflux assays, according to the modified
method described previously [22]. Compounds were dissolved in
DMSO and diluted in 7H9/OADC-supplemented medium at various
sub-inhibitory concentrations (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128
and 1/248 of the MIC). Each concentration of the bonducellin and
compounds (1e19) maintained uniformity throughout the experi-
ment, whereas the EtBr was serially diluted (8, 4, 2, 1, 0.5 and
0.25 mg/L) for MIC determination with and without modulator. A
modulation factor (MF) was used to express the modulating effects
of compounds on MIC of EtBr, where:
4.2.19. 7-Hydroxy-(E)-3-[(3-allyloxyphenyl)methylene]chroman-4-
one (17)
Yield: 39.20 mg, 20.21%; mp: 147e148 ꢂC. 1H NMR (400 MHz,
DMSO-d6):
d
4.63(d, J ¼ 5.1 Hz,2H, H2-100), 5.28(dd,J ¼ 1.4,10.2 Hz,1H,
H-3b00), 5.34 (s, 2H, H-2), 5.42 (dd, J ¼ 1.4,17.3 Hz,1H, H-3a00), 6.06 (m,
1H, H-200), 6.34(d, J ¼ 2.1 Hz,1H, H-8), 6.57 (dd, J ¼ 2.1, 8.7 Hz,1H, H-6),
6.98(d, J ¼ 8.0 Hz,1H, H-60), 6.99(s,1H, H-20), 7.03 (d, J ¼ 8.2 Hz,1H, H-
40), 7.39 (t, J ¼ 8.0 Hz,1H, H-50), 7.65 (s,1H, H-9), 7.76 (d, J ¼ 8.7 Hz,1H,
H-5). 13C NMR (100 MHz, DMSO-d6):
d 67.9, 68.7, 102.9, 111.8, 114.6,
116.4, 116.5, 118.1, 122.9, 129.9, 130.3, 131.7, 132.8, 134.0, 135.8, 135.8,
158.6, 163.1, 165.3, 180.0. IR (KBr, cmꢄ1) 3503, 1660, 1616. HRMS (ESI)
m/z: Calcd for C19H16O4Na [M þ Na]þ 331.0946; found 331.0950.
MICðAntibioticÞ
MF ¼
MICðAntibiotic þ ModulatorÞ
Fractional Inhibitory Concentration Index (FICI) was calculated
by the equation:
4.2.20. 7-Hydroxy-(E)-3-[(4-allyloxyphenyl)methylene]chroman-
4-one (18)
Yield: 40.96 mg, 21.1%; mp: 178e179 ꢂC. 1H NMR (400 MHz,
FICI ¼ FICðAÞ þ FICðBÞ
CD3OD/CDCl3,1:1)
d
4.20(s, 2H, H2-100), 5.32(d, J ¼ 11.0Hz,1H, H-3b00),
5.35 (s, 2H, H2-2), 5.44 (d, J ¼ 17.2 Hz, 1H, H-3a00), 6.07 (m, 1H, H-200),
6.35 (s,1H, H-8), 6.56 (d, J ¼ 8.7 Hz,1H, H-6), 7.01 (d, J ¼ 7.7 Hz, 2H, H-
30 þ H-50), 7.28 (d, J ¼ 7.8 Hz, 2H, H-20 þ H-60), 7.76 (s,1H, H-9), 7.85 (d,
MICðA in presence of BÞ
FICðAÞ ¼
MICðA aloneÞ
J ¼ 8.7 Hz, 1H, H-5). 13C NMR (100 MHz, CD3OD/CDCl3, 1:1):
d 67.7,
MICðB in presence of AÞ
FICðBÞ ¼
68.8, 102.6, 111.2, 114.7, 114.9, 117.6, 127.1, 129.0, 129.7, 131.9, 132.7,
136.7,159.6,163.3, 165.0,181.8. IR (KBr, cmꢄ1) 3399,1616. HRMS (ESI)
m/z: Calcd for C19H16O4Na [M þ Na]þ 331.0946; found 331.0950.
MICðB aloneÞ
Interpretation of FICI was as follows: FICI value of ꢁ0.5 indicates
synergy; ꢀ4.0 indicates antagonism; and 4.0 > FICI > 0.5 indicates
no interaction [17,23].
4.2.21. 7-Hydroxy-(E)-3-[(4-trifluoromethylphenyl)methylene]
chroman-4-one (19)
Yield: 48.21 mg, 24.6%; mp: 198e199 ꢂC. 1H NMR (400 MHz,
4.3.4. EtBr accumulation assay by fluorometric method
CD3OD):
d
5.32 (s, 2H, H2-2), 6.33 (s, 1H, H-8), 6.56 (d, J ¼ 8.8 Hz, 1H,
This assay was performed as per reported methods with some
modification [22]. M. smegmatis was grown in 10 mL 7H9/OADC-
H-6), 7.57 (d, J ¼ 7.6 Hz, 2H, H-30 þ H-50), 7.78 (d, J ¼ 7.6 Hz, 2H, H-

S.K. Roy et al. / European Journal of Medicinal Chemistry 66 (2013) 499e507
507
supplemented medium at 37 ꢂC to an OD600 of 0.8. The culture was
centrifuged at 13,000 rpm in a microfuge tube for 3 min. The su-
pernatant was discarded and the pellet was washed once and
resuspended in phosphate buffer saline (PBS). After adjusting the
OD600 to 0.4, glucose and EtBr were added to yield final concen-
trations of 0.4% and 1 mg/L, respectively to one set of microtubes
containing 1 mL bacterial suspension.
[3] X-Z. Li, L. Zhang, H. Nikaido, Efflux pump-mediated intrinsic drug resistance in
Mycobacterium smegmatis, Antimicrob. Agents Chemother. 48 (2004) 2415e
2423.
[4] L. Rodrigues, J. Ramos, I. Couto, L. Amaral, M. Viveiros, Ethidium bromide
transport across Mycobacterium smegmatis cell-wall: correlation with antibi-
otic resistance, BMC Microbiol. 11 (2011) 35e44.
[5] S. Ramon-Garcia, C. Martin, C.J. Thompson, J.A. Ainsa, Role of the Mycobacte-
rium tuberculosis P55 efflux pump in intrinsic drug resistance, oxidative stress
responses, and growth, Antimicrob. Agents Chemother. 53 (2009) 3675e3682.
[6] P.E.A. da Silva, A. Von Groll, A. Martin, J.C. Palomino, Efflux as a mechanism for
drug resistance in Mycobacterium tuberculosis, FEMS Immunol. Med. Micro-
biol. 63 (2011) 1e9.
Aliquots of 95
mL were distributed to replica sets of 0.2 mL
microtubes, and 5
mL each of known EPI and test compounds as EPI
in case of respective assays, was added. Replica tubes that did not
receive any EPI served as a control. In each accumulation assay, the
positive controls of the reported EPIs such as verapamil, reserpine
and CCCP were dissolved in DMSO and used along with vehicle
control (DMSO). The fluorescence was measured by spectro-
flourimetric microplate reader. The fluorescence at excitation and
emission wavelengths of 530 nm (bandwidth 5 nm) and 600 nm
(bandwidth 10 nm), respectively, was measured at 3 min intervals
for 45 min. Each experiment was repeated three times.
[7] J. Jin, J.Y. Zhang, N. Guo, H. Sheng, L. Li, J.C. Liang, X.L. Wang, Y. Li, M.Y. Liu,
X.P. Wu, Farnesol, a potential efflux pump inhibitor in Mycobacterium smeg-
matis, Molecules 15 (2010) 7750e7762.
[8] S.M. Jachak, S.K. Roy, S. Gupta, P. Ahirrao, S. Gibbons, Small-molecule efflux
pump inhibitors from natural products as a potential source of antimicrobial
agents, in: G. Tegos, E. Mylonakis (Eds.), Antimicrobial Drug Discovery:
Emerging Strategies, CAB International, 2012, pp. 62e76.
[9] R. Gautam, A. Saklani, S.M. Jachak, Indian medicinal plants as a source of
antimycobacterial agents, J. Ethnopharmacol. 110 (2007) 200e234.
[10] S.K. Roy, U.C. Agrahari, R. Gautam, A. Srivastava, S.M. Jachak, Isointricatinol, a
new antioxidant homoisoflavonoid from the roots of Caesalpinia digyna Rot-
tler, Nat. Prod. Res. 26 (2012) 690e695.
[11] S.K. Roy, A. Srivastava, S.M. Jachak, Analysis of homoisoflavonoids in Cae-
salpinia digyna by HPLC-ESI-MS, HPLC and method validation, Nat. Prod.
Commun. 7 (2012) 1189e1192.
[12] T. Al Nakib, V. Bezjak, M.J. Meegan, R. Chandy, Synthesis and antifungal ac-
tivity of some 3-benzylidenechroman-4-ones, 3-benzylidenethiochroman-4-
ones and 2-benzylidene-1-tetralones, Eur. J. Med. Chem. 25 (1990) 455e462.
[13] K. Koch, M.S. Biggers, General preparation of 7-substituted 4-chromanones:
synthesis of a potent aldose reductase inhibitor, J. Org. Chem. 59 (1994)
1216e1218.
[14] V. Siddaiah, C.V. Rao, S. Venkateswarlu, A.V. Krishnaraju, G.V. Subbaraju,
Synthesis, stereochemical assignments, and biological activities of homoiso-
flavonoids, Bioorg. Med. Chem. 14 (2006) 2545e2551.
[15] A. Foroumadi, A. Samzadeh-Kermani, S. Emami, G. Dehghan, M. Sorkhi,
F. Arabsorkhi, M.R. Heidari, M. Abdollahi, A. Shafiee, Synthesis and antioxidant
properties of substituted 3-benzylidene-7-alkoxychroman-4-ones, Bioorg.
Med. Chem. Lett. 17 (2007) 6764e6769.
4.3.5. EtBr efflux assay by fluorometric method
The effectof EPIs onEtBreffluxactivitywasmeasured accordingto
a modification of recently reported fluorescence techniques [22]. The
non-inhibitory concentration of EtBr on working cfu of M. smegmatis
mc2 155 was used. The accumulation of EtBr at 25 ꢂC in the absence of
glucose, caused a higher accumulation without compromising the
cellular viability (4 mg/L, corresponding to one fourth the MIC). The
EtBr-loaded cells were centrifuged at 13,000 rpm in a microfuge for
3min and resuspended in EtBr-free PBS containing 0.4% glucose. After
adjusting the OD600 to 0.4, aliquots of 95
mL were transferred to
replicate 0.2 mL microtubes, and 5 L each of EPI was added. Replica
m
tubes that did not receive any EPI served as a control. In each efflux
assay, the positive controls of the reported EPIs such as verapamil,
reserpine and CCCP were dissolved in DMSO and used along with
vehicle control (DMSO). The EtBr efflux by the cells was monitored in
a spectroflourimetric microplate reader. The fluorescence at excita-
tion and emission wavelengths of 530 nm (bandwidth 5 nm) and
600 nm (bandwidth 10 nm), respectively, was measured at 3 min
intervals for 45 min. Each experiment was repeated three times.
[16] P. Chen, J.-S. Yang, Flavonol galactoside caffeiate ester and homoiso-
flavones from Caesalpinia millettii Hook. et ARN, Chem. Pharm. Bull. 55
(2007) 655e657.
[17] D. Lechner, S. Gibbons, F. Bucar, Plant phenolic compounds as ethidium bro-
mide efflux inhibitors in Mycobacterium smegmatis, J. Antimicrob. Chemother.
62 (2008) 345e348.
[18] B. Groeblacher, O. Kunert, F. Bucar, Compounds of Alpinia katsumadai as
potential efflux inhibitors in Mycobacterium smegmatis, Bioorg. Med. Chem. 20
(2012) 2701e2706.
[19] L. Rodrigues, D. Wagner, M. Viveiros, D. Sampaio, I. Couto, M. Vavra,
W.V. Kern, L. Amaral, Thioridazine and chlorpromazine inhibition of ethidium
bromide efflux in Mycobacterium avium and Mycobacterium smegmatis,
J. Antimicrob. Chemother. 71 (2008) 1076e1082.
Acknowledgements
[20] F.R. Stermitz, P. Lorenz, J.N. Tawara, L.A. Zenewicz, K. Lewis, Synergy in a
medicinal plant: antimicrobial action of berberine potentiated by 50-
methoxyhydnocarpin, a multidrug pump inhibitor, Nat. Acad. Sci. Proc. 97
(2000) 1433e1437.
[21] M. Namikoshi, H. Nakata, T. Saitoh, Homoisoflavonoids from Caesalpinia
sappan, Phytochemistry 26 (1987) 1831e1833.
The authors thank the Director of NIPER and the Director of
IMTECH for providing financial support and necessary facilities to
carry out this work.
[22] S.K. Roy, S. Pahwa, H. Nandanwar, S.M. Jachak, Phenylpropanoids of Alpinia
galanga as efflux pump inhibitors in Mycobacterium smegmatis mc2 155,
Fitoterapia 83 (2012) 1248e1255.
References
[23] European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the
European Society of Clinical Microbiology and Infectious Diseases (ESCMID),
Terminology relating to methods for the determination of susceptibility of
bacteria to antimicrobial agents, Clin. Microbiol. Infect. 6 (2000) 503e508.
[1] E.D. Rossi, J.A. Ainsa, G. Riccardi, Role of mycobacterial efflux transporters in
drug resistance: an unresolved question, FEMS Microbiol. Rev. 30 (2006) 36e52.
[2] B. Marquez, Bacterial efflux systems and efflux pumps inhibitors, Biochimie 87
(2005) 1137e1147.