Ammonium derivatives of chromenones and quinolinones as lead antimicrobial agents

Article abstract of DOI:10.1007/s12039-011-0147-7

A series of novel ammonium derivatives were synthesized and examined for their antimicrobial efficacy. Comparison of antimicrobial spectrum revealed that compounds 9, 11, 16 and 23 had strong potential against pathogens in vitro. Cytotoxicity results showed compound 9 to be least toxic, it is non-toxic to A549 and U87 cells in MTT assay and exhibits marginal toxicity (15-20%) to human erythrocytes at a concentration of 1000 μg/ml as compared to 100% lysis of cells by 31.25 μg/ml of the standard drug amphotericin B. This compound has MIC values in the range of 1.95-31.25 μg/disc in DDA against different pathogens and may considered to be an important lead antimicrobial molecule for further exploration.

Full text of DOI:10.1007/s12039-011-0147-7

J. Chem. Sci. Vol. 124, No. 2, March 2012, pp. 437–449. ꢀc Indian Academy of Sciences.
Ammonium derivatives of chromenones and quinolinones as lead
antimicrobial agents
a
b,c
a
b
SHILPI GUPTA , SEEMA SINGH , ABHA KATHURIA , MANISH KUMAR ,
a,d
b
a
b
SWETA SHARMA , RAM KUMAR , VIRINDER S PARMAR , BHARAT SINGH ,
a
d
b,∗
ANJALI GUPTA , ERIK VAN DER EYCKEN , GAINDA L SHARMA
and
a,∗
SUNIL K SHARMA
Department of Chemistry, University of Delhi, Delhi 110 007, India
Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110 007, India
Department of Biotechnology, Pune University, Pune 411 007, India
Katholieke Universiteit Leuven (K.U.Leuven), Celestijnenlaan 200F, 3001, Leuven, Belgium
a
b
c
d
e-mail: sk.sharma90@gmail.com; drglsharma@hotmail.com
MS received 15 March 2011; revised 14 June 2011; accepted 27 June 2011
Abstract. A series of novel ammonium derivatives were synthesized and examined for their antimicrobial
efficacy. Comparison of antimicrobial spectrum revealed that compounds 9, 11, 16 and 23 had strong potential
against pathogens in vitro. Cytotoxicity results showed compound 9 to be least toxic, it is non-toxic to A549
and U87 cells in MTT assay and exhibits marginal toxicity (15–20%) to human erythrocytes at a concentration
of 1000 μg/ml as compared to 100% lysis of cells by 31.25 μg/ml of the standard drug amphotericin B. This
compound has MIC values in the range of 1.95–31.25 μg/disc in DDA against different pathogens and may
considered to be an important lead antimicrobial molecule for further exploration.
Keywords. Ammonium compounds; antimicrobial activity; chromenones; quinolinones.
1
. Introduction
identification of novel antibiotic classes with activity
against resistant strains is critical to successful therapy
of diseases caused by bacteria.
7
The occurrence of life-threatening infections caused
by pathogenic microorganisms has been a cause of
major concern globally, resulting in high mortality and
Fungal infections including aspergillosis have
emerged as threat to public health in recent years.
Effective therapy of aspergillosis with currently avail-
able drugs is considered to be extremely difficult at
late stages of infection and almost impossible when
1
morbidity. The search for compounds with antimi-
crobial activity has gained tremendous importance
recently, due to the alarming increase in the number
of infections caused by antibiotic-resistant microor-
ganisms. Endemic and opportunistic fungal infections
have become an important problem, along with the
increasing number of patients undergoing immunosup-
pressive therapy. Although it was believed that virtu-
ally all bacterial infections could be treated with avail-
able antibacterial drugs, the drug resistance in bacteria
is fast emerging and deaths following bacterial infec-
tions are on the rise even in patients infected with bacte-
ria that are curable with currently available drugs. This
is because the patients are at risk for infections caused
8
it becomes systemic. At present, amphotericin B
2
and azoles remain the mainstay of systemic therapy9
despite their very severe side effects. Further, there
are reports on the isolation of fungal pathogens that
3–5
10–12
have been resistant to most antifungals.
The suc-
cess of the development of an ideal antifugal drug
relies upon the identification of new molecules with
broad spectrum of activity and novel targets having no
9,13
counterpart in mammalian host.
There have been
studies exploring synthetic compounds and natural
products as potential antifungal agents. Antimycotic
6
by multidrug-resistant pathogens. Consequently, the
14
activity of Datura metel has been demonstrated.
Also in vitro, in vivo and post antifungal effect
15
16
17
of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-
methylethyl pentanoate) isolated from D. metel has
been reported. Chhillar et al. screened a series of
∗
For correspondence
437

438
Shilpi Gupta et al.
synthetic compounds and reported antimicrobial 2.2 General procedure for the synthesis of
activity of dihydropyridines.18
ammonium acyloxy derivatives of chromenones
Many natural and synthetic products consisting of and quinolinones (9–16)
chromenones and quinolinones have been shown to
To an ice cold solution of 11-bromoundecanoic acid
2.25 g, 8.48 mmol) in anhydrous DCM (25 mL) was
display diverse and interesting biological and phar-
macological activities. Our group has been involved
in the synthesis of various analogues of chromen-
(
added oxalyl chloride (2.14 g, 16.96 mmol), the reaction
mixture was stirred under nitrogen for 2 h. The solvent
was then removed in vacuo and the resulting yellow vis-
cous oil was dissolved in THF (20 mL) and the solu-
19,20
21
22
2
-ones,
quinolin-2-ones and chromen-4-ones
to study their pharmacological effects and antiox-
19–22
idant potential.
Further, ammonium compounds
◦
tion was cooled to 5 C. To this, a solution of one equiv-
are known for their antibacterial activity against both
Gram-positive and Gram-negative bacteria, as well as
against some pathogenic species of fungi and proto-
alent chromenones/quinolin-2-ones in THF was added
followed by 1.0 equivalent of anhydrous triethyl amine.
23–28
The reaction mixture was stirred at room temperature
zoa.
The encouraging biological activity results
◦
(
25 C) for about 6 h, the solvent was then removed
of the two classes of heterocyclic compounds and
based on the literature data on ammonium compounds,
we envisioned that chromenones and quinolinones
based ammonium compounds may possibly act as
potent antimicrobial agents. In lieu of this, the present
work was undertaken here; we synthesize a series
of long chain ammonium derivatives of chromenones
and quinolinones and screened them for antimicrobial
activity.
under reduced pressure and the resulting crude prod-
uct was subjected to column chromatography to yield
the corresponding bromo ester (1–8). To the solution
of bromo ester (1–8) in anhydrous acetonitrile (20 mL),
was added triethylamine (5 equivalents) and the reac-
◦
tion mixture was stirred under nitrogen at 60 C for
approximately 48 h. The progress of reaction was mon-
itored on TLC (methanol–chloroform (1:4)). On com-
pletion of the reaction, the solvent was evaporated under
reduced pressure and the resultant product was then
subjected to column chromatography to give the pure
quaternary ammonium compound (9–16) in moderate
to low yields (20–65%) as off white or pale brown solid.
2. Experimental
2.1 General
All the solvents were dried and distilled prior to their
use. Reactions were monitored by precoated TLC plates 2.2a N,N,N-Triethyl-11-(4-methyl-2-oxo-2H-chromen-
Merck silica gel 60F ); the spots were visualized 7-yloxy)-11-oxoundecan-1-aminium bromide (9): The
(
254
either by UV light, or by spraying with 5% alco- product was obtained through column chromatog-
holic FeCl solution. Silica gel (100–200 mesh) was raphy using silica gel in methanol–chloroform (1:9)
3
◦
used for column chromatography. Oxalyl chloride, 11- to give 9 (32%) as a pale white solid. mp = 72 C;
bromo undecanoic acid, dihalo alkanes, and triethy-
lamine were supplied by Spectrochem Pvt. Ltd., India. UV (acetonitrile) λ : 270 and 311 nm; IR (KBr)
R
= 0.54 (silica gel, methanol–chloroform (1:4));
max
f
Potassium carbonate was fused in a muffle furnace νmax: 3468.8, 2977.9, 2923.1, 2851.6, 1764.5, 1724.9,
◦
at 400 C thrice prior to use. Melting points were 1625.2, 1613.9, 1568.1, 1504.0, 1486.5, 1466.5,
recorded in capillaries in sulphuric acid bath and are 1444.8, 1412.9, 1391.6, 1368.5, 1325.6, 1262.6,
uncorrected. Infrared spectra were recorded on Perkin- 1242.5, 1219.1, 1205.4, 1188.0, 1152.4, 1131.0,
1
Elmer FT-IR model 9 spectrophotometer. The H NMR 1111.3, 1094.6, 1071.6, 1019.3, 998.1, 982.1, 918.5,
1
3
−1
and C NMR spectra were recorded on Bruker AC- 879.2, 854.8, 797.3, 579.2, 456.7, 434.3 cm ;
1
4
00 (400 MHz, 100.6 MHz) NMR spectrometer and
Avance-300 (300 MHz, 75.5 MHz) NMR spectrometer 21H, -N(CH CH ) , H-3 - H-8 ), 1.63–1.73 (m, 4H,
using TMS as internal standard. The chemical shift H-2 and H-9 ), 2.39 (s, 3H, C-4 CH ), 2.54 (t, 2H,
H NMR (400 MHz, CDCl ; Me Si): δ 1.26-1.35 (m,
3 4
ꢁ
ꢁ
2
3 3
ꢁ
ꢁ
3
ꢁ
ꢁ
values are on δ scale and the coupling constant val- J = 7.2 hz, H-10 ), 3.23 (t, 2H, J = 7.1 hz, H-1 ),
ues (J) are in Hz. The HRMS spectra were recorded 3.44 (q, 6H, J = 6.9 hz, -N(CH CH ) ), 6.20 (s, 1H,
2
3 3
on Agilent-6210 ES-TOF, JEOL JMX-SX-102A and H-3), 7.01-7.03 (m, 2H, H-6 and H-8), 7.58 (d, 1H, J =
13
Waters LCT Micromass-KC455.
The chromenones and quinolin-2-ones were synthe- 7.95 (-N(CH CH ) ), 18.66 (C-4 CH ), 21.95, 24.56,
9.2 hz, H-5); C NMR (100.6 MHz, CDCl ; Me Si): δ
3 4
2
3
3
3
sized according to literature procedures.20,21,29–31
ꢁ
26.33, 28.74, 28.97, 29.11, 29.44, 29.57, 34.14 (C-2 -

Chromenones and quinolinones as antimicrobial agents
439
ꢁ
ꢁ
ꢁ
C-10 ), 53.38 (-N(CH CH ) ), 57.39 (C-1 ), 110.19 (t, 2H, J = 6.6 hz, H-1 ), 3.50 (q, 6H, J = 6.9 hz,
2
3 3
(
C-8), 114.17 (C-3), 117.60 and 118.08 (C-6 and -N(CH CH ) ), 6.92–7.05 (m, 2H, H-6 and H-8), 7.61
2 3 3
1
3
C-10), 125.41 (C-5), 152.10, 153.04 and 153.92 (d, 1H, J = 9.3 hz, H-5); C NMR (75.5 MHz, CDCl ;
3
ꢁ
ꢁꢁ
(
C-4, C-7 and C-9), 160.41 (C-2) and 171.53 (C-11 ); Me Si): δ 8.07 (-N(CH CH ) ), 14.02 (C-6 ), 14.92
4 2 3 3
+
+
HRMS, m/z: M calculated for C H NO : 444.3114, (C-4 CH ), 22.05, 22.54, 24.65, 26.43, 27.62, 28.63,
27
42
4
3
found 444.3115.
28.82, 28.97, 29.06, 29.18, 29.28, 29.61, 31.61, 34.22
ꢁ
ꢁ
ꢁꢁ
ꢁꢁ
(
C-2 - C-10 and C-1 - C- 5 ), 53.50 (-N(CH CH ) ),
2 3 3
ꢁ
5
7.54 (C-1 ), 109.85 (C-8), 117.87 and 118.47 (C-6
2.2b N,N,N-Triethyl-11-(3-ethyl-4-methyl-2-oxo-2H-
and C-10), 125.25 (C-3), 126.33 (C-5), 145.39 (C-4),
chromen-7-yloxy)-11-oxoundecan-1-aminium bromide 151.98 and 152.48 (C-7 and C-9), 161.41 (C-2) and
ꢁ
+.
+
(
10): The product was obtained through column chro- 171.74 (C-11 ); HRMS, m/z: M and [M+1] calcu-
+
matography using silica gel in methanol–chloroform lated for C33
H
54NO : 528.4053 and 529.4086, found
4
(
6
1:9) to give 10 (22%) as a pale white solid. mp = 528.1099 and 529.1401, respectively.
◦
8–70 C; R = 0.45 (silica gel, methanol–chloroform
f
(
1:4)); UV (acetonitrile) λ : 270 and 315 nm; IR
max
(KBr) νmax: 3419.7, 3054.2, 2926.6, 2855.2, 1756.1, 2.2d N,N,N-Triethyl-11-(3-decyl-4-methyl-2-oxo-2H-
1
1
9
717.3, 1645.3, 1568.1, 1509.5, 1456.2, 1394.1, chromen-7-yloxy)-11-oxoundecan-1-aminium bromide
299.2, 1248.9, 1184.5, 1141.3, 1055.2, 1007.9, 937.9, (12): The product was obtained through column chro-
−1
1
13.5, 798.3, 725.2, 688.1 cm ; H NMR (300 MHz, matography using silica gel in methanol–chloroform
ꢁꢁ
◦
CDCl ; Me Si): δ 1.10 (t, 3H, J = 7.5 hz, H-2 ), (1:9) to give 12 (30%) as a brown solid. mp = 64–65 C;
3
4
ꢁ
ꢁ
1
1
.19-1.50 (m, 21H, -N(CH CH ) and H-3 - H-8 ),
R
f
= 0.6 (silica gel, methanol–chloroform (1:4));
max
2
3
3
ꢁ
ꢁ
.69 – 1.72 (m, 4H, H-2 and H-9 ), 2.46 (s, 3H, C-4 UV (acetonitrile) λ : 275 and 310 nm; IR (KBr)
ꢁ
CH ), 2.58 (t, 2H, J = 7.1 hz, H-10 ), 2.77 (q, 2H, νmax: 3429.1, 3075.6, 2927.1, 2854.9, 2745.1, 2678.2,
3
ꢁꢁ
ꢁ
J = 6.9 hz, H-1 ), 3.22 (t, 2H, J = 6.6 hz, H-1 ), 3.45– 2487.4, 1758.8, 1717.9, 1617.4, 1464.8, 1383.2,
−
1
3
.58 (m, 6H, -N(CH CH ) ), 6.93 (dd, 1H, J = 2.1 1270.1, 1155.6, 1032.9, 927.7, 797.1, 463.4 cm ;
2
3 3
1
and 8.7 hz, H-6), 7.26 (d, 1H, J = 2.1 hz, H-8), 7.68
H NMR (300 MHz, CDCl ; Me Si): δ 0.88 (t, 3H,
3 4
13
ꢁꢁ
(
d, 1H, J = 9.0 hz, H-5); C NMR (75.5 MHz, CDCl ; J = 7.1 hz, H-10 ), 1.28-1.61 (m, 37H, -N(CH CH ) ,
3
2
3 3
ꢁꢁ
ꢁ
ꢁ
ꢁꢁ
ꢁꢁ
ꢁ
Me Si): δ 8.07 (-N(CH CH ) ), 13.41 (C-2 ), 15.00 H-3 - H-8 and H-2 - H-9 ), 2.30 – 2.34 (m, 4H, H-2
4
2
3 3
ꢁꢁ
ꢁ
(
C-4 CH ), 20.21 (C-1 ), 21.98, 22.65, 24.71, 25.28, and H-9 ), 2.41 (s, 3H, C-4 CH ), 2.59 - 2.64 (m, 4H,
3
3
ꢁ
ꢁ
ꢁꢁ
ꢁ
ꢁ
2
6.37, 26.91, 28.97, 31.59, 34.29 (C-2 - C-10 ), 53.50 H-1 and H-10 ), 3.27 (t, 2H, J = 6.9 hz, H-1 ), 3.50
ꢁ
(
-N(CH CH ) ), 57.54 (C-1 ), 108.52 (C-8), 116.37 (q, 6H, J = 6.6 hz, -N(CH CH ) ), 7.05–7.08 (m,
2
3
3
2
3 3
and 118.93 (C-6 and C-10), 125.51 (C-3), 132.46 1H, H-6), 7.29 (s, 1H, H-8), 7.63 (d, 1H, J = 8.5 hz,
13
(C-5), 142.51 (C-4), 151.28 and 152.22 (C-7 and C-9), H-5); C NMR (75.5 MHz, CDCl ; Me Si): δ 8.06
3 4
ꢁ
+.
ꢁꢁ
1
63.31 (C-2) and 172.17 (C-11 ); HRMS, m/z: M
(-N(CH CH ) ), 14.10 (C-10 ), 14.97 (C-4 CH ),
2 3 3 3
+
+
and [M-H] calculated for C H NO : 472.3421 and 22.05, 22.65, 24.71, 26.92, 25.89, 26.45, 27.69, 28.61,
29
46
4
471.3460, found 472.4185 and 471.4941 respectively.
28.73, 29.11, 29.20, 29.34, 29.28, 29.42, 29.58, 31.86,
ꢁ
ꢁ
ꢁꢁ
ꢁꢁ
3
4.06, 34.37 (C-2 - C-10 and C-1 - C-9 ), 53.52
ꢁ
(
-N(CH CH ) ), 57.52 (C-1 ), 109.91 (C-8), 117.91
2
3 3
2.2c N,N,N-Triethyl-11-(3-hexyl-4-methyl-2-oxo-2H-
and 118.53 (C-6 and C-10), 125.30 (C-3), 126.40
chromen-7-yloxy)-11-oxoundecan-1-aminium bromide (C-5), 145.44 (C-4), 152.05 and 152.54 (C-7 and C-9),
ꢁ
+.
(
11): The product was obtained through column chro- 161.49 (C-2) and 171.80 (C-11 ); HRMS, m/z: M
+
matography using silica gel in methanol–chloroform calculated for C37
H
62NO : 584.4700, found 584.4585.
4
(
5
1:9) to give 11 (24%) as a pale white solid. mp =
◦
1–52 C; R = 0.54 (silica gel, methanol–chloroform
f
(
(
1:4)); UV (acetonitrile) λmax: 274 and 313 nm; IR 2.2e N,N,N-Triethyl-11-(4-methyl-2-oxo-1,2-dihy-
KBr) νmax: 3432.1, 3080.2, 2926.6, 2854.0, 2739.1, droquinolin-7-yloxy)-11-oxoundecan-1-aminium bromide
2
1
8
677.9, 2491.8, 1759.6, 1717.3, 1617.4, 1466.6, (13): The product was obtained through column chro-
383.5, 1270.2, 1250.3, 1156.3, 1034.9, 928.1, 883.8, matography using silica gel in methanol–chloroform
−1
1
27.4, 778.1, 736.0, 462.0 cm ; H NMR (300 MHz, (1:9) to give 13 (30%) as a pale white solid. mp =
ꢁ
ꢁ
◦
CDCl ; Me Si): δ 0.89 (t, 3H, J = 7.2 hz, H-6 ), 1.32- 156 C; R = 0.6 (silica gel, methanol–chloroform
3
4
f
ꢁ
ꢁ
ꢁꢁ
1
.51 (m, 29H, -N(CH CH ) , H-3 - H-8 and H-2 - (1:4)); UV (acetonitrile) λ : 275 and 326 nm; IR
max
2
3
3
ꢁꢁ
ꢁ
ꢁ
H-5 ), 1.71–1.73 (m, 4H, H-2 and H-9 ), 2.41 (s, 3H, (KBr) νmax: 3422.2, 2980.4, 2923.9, 2849.0, 1768.5,
C-4 CH ), 2.62–2.66 (m, 4H, H-1 and H-10 ), 3.27 1655.1, 1602.8, 1560.3, 1458.6, 1395.6, 1253.1,
ꢁꢁ
ꢁ
3

440
Shilpi Gupta et al.
1
166.3, 1141.7, 1128.2, 1021.3, 888.5, 813.7, 724.5 phy using silica gel in methanol–chloroform (1:9)
−1
1
and 567.8 cm ; H NMR (DMSO-d , 300 MHz; to give 15 (65%) as a white crystalline solid. mp =
6
◦
Me Si): δ 1.14 (brs, 9H, -N(CH CH ) ), 1.29 (brs, 81–82 C; R = 0.4 (silica gel, methanol–chloroform
4
2
3
3
f
ꢁ
ꢁ
ꢁ
1
9
2H, H-3 - H-8 ), 1.55-1.63 (m, 4H, H-2 and H- (1:4)); UV (acetonitrile) λ : 292 and 301 nm; IR
max
ꢁ
), 2.40 (s, 3H, C-4 CH ), 2.60 (t, 2H, J = 6.6 hz, (KBr) νmax: 2924.3, 2854.7, 1762.0, 1638.5, 1437.1,
3
ꢁ
ꢁ
−1
1
H-10 ), 3.08 (t, 2H, J = 6.9 hz, H-1 ), 3.20 (q, 6H, 1396.1, 1254.4, 1140.2, 1004.0, 832.3 cm ; H NMR
J = 6.6 hz, -N(CH CH ) ), 6.37 (s, 1H, H-3), 6.95 (d, (300 MHz, CDCl ; Me Si): δ 1.28–1.39 (m, 21H,
2
3
3
3
4
ꢁ
ꢁ
1
H, J = 8.7 hz, H-6), 7.03 (s, 1H, H-8), 7.73 (d, 1H, -N(CH CH ) and H-3 - H-8 ), 1.60-1.78 (m, 4H,
2
3
3
13
ꢁ
ꢁ
ꢁ
J = 8.1 hz, H-5) and 11.66 (brs, 1H, NH); C NMR H-2 and H-9 ), 2.61 (t, 2H, J = 7.3 hz, H-10 ), 3.28
ꢁ
(
1
2
DMSO-d , 75.5 MHz; Me Si): δ 7.14 (-N(CH CH ) ), (t, 2H, J = 7.2 hz, H-1 ), 3.50 (q, 6H, J = 6.8 hz,
6
4
2
3 3
8.52 (C-4 CH ), 20.90, 24.21, 25.76, 28.31, 28.46, -N(CH CH ) ), 6.33 (d, 1H, J = 6.0 hz, H-3), 7.15
3
2
3 3
ꢁ ꢁ
8.60, 28.69, 28.72, 33.40 (C-2 - C-10 ), 51.90 (- (d, 1H, J = 8.7 hz, H-6), 7.31 (s, 1H, H-8), 7.88
ꢁ
N(CH CH ) ), 55.94 (C-1 ), 107.87 (C-6), 115.83 and (d, 1H, J = 6.0 hz, H-2), 8.20 (d, 1H, J = 8.7 hz,
2
3 3
13
1
1
17.46 (C-8 and C-10), 120.31 (C-3), 126.15 (C-5), H-5); C NMR (75.5 MHz, CDCl ; Me Si): δ 7.96
3 4
39.52 (C-9), 147.69 (C-4), 151.66 (C-7), 161.73 (C-2) (-N(CH CH ) ), 21.95, 24.54, 24.73, 26.31, 28.79,
2
3 3
ꢁ
+.
ꢁ
ꢁ
and 171.60 (C-11 ); HRMS, m/z: M calculated for 28.97, 29.15, 33.91, 34.16 (C-2 - C-10 ), 53.35
C H O N : 443.3268, found 443.3315.
ꢁ
(-N(CH CH ) ), 57.35 (C-1 ), 111.03 (C-8), 112.93
27
43
3
2
2
3 3
(C-3), 119.50 (C-6), 122.41 and 126.97 (C-5 and
C-10), 152.11 (C-2), 154.30 and 156.89 (C-7 and
ꢁ
C-9), 171.37 (C-11 ) and 176.77 (C-4); HRMS, m/z:
+
+
2
.2f N,N,N-Triethyl-11-(3-ethyl-4-methyl-2-oxo-1,2- [M+H] calculated for C26
H
40NO : 431.2991, found
4
dihydroquinolin-7-yloxy)-11-oxoundecan-1-aminium 431.4231.
bromide (14): The product was obtained through
column chromatography using silica gel in methanol–
chloroform (1:9) to give 14 (33%) as a pale white solid.
◦
mp = 80 C; R = 0.6 (silica gel, methanol–chloroform
f
(
1:4)); UV (acetonitrile) λ : 270 and 328 nm; IR
max
2.2h N,N,N-triethyl-11-(3-hexyl-4-oxo-4H-chromen-
7-yloxy)-11-oxoundecan-1-aminium bromide (16):
(
KBr) νmax: 3379.8, 2929.3, 2855.6, 1757.9, 1642.2,
509.3, 1459.9, 1393.8, 1301.1, 1248.9, 1186.1,
1
1
7
3
1
The product was obtained through column chromatog-
raphy using silica gel in methanol–chloroform (1:9)
156.7, 1141.8, 1110.7, 1055.5, 1008.3, 974.8, 911.7,
−
1
1
◦
93.2, 757.3 and 687.7 cm ; H NMR (DMSO-d ,
6
to give 16 (27%) as a orange solid. mp = 64–65 C;
ꢁꢁ
00 MHz; Me Si): δ 0.99 (t, 3H, J = 7.5 hz, H-2 ),
4
R
= 0.52 (silica gel, methanol–chloroform (1:4));
max
f
ꢁ
.06 (brs, 9H, -N(CH CH ) ), 1.13 (brs, 12H, H-3 -
2
3
3
UV (acetonitrile) λ : 278 and 305 nm; IR (KBr)
ꢁ
ꢁ
ꢁ
H-8 ), 1.54–1.62 (m, 4H, H-2 and H-9 ), 2.34 (s, 3H,
C-4 CH ), 2.58 – 2.62 (m, 4H, H-1 and H-10 ), 3.07
νmax: 3425.1, 2926.4, 2855.1, 1763.1, 1646.5, 1616.9,
ꢁꢁ
ꢁ
−1
3
1440.7, 1396.1, 1228.2, 1167.9, 1112.5, 793.2, cm ;
ꢁ
1
(
-
t, 2H, J = 7.8 hz, H-1 ), 3.19 (q, 6H, J = 6.6 hz,
H NMR (300 MHz, CDCl ; Me Si): δ 0.87 (t, 3H,
3
4
ꢁꢁ
N(CH CH ) ), 6.91 (d, 1H, J = 2.1 and 8.7 hz, H-6),
2
3
3
J = 7.5 hz, H-6 ), 1.17-1.46 (m, 29H, -N(CH CH ) ,
2
3 3
ꢁ ꢁ ꢁꢁ ꢁꢁ ꢁ
6
1
.99 (s, 1H, H-8), 7.75 (d, 1H, J = 8.7 hz, H-5) and
H-3 - H-8 and H-2 - H-5 ), 1.55-1.62 (m, 2H, H-9 ),
13
ꢁ
1.67 (brs, 1H, NH); C NMR (DMSO-d , 75.5 MHz;
6
1.72-1.80 (m, 2H, H-2 ), 2.44 (t, 2H, J = 7.0 hz,
ꢁꢁ
ꢁ
ꢁꢁ
Me Si): δ 7.17 (-N(CH CH ) ), 13.17 (C-2 ), 14.53
(
2
(
4
2
3
3
H-10 ), 2.60 (t, 2H, J = 7.2 hz, H-1 ), 3.26 (t, 2H, J =
ꢁ
ꢁ
ꢁ
C-4 CH ), 19.51 (C-1 ), 20.92, 24.30, 25.78, 26.30,
3
7.2 hz, H-1 ), 3.49 (q, 6H, J = 6.0 hz, -N(CH CH ) ),
2
3 3
ꢁ
ꢁ
8.32, 28.45, 28.59, 28.72, 33.42 (C-2 - C-10 ), 51.95
-N(CH CH ) ), 56.00 (C-1 ), 107.44 (C-6), 115.62
7
.11 (dd, 1H, J = 2.1 and 8.7 hz, H-6), 7.22 (d, 1H,
ꢁ
2
3
3
J = 2.1 hz, H-8), 7.75 (s, 1H, H-2), 8.22 (d, 1H, J =
1
3
and 117.90 (C-8 and C-10), 125.91 (C-3), 132.06 (C-
), 137.84 (C-9), 141.26 (C-4), 150.75 (C-7), 161.45
8
.7 hz, H-5); C NMR (75.5 MHz, CDCl ; Me Si):
3
4
ꢁꢁ
5
δ 8.11 (-N(CH CH ) ), 14.06 (C-6 ), 22.09, 22.56,
2
3 3
ꢁ
+.
(C-2) and 171.60 (C-11 ); HRMS, m/z: M calculated
2
4.68, 26.91, 25.70, 26.43, 28.43, 28.92, 29.08, 29.15,
ꢁ
ꢁ
ꢁꢁ
for C H O N : 471.3581, found 471.3471.
29
47
3
2
29.26, 29.65, 31.59, 34.30 (C-2 - C-10 and C-1 -
C-5 ), 53.54 (-N(CH CH ) ), 57.54 (C-1 ), 110.84
ꢁꢁ
ꢁ
2
3 3
(C-8), 119.14 and 121.59 (C-6 and C-10), 124.90 (C-3),
1
27.17 (C-5), 152.11 (C-2), 154.30 and 156.89 (C-7
ꢁ
2
.2g N,N,N-Triethyl-11-(4-oxo-4H-1-benzopyran-7-
and C-9), 171.52 (C-11 ) and 177.16 (C-4); HRMS,
+.
+
+
4
yloxy)-11-oxoundecan-1-aminium bromide (15): The m/z: M and [M+1] calculated for C H NO :
32
52
product was obtained through column chromatogra- 514.7590, found 514.8330.

Chromenones and quinolinones as antimicrobial agents
441
2.3 General procedure for the synthesis of ammonium 2.3b N,N,N-Triethyl-6-(4-methyl-2-oxo-2H-chromen-
alkoxy derivative of chromen-2-one (20–23)
7-yloxy)hexan-1-aminium bromide (21): The product
was obtained through column chromatography using
To a flask containing 7-hydroxy-4-methyl coumarin
silica gel in methanol–chloroform (1:9) to give 21
◦
(
1.0 g, 5.68 mmol), in acetone (25 mL) and 2.0
equivalent of fused potassium carbonate was added
.2 equivalent of 1-bromo-3-chloropropane/1, 6-
(
R
68%) as a white crystalline solid. mp = 171–172 C;
= 0.62 (silica gel, methanol–chloroform (1:4));
f
1
UV (acetonitrile) λ : 318 nm; IR (KBr) ν : 3413.8,
max
max
dibromohexane/1,10-dibromodecane, the reaction
mixture was refluxed for 24 h. The mixture was then
filtered through celite and the solvent was evapo-
rated in vacuo. The solid residue so obtained was
purified by column chromatography to give corre-
sponding halo alkoxy derivatives of chromen-2-one
2
1
1
1
5
978.0, 2942.1, 2850.9, 1716.1, 1613.5, 1507.9,
458.5, 1417.8, 1388.2, 1369.7, 1347.5, 1278.9,
251.7, 1199.9, 1151.5, 1135.7, 1072.5, 1032.4,
002.7, 982.0, 890.0, 878.0, 840.9, 802.5, 711.2, 589.9,
−1
1
28.9 cm ; H NMR (400 MHz, CDCl ; Me Si):
3
4
δ 1.32 (t, 9H, J = 7.2 hz, -N(CH CH ) ), 1.40-1.55
2
3 3
32
ꢁ
ꢁ
ꢁ
(
(
17–19) as white crystalline solid. A mixture of 17
(m, 4H, H-2 and H-3 ), 1.67-1.79 (m, 4H, H-4 and
1.0 g, 3.96 mmol)/1833 (1.0 g, 2.95 mmol)/19 (1.0 g,
ꢁ
H-5 ), 2.32 (s, 3H, C-4 CH ), 3.29 (t, 2H, J = 6.8 hz,
3
ꢁ
2
.53 mmol), toluene (8 mL), acetone (4 mL), and DMF
H-1 ), 3.44 (q, 6H, J = 6.0 hz, -N(CH CH ) ), 3.94
2
3 3
◦
ꢁ
(0.5 mL) was heated at 50 C. Then trialkyl amine
(
t, 2H, J = 6.6 hz, H-6 ), 6.02 (s, 1H, H-3), 6.67 (d,
(
NEt /NBu ) (2 equivalent) was added to the mixture
3
3
1H, J = 2.4 hz, H-8), 6.78 (dd, 1H, J = 2.4 and
13
in 1 h. The mixture was refluxed for a period of 48 h,
the solvent was evaporated under reduced pressure and
the crude product was purified by flash column chro-
matography to give 20 (400 mg, 32%) or 21 (725 mg,
8
.8 hz, H-6), 7.42 (d, 1H, J = 7.4 hz, H-5); C NMR
(100.6 MHz, CDCl ; Me Si): δ 7.95 (-N(CH CH ) ),
3
4
2
3
3
ꢁ
1
8.55 (C-4 CH ), 21.91, 25.42, 26.01, 28.55 (C-2 -
3
ꢁ
ꢁ
C-5 ), 53.34 (-N(CH CH ) ), 57.31 (C-1 ), 67.98
2
3 3
ꢁ
6
8%) or 22 (860 mg, 66%) or 23 (686 mg, 65%).
(
C-6 ), 101.22 (C-8), 111.46, 112.32 and 113.27 (C-3,
C-6 and C-10), 125.53 (C-5), 152.73 and 154.93 (C-4
+
and C-9), 161.17 (C-7), 161.87 (C-2); HRMS, m/z: M
+
calculated for C H NO : 360.2539, found 360.2537.
22
34
3
2.3a N,N,N-Triethyl-3-(4-methyl-2-oxo-2H-chromen-
7
-yloxy)propan-1-aminium chloride (20): The prod-
2
7
.3c N,N,N-Tributyl-6-(4-methyl-2-oxo-2H-chromen-
-yloxy)hexan-1-aminium bromide (22): The product
uct was obtained through column chromatography
using silica gel in methanol–chloroform (1:9) to give
was obtained through column chromatography using
silica gel in methanol–chloroform (1:9) to give 22
2
2
0 (32%) as a light green colour solid. mp = 221–
◦
23 C; R = 0.27 (silica gel, methanol–chloroform
f
(
66%) as a pale yellow liquid. Rf = 0.76 (silica
(1:4)); UV (acetonitrile) λ : 316 nm; IR (KBr)
max
gel, methanol–chloroform (1:4)); UV (acetonitrile)
λmax: 319 nm; IR (KBr) νmax: 3397.9, 2958.7, 2873.0,
νmax: 3447.9, 3022.8, 3003.7, 2979.4, 2943.2, 2878.5,
1
1
1
7
710.9, 1625.1, 1556.7, 1494.3, 1470.9, 1438.9,
427.6, 1393.6, 1382.9, 1373.2, 1326.2, 1297.1,
197.5, 1154.1, 1076.5, 1008.1, 991.1, 890.9, 857.3,
1715.2, 1613.6, 1557.2, 1471.1, 1388.6, 1294.3,
−1
1
1202.2, 1145.6, 1071.2, 1015.9, 847.9 cm ;
H
−1
1
NMR (400 MHz, CDCl ; Me Si): δ 0.95 (t, 9H,
J
98.5, 750.3, 711.9, 575.7, 544.2, 452.2 cm ; H
3
4
= 7.2 hz, -N(CH ) CH ) ), 1.40-1.41 (m, 6H,
ꢁ
2 2 2 3 3
NMR (400 MHz, DMSO-d ; Me Si): δ 1.20 (t, 9H,
2
4
3 3
6
4
ꢁ
-N(CH ) CH CH ) ), 1.45-1.56 (m, 4H, H-2 and
J = 7.0 hz, -N(CH CH ) ), 2.08-2.15 (m, 2H, H-2 ),
2
3 3
ꢁ
H-3 ), 1.59–1.68 (m, 6H, -N(CH CH CH CH ) ),
1
2
.40 (s, 3H, C-4 CH ), 3.29–3.31 (m, 8H, N(CH CH )
3 3
2
2
2
3 3
3
2
ꢁ
ꢁ
ꢁ
ꢁ
.73–1.80 (m, 4H, H-4 and H-5 ), 2.34 (s, 3H, C-4
and H − 1 ), 4.19 (t, 2H, J = 6.6 hz, H-3 ), 6.23
CH ), 3.32 (t, 8H, J = 7.0 hz, -N(CH CH CH CH ) ,
(
s, 1H, H-3), 6.97 (dd, 1H, J = 2.4 and 8.8 hz,
3
ꢁ
2
ꢁ
2
2
3 3
H-1 ), 3.97 (t, 2H, J = 6.6 hz, H-6 ), 6.05 (s, 1H,
H-3), 6.70 (d, 1H, J = 2.4 hz, H-8), 6.81 (dd, 1H,
J = 2.4 and 8.8 hz, H-6), 7.45 (d, 1H, J = 8.8 hz,
H-6), 7.00 (d, 1H, J = 2.4 hz, H-8), 7.71 (d, 1H,
13
J = 8.8 hz, H-5); C NMR (100.6 MHz, DMSO-d ;
6
Me Si): δ 7.08 (-N(CH CH ) ), 18.07 (C-4 CH ),
4
2
3
3
3
ꢁ
13
ꢁ
H-5);
C
NMR (100.6 MHz, CDCl ; Me Si):
3 4
2
6
1.14 (C-2 ), 52.00 (-N(CH CH ) ), 53.06 (C-1 ),
5.13 (C-3 ), 101.21 (C-8), 111.24, 112.36 and 113.30
2
3 3
ꢁ
δ 13.59 (-N((CH ) CH ) ), 18.58 (C-4 CH ), 19.65
2
3
3
3
3
(-N((CH ) CH CH ) ), 22.23 (-N(CH CH CH CH ) ),
(
(
C-3, C-6 and C-10), 126.50 (C-5), 153.34 and 154.60
C-4 and C-9), 160.01 (C-7), 161.05 (C-2); HRMS,
2
2
2
3
3
2
2
2
3 3
ꢁ
ꢁ
2
4.07, 25.52, 26.06, 28.63 (C-2 - C-5 ), 58.95
+
3
ꢁ
ꢁ
+
(
-N(CH (CH ) CH ) ), 59.13 (C-6 ), 68.08 (C-1 ),
m/z: M calculated for C H NO : 318.2069, found
2 2 2 3 3
19
28
1
01.35 (C-8), 111.54, 112.33 and 113.37 (C-3, C-6
318.2078.

442
Shilpi Gupta et al.
and C-10), 125.57 (C-5), 152.74 and 155.00 (C-4 and Sigma Chemical Company, USA. Discs for testing
+
C-9), 161.25 (C-7), 161.93 (C-2); HRMS, m/z: M
were prepared from Whatman filter paper No. 1 from
Whatman company (USA).
+
calculated for C H NO : 444.3478, found 444.3492.
28
46
3
2
.3d N,N,N-Triethyl-10-(4-methyl-2-oxo-2H-chromen-
2
.4c Antifungal activity assays: The Aspergillus
7-yloxy)decan-1-aminium bromide (23): The product
species and C. albicans were used as model pathogens
to study in vitro antifungal activity of compounds. The
anti-Aspergillus activity was evaluated by disc diffusion
assay (DDA) and microbroth dilution assays (MDA).14
For disc diffusion, various concentrations of the com-
pounds prepared by serial dilution were impregnated
on sterile Whatman paper 1 discs 5 mm in diameter, on
the surface of Sabouraud dextrose agar already inocu-
lated with Aspergillus spores and zone of inhibition was
was obtained through column chromatography using
silica gel in methanol–chloroform (1:9) to give 23
◦
(
65%) as a white crystalline solid. mp = 102–104 C;
R = 0.43 (silica gel, methanol–chloroform (1:4)); UV
f
(acetonitrile) λ : 317 nm; IR (KBr) ν : 3402.89,
max max
3072.1, 2979.1, 2924.6, 2854.1, 1717.8, 1612.0,
1553.2, 1472.4, 1387.5, 1370.7, 1343.9, 1296.2,
1278.5, 1263.3, 1202.9, 1149.7, 1070.9, 1017.3, 978.4,
−1
◦
8
65.1, 800.6, 725.6, 642.0, 566.2, 525.9, 461.2 cm ;
recorded after 48 h of incubation at 37 C. In microbroth
assay, serial dilutions of the compounds in Sabouraud
dextrose broth were prepared in microtitre plates. Each
1
H NMR (400 MHz, CDCl ; Me Si): δ 1.26-1.40
3
4
ꢁ
ꢁ
(m, 21H, H-2 - H-7 , -N(CH CH ) ), 1.63–1.66 (m,
2 3 3
ꢁ
ꢁ
6
2
H, H-8 ), 1.73–1.75 (m, 2H, H-9 ), 2.35 (s, 3H, C-4
well was inoculated with 1 × 10 of Aspergillus spores
ꢁ
CH ), 3.22 (t, 2H, J = 7.0 hz, H-1 ), 3.44 (q, 6H,
3
and observed after 48 h. The minimum concentration
of the compound which visibly inhibited growth of
the fungus was considered as the minimum inhibitory
concentration.
ꢁ
J = 6.6 hz, -N(CH CH ) ), 3.95 (t, 2H, H-10 ), 6.05
2
3 3
(
1
s, 1H, H-3), 6.72 (d, 1H, J = 2.4 hz, H-8), 6.80 (dd,
H, J = 2.4 and 8.8 hz, H-6), 7.45 (d, 1H, J = 8.8 hz,
13
H-5); C NMR (100.6 MHz, CDCl ; Me Si): δ 7.94
3
4
The activity against C. albicans was determined by
34
(
-N(CH CH ) ), 18.59 (C-4 CH ), 21.93, 25.71, 26.33,
2 3 3 3
basic method of Iijima et al. suitably modified by
Chhillar et al. The method used was per cent growth
inhibition assay. Overnight grown culture of C. albi-
cans at 37 C in Saboraud medium was used. Serial dilu-
tions of the compounds were prepared and incubated at
ꢁ
ꢁ
18
28.73, 28.99, 29.02, 29.14, 30.02 (C-2 - C-9 ), 53.36
ꢁ
ꢁ
(
(
-N(CH CH ) ), 57.36 (C-1 ), 68.43 (C-10 ), 101.23
C-8), 111.48, 112.48 and 113.24 (C-3, C-6 and C-10),
2 3 3
◦
1
25.48 (C-5), 152.75 and 155.04 (C-4 and C-9), 161.30
+
◦
(C-7), 162.08 (C-2); HRMS, m/z: M calculated for
37 C for 24 h and OD was measured to determine cell
650
+
C H NO : 416.3165, found 416.3184.
26
42
3
growth. The per cent growth inhibition was calculated
using following formula:
2
.4 Biological activity screening
PGI = 100 − (OD in drug treated well/OD
in control well) × 100.
2.4a Pathogens: Pathogenic strains of Aspergillus,
Candida albicans and bacteria were used. Aspergillus
fumigatus strain (190/96) was a clinical isolate procured
from VP Chest Institute, Delhi (India). Aspergillus
niger (ITCC 4), Aspergillus flavus (ITCC5192) and
Candida albicans (ITCC 4718) were obtained from
Indian Agricultural Research Institute (IARI), New
Delhi, India. Various strains of bacteria namely,
Pseudomonas aeruginosa (MTCCB 741), Staphylo-
coccus epidermidis (MTCCB 435), Salmonella typhi
2
.4d Antibacterial activity: The activity of com-
pounds against pathogenic bacteria was assessed by
MDA and DDA as described by Riffel et al.35 suit-
ably modified in our laboratory by Chaudhary et al.36
Briefly, the inoculum was prepared by growing bacteria
◦
at 37 C for 12–14 h. The OD of overnight culture was
adjusted to 0.09 to 0.10 (equivalent to a 0.5 McFarland
standard) at a transmission wavelength of 600 nm. The
test was performed in 96 well-flat bottom culture plates
(MTCCB 733), Staphylococcus aureus (MTCCB 737)
were procured from Institute of Microbial Technology,
Chandigarh (India).
(Nunc, Nunclon). Pathogen in microwells treated with
various concentrations of the compounds was incubated
at 37 C for 12 h and absorbance at 600 nm was mea-
◦
2.4b Materials: Sabouraud’s dextrose agar and sured to determine the bacterial growth. Kanamycin
Sabouraud’s dextrose broth were purchased from Hi was used in the assay as the standard control drugs.
Media, Mumbai, India. Amphotericin B, DMSO, Tri- An additional control well without any sample but with
ton X-100, Kanamycin and MTT were procured from an equivalent amount of solvent was used as reference

Chromenones and quinolinones as antimicrobial agents
443
in the assay. For disc diffusion, Whatman filter paper 3. Results and discussion
No. 1 discs (5.0 mm in diameter) were impregnated
with dilutions of compounds and incubated at 37 C and
◦
3.1 Chemistry
examined for zone of inhibition.
The synthesis of ammonium acyloxy derivatives of
chromen-2-ones, quinolin-2-ones and chromen-4-ones
is outlined in scheme 1. They were synthesized in two
steps i.e., first the hydroxy chromenone/quinolinones
were treated with bromo acyl chloride in presence of
triethylamine. The bromo acyl chloride in turn was pre-
pared from corresponding bromo acid by treating with
oxalyl chloride. In the next step the bromo acyloxy
chromenones/quinolinones were treated with excess of
triethylamine in anhydrous acetonitrile under refluxing
conditions (scheme 1).
2.4e Haemolytic activity: The in vitro cell cytotox-
icity of the active compounds was evaluated by using
slightly modified haemolytic assay of Latoud et al.
37
Human erythrocytes, collected from apparently healthy
individual were washed and suspended in phosphate
buffer saline (PBS). Erythrocyte suspension (2% v/v)
in PBS was incubated with different concentrations of
◦
compound at 37 C for 1 h. After incubation, cells were
The synthesis of ammonium alkoxy derivatives
pelleted at 3000 g for 10 min. The supernatant was col-
lected and OD450 was determined using a spectropho-
tometer (Spectra Max 384 plus, Molecular Devices).
PBS was used as negative control and Triton X-100
was used for completely lysing the cells. The per cent
haemolysis was calculated using the following formula:
1
7–19 is outlined in scheme 2. They were synthe-
sized by the reaction of terminal dihalo alkanes with
-hydroxy-4-methyl-2H-chromen-2-one in the pres-
7
ence of potassium carbonate and acetone under reflux
conditions. The corresponding ammonium compounds
(
20–23, scheme 2) were then prepared by treating
them with triethyl/tributylamine in a mixture of toluene,
acetone and DMF (in a ratio of 4:4:2) under reflux
conditions.
Percent haemolysis
=
100 − (OD of test/OD of control) × 100.
A total of 12 ammonium chromenones/quinolinones
and their 11 precursors i.e., the corresponding halo
analogs were synthesized, of these compounds 1–16
and 19–23 are novel. All the compounds were fully
characterized on the basis of their physical and spectral
The percent haemolysis was plotted against concentra-
tion of compound to determine the dose cytotoxic to
erythrocytes.
32
33
data. The structures of known compounds 17 and 18
were confirmed by comparison of their melting points
and spectral data with those reported in the literature.
2
.4f MTT assay: The method described by
38
Mosmann (1983) was followed. Cells were main-
tained in RPMI-1640 medium supplemented with
glutamine and fetal calf serum (10% v/v) and were
harvested at the log phase of confluency from the flask.
The assay was performed in 96-well flat bottom tissue
3.2 Antimicrobial activity
4
culture plates (Nunc, Nunclon). The cells (2 × 10 )
in 100.0 μl of media were seeded into each well for The synthesized ammonium derivatives and their halo
overnight. Dilutions of compounds were added and precursors (1–23) were then screened for antimicrobial
◦
incubated at 37 C in atmosphere of 5% (v/v) CO for activity against pathogenic fungi (Aspergillus fumi-
2
24 h. In negative control, equivalent amount of the sol- gatus, Aspergillus flavus, Aspergillus niger, Candida
vent was used in each well. After removing the medium albicans) and bacteria (Staphylococcus aureus, Staphy-
the cells were treated with 10.0 μl of 5 mg/ml (stock) of lococcus epidermidis, Pseudomonas aeruginosa,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Salmonella typhi). The antimicrobial activity screening
bromide (MTT). After adding the dye to the wells, the of all the compounds was performed according to the
◦
14,18,34–36
plate was kept at 37 C in 5% CO for 4 h. The dye established methods described in the literature.
2
was removed from the wells and the cells were lysed
Most of the ammonium compounds screened were
by adding 100.0 μl of DMSO. The wells were read found to possess a broad spectrum of activity
at 540 nm using plate reader (SpectraMax 384 plus, against the pathogens while the halogenated precursors
Molecular Devices). The per cent toxicity with respect (1–8, 17–19) of the ammonium compounds were not
to the negative control was calculated and plotted active against the bacterial pathogens within the range
against concentrations of the compound.
of the concentrations tested. However, six compounds

444
Shilpi Gupta et al.
i
Br
COOH
Br
COCl
8
8
HO
X
O
R
1
X
11'
2'
8
5
1'
10'
8
1
'
O
9
X
O
R
11'
O
O
9
O
R
Br
Et
3
N
Br
7
7
2
iii
2
8
-
8
O
6
3
6
3
10
10
ii
4
5
4
1
-6
9-14
1
/9 X = O; R = H
1
''
R = H / C
2
H
5
/ C
6
H
13
/
2''
C
10
H
21
2/10 X = O; R =
3/11 X = O; R =
1
''
3''
5''
X = O / NH
2''
4''
6''
1
''
3''
5''
7''
9''
4
5
6
/12 X = O; R =
2
''
4''
6''
8''
10''
/13 X = NH; R = H
/14 X = NH; R =
1
''
2
''
HO
O
O
1
O
1
11'
2'
8
5
1'
10'
8
1
'
O
9
11'
O
9
O
C
6
H
13
2
2
Br
3
Et N
7
7
8
iii
Br-
8
O
O
6
6
1
0
3
R
10
3
R
ii
4
5
4
7
-8
15-16
O
O
7
/15 R = H
/16 R =
1
''
3''
5''
8
6
''
2
''
4''
Scheme 1. Synthesis of N,N,N-triethyl-11-(3-alkyl-4-methyl-2-oxo-2H-chromen/
1
,2-dihydro quinolin/4-oxo-4H-chromen-7-yloxy)-11-oxoundecan-1-aminium bro-
◦
mide. (i) (COCl)2, DCM; (ii) NEt3 (1.0 equivalent), CH3CN, 37 C; (iii) NEt3 (5.0
equivalent), CH3CN, 60 C.
◦
i.e., five haloacyloxy chromenones (2–4, 7–8) and two and 1.95 μg/disc in DDA (table 1). The haloacyloxy
haloalkoxy chromenones (15, 16) were found to be derivatives of quinolin-2-ones (5, 6) were found to be
active against most of the fungal strains. The haloacy- completely inactive against all the fungal pathogens.
loxy chromenones (2–4, 7–8) were found to have mod-
An in-depth antimicrobial study of the ammonium
erate activity against all the species of fungi tested, acyloxy derivatives of chromen-2-ones (9–12) showed
while the haloalkoxy derivatives (15, 16) were found to interesting results. These compounds showed a promis-
be active against selective species only, however these ing broad spectrum of activity against most of the
will have more potent activities. This fact was sub- pathogens. Amongst them the compound 11 having
stantiated by the observations that the acyloxy deriva- a C alkyl chain at the C-3 position of pyran ring
6
tives (2–4, 7–8) were active against all the four fungal was strikingly the most active compound, it showed
strains, with compound 3 having the maximum activity remarkably good activity against Aspergillus species
against A. niger at a MIC of 31.25 μg/ml in MDA and i.e., exhibited a MIC of 12.50 μg/ml against A. fumi-
7.81 μg/disc in DDA. While, among the alkoxy deriva- gatus, 7.81 μg/ml against A. niger and 31.25 μg/ml
tives, the compound 18 had a narrow range of activ- against A. flavus in MDA while in DDA the MICs were
ity against only two pathogenic fungi i.e., A. niger and 1.95 μg/disc, 1.95 μg/disc and 15.62 μg/disc, respec-
A. flavus, it was found to be most effective against tively. Its activity against C. albicans was observed
A. niger at a concentration of 15.62 μg/ml in MDA to be 7.81 μg/ml. Also, the compound 11 was found
X
X
m
HO
O
O
X
1'
m
O
O
O
R3N
X
n'
O
O
O
X = Cl / Br
n'
ii
1
'
m
i
17 X = Cl; m = 1; n' = 3'
18 X = Br; m = 4; n' = 6'
19 X = Br; m = 8; n' = 10'
20 R = Et; m = 1; n' = 3'
21 R = Et; m = 4; n' = 6'
22 R = Bu; m = 4; n' = 6'
2
3 R = Et; m = 8; n' = 10'
Scheme 2. Synthesis of N,N,N-triethyl/butyl-3-(4-methyl-2-oxo-2H-chromen-7-
yloxy)alkan-1-aminium halide. (i) K2CO3, Acetone; (ii) NEt3 (5.0 equivalent),
◦
CH3CN, 60 C.

Chromenones and quinolinones as antimicrobial agents
445

446
Shilpi Gupta et al.
to possess significant antibacterial activity. It exhibited six carbon units. The target site for the antimicrobial
a MIC of 15.62 μg/ml in MDA and 7.81 μg/disc in activity is the cell membrane where the antimicrobial
DDA against P. aeruginosa and 7.81 μg/ml in MDA and compound disrupts the ordered structure of the phos-
1.95 μg/disc in DDA against S. typhi, S. epidermidis pholipid bilayer causing leakage of the bacterial mem-
and S. aureus (table 1). The compound 9 that lacks a brane. The hydrophobic interactions due to alkyl groups
substitution at the C-3 position was also observed to at C-3 position such as hexyl might be facilitating this
have a reasonably good antimicrobial activity against process and as a result loss of outer membrane leaves
all the pathogenic species in comparison to compounds the bacteria vulnerable against all external threats. Fur-
1
0 and 12 that have a C and C alkyl chain at the ther exposure to the ammonium compounds disrupts the
2 10
C-3 position of pyran ring respectively (table 1). The inner membrane and lyses the microbe. A detailed study
above results suggested that hexyl is the optimum chain of mechanism is under investigation.
length at C-3 position in ammonium derivatives of
chromen-2-ones to exhibit antimicrobial activity.
Among the ammonium alkoxy derivatives 20–23,
compound 20 having a triethyl ammonium propoxy
Encouraged by these results, the chromen-4-one ether chain at the C-7 position of chromenone skele-
derivative 15–16 were synthesized and screened for the ton was found to be inactive against all the pathogens.
antimicrobial activity. The compound 16 having a hexyl Although on elongation of the alkyl chain to six car-
chain at the C-3 position of the chromen-4-one nucleus bon unit, i.e., in compounds 21 and 22, an improvement
was found to be more active than its unsubstituted coun- in the antimicrobial activity was observed. The small
terpart 15 and showed even better activity than the com- size of alkyl chain in these compounds (20–22) may be
pound 11 (chromen-2-one) against all the pathogenic responsible for their lower activity and thus supports the
strains. It exhibited an MIC of 1.95 μg/ml against A. general hypothesis that the ammonium compounds pos-
fumigatus, 3.90 μg/ml against A. niger and 15.62 μg/ml sessing an aliphatic alkyl chain of less than eight car-
39,40
against A. flavus in MDA, while in DDA the respec- bons are not much active.
tive MIC values were 0.47 μg/disc, 0.95 μg/disc and the increase in the chain length of the polar head group
.81 μg/disc. It was found to be active against C. albi- in the ammonium moiety from two carbon atoms (tri-
We have also noticed that
7
cans at 3.90 μg/ml. It also exhibited good antibacterial ethyl) in compound 21 to four carbon atoms (tributyl)
activities against P. aeruginosa and S. epidermidis at in compound 22 did not exhibit any noticeable change
a MIC of 7.81 μg/ml and 3.90 μg/ml in MDA, while in the antimicrobial activity. A significant enhancement
3.90 μg/disc and 1.95 μg/disc, respectively in DDA in the antimicrobial activity was, however observed for
(table 1). These results further substantiated the fact compound 23 having a C10 alkyl chain. It exhibited
that the antimicrobial activity of chromenones may be a MIC of 25.00 μg/ml against A. fumigatus in MDA.
enhanced by having an alkyl chain of approximately Slightly higher concentration (31.25 μg/ml) of 23 was
1
1
20
00
9
1
1
2
1
6
3
8
6
4
2
0
0
0
0
0
AmpB
0
1
2
3
4
Log [Conc]
Figure 1. Cytotoxicity of active compounds against human erythrocytes by hemolytic assay.

Chromenones and quinolinones as antimicrobial agents
447
1
1
20
00
8
6
4
2
0
0
0
0
0
A549
U87
Concentration [µg/ml]
Figure 2. Cytotoxicity evaluation of compound 9 on viability of mammalian cell lines by MTT assay.
required to completely inhibit the growth of A. niger the cells was plotted with log concentrations of the com-
and A. flavus. The MICs in DDA were 1.95 μg/disc pound. The MTT assay was performed on two mam-
against all the species of Aspergilli, and 7.81 μg/ml malian cell lines, human lung epithelial cells (A549)
against C. albicans. It also showed promising and human glioma cells (U87) and the percent viability
antibacterial activity against two bacterial species, was determined.
i.e., P. aeruginosa and S. epidermidis at a MIC of
The compounds 11 and 16 were found to be
5.62 μg/ml, 3.90 μg/ml in MDA and 7.81 μg/disc and quite toxic to the erythrocytes, a concentration of
.95 μg/disc in DDA, respectively (table 1). 31.25 μg/ml of each lysed all the cells. Compound
The ammonium acyloxy derivatives of quinolin-2- 23 was comparatively less toxic than 11 and 16
1
0
ones (13, 14) were also synthesized and their antimicro- (figure 1). However, amongst all, the compound 9
bial activity was evaluated. Of these, the compound 13 was least toxic, it exhibited only marginal toxicity
was active against only A. fumigatus and C. albicans, (∼15%) at 1000 μg/ml and was non-toxic at 500 μg/ml
the substitution at the C-3 position of the quinolin-2-one (figure 1), thus suggesting it to be safer than the stan-
moiety (14) increased the antimicrobial spectrum.
The antimicrobial potency of these compounds was of erythrocytes at a concentration of 31.25 μg/ml.
compared with the standard antifungal drug, ampho- Given the low haemolytic activity of the compound
dard drug amphotericin B, which caused complete lysis
tericin B and the antibiotic drug kanamycin as con- 9, it was of interest to determine its broader cytotoxicity
trols (table 1). Among all the compounds screened, the against other mammalian cells. Its effect on the viabil-
ammonium derivative 16 showed remarkable activities ity of two mammalian cell lines, human lung epithelial
against all the fungal and bacterial strains. The next cells (A549) and human glioma cell line (U87)
most active compound was 11, followed by 23 and 9.
was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-
,5-diphenyltetrazolium bromide] assay. The results
2
showed that the mammalian cells tolerated the com-
pound up to a concentration of 1000 μg/ml whereas
the standard drug amphotericin B was many fold toxic
3.3 Cytotoxicity studies
(
figure 2).
The four most promising ammonium compounds i.e.,
, 11, 16 and 23 were further investigated for their
cytotoxicity on the viability of human erythrocytes by
9
3
7
hemolytic assay and mammalian cell lines by MTT
4
. Conclusions
38
assay. The serial dilutions of the compounds prepared
in phosphate buffer saline were incubated with erythro- In summary, we have carried out the synthe-
cytes and the effect was studied. The per cent toxicity of sis of a series of novel ammonium derivatives of

448
Shilpi Gupta et al.
chromen-2-ones, quinolin-2-ones and chromen-4-ones,
which showed significant activity against the bacterial
and fungal pathogens tested, paving the way for novel
chromenones and quinilinones to be used as antimicro-
bial agents. Amongst the ammonium compounds syn-
thesized, four compounds viz., N, N, N-triethyl-11-(4-
8. Lass-flörl C, Speth C, Mayr A, Würzner R, Dierich M P,
Ulmer H and Dietrich H 2003 Diagnostic Microbiol.
Infect. Dis. 47 569
9
. Groll A H, De Lucca A J and Walsh T J 1998 Trends
Microbiol. 6 117
1
0. Kauffman C A and Carver P L 1997 Drugs 53
539
methyl-2-oxo-2H-chromen-7-yloxy)-11-oxoundecan- 11. Vandane B H, Dromer F, Improvisi I, Lozano-Chiu M,
Rex J H and Sanglard D 1998 Med. Mycol. 36 119
2. Klepser M E, Ernst E J and Pfaller M A 1997 Trends
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3. Ator M A, Schmidt S J, Adams J L, Dolle R E,
Kruse L I, Frey C L and Barone J M 1992 J. Med. Chem.
35 100
1
-aminium bromide (9) and N,N,N-triethyl-11-(3-
1
hexyl-4-methyl-2-oxo-2H-chromen-7-yloxy)-11-oxo-
undecan-1-aminium bromide (11), N,N,N-triethyl-
1
1
1-(3-hexyl-4-oxo-4H-chromen-7-yloxy)-11-oxoun-
decan-1-aminium bromide (16) and N, N, N-triethyl-
1
0-(4-methyl-2-oxo-2H-chromen-7-yloxy)decan-1- 14. Dabur R and Sharma G L 2002 J. Ethnopharmacol. 80
193
aminium bromide (23) were found to be most
promising. Cytotoxicity study results showed the com-
pounds 11, 16 and 23 to be toxic. Although the potency
of the compound 9 is less than that of the commercial
drugs amphotericin B and kanamycin, it was found
1
5. Dabur R, Chhillar A K, Yadav V, Kamal P K, Gupta J
and Sharma G L 2005 J. Med. Microbiol. 54 549
6. Dabur R, Diwedi S K, Yadav V, Mishra V, Singh R,
Singh H and Sharma G L 2005 Antimicrob. Agents
Chemo. 49 4365
1
to have a broader range of activity than these drugs 17. Dabur R, Mandal T K and Sharma G L 2007 J. Med.
Microbiol. 56 815
among the fungal and bacterial strains tested. Also the
1
8. Chhillar A K, Arya P, Mukherjee C, Kumar P,
Yadav Y, Sharma A K, Yadav V, Gupta J, Dabur R, Jha H
N, Watterson A C, Parmar V S, Prasad A K and Sharma
G L 2006 Bioorg. Med. Chem. 14 973
cytotoxicity studies revealed that the compound 9 was
much safer than the standard antifungal drug ampho-
tericin B. Thus, this compound can be taken up further
for suitable modifications and for the development of 19. Kumar A, Singh B K, Sharma N K, Gyanda K, Jain
more promising analogues having higher therapeutic
index.
S K, Tyagi Y K, Baghel A S, Pandey M K, Sharma S K,
Prasad A K, Jain S C, Rastogi R C, Raj H G, Watterson
A C, Eycken E V and Parmar V S 2007 Eur. J. Med.
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2
2
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0. Kathuria A, Gupta A, Priya N, Singh P, Raj H G,
Prasad A K, Parmar V S and Sharma S K 2009 Bioorg.
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1. Priya N, Gupta A, Chand K, Singh P, Kathuria A,
Raj H G, Parmar V S and Sharma SK 2010 Bioorg. Med.
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2. Kumar S, Singh B K, Pandey A K, Kumar A, Sharma
S K, Raj H G, Prasad A K, Eycken E V, Parmar V S and
Ghosh B 2007 Bioorg. Med. Chem. 15 2952
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Acknowledgements
Financial support from the Defence Research and
Development Organization (DRDO), Govt. of India and
University of Delhi is gratefully acknowledged. We are
also thankful to the Council of Scientific and Industrial
Research (CSIR), New Delhi for awarding SRF to S
Gupta, S Singh, A Kathuria and A Gupta.
2
2
2
4. Huyck C L 1944 Am. J. Pharm. 116 50
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