Quaternary ammonium and amido derivatives of pyranochromenones and chromenones: Synthesis and antimicrobial activity evaluation

Article abstract of DOI:10.1007/s00044-014-1294-4

Infectious diseases and the development of their resistance to antimicrobial agents are alarming issues worldwide and consistent efforts are being made to develop efficient antimicrobial agents. In this perspective, a series of novel ammonium and amide derivatives of pyranocoumarin and coumarin were synthesized and evaluated for their antimicrobial activity. Among them, six compounds exhibited significant activity against gram-positive bacteria Bacillus cereus (MTCC 430) and Bacillus subtilis (MTCC 121) and a gram-negative bacterium Pseudomonas aeruginosa (MTCC 741). The compound N,N,N-triethyl-10-(4,8,8-trimethyl-2-oxo-2,6,7,8-tetrahydropyrano[3,2-g]chromen-10-yloxy)decan-1-aminium bromide (16) exhibited the highest antibacterial activity with MIC value of 5 μg/ml in MDA. Compounds 17 and 18 exhibited modest antifungal activity with MIC of 6.25 μg/ml against Trichophyton rubrum (clinical isolate) in MDA. Haemolytic assay results demonstrated that three out of six compounds were safe at their respective MIC values. These results provide insights for further optimization of the scaffolds for designing the next generation of compounds as lead antimicrobial agents.

Full text of DOI:10.1007/s00044-014-1294-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1294-4
ORIGINAL RESEARCH
Quaternary ammonium and amido derivatives
of pyranochromenones and chromenones: synthesis
and antimicrobial activity evaluation
•
Suchita Prasad Shiv Kumar Bipul Kumar Abhishek Kumar Singh
•
•
•
•
Hemant K. Gautam Sunil K. Sharma
Received: 9 July 2014 / Accepted: 30 October 2014
Ó Springer Science+Business Media New York 2014
Abstract Infectious diseases and the development of
their resistance to antimicrobial agents are alarming issues
worldwide and consistent efforts are being made to develop
efficient antimicrobial agents. In this perspective, a series
of novel ammonium and amide derivatives of pyrano-
coumarin and coumarin were synthesized and evaluated for
their antimicrobial activity. Among them, six compounds
exhibited significant activity against gram-positive bacteria
Bacillus cereus (MTCC 430) and Bacillus subtilis (MTCC
121) and a gram-negative bacterium Pseudomonas aeru-
ginosa (MTCC 741). The compound N,N,N-triethyl-
10-(4,8,8-trimethyl-2-oxo-2,6,7,8-tetrahydropyrano[3,2-g]
chromen-10-yloxy)decan-1-aminium bromide (16) exhib-
ited the highest antibacterial activity with MIC value of
5 lg/ml in MDA. Compounds 17 and 18 exhibited modest
antifungal activity with MIC of 6.25 lg/ml against
Trichophyton rubrum (clinical isolate) in MDA. Haemo-
lytic assay results demonstrated that three out of six
compounds were safe at their respective MIC values.
These results provide insights for further optimization of
the scaffolds for designing the next generation of com-
pounds as lead antimicrobial agents.
Keywords Antimicrobial activity ꢀ Chromen-2-ones ꢀ
Haemolytic assay ꢀ Pyranochromen-2-ones ꢀ
Quaternary ammonium compounds
Introduction
Infectious diseases, a clinically evident transmissible or
communicable diseases caused by a pathogenic biological
agent (bacterium, fungus, virus or parasite) that enters the
body of a host organism and multiplies, have resulted in an
increase in morbidity and mortality, and thus have emerged
as a global health issue (Khloya et al., 2013). They have
plagued humans throughout history and are the second
leading cause of death worldwide following heart disease
(www.bcm.edu/molvir/id 2013). Though, the development
of antimicrobial drugs represent one of the most important
advances in therapeutics that has improved the quality of life
and advances in many other areas of medicine such as
cancer chemotherapy, organ transplantation and major sur-
gery etc., (CDC, 2013), infectious diseases still remain a
very serious threat. The development of drugs for the control
and eradication of infectious disease poses unique chal-
lenges as the very use of potent antibiotics creates newer
resistant strains and fosters their spread. The loss of effec-
tive antibiotic treatments due to drug resistance has not only
crippled the ability to fight routine infectious diseases, but
has also vulnerably increased the complications of immu-
nosuppressive therapy such as joint replacements, organ
transplants, cancer therapy, asthma and rheumatoid arthritis
(Projan and Bradford, 2007). According to the recent stud-
ies, a highly potent antimicrobial agent when applied with
appropriate dosage regime not only abrogates bacterial
growth but also minimises the probability of bacterial
resistance formation (Bonde and Gaikwad, 2004).
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-1294-4) contains supplementary
material, which is available to authorized users.
S. Prasad ꢀ S. Kumar ꢀ A. K. Singh ꢀ S. K. Sharma (&)
Department of Chemistry, University of Delhi, Delhi 110007,
India
e-mail: sk.sharma90@gmail.com
B. Kumar ꢀ H. K. Gautam (&)
CSIR-Institute of Genomics and Integrative Biology, Sukhdev
Vihar, Mathura Road, Delhi 110025, India
e-mail: hemant@igib.res.in
123

Med Chem Res
Infections by pathogenic fungal species too have been
recognised as an emerging challenge despite recent
advances in antifungal chemotherapeutic regimen (Andes
et al., 2009; Saral, 1991; McNeil et al., 2001). Ampho-
tericin B has been the drug of choice but at the same
time, it suffers from severe drawbacks such as toxicity,
immunosuppression and non-availability of an absorbable
oral form (Peacock et al., 1993; Fischer et al., 1989).
Also some synthetic antifungals have been reported to
induce serious side effects in human beings which limit
their use as drugs (Goa and Barradell, 1995; Gearhart,
1994; Lazar and Wilkner, 1990). Further, literature
reports reveal the development of resistance in fungi to
most available drugs (Guinet et al., 1983; Ghannoum and
Rice, 1999; Cuenca-Estrella et al., 2000). Thus, the con-
tinued commitment to the arduous tasks involved in the
discovery of novel antimicrobials with activity against
resistant strains remains critically important in order to
meet a perpetual change.
hot spot in the development of biologically active mole-
cules because of their high efficiency active features and
broad spectrum bioactivities, such as antifungal (Kim et al.,
2010; Fu et al., 2010; Kushwaha et al., 2011), antituber-
culosis, antimicrobial, anticonvulsant, analgesic, anti-
inflammatory, insecticidal and antitumor properties (Ku-
shwaha et al., 2011; Cheptea et al., 2013). Taking the
cognizance of anticipated antimicrobial activities of cou-
marins and amides and also in search of novel antimicro-
bials with higher therapeutic index, we tried to incorporate
amide groups in the coumarin scaffold and evaluated them
for their antimicrobial efficacy.
Results and discussion
Chemistry
A series of novel ammonium alkoxy and alkyl derivatives
of pyranochromen-2-ones (14–18) were synthesized by
reaction of corresponding bromo alkoxy and bromo alkyl
pyranochromenones (9–13) with excess of triethylamine in
anhydrous acetonitrile under refluxing conditions
(Scheme 1) (Gupta et al., 2012).
Quaternary ammonium compounds (QACs), known
bioactive substances, have been widely used as antiseptics
and disinfectants (Demberelnyamba et al., 2004). These
compounds have been reported to possess antibacterial
activity along with activity against some pathogenic spe-
cies of fungi and protozoa (Thorsteinsson et al., 2003). The
antimicrobial action of QACs involves perturbation of
cytoplasmic and outer membrane lipid bilayers through
interaction of the positively charged quaternary nitrogen
with the polar head groups of acidic phospholipids and that
of hydrophobic tail with the hydrophobic membrane core
causing the rearrangement of the membrane and the sub-
sequent leakage of intracellular constituents and ultimately
cell death (Ioannou et al., 2007). Earlier, our research
group has synthesized ammonium derivatives of chrome-
nones and quinolinones as promising antimicrobial agents
(Gupta et al., 2012; Singh et al., 2012). Pyranocoumarins, a
widely distributed class of chromen-2-ones across the plant
kingdom, contains a pyran scaffold annelated to the
benzopyran-2-one moiety. It displays myriad pharmaco-
logical activities such as antifungal (Magiatis et al., 1998),
anticancer (Magiatis et al., 1998), anti-HIV (McKee et al.,
1996), anti-inflammatory (Peng-Jiu et al., 2012), antibac-
terial (Melliou et al., 2005) and photoactive drugs for skin
disorders (Mali et al., 1995). In this context and in an
attempt to explore chemical diversity space around pyr-
anocoumarin, we tried to blend the two pharmacophores
(pyranocoumarin and QACs) in one unit and screened them
for antimicrobial activity.
The desired precursors of quaternary ammonium com-
pounds i.e. compounds 9–13 were obtained from 3-(x-
bromoalkyl)-7,8-dihydroxy-4-methyl-chromen-2-ones (7/
8) and 10-hydroxy-4,8,8-trimethyl-chromen-2-ones (6),
which in turn were synthesized from pyrogallol (4). In the
case of bromo alkoxy pyranochromen-2-ones (11–13),
firstly, pyrogallol (4) was made to undergo condensation
with 2-methylbuta-l,3-diene (isoprene) in the presence of
orthophosphoric acid and using xylene as a solvent to get
2,2-dimethylchroman-7,8-diol (5) (Ahluwalia et al., 1982).
The self-condensation of isoprene, the chief difficulty
encountered while working with dienes, was minimised by
slow addition of isoprene to a stirred solution of pyrogallol
in xylene and orthophosphoric acid. Condensation of iso-
prene with phenols catalysed by acids may be regarded as
the chemical equivalent of proposed biogenetic pathways
(Ahluwalia et al., 1982). 10-hydoxy-4,8,8-trimethyl-7,8-
dihydropyrano[3,2-g]chromen-2(6H)-one (6) was synthe-
sized in quantitative yields by Pechmann condensation of 5
with ethyl acetoacetate in the presence of sulphuric acid
(Ahluwalia and Jolly 1982). The reaction of terminal dib-
romoalkanes with 6 yielded the corresponding 10-(x-bro-
moalkoxy)-4,8,8-trimethyl-7,8-dihydropyrano[3,2-g]chro-
men-2(6H)-ones (11–13) using potassium carbonate as
base and DMF as solvent (Schemes 1 and 2) (Gupta et al.,
2012).
Coumarins, a pharmacophore of considerable biological
importance have been reported to exert notably antibacte-
rial as well as antifungal activity (Muratovic et al., 2013;
Appendino et al., 2004). On the other hand, amide deriv-
atives found ubiquitously in nature have become a research
However, in order to form bromo alkyl pyranochrome-
nones (9/10), a different protocol was employed wherein
pyrogallol (4) was initially condensed with ethyl 2-acetyl-
123

Med Chem Res
2"
1"
N
Br
n
1
Br
n
1'
11' O
1'
O
O
9
9'
O 13'
1
10'
10
2'
2
O
O 13
11 O
O
8'
7'
8
i
6
ii
3'
3
7
14'
6'
12'
14
6
12
5'
4'
5
4
OH
14 n = 1
15 n = 5
16 n = 9
11 n = 1
12 n = 5
13 n = 9
O
O
O
R1
6 R1 = H
9 R1 = n-C6H12Br
10 R1 = n-C10H20Br
OH
1'
9'
O
2"
10'
2'
11' O
O
13'
14'
8'
7'
1"
iii
3'
9/10
17 n = 4
18 n = 8
N
12'
n
4'
6'
1
5'
Br
Scheme 1 Synthesis of quaternary ammonium pyranocoumarin derivatives; Reagents and conditions: i K2CO3, Br(CH2)nBr, DMF, RT, 15 h; ii
NEt3 (5.0 equivalent), CH3CN, 60 °C, 96 h; iii NEt3 (5.0 equivalent), CH3CN, 60 °C, 96 h
Scheme 2 Synthesis of
pyranocoumarin derivatives;
Reagents and conditions:
i H3PO4, isoprene, xylene, RT,
12 h; ii Ethyl acetoacetate,
conc. H2SO4, RT, 6 h; iii Ethyl
2-acetyl-x-bromoalkanoates,
conc. H2SO4, RT, 6 h; iv
2-Methyl-3-buten-2-ol, p-TSA,
toluene, reflux, 30 h
OH
OH
OH
1
8
2
HO
7
O
O
HO
OH
O
OH
iii
i
3
Br
6
n
4
5
1'
7 n = 4
8 n = 8
4
5
iv
ii
OH
OH
1
9
O
10
13
2
8
11 O
O
O
O
O
3
Br
7
12
14
n
5
4
1'
6
9 n = 4
10 n = 8
6
Ethyl 2-acetyl-x-bromoalkanoates (2/3) used in the reac-
tion was in turn synthesized from ethyl acetoacetate (1) and
dibromoalkanes under reflux conditions (Scheme 3).
Nuclear prenylation of 7 and 8 was achieved using
2-methyl-3-buten-2-ol in the presence of p-toluene sul-
phonic acid, which undergoes in situ cyclization to afford
corresponding pyranochromenones (9/10) (Scheme 2)
(Kathuria et al., 2011).
O
O
O
O
1"
2
i
1
2'
1'
O
O
2"
3
n
Br
2 n = 4
3 n = 8
1
Scheme 3 Synthesis of ethyl 2-acetyl-x-bromoalkanoates; Reagents
and conditions: i NaH, Br(CH2)nBr, THF, reflux, 30 h
The synthesis of (coumarinyloxy)acetamide derivatives
(32–47) is outlined in Scheme 4. 7-Hydroxy-4-meth-
ylcoumarins (23–25) were prepared via Pechmann con-
densation of resorcinol (19) and ethyl acetoacetate as well
as alkylated ethyl acetoacetate with drop wise addition of
x-bromoalkanoates (2/3) under Pechmann reaction condi-
tions to yield 3-(x-bromoalkyl)-7,8-dihydroxy-4-methyl-
chromen-2-ones (7/8) (Scheme 2) (Kathuria et al., 2011).
123

Med Chem Res
O
O
O
O
OH
HO
OH
iii
ii
i
R3
MeO
MeO
R1
R2
22 R1 = H; R2 = OH; R3 = H
19
21
20
23 R1 = 7-OH; R2 = CH3; R3 = H
24 R1 = 7-OH; R2 = CH3; R3 = C2H5
25 R1 = 7-OH; R2 = CH3; R3 = n-C6H13
26 R1 = 6-OH; R2 = CH3; R3 = H
iv
1'
O
8'
2'
O
O
9'
O
7'
6'
R1
v
R3
R3
10'
3'
R1
4'
R2
5'
R2
27 R1 = H; R2 = OCH2COOEt; R3 = H
32/33 R1 = H; R2 = OCH2CONHR4; R3 = H
28 R1 = 7-OCH2COOEt; R2 = CH3; R3 = H
34-36 R1 = 7-OCH2CONHR4; R2 = CH3; R3 = H
37-41 R1 = 7-OCH2CONHR4; R2 = CH3; R3 = C2H5
42-44 R1 = 7-OCH2CONHR4; R2 = CH3; R3 = n-C6H13
45-47 R1 = 6-OCH2CONHR4; R2 = CH3; R3 = H
29 R1 = 7-OCH2COOEt; R2 = CH3; R3 = C2H5
30 R1 = 7-OCH2COOEt; R2 = CH3; R3 = n-C6H13
31 R1 = 6-OCH2COOEt; R2 = CH3; R3 = H
H
2
2"'
4"'
6"'
2"'
R1/R2 =
R3 =
1
N
O
R4
1"'
3"'
5"'
1"'
3""
O
4""
2"
4"
6"
8"
R4 =
2""
40
1""
3"
7"
1"
5"
2""
2""
1""
2"
R4 =
R4 =
2""
2"
1"
32/34/37/42/45
33
4""
N
R4 =
1""
35/38
36/39
N
1""
1"
3"
2"
R4 =
R4 =
3""
1""
3"
N
41/43/46
44/47
1""
1"
3"
2""
1""
2"
4"
1""
6"
2"
R4 =
2"
N
OH
1"
3"
5"
1"
1"
1""
Scheme 4 Synthesis of (coumarinyloxy)acetamide derivatives;
Reagents and conditions: Ethyl 2-alkylacetoacetates, Conc.
H2SO4, RT, 12 h; ii Ethyl acetoacetate, H2SO4–C2H5OH (7:3), RT,
24 h; iii HBr–AcOH (7:3), 110 °C, 24 h; iv Ethyl bromoacetate,
K2CO3, DMF, RT, 12 h; v R5NH2, n-butanol, RT, 48 h
i
sulphuric acid acting as catalyst (Pechmann and Duisberg,
1883; Kathuria et al., 2009). On the other hand,
4-methoxyphenol (20) was used as the starting material in
order to obtain 6-hydroxy-4-methyl-2H-chromen-2-one
(26) (Kathuria et al., 2011). Synthesis of alkylated
b-ketoester was achieved according to the earlier published
procedure from our group (Kathuria et al., 2009). Fur-
thermore, to have positional variations in the synthesized
molecules, commercially available 4-hydroxy-2H-chro-
men-2-one (22) was used. The key intermediates (27–31)
were obtained from corresponding hydroxycoumarins (22–
26) on treatment with ethyl bromoacetate (Fonseca et al.,
2012), which further on reaction with alkylamines, N,N-
dialkylaminoalkanes and ethanolamine and using n-butanol
as solvent afforded the desired coumarin amides in almost
quantitative yields.
The structural characterization of all the synthesized
compounds was achieved using various spectroscopic
techniques. The structures of known compounds were
confirmed by comparison of their melting point and spec-
tral data with those reported in the literature.
Biology
Antimicrobial activity
The antibacterial screening of all the synthesized quaternary
ammonium pyranocoumarins (14–18) and (coumarinyloxy)
123

Med Chem Res
Table 1 Zone of inhibition and minimum inhibitory concentration of active compounds 16–18, 38, 42, 43 and their precursors 9, 10, 13, 29, 30
against pathogenic microbial test strains using gentamicin and amphotericin B as positive controls
Compounds
B. cereus
B. subtilis
P. aeruginosa
T. rubrum
ZIa (mm)
MICb (lg/ml)
ZIa (mm)
MICb (lg/ml)
ZIa (mm)
MICb (lg/ml)
ZIa (mm)
MICb (lg/ml)
c
c
c
c
9
–
200
200
200
5
–
200
200
200
5
–
200
200
200
5
–
550
475
450
50
c
c
–
c
c
10
13
16
17
18
29
30
38
42
43
a
–
–
–
c
c
–
c
c
–
–
–
24
15
20
24
14
16
26
13
18
14
21
21
20
10
40
6.25
6.25
475
550
100
500
500
5
10
10
c
c
–
c
c
–
200
200
80
200
200
100
20
–
200
200
80
–
c
c
–
c
c
–
–
–
16
19
15
11
17
14
14
16
14
12
11
11
20
40
40
40
40
Diameter of zone of inhibition (mm) including disc diameter of 6 mm; Concentration of compound = 250 lg/disc
b
c
Minimum inhibitory concentration
No activity at the tested concentration. All the experiments were carried out in triplicate
acetamides (32–47) was carried out against a broad range of
B. subtilis wherein the inhibition zone decreased to 18 and
16 mm, respectively. However, on decreasing the carbon
chain length from ten to six in case of both the ammonium
alkoxy (15) and alkyl (17) derivatives of pyranochromen-2-
ones, a downward trend in the activity profile was observed.
Though ammonium alkyl derivative (17) showed a downfall
of zone of inhibition from 20 to 15 mm, 16 to 14 mm and 18
to 13 mm for B. cereus, B. subtilis and P. aeruginosa,
respectively; however, this decrease in the activity for the
corresponding alkoxy derivative (15) was much more abrupt
i.e. from 24 to 11 mm and 26 to 11 mm for B. cereus and P.
aeruginosa, respectively. Furthermore, the activity was
completely lost against B. subtilis on reducing the size of
alkyl chain from ten carbons (16) to six carbons (15) in case
of ammonium alkoxy derivatives (Table 1 and Table S1).
In order to further study the antimicrobial activity of
newer derivatives of coumarins, we synthesized a series of
novel coumarin derivatives by incorporating acetamido
functionality on coumarin skeleton and screened them for
their antimicrobial efficacy. From the antibacterial activity
screening results, it could be established that the substitu-
tion at C-3 position of coumarin with long alkyl chain
along with the use of longer N,N-diaminoalkanes for am-
idation was important for a molecule to possess activity
against pathogenic bacteria. This fact was substantiated by
the observation that compounds 38, 42 and 43 led to the
inhibition of growth of B. cereus; B. subtilis; P. aeruginosa
by creating a zone of 16, 19 and 15 mm; 11, 17 and
14 mm; 14, 16 and 14 mm, respectively (Table 1 and
Table S1, supporting information).
pathogenic microbial test strains using disc diffusion assay with
some modifications (Pathak et al., 2013; Yadav et al., 2014). To
evaluate the antimicrobial activities against six pathogenic bac-
terial strains namely Bacillus cereus, Bacillus subtilis, Pseudo-
monas aeruginosa, Escherichia coli, Salmonella typhimurium
and Klebsiella pneumoniae, disc diffusion assay was performed
at a concentration of 250 lg/disc (Table S1 (supporting infor-
mation)). The screening results revealed that six compounds
(16–18/38/42/43) exhibited excellent to moderate activity
against two gram-positive bacteria (B. cereus and B. subtilis)and
a gram-negative bacterium (P. aeruginosa). All of these com-
pounds developed a zone of inhibition equal to or greater than
12 mm, the cut-off value for a molecule to be considered active.
However, noneof the compoundswere found to be active against
the other three tested gram-negative bacteria viz. E. coli, S. ty-
phimurium and K. pneumoniae. Quaternary ammonium deriv-
atives were found to possess excellent activity against B. cereus,
B. subtilis and P. aeruginosa wherein three out of five quaternary
ammonium derivatives inhibited the growth of bacteria up to a
diameter of 12 mm or more. N,N,N-Triethyl-10-(4,8,8-tri-
methyl-2-oxo-2,6,7,8-tetrahydropyrano[3,2-g]chromen-10-
yloxy)decan-1-aminium bromide (16) developed the maxi-
mum zone of inhibition of 24, 24 and 26 mm against B.
cereus, B. subtilis and P. aeruginosa, respectively, followed
by compound 18 bearing quaternary ammonium group with
a ten carbon spacer at C-3 position of pyranocoumarin. It led
to the inhibition of bacterial growth up to a diameter of
20 mm in case of B. cereus. On the other hand, a slight
decrease in the activity was observed for P. aeruginosa and
123

Med Chem Res
The precursors (9/10/13/29/30) of the shortlisted active
molecules (16–18/38/42/43) were also screened against the
bacterial strains B. cereus, B. subtilis and P. aeruginosa.
However, none of the precursors were found to be active
against the bacterial pathogens at the tested concentration
(250 lg/disc). In order to have further insights on the
antimicrobial action of these compounds, the antifungal
activity screening was carried out using Trichophyton ru-
brum as a model pathogen. Compounds 17 and 18 bearing
quaternary ammonium group at C-3 position of the pyran
ring and a hydroxyl group at C-10 position exhibited
excellent antifungal activity by inhibiting the growth of the
fungus up to a diameter of 21 mm. Also compound 38
having an ethyl substituent at C-3 position of the pyran ring
had a zone of inhibition (13 mm) higher than that for
compounds 42 and 43 (11 mm) bearing a hexyl chain at the
C-3 position of the pyran ring. Thus, it could be concluded
that in contrast to antibacterial activity results, the size of
alkyl chain is not an important criteria for a molecule to
exhibit activity against pathogenic fungi. However, pre-
sence of hydroxyl group along with quaternary ammonium
functionality has been found to be critical for a compound
to possess significant antifungal activity (Table 1).
Fig. 1 Haemolytic assay of compounds 16, 17, 38 and 43 against
human erythrocytes
value much higher than that of the active compounds
itself (Table 1).
Haemolytic study
Furthermore, in order to have an in-depth understanding
of factors that affect the growth inhibition process of var-
ious bacterial strains and fungi, minimum inhibitory con-
centration i.e. MIC was evaluated using microbroth
dilution assay (Gupta et al., 2012; Pathak et al., 2013;
Yadav et al., 2014). Table 1 shows the MIC values of the
active compounds along with their precursors. Compound
16 which developed the maximum zone of inhibition
against Bacillus cereus, Bacillus subtilis and Pseudomonas
aeruginosa inhibited the growth of these pathogenic bac-
teria at a concentration of 5 lg/ml. N,N,N-Triethyl-10-(10-
hydroxy-4,8,8-trimethyl-2-oxo-2,6,7,8-tetrahydropyr-
ano[3,2-g]chromen-3-yl)decan-1-aminium bromide (18)
exhibited MIC of 5 lg/ml against B. cereus, 10 lg/ml
against B. subtilis and P. aeruginosa. However, on
decreasing the size of alkyl chain from ten carbons (18) to
six carbons (17), MIC value increased and a concentration
of 20, 10 and 40 lg/ml was sufficient to inhibit the growth
of B. cereus, B. subtilis and P. aeruginosa, respectively.
In the case of (coumarinyloxy)acetamides (38/42/43),
compound 42 bearing a hexyl chain and N,N-diethyl-
amino group was found to possess the lowest MIC value
of 20, 20 and 40 lg/ml against B. cereus, B. subtilis and
P. aeruginosa, respectively. Antifungal activity results
revealed that the compounds 17 and 18 (bearing hydroxyl
groups), which developed the maximum zone of inhibi-
tion had the lowest MIC value of 6.25 lg/ml amongst the
various tested compounds. Furthermore, the precursors of
all the shortlisted active compounds exhibited an MIC
In order to shed light on toxicity, the promising compounds
(16–18/38/42/43) were investigated for the viability of
human erythrocytes by haemolytic assay (Ciornei et al.,
2005; Dobrovolskaia et al., 2008) at various multiples of
their MIC values. Compound 16, the most potent antibac-
terial compound was found to be non-haemolytic with
marginal toxicity of 20 % even up to concentration 20
times of its MIC value (5 lg/ml, the MIC in case of bac-
terial pathogens). Also it could be established that 16 was
non-toxic at the MIC for the tested fungal strain T. rubrum.
However, the least active antibacterial compound (38) was
found to be much safer as it exhibited 13 and 11 %
haemolysis at 25 X MIC, the MIC for B. subtilis, T. rubrum
and B. cereus, P. aeruginosa, respectively. The haemolysis
results for compound 17 provided a vivid picture that the
human RBCs were negligibly lysed at various MICs of 17.
RBCs were lysed by less than 18 % at the MIC for P.
aeruginosa, while a lysis of 9 % and less was observed at
the MIC for B. cereus, B. subtilis and T. rubrum. In addi-
tion to this, N-(3-(dimethylamino)propyl)-2-((3-hexyl-4-
methyl-2-oxo-2H-chromen-7-yl)oxy)
acetamide
(43)
exhibited haemolysis of 7 % at the MICs for pathogenic
bacterial strains, while 34–35 % cells were lysed at the
MIC for T. rubrum. Compound 18 was found to be the
most toxic to the erythrocytes. It caused 80 % lysis of the
cells at its MIC for B. subtilis and P. aeruginosa. On the
other hand, 42 % cells were lysed at the MIC for
123

Med Chem Res
P. aeruginosa in case of compound 42 (Fig. 1 and Table S2
(supporting information)).
by UV light or by spraying with 5 % alcoholic FeCl3
solution. Silica gel (100–200 mesh) was used for column
chromatography. All of the chemicals and reagents were
procured from Spectrochem Pvt. Ltd., India and Sigma-
Aldrich Chemicals Pvt. Ltd., USA. Melting points were
measured on a Buchi M-560 equipment and are uncor-
rected. Infrared spectra were recorded on Perkin-Elmer
SPECTRUM BX FTIR and SPECTRUM XIFT IR spec-
Conclusions
In summary, a total of twenty one compounds including
five quaternary ammonium derivatives and sixteen (cou-
marinyloxy)acetamides were synthesized and fully char-
acterized by 1H NMR, 13C NMR, UV, FTIR and high
resolution mass spectroscopy (HRMS). Of these, twenty
compounds i.e. 14–18, 32–35 and 37–47 are novel.
Although compound 36 is known in literature (Wang
et al., 2011), its complete spectral data were not reported.
Herein, we have reported the spectral data for all the
compounds in the experimental section. All of the com-
pounds synthesized were evaluated for their antibacterial
activity against both gram-positive (B. cereus and B.
subtilis) and gram-negative bacteria (P. aeruginosa,
E. coli, S. typhimurium and K. pneumoniae). The anti-
bacterial activity results showed that six compounds (16–
18/38/42/43) exhibited significant activity against both,
the gram-positive bacteria B. cereus and B. subtilis and a
gram-negative bacterium P. aeruginosa. These com-
pounds were further screened against a fungal strain T.
rubrum along with the investigation of their MIC values.
Out of the various compounds screened, compound 16, a
C10 alkyl chain containing quaternary ammonium deriv-
ative was found to be the most potent against bacterial
growth with an MIC value of 5 lg/ml, while compounds
17 and 18 (quaternary ammonium compounds with
hydroxyl groups) exhibited modest anti-Trichopyhton
activity (MIC: 6.25 lg/ml). The haemolytic assay results
revealed that out of six compounds i.e. 16–18/38/42/43
shortlisted for potent antibacterial activity, three com-
pounds (16/17/38) were found to be reasonably safe at
their respective MIC values. Compound 43 too had neg-
ligible toxicity at the MIC for the bacterial strains and a
lysis of 34–35 % was observed at the MIC for the fungus.
These results can be used for the design and synthesis of
newer pharmacophores with improved antimicrobial
activity.
1
trophotometer. The H and 13C NMR spectra were recor-
ded on Jeol-400 (400 MHz, 100.5 MHz) NMR
spectrometer using tetramethylsilane as internal standard.
The chemical shift values are on a d scale and the coupling
constant values (J) are in Hertz. The UV data were
recorded on Cary 300 UV–Vis spectrophotometer, Agilent
Technologies. The HRMS data were recorded on Waters
(Micromass) LCT 1 using ESI TOF MS technique.
General procedure for the synthesis of ammonium alkoxy
derivatives of pyranochromen-2-ones (14–16)
To a stirred solution of 10-hydroxy-4,8,8-trimethyl-7,8-
dihydropyrano[3,2-g]chromen-2(6H)-one
(6)
(1 g,
3.84 mmol) in anhydrous DMF (50 ml), fused potassium
carbonate (2.2 equivalent) was added and the solution
stirred at room temperature for 30 min followed by slow
addition of 1,2-dibromoethane/1,6-dibromohexane/1,10-
dibromodecane (1.1 equivalent). The resulting reaction
mixture was stirred at room temperature for 24 h. The
progress of the reaction was monitored on TLC. On com-
pletion of the reaction, the solvent was removed under
reduced pressure and the solid so obtained was dissolved in
chloroform (50 ml). The chloroform layer was washed
with water (2 9 30 ml) and then dried over anhydrous
sodium sulphate. The solvent was then removed in vacuo in
a rotary evaporator and the resulting crude product sub-
jected to column chromatography to give corresponding
bromo alkoxy derivative of pyranochromen-2-one (11–13).
The bromo compound (11–13) was then converted to
quaternary ammonium derivative (14–16) by dissolving it
in acetonitrile (1 g in 50 ml) followed by the addition of
triethylamine (5 equivalent) and stirring the reaction mix-
ture under nitrogen at 60 °C for 96 h. The progress of
reaction was monitored using TLC. After completion of the
reaction, the solvent was evaporated under reduced pres-
sure and the resultant product was then subjected to column
chromatography to give the pure quaternary ammonium
compound (14–16).
Experimental section
Chemistry
Materials and methods
10-(2-Bromoethoxy)-4,8,8-trimethyl-7,8-dihydropyrano[3,2-g]
chromen-2(6H)-one (11) The reaction of 10-hydroxy-
4,8,8-tr imethyl-7,8-dihydropyrano[3,2-g]chromen-2(6H)-
one (6) (1 g, 3.84 mmol) with 1,2-dibromoethane (0.79 g,
The organic solvents were dried and distilled prior to their
use. Reactions were monitored by precoated TLC plates
(Merck silica gel 60F254); the spots were visualized either
123

Med Chem Res
1
4.22 mmol) gave the title compound 11 as a light brown
solid (1.27 g, 90 %) by following the general procedure;
m.p.: 117.9–118.7 °C; Rf: 0.30 (ethyl acetate:petroleum
ether: 3:7); UV (MeOH) kmax: 259 and 327 nm; IR (KBr)
74 cm-1; H NMR (400 MHz, CDCl3): d 1.24–1.43 (m,
16H, H-40–H-80 and 29 C-8 CH3), 1.46–1.55 (m, 2H,
H-30), 1.73–1.88 (m, 6H, H-7, H-20 and H-90), 2.35 (s, 3H,
C-4 CH3), 2.84 (t, 2H, J = 6.59 Hz, H-6), 3.40 (t, 2H, J =
6.59 Hz, H-100), 4.06 (t, 2H, J = 6.59 Hz, H-10), 6.09 (s,
1H, H-3), 7.02 (s, 1H, H-5) ppm; 13C NMR (100.5 MHz
CDCl3): d 18.93 (C-4 CH3), 22.55 (C-6), 27.08 (29 C-8
CH3), 26.12, 28.34, 28.93, 29.57, 29.68, 30.28, 32.58 (C-
20–C-90), 33.01 (C-7), 34.28 (C-100), 74.12 (C-10), 75.82
(C-8), 112.13 (C-3), 113.42 (C-12), 118.45 (C-14), 118.97
(C-5), 135.12 (C-10), 146.56 (C-11), 150.94 (C-13), 152.68
(C-4), 161.29 (C-2) ppm; HRMS: m/z [M ? Na]? Calcu-
lated for C25H35BrO4: 501.1617; found: 501.1604.
m
max: 2976.44, 2944.92, 1716.10 (C=O), 1609.35, 1568.01,
1395.70, 1105.82, 1056.20, 843.74, 754.67, 665.33 cm-1
;
1H NMR (400 MHz, CDCl3): d 1.41 (s, 6H, 29 C-8 CH3),
1.86 (t, 2H, J = 6.59 Hz, H-7), 2.36 (s, 3H, C-4 CH3), 2.85
(t, 2H, J = 6.59 Hz, H-6), 3.65 (t, 2H, J = 7.32 Hz, H-20),
4.36 (t, 2H, J = 7.32 Hz, H-10), 6.11 (s, 1H, H-3), 7.06 (s,
1H, H-5) ppm; 13C NMR (100.5 MHz CDCl3): d 18.94 (C-
4 CH3), 22.49 (C-6), 27.07 (29 C-8 CH3), 29.71 (C-20), 32.
54 (C-7), 73.13 (C-10), 76.28 (C-8), 112.24 (C-3), 113.41
(C-12), 118.62 (C-14), 119.70 (C-5), 134.10 (C-10), 146.25
(C-11), 150.62 (C-13), 152.69 (C-4), 160.99 (C-2) ppm;
HRMS: m/z [M ? Na]? Calculated for C17H19BrO4: 389.
0364; found: 389.0373.
N,N,N-Triethyl-2-((4,8,8-trimethyl-2-oxo-2,6,7,8-tetrahy-
dropyrano[3,2-g]chromen-10-yl)oxy)ethanaminium bro-
mide (14) The reaction of 10-(2-bromoethoxy)-4,8,8-
trimethyl-7,8-dihydropyrano[3,2-g]chromen-2(6H)-one
(11) (1 g, 2.72 mmol) with triethylamine (1.18 g, 13.
60 mmol) gave the title compound 14 as an off-white
solid (0.89 g, 70 %) by following the general proce-
dure; m. p.: 193.8–195.3 °C; Rf: 0.58 (MeOH:CHCl3: 1:
10-((6-Bromohexyl)oxy)-4,8,8-trimethyl-7,8-dihydropyr-
ano[3,2-g]chromen-2(6H)-one (12) The reaction of
10-hydroxy-4,8,8-trimethyl-7,8-dihydropyrano[3,2-g]chro-
men-2(6H)-one (6) (1 g, 3.84 mmol) with 1,6-dibromo-
hexane (1.03 g, 4.22 mmol) gave the title compound 12 as
a light brown solid (1.44 g, 89 %) by following the general
procedure; m. p.: 61.0–61.9 °C; Rf: 0.40 (ethyl acetate:
petroleum ether: 3:7); UV (MeOH) kmax: 260 and 329 nm;
IR (KBr) mmax: 2974.00, 2935.02, 1724.94 (C=O), 1610.76,
1568.49, 1399.08, 1109.93, 1057.34, 865.80, 771.70, 709.
4); UV (MeOH) kmax: 258 and 327 nm; IR (KBr) mmax
3410.62, 2973.07, 2926.76, 1720.42 (C=O), 1610.65,
:
1569.44, 1388.62, 1367.85, 1105.26, 1061.05, 999.46,
893.14, 834.17, 782.60, 583.60 cm-1
;
1H NMR
(400 MHz, CDCl3): d 1.38 (s, 6H, 29 C-80 CH3), 1.45
(t, 9H, J = 6.59 Hz, H-200), 1.84 (t, 2H, J = 6.59 Hz,
H-70), 2.35 (s, 3H, C-40 CH3), 2.82 (t, 2H, J = 6.59 Hz,
H-60), 3.77 (q, 6H, J = 7.32 Hz, H-100), 4.15 (t, 2H,
J = 4.39 Hz, H-1), 4.40 (t, 2H, J = 4.39 Hz, H-2), 6.06
(s, 1H, H-30), 7.09 (s, 1H, H-50) ppm; 13C NMR (100.
5 MHz CDCl3): d 8.44 (C-200), 18.93 (C-40 CH3), 22.29
(C-60), 27.10 (29 C-80 CH3), 32.31 (C-70), 54.57 (C-100),
57.48 (C-1), 67.03 (C-2), 77.40 (C-80), 111.91 (C-30),
113.32 (C-120), 119.14 (C-140), 120.65 (C-50), 132.92
(C-100), 145.91 (C-110), 150.64 (C-130), 153.19 (C-40),
160.43 (C-20) ppm; HRMS: m/z [M]? Calculated for
C23H34NO4?: 388.2488; found: 388.2478.
1
02 cm-1; H NMR (400 MHz, CDCl3): d 1.39 (s, 6H, 29
C-8 CH3), 1.47–1.65 (m, 4H, H-30 and H-40), 1.75–1.93 (m,
6H, H-20, H-50 and H-7), 2.35 (s, 3H, C-4 CH3), 2.84 (t, 2H,
J = 6.59 Hz, H-6), 3.43 (t, 2H, J = 6.59 Hz, H-60), 4.06 (t,
2H, J = 6.59 Hz, H-10), 6.08 (s, 1H, H-3), 7.02 (s, 1H,
H-5) ppm; 13C NMR (100.5 MHz CDCl3): d 18.90 (C-4
CH3), 22.50 (C-6), 25.33 (C-30/C-40), 27.06 (29 C-8 CH3),
28.10 (C-30/C-40), 30.06 (C-20), 32.54 (C-50), 32.94 (C-7),
34.22 (C-60), 73.72 (C-10), 75.86 (C-8), 112.08 (C-3), 113.
39 (C-12), 118.51 (C-14), 119.06 (C-5), 134.97 (C-10),
146.48 (C-11), 150.87 (C-13), 152.73 (C-4), 161.21 (C-2)
ppm; HRMS: m/z [M ? Na]? Calculated for C21H27BrO4:
445.0990; found: 445.1006.
N,N,N-Triethyl-6-((4,8,8-trimethyl-2-oxo-2,6,7,8-tetrahy-
dropyrano[3,2-g]chromen-10-yl)oxy)hexan-1-aminium
bromide (15) The reaction of 10-((6-bromohexyl)oxy)-
4,8,8-trimethyl-7,8-dihydropyrano[3,2-g]chromen-2(6H)-
one (12) (1 g, 2.36 mmol) with triethylamine (1.20 g, 11.
80 mmol) gave the title compound 15 as an off-white solid
(0.93 g, 75 %) by following the general procedure; m. p.:
148.0–149.3 °C; Rf: 0.58 (MeOH:CHCl3: 1:4); UV
(MeOH) kmax: 259 and 329 nm; IR (KBr) mmax: 3468.46,
3407.60, 2930.43, 2919.42, 1722.35 (C=O), 1607.89, 1452.
32, 1395.06, 1370.45, 1107.23, 1061.77, 926.06, 865.70,
10-((10-Bromodecyl)oxy)-4,8,8-trimethyl-7,8-dihydropyr-
ano[3,2-g]chromen-2(6H)-one (13) The reaction of
10-hydroxy-4,8,8-trimethyl-7,8-dihydropyrano[3,2-g]chro-
men-2(6H)-one (6) (1 g, 3.84 mmol) with 1,10-dibromo-
decane (1.26 g, 4.22 mmol) gave the title compound 13 as
a dark brown solid (1.65 g, 90 %) by following the general
procedure; m. p.: 45.5–46.0 °C; Rf: 0.45 (ethyl acetate:
petroleum ether: 3:7); UV (MeOH) kmax: 260 and 328 nm;
IR (KBr) mmax: 2928.31, 2854.32, 1725.14 (C=O), 1610.18,
1569.04, 1398.99, 1109.01, 1058.51, 865.10, 771.51, 709.
847.21, 564.86 cm-1; H NMR (400 MHz, CDCl3): d 1.
32–1.42 (m, 15H, 29 C-80 CH3 and 39 H-200), 1.52–1.61
1
123

Med Chem Res
(m, 2H, H-3), 1.70–1.88 (m, 8H, H-2, H-4, H-5 and H-70),
2.36 (s, 3H, C-40 CH3), 2.84 (t, 2H, J = 6.59 Hz, H-60), 3.
32–3.39 (m, 2H, H-1), 3.49 (q, 6H, J = 7.32 Hz, 39 H-100),
4.02–4.06 (m, 2H, H-6), 6.03 (s, 1H, H-30), 7.04 (s, 1H,
30 h. The reaction was monitored by TLC (10 % ethyl
acetate–petroleum ether). On completion of the reaction,
ethyl acetate was added and the solution was then filtered.
The filtrate was washed with water, the organic layer dried
over anhydrous sodium sulphate and the solvent evapo-
rated. The crude product so obtained was subjected to
column chromatography to get the desired product (2/3)
using ethyl acetate–petroleum ether (1:49) as eluent.
13
H-50) ppm; C NMR (100.5 MHz CDCl3): d 8.10 (C-200),
18.93 (C-40 CH3), 22.03, 22.43, 25.51, 25.77, 29.67 (C-2–
C-5 and C-60), 27.02 (29 C-80 CH3), 32.45 (C-70), 53.49
(C-100), 57.42 (C-1), 73.10 (C-6), 76.07 (C-80), 111.69 (C-
30), 113.29 (C-120), 118.82 (C-140), 119.31 (C-50), 134.75
(C-100), 146.21 (C-110), 150.97 (C-130), 153.31 (C-40), 161.
31 (C-20) ppm; HRMS: m/z [M]? Calculated for
C27H42NO4?: 444.3114; found: 444.3115.
Ethyl 2-acetyl-8-bromooctanoate (2) The reaction of
ethyl 3-oxobutanoate (1) (5 g, 38.42 mmol) with 1,6-
dibromohexane (10.31 g, 42.26 mmol) gave the title
compound 2 as a pale yellow coloured oil (6.19 g, 55 %)
by following the general procedure; Rf: 0.40 (ethyl acetate:
petroleum ether: 1:9); IR (KBr) mmax: 2927.81, 2855.37,
1741.54 (COO–), 1717.59 (C=O), 1465.15, 1357.82, 1241.
N,N,N-Triethyl-10-((4,8,8-trimethyl-2-oxo-2,6,7,8-tetrahy-
dropyrano[3,2-g]chromen-10-yl)oxy)decan-1-aminium
bromide (16) The reaction of 10-((10-bromodecyl)oxy)-
4,8,8-trimethyl-7,8-dihydropyrano[3,2-g]chromen-2(6H)-
one (13) (1 g, 2.09 mmol) with triethylamine (1.06 g, 10.
45 mmol) gave the title compound 16 as a light brown low
melting solid (0.91 g, 75 %) by following the general
procedure; Rf: 0.41 (MeOH:CHCl3: 1:4); UV (MeOH)
1
75, 1150.40, 1025.29, 859.31, 722.35, 644.01 cm-1; H
NMR (400 MHz, CDCl3): d 1.21–1.43 (m, 9H, H-4–H-6
and H-200), 1.73–1.85 (m, 4H, H-3 and H-7), 2.18 (s, 3H,
H-20), 3.36 (t, 3H, J = 6.96 Hz, H-2 and H-8), 4.16 (q, 2H,
J = 7.32 Hz, H-100) ppm; 13C NMR (100.5 MHz CDCl3):
d 14.24 (C-200), 27.30, 27.94, 28.11, 28.58, 28.90, 32.72 (C-
3–C-7 and C-20), 33.91 (C-8), 59.93 (C-2), 61.44 (C-100),
169.97 (C-1), 203.32 (C-10) ppm.
k
max: 260 and 328 nm; IR (KBr) mmax: 3412.46, 2933.91,
1718.29 (C=O), 1609.88, 1568.95, 1399.37, 1108.57, 1054.
36, 937.64, 887.63, 792.37, 585.05 cm-1 1H NMR
;
(400 MHz, CDCl3): d 1.30–1.43 (m, 23H, H-4–H-7, 29
C-80 CH3 and 39 H-200), 1.46–1.57 (m, 2H, H-3), 1.67–1.
87 (m, 8H, H-2, H-8, H-9 and H-70), 2.36 (s, 3H, C-40
CH3), 2.84 (t, 2H, J = 6.59 Hz, H-60), 3.21–3.28 (m, 2H,
H-1), 3.50 (q, 6H, J = 7.32 Hz, 39 H-100), 4.05 (t, 2H,
J = 6.59 Hz, H-100), 6.07 (s, 1H, H-30), 7.03 (s, 1H, H-50)
Ethyl 2-acetyl-12-bromododecanoate (3) The reaction of
ethyl 3-oxobutanoate (1) (5 g, 38.42 mmol) with 1,10-
dibromodecane (12.68 g, 42.26 mmol) gave the title
compound 3 as a pale yellow coloured oil (8.05 g, 60 %)
by following the general procedure; Rf: 0.45 (ethyl acetate:
petroleum ether: 1:9); UV (MeOH) kmax: 259 nm; IR
(KBr) mmax: 2927.70, 2855.22, 1742.93 (COO–), 1717.80
(C=O), 1459.09, 1358.00, 1241.22, 1150.28, 1026.33, 858.
13
ppm; C NMR (100.5 MHz CDCl3): 8.18 (C-200), 18.93
(C-40 CH3), 27.03 (29 C-80 CH3), 22.01, 22.47, 25.93, 26.
42, 29.04, 29.12, 29.28, 29.30, 30.17 (C-2–C-9 and C-60),
32.48 (C-70), 53.60 (C-100), 57.64 (C-1), 73.97 (C-10), 75.
92 (C-80), 111.87 (C-30), 113.32 (C-120), 118.62 (C-140),
119.11 (C-50), 134.93 (C-100), 146.38 (C-110), 150.92 (C-
130), 153.03 (C-40), 161.36 (C-20) ppm; HRMS: m/z [M]?
Calculated for C31H50NO4?: 500.3740; found: 500.3753.
1
78, 772.26, 643.73 cm-1; H NMR (400 MHz, CDCl3): d
1.20–1.30 (m, 15H, H-5–H-10 and H-200), 1.35-1.43 (m,
2H, H-4), 1.78–1.87 (m, 4H, H-3 and H-11), 2.20 (s, 3H,
H-20), 3.34–3.41 (m, 3H, H-2 and H-12), 4.18 (q, 2H, J =
7.32 Hz, H-100) ppm; 13C NMR (100.5 MHz CDCl3): d 14.
25 (C-200), 27.50, 28.28, 28.32, 28.85, 29.37, 29.43, 29.48,
32.95 (C-3–C-11 and C-20), 34.13 (C-12), 60.08 (C-2), 61.
39 (C-100), 170.08 (C-1), 203.47 (C-10) ppm; HRMS: m/z
[M ? Na]? Calculated for C16H29BrO3: 371.1198; found:
371.1198.
General procedure for the synthesis of ammonium alkyl
derivatives of pyranochromen-2-ones (17/18)
Synthesis of ethyl 2-acetyl-x-bromoalkanoates (2/
3) Sodium hydride (1.7 equivalent) was added to a cold
solution (0 °C) of ethyl 3-oxobutanoate (1) (5 g, 38.
42 mmol) in THF (15 ml) and the reaction mixture was
heated at 60 °C for 2 h. The contents of the flask were then
allowed to attain room temperature, further, addition of
1,6-dibromohexane/1,10-dibromodecane (1.1 equivalent)
dissolved in THF was carried out under ice cold conditions.
The resultant mixture thus obtained was heated at 80 °C for
Synthesis of 3-(x-bromoalkyl)-7,8-dihydroxy-4-methyl-2H-
chromen-2-ones (7/8) A mixture of pyrogallol (4) (3 g,
23.78 mmol) and 2/3 (1.1 equivalent) was taken in a round
bottom flask and the mixture was cooled to 0 °C. Conc.
H2SO4 (10 ml) was then added dropwise and the temper-
ature of the reaction mixture was maintained at 0 °C. The
reaction mixture was then stirred at room temperature for
6 h. On completion of the reaction, the contents of the flask
123

Med Chem Res
were poured over ice. The solid so obtained (7/8) was fil-
tered, washed with water, dried under vacuum and purified
by column chromatography.
reaction was monitored using TLC. On completion of the
reaction, the solvent was removed in vacuo and the solid
obtained was dissolved in chloroform (50 ml). The chlo-
roform layer was washed first with sodium hydroxide
solution (1 N, 2 9 50 ml) and then with brine solution
(20 ml). The organic layer was then dried over anhydrous
sodium sulphate. The solvent was removed under reduced
pressure and purified by column chromatography to give
the desired product (9/10).
3-(6-Bromohexyl)-7,8-dihydroxy-4-methyl-2H-chromen-2-
one (7) The reaction of pyrogallol (4) (3 g, 23.78 mmol)
with ethyl 2-acetyl-8-bromooctanoate (2) (7.67 g, 26.
16 mmol) gave the title compound 7 as a white solid (6.
59 g, 78 %) by following the general procedure; m. p.:
138.0–138.7 °C; Rf: 0.30 (MeOH:CHCl3: 1:19); UV
(MeOH) kmax: 262 and 323 nm; IR (KBr) mmax: 3422.44
(OH), 2929.20, 1671.08 (C=O), 1610.19, 1583.60, 1379.
83, 1306.96, 1090.04, 1053.73, 955.29, 814.43, 776.
3-(6-Bromohexyl)-10-hydroxy-4,8,8-trimethyl-7,8-dihydro-
pyrano[3,2-g]chromen-2(6H)-one (9) The reaction of
3-(6-bromohexyl)-7,8-dihydroxy-4-methyl-2H-chromen-2-
one (7) (2 g, 5.63 mmol) with 2-methyl-3-buten-2-ol (0.
73 g, 8.45 mmol) gave the title compound 9 as an off-
white solid (1.55 g, 65 %) by following the general pro-
cedure; m. p.: 123.8–124.2 °C; Rf: 0.30 (ethyl acetate:
petroleum ether: 3:7); UV (MeOH) kmax: 265 and 327 nm;
IR (KBr) mmax: 3380.20 (OH), 2924.18, 1675.32 (C=O),
1
52 cm-1; H NMR (400 MHz, CDCl3): d 1.38–1.58 (m,
6H, H-20–H-40), 1.82-1.91 (m, 2H, H-50), 2.38 (s, 3H, C-4
CH3), 2.63 (t, 2H, J = 7.63 Hz, H-10), 3.40 (t, 2H, J = 7.
02 Hz, H-60), 6.90 (d, 1H, J = 8.54 Hz, H-6), 7.09 (d, 1H,
J = 8.54 Hz, H-5) ppm; 13C NMR (100.5 MHz CDCl3): d
15.21 (C-4 CH3), 27.61, 28.17, 28.74, 28.94, 29.57, 29.61,
29.80 (C-10–C-40), 32.90 (C-50), 34.08 (C-60), 112.03 (C-6),
114.47 (C-10), 116.28 (C-5), 123.08 (C-3), 130.37 (C-8),
141.01 (C-4), 146.52 (C-7), 147.75 (C-9), 161.70 (C-2)
ppm; HRMS: m/z [M ? Na]? Calculated for C16H19BrO4:
377.0364; found: 377.0371.
1458.46, 1388.78, 1121.13, 1032.89, 930.27, 781.64 cm-1
;
1H NMR (400 MHz, CDCl3): d 1.36–1.57 (m, 12H, H-20–
H-40 and 29 C-8 CH3), 1.82-1.90 (m, 4H, H-50 and H-7), 2.
35 (s, 3H, C-4 CH3), 2.63 (t, 2H, J = 7.93 Hz, H-10), 2.84
(t, 2H, J = 6.71 Hz, H-6), 3.40 (t, 2H, J = 6.71 Hz, H-60),
5.60 (brs, 1H, OH), 6.87 (s, 1H, H-5) ppm; 13C NMR (100.
5 MHz CDCl3): d 15.08 (C-4 CH3), 22.32 (C-6), 27.01 (29
C-8 CH3), 27.64 (C-10), 28.18, 28.78, 28.91 (C-20–C-40),
32.91 (C-50 and C-7), 34.13 (C-60), 76.39 (C-8), 114.06 (C-
12), 114.90 (C-5), 117.65 (C-14), 123.62 (C-3), 131.98 (C-
10), 139.00 (C-11), 143.47 (C-4), 146.55 (C-13), 161.55
(C-2) ppm; HRMS: m/z [M ? Na]? Calculated for
C21H27BrO4: 445.0990; found: 445.1001.
3-(10-Bromodecyl)-7,8-dihydroxy-4-methyl-2H-chromen-
2-one (8) The reaction of pyrogallol (4) (3 g, 23.
78 mmol) with ethyl 2-acetyl-12-bromododecanoate (3) (9.
14 g, 26.16 mmol) gave the title compound 8 as an off-
white solid (7.73 g, 79 %) by following the general pro-
cedure; m. p.: 112.9–113.2 °C; Rf: 0.35 (MeOH:CHCl3: 1:
19); UV (MeOH) kmax: 262 and 324 nm; IR (KBr) mmax
:
3431.60 (OH), 2927.00, 1670.58 (C=O), 1610.35, 1581.83,
1379.22, 1307.47, 1089.07, 1055.91, 954.26, 811.76, 776.
1
3-(10-Bromodecyl)-10-hydroxy-4,8,8-trimethyl-7,8-dihy-
dropyrano[3,2-g]chromen-2(6H)-one (10) The reaction
of 3-(10-bromodecyl)-7,8-dihydroxy-4-methyl-2H-chro-
men-2-one (8) (2 g, 4.86 mmol) with 2-methyl-3-buten-2-
ol (0.63 g, 7.29 mmol) gave the title compound 10 as an
off-white solid (1.46 g, 63 %) by following the general
procedure; m. p.: 87.7–88.1 °C; Rf: 0.40 (ethyl acetate:
petroleum ether: 3:7); UV (MeOH) kmax: 266 and 329 nm;
IR (KBr) mmax: 3385.74 (OH), 2927.15, 2854.00, 1704.54
(C=O), 1622.97, 1580.91, 1466.46, 1390.52, 1121.57,
15, 526.67 cm-1; H NMR (400 MHz, CDCl3): d 1.23–1.
54 (m, 14 H, H-20–H-80), 1.82–1.89 (m, 2H, H-90), 2.38 (s,
3H, C-4 CH3), 2.62 (t, 2H, J = 8.05 Hz, H-10), 3.40 (t, 2H,
J = 6.59 Hz, H-100), 6.90 (d, 1H, J = 8.05 Hz, H-6), 7.09
(d, 1H, J = 8.05 Hz, H-5) ppm; 13C NMR (100.5 MHz
CDCl3): d 15.12 (C-4 CH3), 27.60, 28.20, 28.87, 29.54, 29.
57, 29.61, 29.80 (C-10–C-80), 32.95 (C-90), 34.21 (C-100),
112.23 (C-6), 114.40 (C-10), 115.95 (C-5), 122.89 (C-3),
130.71 (C-8), 141.19 (C-4), 146.97 (C-7), 148.12 (C-9),
162.50 (C-2) ppm; HRMS: m/z [M ? Na]? Calculated for
C20H27BrO4: 433.0990; found: 433.0980.
1054.72, 937.24, 783.79 cm-1
;
1H NMR (400 MHz,
CDCl3): d 1.24-1.55 (m, 20H, H-20–H-80 and 29 C-8 CH3),
1.80–1.90 (m, 4H, H-7 and H-90), 2.34 (s, 3H, C-4 CH3), 2.
61 (t, 2H, J = 8.05 Hz, H-10), 2.84 (t, 2H, J = 6.59 Hz,
H-6), 3.40 (t, 2H, J = 6.59 Hz, H-100), 6.86 (s, 1H, H-5)
ppm, 13C NMR (100.5 MHz CDCl3): d 14.98 (C-4 CH3),
22.23 (C-6), 26.93 (29 C-8 CH3), 27.69 (C-10), 28.22, 28.
81, 28.93, 29.46, 29.54, 29.71 (C-20–C-80), 32.79 (C-90),
32.90 (C-7), 34.18 (C-100), 76.28 (C-8), 114.00 (C-12),
114.81 (C-5), 117.56 (C-14), 123.74 (C-3), 131.87 (C-10),
Synthesis of 3-(x-bromoalkyl)-10-hydroxy-4,8,8-trimethyl-
7,8-dihydropyrano[3,2-g]chromen-2(6H)-ones (9/10) To
a stirred solution of 3-(x-bromoalkyl)-7,8-dihydroxy-4-
methyl-2H-chromen-2-ones (7/8) (1.0 g) and p-toluene-
sulfonic acid monohydrate (1 equivalent) in toluene was
added 1.5 equivalent of 2-methyl-3-buten-2-ol and the
reaction mixture refluxed for 30 h. The progress of the
123

Med Chem Res
138.88 (C-11), 143.34 (C-4), 146.40 (C-13), 161.48 (C-2)
ppm; HRMS: m/z [M ? Na]? Calculated for C25H35BrO4:
501.1617; found: 501.1611.
1466.10, 1389.38, 1369.68, 1121.52, 1056.00, 937.94, 783.
36, 752.11 cm-1; 1H NMR (400 MHz, CDCl3): d 1.20–1.50
(m, 29H, H-3–H-9, 2 x C-8 CH3 and 39 H-200), 1.63-1.72
(m, 2H, H-2), 1.83 (t, 2H, J = 6.59 Hz, H-70), 2.32 (s, 3H,
C-40 CH3), 2.57 (t, 2H, J = 8.05 Hz, H-10), 2.81 (t, 2H,
J = 6.59 Hz, H-60), 3.20–3.30 (m, 2H, H-1), 3.45–3.55 (m,
6H, H-100), 6.00 (brs, 1H, OH), 6.85 (s, 1H, H-50) ppm; 13C
NMR (100.5 MHz CDCl3): d 8.15 (C-200), 15.13 (C-40
CH3), 22.06 (C-60), 22.30 (C-10), 27.01 (2 x C-8 CH3), 26.
40, 27.43, 28.72, 28.92, 29.01, 29.23, 29.84, (C-2–C-9), 32.
87 (C-70), 53.60 (C-100), 57.64 (C-1), 76.44 (C-80), 114.11
(C-120), 114.87 (C-50), 117.67 (C-140), 123.60 (C-30), 132.
09 (C-100), 139.01 (C-110), 143.60 (C-40), 146.84 (C-130),
161.86 (C-20) ppm; HRMS: m/z [M]? Calculated for
C31H50NO4?: 500.3740; found: 500.3753.
Synthesis of N,N,N-triethyl-x-(10-hydroxy-4,8,8-trimethyl-
2-oxo-2,6,7,8-tetrahydropyrano[3,2-g]chromen-3-yl)al-
kan-1-aminium bromide (17/18) Triethylamine (5 equiv-
alents) was added to a solution of 9 (1 g, 2.36 mmol)/10
(1 g, 2.08 mmol) in anhydrous acetonitrile (50 ml) and the
reaction mixture was stirred under nitrogen at 60 °C for
96 h. The progress of reaction was monitored using TLC.
After completion of the reaction, the solvent was evapo-
rated under reduced pressure, followed by column chro-
matography of the resultant product to give the pure
quaternary ammonium compound (17/18) in moderate
yields.
General procedure for the synthesis
N,N,N-Triethyl-6-(10-hydroxy-4,8,8-trimethyl-2-oxo-2,6,7,8-
tetrahydropyrano[3,2-g]chromen-3-yl)hexan-1-aminium
bromide (17) The reaction of 3-(6-bromohexyl)-10-
hydroxy-4,8,8-trimethyl-7,8-dihydropyrano[3,2-g]chro-
men-2(6H)-one (9) (1 g, 2.36 mmol) with triethylamine
(1.20 g, 11.80 mmol) gave the title compound 17 as a
brown low melting solid (0.73 g, 70 %) by following
the general procedure; Rf: 0.38 (MeOH:CHCl3: 1:4);
of (coumarinyloxy)acetamide derivatives (32–47)
To a stirred solution of (coumarinyloxy)acetate (27–31)
(1 g) in n-butanol (20 ml) was added aminoalkane/N,N-
diaminoalkane (4 equivalent) and the reaction was stir-
red at room temperature for 48 h. The progress of
reaction was monitored on TLC and on completion of the
reaction, the solvent was removed in vacuo. The solid so
obtained was washed with hexane (3 9 20 ml) and
crystallised from ethanol/ethyl acetate–petroleum ether
to give (coumarinyloxy)acetamides as off-white/white/
brown solid.
UV (MeOH) kmax: 264 and 329 nm; IR (KBr) mmax
3387.59, 2927.26, 2854.88, 1698.02 (C=O), 1619.60,
:
1579.03, 1467.63, 1390.63, 1370.34, 1122.01, 1054.68,
938.90, 753.43, 660.97 cm-1
;
1H NMR (400 MHz,
CDCl3): d 1.16.1.44 (m, 23H, H-2–H-5, 29 C-8 CH3
and 39 H-200), 1.80 (t, 2H, J = 6.59 Hz, H-70), 2.27 (s,
3H, C-40 CH3), 2.51 (t, 2H, J = 7.69 Hz, H- 6), 2.78 (t,
2H, J = 6.59 Hz, H- 60), 3.21-3.30 (m, 2H, H-1) 3.40–3.
50 (m, 6H, H-100), 6.81 (s, 1H, H-50) ppm; 13C NMR
(100.5 MHz CDCl3): d 8.15 (C-200), 15.16 (C-40 CH3),
21.74 (C-60), 22.27 (C-6), 27.01 (2 x C-8 CH3), 25.98,
27.09, 28.17, 28.63 (C-2– C-5), 32.87 (C-70), 53.56 (C-
100), 57.65 (C-1), 76.52 (C-80), 114.04 (C-120), 115.04
(C-50), 117.76 (C-140), 123.07 (C-30), 132.06 (C-100),
138.98 (C-110), 143.66 (C-40), 147.23 (C-130), 161.85
(C-20) ppm; HRMS: m/z [M]? Calculated for
C27H42NO4?: 444.3114; found: 444.3118.
N-(3-(Diethylamino)propyl)-2-((2-oxo-2H-chromen-4-yl)
oxy)acetamide (32) The reaction of ethyl 2-((2-oxo-2H-
chromen-4-yl)oxy)acetate (27) (1 g, 4.03 mmol) with
N1,N1-diethylpropane-1,3-diamine (2.09 g, 16.12 mmol)
gave the title compound 32 as a light brown solid (0.92 g,
69 %) by following the general procedure; m. p.: 105.
4–106.1 °C; Rf: 0.42 (MeOH:CHCl3: 1:49); UV (MeOH)
kmax: 266 nm; IR (KBr) mmax: 3293.20 (N–H str), 2966.72,
1717.49 (C=O), 1654.36 (NHCO–), 1623.56, 1549.98,
1248.95, 1112.80, 1018.36, 934.79, 855.76, 769.16, 605.
22 cm-1; 1H NMR (400 MHz, CDCl3): d 0.93 (t, 6H, J =
7.32 Hz, H-20000), 1.67–1.73 (m, 2H, H-200), 2.42 (q, 4H,
J = 7.32, Hz, H-10000), 2.53 (t, 2H, J = 5.86 Hz, H-300), 3.
51–3.47 (m, 2H, H-100), 4.65 (s, 2H, H-2), 5.70 (s, 1H,
H-30), 7.28–7.36 (m, 2H, H-60 and H-80), 7.58–7.62 (m, 1H,
H-70), 7.85–7.87 (m, 1H, H-50), 8.30 (brs, 1H, NH) ppm;
N,N,N-Triethyl-10-(10-hydroxy-4,8,8-trimethyl-2-oxo-2,6,7,8-
tetrahydropyrano[3,2-g]chromen-3-yl)decan-1-aminium bro-
mide (18) The reaction of 3-(10-Bromodecyl)-10-
hydroxy-4,8,8-trimethyl-7,8-dihydropyrano[3,2-g]chromen-
2(6H)-one (10) (1 g, 2.09 mmol) with triethylamine
(1.06 g, 10.45 mmol) gave the title compound 18 as a
brown solid (0.83 g, 69 %) by following the general pro-
cedure; m. p.: 83.6–84.9 °C; Rf: 0.40 (MeOH:CHCl3: 1:4);
UV (MeOH) kmax: 266 and 328 nm; IR (KBr) mmax: 3385.
86, 2929.96, 2857.37, 1701.41 (C=O), 1617.97, 1578.18,
13
C NMR (100.5 MHz, CDCl3): d 11.28 (C-20000), 25.20 (C-
200), 40.12 (C-100), 47.14 (C-10000), 52.00 (C-300), 67.80 (C-2),
91.83 (C-30), 114.99 (C-60), 116.94 (C-80), 122.77 (C-100),
123.80 (C-70), 132.84 (C-50), 153.31 (C-90), 162.10, 164.26
and 165.25 (C-20, C-1 and C-40) ppm; HRMS: m/z
[M ? Na]? Calculated for C18H24N2O4: 355.1634; found:
355.1634.
123

Med Chem Res
N-(2-Ethylhexyl)-2-((2-oxo-2H-chromen-4-yl)oxy)acetam-
ide (33) The reaction of ethyl 2-((2-oxo-2H-chromen-4-
yl)oxy)acetate (27) (1 g, 4.03 mmol) with 2-ethylhexan-1-
amine (2.08 g, 16.12 mmol) gave the title compound 33 as
a white solid (1 g, 75 %) by following the general proce-
dure; m. p.: 123.6–124.3 °C; Rf: 0.32 (MeOH:CHCl3: 1:
49); UV (MeOH) kmax: 266 nm; IR (KBr) mmax: 3349.29
(N–H str), 3087.88, 2929.24, 1718.03 (C=O), 1654.64
(NHCO–), 1624.09, 1545.03, 1248.32, 1113.84, 1025.39,
937.60, 859.31, 771.85, 594.68 cm-1; 1H NMR (400 MHz,
CDCl3): d 0.89 (t, 3H, J = 5.86 Hz, H-600), 0.91 (t, 3H,
J = 8.05 Hz, H-20000), 1.28–1.38 (m, 8H, H-300– H-500 and
H-10000), 1.48–1.54 (m, 1H, H-200), 3.32–3.35 (m, 2H, H-100),
4.64 (s, 2H, H-2), 5.70 (s, 1H, H-30), 6.32 (brs, 1H, NH), 7.
30–7.36 (m, 2H, H-60 and H-80), 7.58–7.61 (m, 1H, H-70),
7.75–7.77 (m, 1H, H-50) ppm; 13C NMR (100.5 MHz,
CDCl3): d 10.85 (C-600), 13.98 (C-20000), 22.95 (C-500), 24.30
(C-10000), 28.77 (C-400), 31.02 (C-300), 39.25 (C-200), 42.06
(C-100), 67.57 (C-2), 92.00 (C-30), 114.86 (C-80), 117.13 (C-
60), 122.26 (C-100), 124.15 (C-70), 132.93 (C-50), 153.34
(C-90), 161.92, 163.83 and 165.41 (C-20, C-1 and C-40)
ppm; HRMS: m/z [M ? Na]? Calculated for C19H25NO4:
354.1681; found: 354.1688.
15.24 mmol) gave the title compound 35 as a white solid
(1.21 g, 79 %) by following the general procedure; m. p.:
129.5–130.8 °C; Rf: 0.41 (MeOH:CHCl3: 1:49); UV
(MeOH) kmax: 290 and 317 nm; IR (KBr) mmax: 3383.32
(N–H str), 2954.60, 1708.46 (C=O), 1666.97 (NHCO–),
1629.14, 1545.91, 1153.54, 1081.08, 845.86, 595.42 cm-1
;
1H NMR (400 MHz, CDCl3): d 0.79–0.85 (m, 6H, H-40000),
1.16–1.27 (m, 4H, H-30000), 1.31–1.40 (m, 4H, H-20000), 1.
61–1.69 (m, 2H, H-200), 2.31–2.37 (m, 4H, H-10000), 2.39 (s,
3H, C-40 CH3), 2.46–2.52 (m, 2H, H-300), 3.40–3.47 (m,
2H, H-100), 4.51 (s, 2H, H-2), 6.15 (s, 1H, H-30), 6.81–6.88
(m, 2H, H-80 and H-60), 7.51–7.55 (m, 1H, H-50), 8.27 (brs,
1H, NH) ppm; 13C NMR (100.5 MHz, CDCl3): d 14.20 (C-
40000), 18.80 (C-40 CH3), 20.88 (C-30000), 25.60 (C-200), 29.05
(C-20000), 40.02 (C-100), 53.70 (C-300), 54.29 (C-10000), 67.86
(C-2), 102.78 (C-80), 111.64 (C-60), 112.86 (C-30), 114.74
(C-100), 126.05 (C-50), 152.36 (C-40), 155.23 and 160.39
(C-90 and C-70), 161.04 and 167.01 (C-20 and C-1) ppm;
HRMS: m/z [M ? Na]? Calculated for C23H34N2O4: 425.
2416; found: 425.2402.
N-(2-Hydroxyethyl)-2-((4-methyl-2-oxo-2H-chromen-7-yl)
oxy)acetamide (36) (Wang et al., 2011) The reaction of
ethyl
2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetate
N-(3-(Diethylamino)propyl)-2-((4-methyl-2-oxo-2H-chro-
men-7-yl)oxy)acetamide (34) The reaction of ethyl 2-((4-
methyl-2-oxo-2H-chromen-7-yl)oxy)acetate (28) (1 g, 3.
81 mmol) with N1,N1-diethylpropane-1,3-diamine (1.98 g,
15.24 mmol) gave the title compound 34 as a white solid
(1.08 g, 82 %) by following the general procedure; m. p.:
106.2–107.7 °C; Rf: 0.40 (MeOH:CHCl3: 1:49); UV
(MeOH) kmax: 292 and 318 nm; IR (KBr) mmax: 3255.61
(N–H str), 2956.53, 1730.14 (C=O), 1679.02 (NHCO–),
1617.18, 1527.43, 1276.70, 1158.08, 1073.82, 846.35, 735.
(28) (1 g, 3.81 mmol) with 2-aminoethanol (0.97 g, 15.
24 mmol) gave the title compound 36 as a white solid (0.
68 g, 65 %) by following the general procedure; m. p.:
188.2–188.8 °C; Rf: 0.25 (MeOH:CHCl3: 1:49); UV
(MeOH) kmax: 291 and 318 nm; IR (KBr) mmax: 3396.13
(O–H str), 3309.01 (N–H str), 2922.84, 1700.11 (C=O),
1659.28 (NHCO–), 1615.46, 1515.50, 1285.30, 1146.79,
991.46, 853.63, 754.94, 505.12 cm-1; 1H NMR (400 MHz,
DMSO-d6): d 2.38 (s, 3H, C-40 CH3), 3.18-3.23 (m, 2H,
H-100), 3.40–3.46 (m, 2H, H-200), 4.60 (s, 2H, H-2), 4.70–4.
75 (m, 1H, OH), 6.21 (s, 1H, H-30), 6.95 (d, 1H, J = 2.
92 Hz, H-80), 6.99 (dd, 1H, J = 2.96 and 8.80 Hz, H-60), 7.
69 (d, 1H, J = 8.80 Hz, H-50), 8.11 (brs, 1H, NH) ppm;
13C NMR (100.5 MHz, DMSO-d6): d 18.13 (C-40 CH3),
41.23 (C-100), 59.60 (C-200), 67.14 (C-2), 101.65 (C-80),
111.43 (C-60), 112.44 (C-30), 113.60 (C-100), 126.51 (C-50),
153.37 (C-40), 154.50 and 160.07 (C-90 and C-70), 160.66
and 167.01 (C-20 and C-1) ppm; HRMS: m/z [M ? Na]?
Calculated for C14H15NO5: 300.0848; found: 300.0852.
1
40, 589.81 cm-1; H NMR (400 MHz, CDCl3): d 1.00 (t,
6H, J = 7.32 Hz, H-20000), 1.65–1.73 (m, 2H, H-200), 2.40
(d, 3H, J = 1.48 Hz, C-40 CH3), 2.48–2.56 (m, 6H, H-10000
and H-300), 3.43–3.49 (m, 2H, H-100), 4.52 (s, 2H, H-2), 6.16
(d, 1H, J = 1.48 Hz, H-30), 6.83 (d, 1H, J = 2.20 Hz,
H-80), 6.87 (dd, 1H, J = 2.92 and 8.80 Hz, H-60), 7.54 (d,
1H, J = 8.76 Hz, H-50), 8.47 (brs, 1H, NH) ppm; 13C
NMR (100.5 MHz, CDCl3): d 11.34 (C-20000), 18.64 (C-40
CH3), 25.11 (C-200), 39.66 (C-100), 46.92 (C-10000), 52.21 (C-
300), 67.58 (C-2), 102.58 (C-80), 111.44 (C-60), 112.66 (C-
30), 114.57 (C-100), 125.94 (C-50), 152.23 (C-40), 155.04
and 160.16 (C-90 and C-70), 160.93 and 166.89 (C-20 and
C-1) ppm; HRMS: m/z [M ? Na]? Calculated for
C19H26N2O4: 369.1790; found: 369.1789.
N-(3-(Diethylamino)propyl)-2-((3-ethyl-4-methyl-2-oxo-2H-
chromen-7-yl)oxy)acetamide (37) The reaction of ethyl
2-((3-ethyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetate
(29) (1 g, 3.44 mmol) with N1,N1-diethylpropane-1,3-dia-
mine (1.79 g, 13.76 mmol) gave the title compound 37 as a
white solid (1.04 g, 81 %) by following the general pro-
cedure; m. p.: 122.1–122.9 °C; Rf: 0.40 (MeOH:CHCl3: 1:
N-(3-(Dibutylamino)propyl)-2-((4-methyl-2-oxo-2H-chro-
men-7-yl)oxy)acetamide (35) The reaction of ethyl 2-((4-
methyl-2-oxo-2H-chromen-7-yl)oxy)acetate (28) (1 g, 3.
81 mmol) with N1,N1-dibutylpropane-1,3-diamine (2.83 g,
49); UV (MeOH) kmax: 291 and 318 nm; IR (KBr) mmax
:
3375.70 (N–H str), 2964.89, 1712.57 (C=O), 1661.90
123

Med Chem Res
(NHCO–), 1618.23, 1545.43, 1161.61, 1096.47, 872.25,
778.04, 596.54 cm-1; 1H NMR (400 MHz, CDCl3): d 1.00
(t, 6H, J = 7.32 Hz, H-20000), 1.13 (t, 3H, J = 7.32 Hz,
H-2000), 1.65-1.72 (m, 2H, H-200), 2.38 (s, 3H, C-40 CH3), 2.
48–2.55 (m, 6H, H-10000 and H-300), 2.66 (q, 2H, J = 7.
32 Hz, H-1000), 3.43–3.47 (m, 2H, H-100), 4.50 (s, 2H, H-2),
6.80 (d, 1H, J = 2.20 Hz, H-80), 6.84 (dd, 1H, J = 2.20
and 8.80 Hz, H-60), 7.53 (d, 1H, J = 8.80 Hz, H-50), 8.44
(brs, 1H, NH) ppm; 13C NMR (100.5 MHz, CDCl3): d 11.
61 (C-20000), 13.26 (C-2000), 14.73 (C-40 CH3), 21.02 (C-1000),
25.38 (C-200), 39.88 (C-100), 47.13 (C-10000), 52.45 (C-300),
67.77 (C-2), 102.47 (C-80), 111.35 (C-60), 115.52 (C-100),
125.91 (C-30), 125.95 (C-50), 145.62 (C-40), 153.64 and
159.29 (C-90 and C-70), 161.87 and 167.23 (C-20 and C-1)
ppm; HRMS: m/z [M ? Na]? Calculated for C21H30N2O4:
397.2103; found: 397.2093.
UV (MeOH) kmax: 292 and 318 nm; IR (KBr) mmax: 3402.
94 (O–H str), 3302.37 (N–H str), 2959.90, 2914.37, 1692.
92 (C=O), 1649.99 (NHCO–), 1609.72, 1553.78, 1258.79,
1181.98, 1095.72, 863.67, 779.60, 600.59 cm-1; 1H NMR
(400 MHz, DMSO-d6): d 1.02 (t, 3H, J = 7.32 Hz,
H-2000), 2.36 (s, 3H, C-40 CH3), 2.54 (q, 2H, J = 7.32 Hz,
H-1000), 3.18–3.22 (m, 2H, H-100), 3.42 (brm, 2H, H-200), 4.
58 (s, 2H, H-2), 4.74 (brs, 1H, OH), 6.92 (d, 1H, J = 2.
92 Hz, H-80), 6.97 (dd, 1H, J = 2.20 and 8.80 Hz, H-60),
7.70 (d, 1H, J = 8.76 Hz, H-50), 8.11 (brs, 1H, NH) ppm;
13C NMR (100.5 MHz, DMSO-d6): d 12.95 (C-2000), 14.35
(C-40 CH3), 20.22 (C-1000), 41.23 (C-100), 59.61 (C-200), 67.
13 (C-2), 101.35 (C-80), 112.33 (C-60), 114.16 (C-100),
124.02 (C-30), 126.36 (C-50), 146.42 (C-40), 152.88 and
159.68 (C-90 and C-70), 160.72 and 167.09 (C-20 and C-1)
ppm; HRMS: m/z [M ? Na]? Calculated for C16H19NO5:
328.1161; found: 328.1151.
N-(3-(Dibutylamino)propyl)-2-((3-ethyl-4-methyl-2-oxo-
2H-chromen-7-yl)oxy)acetamide (38) The reaction of
ethyl 2-((3-ethyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)
acetate (29) (1 g, 3.44 mmol) with N1,N1-dibutylpropane-
1,3-diamine (2.56 g, 13.76 mmol) gave the title compound
38 as a white solid (1.23 g, 83 %) by following the general
procedure; m. p.: 121.3–130.0 °C; Rf: 0.42 (MeOH:CHCl3:
2-((3-Ethyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N-octy-
lacetamide (40) The reaction of ethyl 2-((3-ethyl-4-
methyl-2-oxo-2H-chromen-7-yl)oxy)acetate (29) (1 g, 3.
44 mmol) with octan-1-amine (1.77 g, 13.76 mmol) gave
the title compound 40 as a white solid (1.02 g, 79 %) by
following the general procedure; m. p.: 140.2–141.0 °C;
Rf: 0.41 (MeOH:CHCl3: 1:49); UV (MeOH) kmax: 293 and
318 nm; IR (KBr) mmax: 3372.19 (N–H str), 2927.59, 1709.
18 (C=O), 1664.71 (NHCO–), 1618.08, 1559.72, 1154.82,
1:49); UV (MeOH) kmax: 292 and 318 nm; IR (KBr) mmax
:
3371.69 (N–H str), 2957.40, 1712.48 (C=O), 1661.03
(NHCO–), 1618.49, 1546.92, 1160.04, 1095.98, 870.93,
777.91, 599.61 cm-1; 1H NMR (400 MHz, CDCl3): d 0.82
(t, 6H, J = 7.32 Hz, H-40000), 1.12 (t, 3H, J = 7.36 Hz,
H-2000), 1.16–1.26 (m, 4H, H-30000), 1.32–1.39 (m, 4H,
H-20000), 1.63–1.69 (m, 2H, H-200), 2.34 (t, 4H, J = 8.08 Hz,
H-10000), 2.37 (s, 3H, C-40 CH3), 2.50 (t, 2H, J = 6.24 Hz,
H-300), 2.65 (q, 2H, J = 7.32 Hz, H-1000), 3.41–3.47 (m, 2H,
H-100), 4.51 (s, 2H, H-2), 6.80 (d, 1H, J = 2.20 Hz, H-80),
6.84 (dd, 1H, J = 2.92 and 8.76 Hz, H-60), 7.52 (d, 1H,
J = 8.80 Hz, H-50), 8.26 (brs, 1H, NH) ppm; 13C NMR
(100.5 MHz, CDCl3): d 13.22 (C-2000), 14.20 (C-40000), 14.70
(C-40 CH3), 20.87 (C-30000), 21.00 (C-1000), 25.60 (C-200), 28.
91 (C-20000), 39.91 (C-100), 53.58 (C-300), 54.24 (C-10000), 67.
82 (C-2), 102.43 (C-80), 111.44 (C-60), 115.51 (C-100), 125.
87 (C-30), 125.92 (C-50), 145.57 (C-40), 153.64 and 159.33
(C-90 and C-70), 161.82 and 167.27 (C-20 and C-1) ppm;
HRMS: m/z [M ? Na]? Calculated for C25H38N2O4: 453.
2729; found: 453.2729.
1096.29, 872.47, 715.80, 601.06 cm-1
;
1H NMR
(400 MHz, CDCl3): d 0.86 (t, 3H, J = 6.6 Hz, H-800), 1.13
(t, 3H, J = 7.36 Hz, H-2000), 1.21–1.32 (m, 10H, H-700–H-
300), 1.51-1.58 (m, 2H, H-200), 2.38 (s, 3H, C-40 CH3), 2.66
(q, 2H, J = 7.32 Hz, H-1000), 3.31–3.38 (m, 2H, H-100), 4.52
(s, 2H, H-2), 6.54 (brs, 1H, NH), 6.83 (d, 1H, J = 2.96 Hz,
H-80), 6.86 (dd, 1H, J = 2.16 and 8.80 Hz, H-60), 7.54 (d,
1H, J = 8.80 Hz, H-50) ppm; 13C NMR (100.5 MHz,
CDCl3): d 13.25 (C-800), 14.26 (C-2000), 14.74 (C-40 CH3),
21.04 (C-1000), 22.79 (C-700), 27.02 (C-600), 29.34 and 29.36
(C-500 and C-400), 29.68 (C-300), 31.92 (C-200), 39.37 (C-100),
67.62 (C-2), 102.49 (C-80), 111.46 (C-60), 115.72 (C-100),
125.99 (C-30), 126.07 (C-50), 145.58 (C-40), 153.62 and
158.91 (C-90 and C-70), 161.81 and 167.31 (C-20 and C-1)
ppm; HRMS: m/z [M ? Na]? Calculated for C22H31NO4:
396.2151; found: 396.2162.
N-(3-(Dimethylamino)propyl)-2-((3-ethyl-4-methyl-2-oxo-
2H-chromen-7-yl)oxy)acetamide (41) The reaction of
ethyl 2-((3-ethyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)
acetate (29) (1 g, 3.44 mmol) with N1,N1-dimethylpropane-
1,3-diamine (1.40 g, 13.76 mmol) gave the title compound
41 as a white solid (0.95 g, 80 %) by following the general
procedure; m. p.: 136.6–137.5 °C; Rf: 0.35 (MeOH:CHCl3::
2-((3-Ethyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N-(2-
hydroxyethyl)acetamide (39) The reaction of ethyl
2-((3-ethyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetate
(29) (1 g, 3.44 mmol) with 2-aminoethanol (0.84 g, 13.
76 mmol) gave the title compound 39 as an off-white
solid (0.65 g, 62 %) by following the general procedure;
m. p.: 192.3–193.1 °C; Rf: 0.25 (MeOH:CHCl3: 1:49);
1:49); UV (MeOH) kmax: 290 and 317 nm; IR (KBr) mmax
:
123

Med Chem Res
3375.80 (N–H str), 2966.80, 1713.47 (C=O), 1666.42
(NHCO–), 1618.60, 1549.02, 1159.70, 1095.12, 871.43,
777.79, 599.98 cm-1; 1H NMR (400 MHz, CDCl3): d 1.12
(t, 3H, J = 7.32 Hz, H-2000), 1.65–1.71 (m, 2H, H-200), 2.18
(s, 6H, H-10000), 2.38 (s, 3H, C-40 CH3), 2.40 (t, 2H, J = 5.
84 Hz, H-300), 2.65 (q, 2H, J = 7.32 Hz, H-1000), 3.41–3.47
(m, 2H, H-100), 4.50 (s, 2H, H-2), 6.79 (d, 1H, J = 2.92 Hz,
H-80), 6.83 (dd, 1H, J = 2.92 and 8.80 Hz, H-60), 7.53 (d,
1H, J = 8.80 Hz, H-50), 8.31 (brs, 1H, NH) ppm; 13C NMR
(100.5 MHz, CDCl3): d 13.24 (C-2000), 14.72 (C-40 CH3), 21.
01 (C-1000), 25.59 (C-200), 39.55 (C-100), 45.48 (C-10000), 58.99
(C-300), 67.66 (C-2), 102.36 (C-80), 111.34 (C-60), 115.50 (C-
100), 125.89 (C-30), 125.95 (C-50), 145.61 (C-40), 153.64 and
159.29 (C-90 and C-70), 161.82 and 167.31 (C-20 and C-1)
ppm; HRMS: m/z [M ? Na]? Calculated for C19H26N2O4:
369.1790; found: 369.1789.
(MeOH:CHCl3: 1:49); UV (MeOH) kmax: 318 nm; IR
(KBr) mmax: 3373.96 (N–H str), 2923.56, 1709.94 (C=O),
1662.74 (NHCO–), 1618.85, 1550.06, 1430.12, 1285.53,
1
1162.34, 1090.23, 871.91, 780.88, 601.50 cm-1; H NMR
(400 MHz, CDCl3): d 0.88 (t, 3H, J = 6.96 Hz, H-6000), 1.
29–1.31 (m, 4H, H-4000 and H-5000), 1.36–1.41 (m, 2H,
H-3000), 1.47–1.54 (m, 2H, H-2000), 1.66–1.70 (m, 2H, H-200),
2.18 (s, 6H, H-10000), 2.38 (s, 3H, C-40 CH3), 2.39 (t, 2H,
J = 5.86 Hz, H-300), 2.62 (t, 2H, J = 8.05 Hz, H-1000),
3.42–3.47 (m, 2H, H-100), 4.51 (s, 2H, H-2), 6.80 (d, 1H,
J = 2.20 Hz, H-80), 6.83 (dd, 1H, J = 2.20 and 8.79 Hz,
H-60), 7.53 (d, 1H, J = 8.79 Hz, H-50), 8.33 (brs, 1H, NH)
13
ppm; C NMR (100.5 MHz, CDCl3): d 14.02 (C-6000), 14.
79 (C-40 CH3), 22.56 (C-5000), 25.43 (C-200), 27.53 (C-4000),
28.70 (C-3000), 29.31 (C-2000), 31.64 (C-1000), 39.43 (C-100),
45.34 (C-10000), 58.86 (C-300), 67.46 (C-2), 102.15 (C-80),
111.06 (C-60), 115.30 (C-100), 124.57 (C-30), 125.73 (C-50),
145.56 (C-40), 153.43 and 159.06 (C-90 and C-70), 161.74
and 167.08 (C-20 and C-1) ppm; HRMS: m/z [M ? Na]?
Calculated for C23H34N2O4: 425.2416; found: 425.2402.
N-(3-(Diethylamino)propyl)-2-((3-hexyl-4-methyl-2-oxo-
2H-chromen-7-yl)oxy)acetamide (42) The reaction of
ethyl 2-((3-hexyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)
acetate (30) (1 g, 2.88 mmol) with N1,N1-diethylpropane-
1,3-diamine (1.50 g, 11.52 mmol) gave the title compound
42 as a white solid (1 g, 81 %) by following the general
procedure; m. p.: 115.6–117.2 °C; Rf: 0.48 (MeOH:CHCl3:
1:49); UV (MeOH) kmax: 319 nm; IR (KBr) mmax: 3370.84
(N–H str), 2922.59, 1710.76 (C=O), 1662.03 (NHCO–),
1618.44, 1549.66, 1426.54, 1286.31, 1160.00, 1088.95,
781.66, 599.06 cm-1; 1H NMR (400 MHz, CDCl3): d 0.86
(t, 3H, J = 6.87 Hz, H-6000), 0.98 (t, 6H, J = 7.33 Hz,
H-20000), 1.27–1.31 (m, 4H, H-4000 and H-5000), 1.33–1.40 (m,
2H, H-3000), 1.45–1.53 (m, 2H, H-2000), 1.63–1.69 (m, 2H,
H-200), 2.36 (s, 3H, C-40 CH3), 2.47 (q, 4H, J = 7.33 Hz,
H-10000), 2.51 (t, 2H, J = 5.95 Hz, H-300), 2.61 (t, 2H, J = 7.
33 Hz, H-1000), 3.42–3.46 (m, 2H, H-100), 4.49 (s, 2H, H-2),
6.79 (d, 1H, J = 2.29 Hz, H-80), 6.68 (dd, 1H, J = 2.29
and 8.70 Hz, H-60), 7.52 (d, 1H, J = 8.70 Hz, H-50), 8.47
(brs, 1H, NH) ppm; 13C NMR (100.5 MHz, CDCl3): d 11.
52 (C-20000), 14.04 (C-6000), 14.80 (C-40 CH3), 22.57 (C-5000),
25.19 (C-200), 27.53 (C-4000), 28.71 (C-3000), 29.31 (C-2000),
31.65 (C-1000), 39.82 (C-100), 46.95 (C-10000), 52.32 (C-300),
67.55 (C-2), 102.26 (C-80), 111.04 (C-60), 115.30 (C-100),
124.56 (C-30), 125.73 (C-50), 145.59 (C-40), 153.41 and
159.03 (C-90 and C-70), 161.79 and 166.96 (C-20 and C-1)
ppm; HRMS: m/z [M ? Na]? Calculated for C25H38N2O4:
453.2729; found: 453.2729.
N-(2-(Dimethylamino)ethyl)-2-((3-hexyl-4-methyl-2-oxo-
2H-chromen-7-yl)oxy)acetamide (44) The reaction of
ethyl 2-((3-hexyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)
acetate (30) (1 g, 2.88 mmol) with N1,N1-dimethylethane-
1,2-diamine (1.01 g, 11.52 mmol) gave the title compound
44 as a white solid (0.89 g, 79 %) by following the general
procedure; m. p.: 133.4–135.0 °C; Rf: 0.48 (MeOH:CHCl3:
1:49); UV (MeOH) kmax: 319 nm; IR (KBr) mmax: 3372.39
(N–H str), 2923.11, 1709.01 (C=O), 1661.57 (NHCO–),
1619.11, 1552.59, 1430.03, 1287.14, 1160.80, 1089.68,
869.87, 781.53, 602.82, 533.51 cm-1; 1H NMR (400 MHz,
CDCl3): d 0.86 (t, 3H, J = 7.32 Hz, H-6000), 1.28–1.31 (m,
4H, H-4000 and H-5000), 1.35–1.46 (m, 2H, H-3000), 1.47–1.54
(m, 2H, H-2000), 2.22 (s, 6H, H-10000), 2.37 (s, 3H, C-40 CH3),
2.43 (t, 2H, J = 5.86 Hz, H-200), 2.62 (t, 2H, J = 8.05 Hz,
H-1000), 3.39–3.43 (m, 2H, H-100), 4.53 (s, 2H, H-2), 6.83 (d,
1H, J = 2.20 Hz, H-80), 6.87 (dd, 1H, J = 2.20 and 8.
75 Hz, H-60), 7.04 (brs, 1H, NH), 7.52 (d, 1H, J = 8.
75 Hz, H-50) ppm; 13C NMR (100.5 MHz, CDCl3): d 14.10
(C-6000), 14.78 (C-40 CH3), 22.55 (C-5000), 27.52 (C-4000), 28.
68 (C-3000), 29.30 (C-2000), 31.63 (C-1000), 36.46 (C-100), 45.
14 (C-10000), 57.69 (C-200), 67.49 (C-2), 102.21 (C-80), 111.
49 (C-60), 115.39 (C-100), 124.63 (C-30), 125.66 (C-50),
145.54 (C-40), 153.39 and 158.85 (C-90 and C-70), 161.70
and 167.21 (C-20 and C-1) ppm; HRMS: m/z [M ? Na]?
Calculated for C22H32N2O4: 411.2260; found: 411.2260.
N-(3-(Dimethylamino)propyl)-2-((3-hexyl-4-methyl-2-oxo-
2H-chromen-7-yl)oxy)acetamide (43) The reaction of
ethyl 2-((3-hexyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)
acetate (30) (1 g, 2.88 mmol) with N1,N1-dimethylpro-
pane-1,3-diamine (1.17 g, 11.52 mmol) gave the title
compound 43 as a white solid (0.93 g, 80 %) by following
the general procedure; m. p.: 136.0–137.1 °C; Rf: 0.47
N-(3-(Diethylamino)propyl)-2-((4-methyl-2-oxo-2H-chro-
men-6-yl)oxy)acetamide(45) The reaction of ethyl 2-((4-
methyl-2-oxo-2H-chromen-6-yl)oxy)acetate (31) (1 g, 3.
81 mmol) with N1,N1-diethylpropane-1,3-diamine (1.98 g,
123

Med Chem Res
15.24 mmol) gave the title compound 45 as a white solid
(1 g, 76 %) by following the general procedure; m. p.: 107.
2–108.9 °C; Rf: 0.44 (MeOH:CHCl3: 1:49); UV (MeOH)
kmax: 272 nm; IR (KBr) mmax: 3483.31 (N–H str), 3266.03,
2831.81, 1711.00 (C=O), 1652.01 (NHCO–), 1573.45, 1552.
15, 1431.37, 1246.74, 1168.79, 1060.91, 930.85, 837.95,
607.85, 548.75 cm-1; 1H NMR (400 MHz, CDCl3): d 2.20
(s, 6H, H-10000), 2.39 (s, 3H, C-40 CH3), 2.43 (t, 2H, J = 5.
95 Hz, H-200), 3.38–3.43 (m, 2H, H-100), 4.52 (s, 2H, H-2), 6.
29 (s, 1H, H-30), 7.04 (d, 1H, J = 2.75 Hz, H-50), 7.08 (brs,
1H, NH), 7.14 (dd, 1H, J = 2.75 and 9.16 Hz, H-70), 7.27 (d,
1H, J = 9.16 Hz, H-80) ppm; 13C NMR (100.5 MHz,
CDCl3): d 18.64 (C-40 CH3), 36.45 (C-100), 45.15 (C-10000), 57.
78 (C-200), 68.02 (C-2), 108.77 (C-50), 115.78 (C-30), 118.27
(C-70), 119.46 (C-80), 120.55 (C-100), 148.54, 151.69 and
153.61 (C-90, C-60 and C-40), 160.63 and 167.62 (C-20 and
C-1) ppm; HRMS: m/z [M ? Na]? Calculated for
C16H20N2O4: 327.1321; found: 327.1331.
k
max: 318 nm; IR (KBr) mmax: 3249.59 (N–H str), 2956.25,
1728.41 (C=O), 1678.24 (NHCO–), 1615.95, 1531.30,
1393.28, 1275.85, 1157.55, 1073.27, 846.08, 589.64 cm-1
;
1H NMR (400 MHz, CDCl3): d 0.98 (t, 6H, J = 7.33 Hz,
H-20000), 1.63–1.69 (m, 2H, H-200), 2.39 (d, 3H, J = 0.
92 Hz, C-40 CH3), 2.48 (q, 4H, J = 7.33 Hz, H-10000), 2.51
(t, 2H, J = 5.95 Hz, H-300), 3.42–3.47 (m, 2H, H-100), 4.51
(s, 2H, H-2), 6.16 (d, 1H, J = 0.92 Hz, H-30), 6.28 (d, 1H,
J = 2.75 Hz, H-50), 6.85 (dd, 1H, J = 2.75 Hz and 8.
70 Hz, H-70), 7.53 (d, 1H, J = 8.70 Hz, H-80), 8.46 (brs,
1H, NH) ppm; 13C NMR (100.5 MHz, CDCl3): d 11.53 (C-
20000), 18.62 (C-40 CH3), 25.19 (C-200) 39.8 (C-100), 46.98 (C-
10000), 52.40 (C-300), 67.60 (C-2), 102.62 (C-50), 111.33 (C-
30), 112.57 (C-70), 125.93 (C-80 and C-100), 152.20, 155.04
and 160.16 (C-90, C-60 and C-40), 160.90 and 166.79 (C-20
and C-1) ppm; HRMS: m/z [M ? Na]? Calculated for
C19H26N2O4: 369.1790; found: 369.1789.
Biology
Pathogens
Various pathogenic strains of bacteria namely, Bacillus
cereus (MTCC 430), Bacillus subtilis (MTCC 121),
Escherichia coli (MTCC 1586), Pseudomonas aeruginosa
(MTCC 741), Klebsiella pneumoniae (MTCC 3384), Sal-
monella enterica typhimurium (MTCC 98) were procured
from Institute of Microbial Technology, Chandigarh
(India). Trichophyton rubrum was a clinical isolate
obtained from Sir Ganga Ram Hospital, Delhi (India).
N-(3-(Dimethylamino)propyl)-2-((4-methyl-2-oxo-2H-chro-
men-6-yl)oxy)acetamide (46) The reaction of ethyl 2-((4-
methyl-2-oxo-2H-chromen-6-yl)oxy)acetate (31) (1 g, 3.
81 mmol) with N1,N1-dimethylpropane-1,3-diamine (1.
55 g, 15.24 mmol) gave the title compound 46 as a white
solid (0.89 g, 73 %) by following the general procedure; m.
p.: 119.7–118.8 °C; Rf: 0.46 (MeOH:CHCl3: 1:49); UV
(MeOH) kmax: 272 nm; IR (KBr) mmax: 3467.99 (N–H str),
2815.93, 1715.96 (C=O), 1648.52 (NHCO–), 1574.59, 1432.
04, 1245.18, 1173.60, 1058.26, 929.85, 838.82, 610.
28 cm-1; 1H NMR (400 MHz, CDCl3): d 1.63–1.69 (m, 2H,
H-200), 2.15 (s, 6H, H-10000), 2.23 (t, 2H, J = 6.40 Hz, H-300),
2.39 (d, 3H, J = 0.92 Hz, C-40 CH3), 3.39–3.44 (m, 2H,
H-100), 4.49 (s, 2H, H-2), 6.29 (d, 1H, J = 0.92 Hz, H-30), 7.
00 (d, 1H, J = 2.75 Hz, H-50), 7.08 (dd, 1H, J = 2.75 and 9.
16 Hz, H-70), 7.27 (d, 1H, J = 9.16 Hz, H-80) 8.15 (brs, 1H,
Materials
Mueller-Hinton agar, Nutrient broth, Sabouraud dextrose
agar, Sabouraud dextrose broth, sterile filter paper discs
(6 mm) were procured from HiMedia, Mumbai, India.
Gentamicin, Iodonitrotetrazolium chloride (INT), Ampho-
tericin B, Triton X-100 and DMSO were purchased from
Sigma-Aldrich Chemicals Pvt. Ltd., USA.
13
NH) ppm; C NMR (100.5 MHz, CDCl3): d 18.63 (C-40
CH3), 25.66 (C-200) 39.13 (C-100), 45.33 (C-10000), 58.55 (C-
300), 67.86 (C-2), 108.28 (C-50), 115.77 (C-30), 118.31 (C-70),
119.21 (C-80), 120.55 (C-100), 148.43, 151.62 and 153.76 (C-
90, C-60 and C-40), 160.61 and 167.47 (C-20 and C-1) ppm;
HRMS: m/z [M ? Na]? Calculated for C17H22N2O4: 341.
1477; found: 341.1490.
Antibacterial activity assay
The inhibitory potency of bacterial growth by the synthe-
sized compounds was determined using Kirby-Bauer disc
susceptibility test with some minor modifications (Pathak
et al., 2013; Yadav et al., 2014). The samples at a concen-
tration of 250 lg/disc were impregnated on a 6 mm sterile
filter paper discs placed in the respective grid of each Mu-
eller-Hinton agar plates already seeded with the target test
organisms and then kept at 37 °C for overnight incubation.
Gentamicin was used in the assay as a positive control. The
zone of inhibition was recorded using Himedia Hiantibiotic
Zone Scale. All the assays were carried out in triplicates.
N-(2-(Dimethylamino)ethyl)-2-((4-methyl-2-oxo-2H-chro-
men-6-yl)oxy)acetamide (47) The reaction of ethyl 2-((4-
methyl-2-oxo-2H-chromen-6-yl)oxy)acetate (31) (1 g, 3.
81 mmol) with N1,N1-dimethylethane-1,2-diamine (1.37 g,
15.24 mmol) gave the title compound 47 as a white solid (0.
87 g, 75 %) by following the general procedure; m. p.: 116.
5–117.4 °C; Rf: 0.41 (MeOH:CHCl3: 1:49); UV (MeOH)
123

Med Chem Res
Quantitative evaluation of antibacterial activity was per-
formed by estimating the minimum inhibitory concentration
(MIC) of the compounds using micro-well serial dilution
method with some minor modifications as described previ-
ously by Pathak and co-workers (Pathak et al., 2013). The
bacterial strains were cultured overnight at 37 °C in nutrient
broth and used as an inoculum. The wells of 96-well flat-
bottom culture plates with various dilutions of the com-
pounds (2–0.0001 mg/ml) were treated with 50 ll of mid-
log phase inoculums (OD600 = 0.4–0.6) of the relevant
culture and incubated at 37 °C for overnight using genta-
micin as a positive control. Absorbance at 600 nm was
measured. An additional control well containing an equiv-
alent amount of solvent without any sample was used as a
negative control. As an indicator of bacterial growth, 50 ll
of 0.25 mg/ml iodonitrotetrazolium chloride (INT) was
added to the wells and incubated at 37 °C for 30 min. The
pervasiveness of live bacterial cells was indicated by sig-
nificant intensity of red colour formation in microtiter plate,
while clear solution signified the suppression of bacterial
growth. All the assays were performed in triplicate.
Haemolytic activity assay
In order to have an insight of the in vitro human RBC
toxicity, the active compounds were subjected to hae-
molytic assay as described by Ciornei et al., 2005 and
Dobrovolskaia et al., 2008 with some minor modifica-
tions. Fresh human erythrocytes washed and centrifuged
with phosphate buffer saline (PBS) thrice, were suspended
in PBS (pH 7.4) to make 4 % (v/v) solution. A 100 ll of
cell suspension, added to each well of 96-well flat-bottom
tissue culture plate along with 100 ll of compounds of
different concentrations, were incubated for 1 h at 37 °C
and centrifuged at 1000 g for 5 min. Aliquots (100 ll) of
supernatant were transferred to fresh 96-well microtitre
plate and OD540 was recorded using ELISA plate reader
(Bio-Teck Instruments, Inc). The absorbance of erythro-
cyte suspension treated with Triton X-100 was taken as
100 % haemolysis and used as positive control. On the
other hand, cell suspension treated with PBS was used as
a negative control. Percentage of haemolysis was calcu-
lated using the following formula:
ꢀ
ꢁ
ðOD540 nm of Sample ꢁ OD540 nm of PBSÞ
% Haemolysis ¼
ꢂ 100:
ðOD540 nm of 0:1 % Triton X ꢁ 100 ꢁ OD540 nm of PBSÞ
Acknowledgments We gratefully acknowledge the financial assis-
tance from Defence Research Development Organization (DRDO,
Delhi), University of Delhi for the DU-DST PURSE Grant and
Council of Scientific and Industrial Research (CSIR) task force pro-
ject (BSC0302). Also we are thankful to CSIR, New Delhi, for pro-
viding fellowships to Suchita Prasad and Shiv Kumar.
Antifungal activity assay
The in vitro antifungal screening of compounds was
achieved using Trichophyton rubrum as a model patho-
gen. The antifungal activity was assessed by disc diffu-
sion assay (DDA) and percent growth inhibition assay
(PGIA) with some minor modifications (Gupta et al.,
2012). For disc diffusion, sterile filter paper discs, 6 mm
in diameter, placed on the surface of Sabouraud dextrose
agar already seeded with the isolated conidia of target test
organism, were impregnated with the samples at a con-
centration of 250 lg/disc. The zone of inhibition was
examined after 72 h of incubation at 30 °C. In percent
growth inhibition assay, the wells in microtitre plate,
treated with the dilutions of compounds (2-0.0001 mg/ml)
and inoculated with 1 3 106 of conidia isolated from T.
rubrum, were incubated at 30 °C for 72 h and OD650 was
measured to determine cell growth. Amphotericin B and
an equivalent amount of solvent were used as positive and
negative controls, respectively.
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