Synthesis, characterization, antimicrobial and biofilm inhibitory activities of new N-oxide esters

Article abstract of DOI:10.1007/s00044-017-1885-y

Novel N-oxide esters were synthesized from tertiary amine esters, which in turn were obtained from the reaction of 11-bromoundecanoic acid and different alkyl amines. The synthesized N-oxide esters were characterized by fourier transform infrared, proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and mass spectral analysis. The synthetic route involved three steps; initially, 11-bromoundecanoic acid was converted into methyl 11-bromoundecanoate, which was further converted into tertiary amine (mono and di) esters by reacting with different aliphatic amines (hexyl, dodecyl, octadecyl, dioctyl, and dicyclohexyl amine). Finally, the obtained amine esters were converted into N-oxide esters by treating with m-chloroperbenzoic acid. The synthesized N-oxide esters (4a–e) were studied for their antimicrobial and anti-biofilm activities. Among all the synthesized N-oxide esters, compounds 4a and e showed good antimicrobial activity especially towards pathogenic gram-positive bacterial strains with minimum inhibitory concentration values in the range of 7.8–31.2 μg mL^?1. The compounds which exhibited antimicrobial activity were also effective in anti-biofilm activity and it was found that the compound 4e exhibited promising biofilm inhibitory activity with IC₅₀ value of 6.4 μg mL^?1 against Bacillus subtilis MTCC 121. Further, compound 4e showed increased levels of intracellular reactive oxygen species accumulation, which may be contributing to the bactericidal activity in B. subtilis MTCC 121.

Full text of DOI:10.1007/s00044-017-1885-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1885-y
ORIGINAL RESEARCH
Synthesis, characterization, antimicrobial and biofilm inhibitory
activities of new N-oxide esters
Sathyam Reddy Yasa1 Shiva Shanker Kaki1 Y. Poornachandra2
●
●
●
C. Ganesh Kumar2 Vijayalakshmi Penumarthy1
●
Received: 2 January 2017 / Accepted: 22 March 2017
© Springer Science+Business Media New York 2017
Abstract Novel N-oxide esters were synthesized from ter-
tiary amine esters, which in turn were obtained from the
reaction of 11-bromoundecanoic acid and different alkyl
amines. The synthesized N-oxide esters were characterized
by fourier transform infrared, proton nuclear magnetic
resonance, carbon-13 nuclear magnetic resonance, and mass
spectral analysis. The synthetic route involved three steps;
initially, 11-bromoundecanoic acid was converted into
methyl 11-bromoundecanoate, which was further converted
into tertiary amine (mono and di) esters by reacting with
different aliphatic amines (hexyl, dodecyl, octadecyl, dioc-
tyl, and dicyclohexyl amine). Finally, the obtained amine
esters were converted into N-oxide esters by treating with
m-chloroperbenzoic acid. The synthesized N-oxide esters
(4a–e) were studied for their antimicrobial and anti-biofilm
activities. Among all the synthesized N-oxide esters, com-
pounds 4a and e showed good antimicrobial activity espe-
cially towards pathogenic gram-positive bacterial strains
with minimum inhibitory concentration values in the range
of 7.8–31.2 μg mL−1. The compounds which exhibited
antimicrobial activity were also effective in anti-biofilm
activity and it was found that the compound 4e exhibited
promising biofilm inhibitory activity with IC50 value of
6.4 μg mL−1 against Bacillus subtilis MTCC 121. Further,
compound 4e showed increased levels of intracellular
reactive oxygen species accumulation, which may be
contributing to the bactericidal activity in B. subtilis
MTCC 121.
●
●
Keywords Amine esters Lipophilic Antimicrobial
●
●
activity Biofilm inhibitory activity 11-Bromoundecanoic
●
acid N-Oxide esters
Introduction
Amine oxides represent a group of chemical compounds
resulting from tertiary amines and contain a strongly
polarized N–O bond (Linton 1940; Bonetti et al. 1970).
Many naturally occurring or chemically synthesized amine
oxides are known antimetabolites, chemotherapeutic, psy-
chotropic, and cancerostatic drugs (Bickel 1969; Lindner
1964). Amine oxides (N-oxides) occupy a significant role in
the field of synthetic and medicinal chemistry due to their
unique property to exist as stable free radicals. They have
potential applications as spin labels in biochemistry (Miller
1979), as probes in materials, as antioxidants in the pre-
vention of erythrocyte membrane lipid oxidation (Kleszc-
zynska et al. 2005), magnetic resonance imaging (Brik et al.
1990), electron spin resonance imaging (Kuppusamy et al.
1995), and in polymer synthesis (Hawker 1996). The N-
alkyl amine oxides of heterocyclic amines are reported to be
used successfully in different areas of agriculture (Cerecetto
et al. 2000). Along with these physicochemical properties,
they are reported to possess significant antimicrobial
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1885-y) contains supplementary material,
which is available to authorized users.
* Vijayalakshmi Penumarthy
pvl@iict.res.in
1
Centre for Lipid Research, CSIR-Indian Institute of Chemical
Technology (CSIR-IICT), Telangana, Hyderabad 500007, India
2
Medicinal Chemistry and Pharmacology Division, CSIR-Indian
Institute of Chemical Technology (CSIR-IICT), Telangana,
Hyderabad 500007, India

Med Chem Res
activity (Mlynarefk et al. 1975; Subik et al. 1977). Some of
the studies revealed that amine oxides exhibit prominent
antimicrobial activity when used separately or in combi-
nation with betaines (Devinsky et al. 1990; Mlynarcik et al.
1978; Corner et al. 1988). In addition, they are also reported
to have applications as amphiphilic surfactants (Imae et al.
1986; Rathman and Christian 1990). The anti-bactericidal
and anti-biofilm activities of N-alkyl-N,N-dimethyl amine
oxides have shown that they are effective on several
microorganisms (Walsh et al. 2003; Fraud et al. 2005).
Reported studies showed that N-oxides possess biological
activities, which were studied in a model where interaction
of N-oxides with model biological membranes was inves-
tigated (Kleszczynska et al. 2005; Kleszczynska et al.
2006).
Literature reports suggest that lipophilic N-oxide com-
pounds with chain lengths ranging from C8 to C18 have
shown good biological activity. However, very few studies
have been reported with lipophilic chain lengths (Birnie
et al. 2000). Hence, in the present study, we describe the
synthesis and antibacterial activity of novel lipophilic N-
oxides including their biofilm inhibitory studies. The
synthesis of novel lipophilic N-oxides starting from 11-
bromoundecanoic acids was carried out in three steps, and
all the intermediates and products were characterized by
spectral studies.
spectrometer using CHCl3 and KBr. All 1H and 13C spectra
were recorded on 500 MHz (Varian) and 125 MHz
(Brucker) spectrometers, respectively. High-resolution mass
spectrometry (HRMS) data were recorded on a Thermo
Scientific Exactive Orbitrap mass spectrometer (Germany)
and are given in mass units (m/z). Electrospray ionisation
mass spectrometry (ESI-MS) spectra were recorded on
Waters (Model: Q STAR XL, Applied Biosystems, USA)
Mass Spectrometer equipped with an Electrospray Ioniza-
tion source.
General procedure for the synthesis of dimethyl 11,11′-
(alkylazanediyl) diundecanoates (3a–c)
Synthesis of dimethyl 11,11′-(alkylazanediyl) diundecano-
ates was performed by reported method (Yasa et al. 2016).
Initially, 11-bromo undecanoic acid was converted into
methyl 11-bromoundecanoate (11-BUME). Further, 11-
BUME (1 eq) and amine (0.55 eqs) were dissolved in DMF
solvent and stirred for 15 min, followed by the addition of
anhydrous K2CO3 catalyst (2 eqs to 11-BUME). The reac-
tion temperature was raised to 110 °C and continued for 12
h until complete conversion of starting material, 11-BUME
as shown in Scheme 1. The progress of the reaction was
monitored by TLC using solvent system of CHCl3 and
methanol (9:1 v/v). The crude reaction product was filtered
to separate K2CO3 followed by washing with water and
extraction with ethyl acetate. Pure dimethyl 11,11′-(alkyla-
zanediyl) diundecanoates (3a–c) was separated from crude
product by silica gel (60–120 mesh) column chromato-
graphy using chloroform and methanol (99:1, v/v) solvent
system as eluent. Isolated yields were in the range of
88–93%.
Materials and methods
Materials
The raw materials needed for the synthesis of N-oxide
esters, such as hexyl, dodecyl, octadecyl, dioctyl, dicyclo-
hexyl amines, and 11-bromoundecanoic acid were pur-
chased from Sigma-Aldrich Chemicals (USA). Anhydrous
potassium carbonate (K2CO3), p-toluenesulfonic acid,
dimethylformamide (DMF), m-chloroperbenzoic acid
(MCPBA) and other solvents were purchased from S.D Fine
chemicals, Mumbai (India). All solvents and chemicals
were of reagent grade and used directly without further
purification. Silica gel (60–120 mesh) for column chroma-
tography was purchased from Acme Synthetic Chemicals,
Mumbai, India. Precoated thin-layer chromatography (TLC)
plates were purchased from Merck, Darmstadt, Germany.
All microbial strains were obtained from Microbial Type
Culture Collection (MTCC) and Gene Bank, Chandigarh
(India).
General procedure for the synthesis of methyl 11-
(dialkylamino) undecanoates (3d–e)
Synthesis of methyl 11-(dialkylamino) undecanoates was
performed employing reported method (Yasa et al. 2016).
11-BUME (1.0 eq) and dialkylamine (1.1 eqs) were dis-
solved in DMF solvent and stirred for 15 min, followed by
the addition of K2CO3 (2 eqs to 11-BUME) and continued
the reaction for 12 h at 110 °C as shown in Scheme 1. The
progress of the reaction was monitored by TLC using
CHCl3 and Methanol (9:1 v/v) as a solvent system. The
crude reaction product was worked up by filtering K2CO3
followed by washing with water and extracted by ethyl
acetate, and was dried by passing through anhydrous
sodium sulphate followed by removal of solvent by rotary
evaporator. Pure methyl 11-(dialkylamino) undecanoate (3d
and e) was separated from crude reaction product by col-
umn chromatography using silica gel (60–120 mesh) with
Analytical methods
IR spectra were recorded on a Perkin Elmer (Model:
Spectrum BX) fourier transform infrared (FT-IR)

Med Chem Res
Scheme 1 Schematic
presentation for the chemical
synthesis of N-oxide esters
Br
Br
OH
O
(1)
11-bromo undecanoic acid
Methanol, PTSA, reflux, 12h
O
O
(2)
11-bromo methylundecanoate
R-NH2 (0.55 eq),
K2CO3, 110 °C,
12h
R1-NH-R1 (1.1 eq),
K2CO3, 110 °C,
12h
O
O
R1
O
O
R1
N
O
O
R N
o
3d-e)
N,N-dialkyl-3 -amine monoester (
o
(3a-c)
N-alkyl-3 - amine diester
MCPBA, rt, 6h
MCPBA, rt, 6h
R1
R1
N
O
O
O
O
O
R N
O
4d-e)
N,N-dialkyl N-monoester N-oxide (
O
O
1
3d
4d
R = Octyl group -
and
3e
1
4a-c)
N-alkyl N,N-diester N-oxide (
4e
R = Cyclohexyl ring - and
3a
4a
R = Hexyl group - and
3b
4b
R = Dodecyl group -
R = Octadecyl group - and
and
3c
4c
chloroform and methanol (98:2, v/v) as eluent. Isolated
yields were in the range of 95–96%.
traces of unreacted amine was removed by silica gel
(60–120 mesh) column chromatography with chloroform:
methanol (96:4 v/v) as eluent. The final products N-oxide
esters were eluted out by chloroform: methanol (80:20; v/v).
The synthesized N-oxide esters (4a–e) yields were obtained
in the range of 93–96% and characterized by spectral
techniques like FT-IR, (1H & 13C)-NMR and mass spectral
data (ESI-MS and HRMS).
General procedure for the synthesis of N,N-bis(11-
methoxy-11-oxoundecyl) alkyl-1-amine oxides (4a–c)
and 11-methoxy-N,N-dialkyl-11-oxoundecan-1-amine
oxides (4d–e)
A reported protocol was followed for N-oxidation of
dimethyl 11,11′-(alkylazanediyl) diundecanoates (3a–c) and
methyl 11-(dialkylamino) undecanoate (3d and e) to convert
them into respective N-oxides (Cymerman Craig and Pur-
ushothaman 1970). A 25-mL two-neck round bottom flask
was charged with amino esters (2.0 mmol) and dissolved in
CHCl3 (5.0 mL) followed by addition of a solution of
MCPBA (2.4 mmol) in CHCl3 at 0–5 °C temperature as
shown in Scheme 1. The reaction was further continued for
6 h at room temperature until completion of the reaction. At
the end of the reaction, the reaction mixture was passed
through a column of basic alumina (100–200 mesh, ca. 20
times the weight of the combined starting materials), and
Characterization of synthesized N-oxide esters (4a–e)
N,N-bis(11-methoxy-11-oxoundecyl)hexa-1-amine oxide
(4a)
Thick brown liquid; yield 93.5%; IR (Neat, cm−1): 2928,
1
2857, 1738, 1556, 1453, 1365, 1172; H-NMR (500 MHz,
CDCl3): δ/ppm 3.67 (6H, s), 3.25–3.12 (6H, m), 2.30 (4H, t,
J = 7.55 Hz), 1.80–1.70 (4H, m), 1.66–1.56 (6H, m),
1.39–1.22 (30H, m), 0.89 (3H, t, J = 6.71 Hz); 13C-NMR
(CDCl3, 125): δ/ppm 174.23, 65.23, 51.38, 34.01, 31.39,
29.29, 29.21, 29.11, 29.03, 26.66, 26.33, 24.85, 22.92,

Med Chem Res
22.41, 13.87; ESI-MS (m/z) (M + H)+ (C30H59NO5 + H)+
514.55; HRMS-ESI (m/z) calculated 514.4466, found (M)+
514.4459
(CDCl3, 125): δ/ppm 174.25, 72.92, 59.89, 51.39, 34.01,
33.71, 29.63, 29.30, 29.11, 29.03, 28.82, 27.64, 27.14,
26.95, 25.94, 25.75, 25.34, 24.84, 24.32; ESI-MS (m/z) (M
+ H)+ (C24H45NO3+H)+ 396.52; HRMS-ESI (m/z) calcu-
lated 396.3472, found (M)+ 396.3466.
N,N-bis(11-methoxy-11-oxoundecyl)dodecan-1-amine oxide
(4b)
Antimicrobial activity assay
Thick brown liquid; yield 94.0%; IR (Neat, cm−1): 2926,
2856, 1737, 1598, 1455, 1365, 1167; H-NMR (500 MHz,
1
Antimicrobial activity of the synthesized N-oxide esters was
determined using well diffusion method, against a different
pathogenic reference strains such as Micrococcus luteus
MTCC 2470, Bacillus subtilis MTCC 121, Staphylococcus
aureus MTCC 96, S. aureus MLS16 MTCC 2940, Kleb-
siella planticola MTCC 530, Escherichia coli MTCC 739,
Pseudomonas aeruginosa MTCC 2453 and Candida albi-
cans MTCC 3017 procured from the MTCC, CSIR-Institute
of Microbial Technology, Chandigarh, India (Amsterdam
1996). The pathogenic reference strains were seeded on the
surface of the media petri plates, containing Muller–Hinton
agar with 0.1 mL of earlier prepared microbial suspensions
individually containing 1.5 × 108 cfu mL−1 (equal to 0.5
McFarland). Wells of 6.0 mm diameter were prepared in the
media plates using a cork borer and the synthesized N-oxide
esters at a dose range of 125–0.9 μg per well were added in
each well under sterile conditions in a laminar air flow
chamber. Standard antibiotic solution of ciprofloxacin
(bacterial strains) and miconazole (Candida strains) at a
dose range of 125–0.9 μg per well, and the well containing
methanol served as positive and negative controls, respec-
tively. The plates were incubated for 24 h at 37 °C for
bacterial strains and 30 °C for different Candida strains, and
the well containing the least concentration showing the
inhibition zone was considered as the minimum inhibitory
concentration (MIC). All experiments were carried out in
duplicates and mean values are represented.
CDCl3): δ/ppm 3.67 (6H, s), 3.33–3.17 (6H, m), 2.30 (4H, t,
J = 7.55 Hz), 1.80–1.68 (4H, m), 1.66–1.56 (6H, m),
1.38–1.22 (42H, m), 0.88 (3H, t, J = 6.71 Hz); 13C-NMR
(CDCl3, 125): δ/ppm 174.19, 64.76, 51.34, 33.98, 31.81,
29.52, 29.41, 29.34, 29.25, 29.18, 29.08, 29.00, 26.56,
24.81, 22.82, 22.58, 14.02; ESI-MS (m/z) (M + H)+
(C36H71NO5+H)+ 598.82; HRMS-ESI (m/z) calculated
598.5405, found (M)+ 598.5391.
N,N-bis(11-methoxy-11-oxoundecyl)octadecan-1-amine
oxide (4c)
Thick brown liquid; yield 93.0%; IR (Neat, cm−1): 2925,
1
2855, 1738, 1602, 1457, 1365, 1170; H-NMR (500 MHz,
CDCl3): δ/ppm 3.67 (6H, s), 3.24–3.09 (6H, m), 2.30 (4H, t,
J = 7.47 Hz), 1.80–1.69 (4H, m), 1.66–1.56 (6H, m),
1.38–1.20 (54H, m), 0.88 (3H, t, J = 6.94 Hz); 13C-NMR
(CDCl3, 125): δ/ppm 174.16, 65.32, 51.32, 33.96, 31.82,
29.60, 29.41, 29.36, 29.26, 29.21, 29.08, 28.99, 26.66,
24.81, 22.91, 22.59, 14.02; ESI-MS (m/z) (M)+
(C42H83NO5+H)+ 683.19; HRMS-ESI (m/z) calculated
682.6344, found (M)+ 682.6336.
11-methoxy-N,N-dioctyl-11-oxoundecan-1-amine oxide (4d)
Thick brown liquid ; yield 96.0%; IR (Neat, cm−1): 2928,
1
2857, 1738, 1562, 1452, 1365, 1170; H-NMR (500 MHz,
CDCl3): δ/ppm 3.67 (3H, s), 3.30 (6H, t, J = 8.31 Hz), 2.30
(2H, t, J = 7.55 Hz), 1.79–1.68 (6H, m), 1.65–1.57 (2H, m),
1.38–1.22 (32H, m), 0.88 (6H, t, J = 6.94 Hz); 13C-NMR
(CDCl3, 125): δ/ppm 174.23, 64.63, 51.38, 34.02, 31.62,
29.64, 29.28, 29.19, 29.16, 29.12, 29.02, 26.56, 24.85,
22.80, 22.53, 14.00; ESI-MS (m/z) (M + H)+ (C28H57NO3
+ H)+ 456.58; HRMS-ESI (m/z) calculated 456.4411,
found (M)+ 456.4393.
Minimum bactericidal concentration (MBC)
Bactericidal assay was performed in sterile 2.0 mL micro-
fuge tubes against a panel of pathogenic bacterial strains,
including M. luteus MTCC 2470, S. aureus MTCC 96, S.
aureus MLS-16 MTCC 2940, B. subtilis MTCC 121, E.
coli MTCC 739, P. aeruginosa MTCC 2453 and K. plan-
ticola MTCC 530, which were cultured overnight in
Mueller Hinton broth (NCCLS 2000). Serial dilutions of
test compounds were prepared in Mueller–Hinton broth
using different concentrations ranging from 0 to 125 μg
mL−1. To the test compounds, 100 μl of overnight cultured
bacterial suspensions were added to reach a final con-
centration of 1.5 × 108 cfu mL−1 (equal to 0.5 McFarland)
and incubated at 37 °C for 24 h. After 24 h incubation, the
MBC was determined by sampling 10 μL of suspension
from the tubes onto Mueller–Hinton agar plates and were
11-methoxy-N,N-dicyclohexyl-11-oxoundecan-1-amine
oxide (4e)
Thick brown liquid; yield 95.5%; IR (Neat, cm−1): 2929,
1
2857, 1737, 1559, 1451, 1367, 1172; H-NMR (500 MHz,
CDCl3): δ/ppm 3.67 (3H, s), 3.54–3.44 (2H, m), 3.39 (2H, t,
J = 8.54 Hz), 2.30 (2H, t, J = 7.47 Hz), 2.10–1.81 (6H, m),
1.73–1.55 (10H, m), 1.40–1.11 (20H, m); 13C-NMR

Med Chem Res
incubated for 24 h at 37 °C to observe the growth of test
organisms. MBC is the lowest concentration of a compound
required to kill a particular bacterium. All the experiments
were carried in duplicates.
Results and discussion
Synthesis of N-oxide esters
The synthesis of N-oxide esters (4a–e) was performed in three
steps as shown in Scheme 1. Initially, 11-bromoundecanoic
acid was converted into methyl 11-BUME followed by the
synthesis of tertiary amino esters (3a–e) using different ali-
phatic amines like hexyl, dodecyl, octadecyl, dicyclohexyl,
and dioctyl amines, and methyl 11-BUME (2) in the presence
of base catalyst K2CO3 at 110 °C for 12 h. The yields of the
isolated amino esters were depending on the equivalent ratio of
starting materials, such as methyl 11-bromoundecanote (1 eq)
and amine (0.55 eq) and found to be obtained in the range of
83–96% yields. In the final step, the formed tertiary amino
esters were converted in to N-oxides by reacting with an
oxidizing reagent, MCPBA to obtain good yields (94–96%).
The synthesized N-oxide esters (4a–e) showed in Fig. 1
were characterized by spectral techniques like FT-IR, proton
nuclear magnetic resonance (1H-NMR), carbon-13 nuclear
magnetic resonance (13C-NMR) and mass data (ESI-MS and
HRMS). In the FT-IR spectra, the compound (4a) showed a
strong absorption band at 1549 cm−1 indicating the asym-
metric stretching, and at 1365 cm−1 shows the symmetric
stretching mode of strong polarized bond –N‒O, a strong
band at 1742 cm−1 indicates the carbonyl functionality of
ester group and the strong band at 2928 cm−1 shows the
Biofilm inhibition crystal violet assay
The synthesized N-oxide esters were screened in sterile 96-
well polystyrene microtiter plates using the modified biofilm
inhibition assay, against a panel of pathogenic bacterial strains
including S. aureus MTCC 96, S. aureus MLS16 MTCC 2940
and B. subtilis MTCC 121, which were cultured overnight in
tryptone soy broth (supplemented with 0.5% glucose) (Kamal
et al. 2015). The test compounds of predetermined con-
centrations ranging from 0 to 125 μg mL−1 were mixed with
the bacterial suspensions having an initial inoculum con-
centration of 5 × 105 cfu mL−1. Aliquots of 100 μL were
distributed in each well and then incubated at 37 °C for 24 h
under static conditions. The medium was then discarded and
washed with phosphate buffered saline to remove the non-
adherent bacteria. Each well of the microtiter plate was
stained with 100 μL of 0.1% crystal violet solution followed
by 30-min incubation at room temperature. Later the crystal
violet solution from the plates was discarded, thoroughly
washed with distilled water for three to four times and air
dried at room temperature. The crystal violet stained biofilm
was solubilized in 95% ethanol (100 μL) and the absorbance
was recorded at 540 nm using TRIAD multimode reader
(Dynex Technologies, Inc, Chantilly, VA, USA). Blank
wells were employed as background check. The inhibition
data were interpreted from the dose-response curves, where
IC50 value is defined as the concentration of inhibitor
required to inhibit 50% of biofilm formation under the
above assay conditions. All the experiments were carried
out in triplicates and the values are indicated as mean S.D
1
alkyl chain (–C‒H) stretching vibration. In H-NMR spec-
tra, a mutiplet at δ 3.25–3.12 ppm indicates the six protons
on carbons adjacent to highly polar nitrogen (O‒N‒(CH2)3)
and a triplet at δ 2.30 ppm showed two carbon protons
adjacent to carbonyl of ester functional group. Mass spectral
studies (ESI-HRMS) also showed that the base peak at
514.4459 (C30H59NO5 + H)+ corresponding to the mole-
cular weight of the compound, which further confirmed the
formation of desired product, final compound 4a.
O
O
Fig. 1 Molecular structures of
the synthesized new N-oxide
esters
N
O
O
N
n
O
O
O
O
N N
N N
N
O
O
O
N N

Med Chem Res
Antimicrobial activity
the N-oxides like, N-alkyl-N,N-dimethylamine oxides with
long alkyl chain or above C14 carbon chain did not show
antimicrobial activity (Birnie et al. 2000; Subik et al.
1977).
The synthesized amine esters (3a–e) and their corre-
sponding N-oxides (4a–e) were screened for their anti-
microbial activity against both gram-positive as well as
gram-negative bacterial strains along with a fungal strain,
Candida albicans. The antimicrobial activity results of the
compounds (3a–e) were reported by us in previous study
(Yasa et al. 2016). In the present study, we report the
antimicrobial activity of the compounds (4a–e) and the
results to this regard are shown in Tables 1 and 2. Some of
the compounds (4a, 4d and 4e) exhibited good anti-
bacterial activity with (MIC) values ranging between
7.8–31.2 μg mL−1 and MBC values ranging between
15.6–62.5 μg mL−1. All the synthesized N-oxide esters did
not exhibit antifungal activity on Candida albicans MTCC
The results from the present investigation showed that
the compound 4a, (N,N-bis(11-methoxy-11-oxoundecyl)
hexa-1-amine oxide) prepared from relatively shorter chain
aliphatic amine i.e., hexyl amine and 11-BUME showed
good antibacterial activity on Gram-positive bacteria with
MIC values ranging between 7.8–31.2 μg mL−1 and the
MBC values were ranged between 15.6–62.5 μg mL−1
.
More importantly, the compound 4e (11-methoxy-N,N-
dicyclohexyl-11-oxoundecan-1-amine oxide) synthesized
using dicyclohexyl amine and 11-BUME showed good
antimicrobial activity with MIC values ranging between
7.8–15.6 μg mL−1 and MBC values ranged between
15.6–62.5 μg mL−1. The compound 4d (11-methoxy-N,N-
dioctyl-11-oxoundecan-1-amine oxide) also showed mod-
erate antimicrobial activity against gram-positive bacteria
with MIC values of 31.2 μg mL−1 on all tested strains.
However, all the synthesized N-oxide esters (4a–e) did not
show antimicrobial activity on gram-negative bacteria.
3017 even up to the tested concentration of 125 μg mL−1
.
The synthesized compounds having long alkyl chains i.e.,
dodecyl/C12 carbon chain (4b) and octadecyl/C18 carbon
chain (4c) did not show any antimicrobial activity on the
tested strains up to the maximum tested concentration of
125 μg mL−1. Earlier literature reports also suggest that
Table 1 Antimicrobial activity
of synthesized amine esters (3a–e)
and N-oxide esters (4a–e)
Test compound
MIC (µg/mL)
aS. a.
aB. s.
aS. m.
aM. l.
bK. p.
bE. c.
bP. a.
cC. a.
4a
31.2
>125
>125
31.2
15.6
–
31.2
>125
>125
31.2
7.8
7.8
31.2
>125
>125
31.2
15.6
–
>125
>125
>125
>125
>125
–
>125
>125
>125
>125
>125
–
>125
>125
>125
>125
>125
–
–
4b
>125
>125
31.2
15.6
–
–
4c
–
4d
–
4e
–
Miconazole
Ciprofloxacin
–
7.8
–
0.9
0.9
0.9
0.9
0.9
0.9
0.9
MIC and the values are mean of three determinations
S. a., S. aureus MTCC 96; B. s., B. subtilis MTCC 121; S. m., S. aureus MTCC 2940; M. l., M. luteus
MTCC 2470; K. p., K. planticola MTCC 530; E. c., E. coli MTCC 739; P. a., P. aeruginosa MTCC 2453; C.
a., Candida albicans MTCC 3017
a
Gram-positive bacteria
b
Gram-negative bacteria
c
fungus
Table 2 MBC of synthesized
compounds
Test compound MBC (µg/mL)
S. aureus MTCC B. subtilis MTCC
S. aureus MLS16 MTCC M. luteus MTCC
96
121
2940
2470
4a
125
62.5
31.2
0.9
62.5
125
15.6
1.9
31.2
125
31.2
0.9
62.5
62.5
62.5
1.9
4d
4e
Ciprofloxacin
MBC and the values are mean of three determinations.

Med Chem Res
Table 3 Biofilm inhibition
assay of synthesized compounds
Test compound IC50 values (μg/mL)
S. aureus MTCC
B. subtilis MTCC
S. aureus MLS16
M. luteus MTCC
96
121
MTCC 2940
2470
4a
32.4 0.25
25.9 0.42
10.2 0.36
0.8 0.12
19.3 0.43
30.4 0.38
6.4 0.16
0.6 0.08
15.6 0.12
19.8 0.25
11.1 0.31
0.5 0.11
29.8 0.52
22.6 0.48
8.8 0.29
0.6 0.11
4d
4e
Ciprofloxacin
Biofilm inhibitory activity
10000
1000
100
10
Control
4e
Ciprofloxacin
Biofilms are complex communities of bacteria that exist in a
self-produced matrix of polysaccharides, proteins, and
extracellular DNA, which are responsible for the growing
resistance in bacteria towards antibiotics (Olsen 2015).
Several clinically major pathogens producing biofilms are
implicated in more than 80% of the bacterial infections
(Davies 2003). Therefore, the synthesized N-oxide esters
(4a–e) were studied for their anti-biofilm activity against
four bacterial strains, namely S. aureus MTCC 96, B.
subtilis MTCC 121, S. aureus MLS16 MTCC 2940 and M.
luteus MTCC 2470. The results on the biofilm inhibitory
activity for the synthesized N-oxide esters are shown in
Table 3, which suggests that these compounds exhibited
good to moderate anti-biofilm activity against all the four
tested strains. Particularly, compound 4e exhibited good
anti-biofilm activity with IC50 values of 10.2, 6.4, 11.1, and
8.8 μg mL−1 against S. aureus MTCC 96, B. subtilis MTCC
121, S. aureus MLS16 MTCC 2940, and M. luteus MTCC
2470, respectively. Other compounds, such as 4a (IC50
values ranged between 15.6–32.4 μg mL−1 and 4d
(19.8–30.4 μg mL−1) showed moderate biofilm inhibitory
activity on all the tested bacterial strains. It is important to
note that the ability to inhibit biofilm formation of these
compounds to a maximum extent is in coherence with the
antimicrobial activity against the respective strain. Earlier
literature reports also supports the fact that N-oxide type of
compounds like N-alkyl-N,N-dimethyl amine oxides
exhibited anti-biofilm inhibitory activity (Fraud et al. 2005).
Literature reports show that reactive oxygen species
(ROS) is an important inducer of apoptosis in bacteria,
which may cause oxidative damage to cellular compounds
and lead to cellular dysfunction or cell death (Madeo et al.
1999). Thus, to explain whether oxidative stress is involved
in the apoptotic cell death, the intracellular ROS accumu-
lation within the cells of mature biofilms was measured
using the fluorescent probe 2′,7′-dichlorofluorescein-diace-
tate (DCFH-DA), a general ROS fluorescent probe. The
DCFH-DA dye conversion mainly depends on the metabo-
lically active cells in the biofilm. DCFH-DA is deacetylated
in the cells, where it can react quantitatively with intracel-
lular radicals (mainly H2O2) to get converted to its
1
2
4
6
8
Concentration (µg/mL)
Fig. 2 Intracellular ROS accumulation B. subtilis MTCC 121 of the
compound 4e
fluorescent product (dichlorofluorescein), which is retained
within the cells and thus provides an index of oxidation in
the cell cytosol. The intracellular ROS accumulation in the
biofilms of B. subtilis MTCC 121 was measured using
DCFHDA dye, in which the dye conversion depends on the
metabolically active cells in the biofilm (Kumar and Poor-
nachandra 2015). After treatment with the compounds, 11-
methoxy-N,N-dicyclohexyl-11-oxoundecan-1-amine oxide
(4e) and Ciprofloxacin, a significant increase in the accu-
mulation of ROS levels was observed in B. subtilis MTCC
121 as shown in Fig. 2. At a concentration of 2 µg/mL, the
compound 4e-treated biofilms showed increased levels of
intracellular ROS accumulation, which was closer to the
standard drug ciprofloxacin, and it showed a similar trend till
a higher concentration of 8 µg/mL. Further, the ROS mea-
surements were also carried out separately in both sessile
cells and in the supernatant. The ROS induced increase in
fluorescence was observed only for the sessile cells, sug-
gesting that the ROS accumulation is of intracellular origin.
This accumulated ROS may be contributing to the observed
bactericidal activity. Oxygen-containing free radical mole-
cules and their precursors formed in biological systems are
collectively termed as ROS, comprising of superoxides
(O2−), peroxides (H2O2 and ROOH) and free radicals (HO
and RO). Some of the bactericidal drugs stimulate free
radical formation via the Fenton reaction (Kohanski et al.
2007). Recent studies suggest that when bacteria is under

Med Chem Res
Dixon SJ, Stockwell BR (2013) The role of iron and reactive oxygen
species in cell death. Nat Chem Biol 10:9–17
Fraud S, Maillard JY, Kaminski MA, Hanlon GW (2005) Activity of
amine oxide against biofilms of Streptococcus mutans: a potential
biocide for oral care formulations. J Antimicrob Chemother
56:672–677
Hawker CJ (1996) Advances in ‘living’ free-radical polymerization:
architectural and structural control trends. Polym Sci 4:183–186
Imae T, Araki H, Ikeda S (1986) The absorption spectra and the
micelle species of dimethyloleylamine oxide in aqueous solu-
tions. Colloids Surf 17:221–228
oxidative stress, these free radicals contribute to arrest in cell
growth or result in damage of the specific essential meta-
bolic enzymes (cellular respiratory chain), disrupting cellular
membrane, and DNA damage ultimately causing cell lysis
and death (Dixon and Stockwell 2013).
Conclusions
In conclusion, we have synthesized five novel N-oxide
esters (4a–e) in facile and convenient chemical synthetic
route with good yields. The synthesized N-oxide esters were
evaluated for antimicrobial and anti-biofilm activities. The
results suggest that compounds 4a (hexyl) and 4e (dicy-
clohexyl) showed good antibacterial activity along with
good biofilm inhibitory activity. We observed that the
synthesized N-oxide esters (4b and c) with more than C12
alkyl chain did not exhibit any antibacterial activity. Finally,
we concluded that some of the synthesized N-oxide esters
find use as antimicrobial and anti-biofilm agents.
Kamal A, Rahim A, Riyaz S, Poornachandra Y, Balakrishna M,
Kumar CG, Hussaini SMA, Sridhar B, Machiraju PK (2015)
Regioselective synthesis, antimicrobial evaluation and theoretical
studies of 2-styryl quinolines. Org Biomol Chem 13:1347–1357
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A common mechanism of cellular death induced by bactericidal
antibiotics. Cell 130:797–810
Kumar CG, Poornachandra
Y (2015) Biodirected synthesis of
Acknowledgements One of the authors
S R Yasa gratefully
Miconazole-conjugated bacterial silver nanoparticles and their
application as antifungal agents and drug delivery vehicles.
Colloids Surf B Biointerfaces 125:110–119
acknowledges the Department of Biotechnology (DBT), New Delhi
for the financial assistance under sponsored project and Director,
CSIR-IICT for providing the facilities.
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mization of three-dimensional spatial EPR imaging for biological
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Conflict of interest The authors declare that they have no competing
interests.
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