Synthesis, characterization, antimicrobial and biofilm inhibitory studies of new esterquats

Article abstract of DOI:10.1016/j.bmcl.2016.03.002

Novel esterquats (monoesterquats and diesterquats) were synthesized from 11-bromo undecanoic acid (11-BUA) and different alkyl amines. The prepared compounds were characterized by FT-IR, ¹H NMR, ¹³C NMR and mass spectral analysis. 11-BUA was converted into methyl 11-bromo undecanoate which was further converted into amine ester (amine monoester and diester) by reacting with different aliphatic amines (hexyl, dodecyl, octadecyl, dioctyl and dicyclohexyl amine). Finally, the obtained amine esters were converted into esterquats (monoesterquat and diesterquat) by reacting with methyl iodide followed by ion exchange to afford chloride counter ion esterquats (5a-h). The synthesized esterquat products were studied for their antimicrobial and biofilm inhibitory activities. Among all the compounds, amine ester 3a and esterquat 5d showed potent antimicrobial activity towards pathogenic Gram-positive bacterial strains with minimum inhibitory concentration (MIC) values in the range of 3.9-15.6 μg mL^-1 and 1.9-7.8 μg mL^-1, respectively. The esterquat 5d also showed promising antifungal activity against Candida albicans MTCC 3017, Candida albicans MTCC 4748 and Candida aaseri MTCC 1962 strains with MIC value of 7.8 μg mL^-1 which was identical to standard Miconazole. The compounds which exhibited antimicrobial activity were also effective in anti-biofilm activity and it was found that compound 5d exhibited excellent biofilm inhibitory activity with IC₅₀ value of 0.9 μg mL^-1 against Staphylococcus aureus MLS16 MTCC 2940.

Full text of DOI:10.1016/j.bmcl.2016.03.002

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1978–1982
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, characterization, antimicrobial and biofilm inhibitory
studies of new esterquats
a
a
b
b
Sathyam Reddy Yasa , Shiva Shanker Kaki , Y. Poornachandra , C. Ganesh Kumar ,
a,
⇑
Vijayalakshmi Penumarthy
a
Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500007, Telangana, India
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500007, Telangana, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel esterquats (monoesterquats and diesterquats) were synthesized from 11-bromo undecanoic acid
Received 30 December 2015
Revised 16 February 2016
Accepted 1 March 2016
Available online 3 March 2016
11-BUA) and different alkyl amines. The prepared compounds were characterized by FT-IR, 1H NMR,
(
13
C NMR and mass spectral analysis. 11-BUA was converted into methyl 11-bromo undecanoate which
was further converted into amine ester (amine monoester and diester) by reacting with different alipha-
tic amines (hexyl, dodecyl, octadecyl, dioctyl and dicyclohexyl amine). Finally, the obtained amine esters
were converted into esterquats (monoesterquat and diesterquat) by reacting with methyl iodide followed
by ion exchange to afford chloride counter ion esterquats (5a–h). The synthesized esterquat products
were studied for their antimicrobial and biofilm inhibitory activities. Among all the compounds, amine
ester 3a and esterquat 5d showed potent antimicrobial activity towards pathogenic Gram-positive bac-
Keywords:
Aliphatic amines
Amine esters
Esterquats
Antimicrobial activity
Biofilm inhibitory activity
ꢀ
1
terial strains with minimum inhibitory concentration (MIC) values in the range of 3.9–15.6
l
g mL and
g mL , respectively. The esterquat 5d also showed promising antifungal activity against
Candida albicans MTCC 3017, Candida albicans MTCC 4748 and Candida aaseri MTCC 1962 strains with
ꢀ1
1.9–7.8
l
1
1-Bromoundecanoic acid
ꢀ1
MIC value of 7.8
lg mL which was identical to standard Miconazole. The compounds which exhibited
antimicrobial activity were also effective in anti-biofilm activity and it was found that compound 5d
ꢀ1
exhibited excellent biofilm inhibitory activity with IC50 value of 0.9 lg mL against Staphylococcus aur-
eus MLS16 MTCC 2940.
Ó 2016 Elsevier Ltd. All rights reserved.
The amine moiety has played a central role in chemotherapeutics
of numerous diseases due to its unique biological properties.
cellular content.15,16 Another related class of soft antimicrobial
amphiphilic compounds are esterquats (EQs) which have been
shown to exhibit good antibacterial activity. The studies suggest
that modification of head groups had a positive effect on the antimi-
1
,2
Nitrogen containing fatty acid derivatives like long chain aza, aziri-
dine and heterocyclic based fatty esters, and quaternary ammonium
compounds are important chemical entities synthesized and
broadly used as antimicrobial agents.3 Quaternary ammonium
compounds (QACs) are ubiquitous in nature and find application
in surfactants, antimicrobials, disinfectants and dyes.6 QACs are
widely used as bioactive agents and biocides that possess antimicro-
bial effect against a broad range of microorganisms and function
over a wide range of pH and find use in domestic, industrial, agricul-
tural, and medical applications such as wood preservatives, pesti-
1
7,18
crobial activity.
EQs were obtained via reactions between alky-
–5
lation agents and tertiary amines synthesized from fatty acid and
1
9
methyldiethanolamine (MDEA) or triethanolamine. EQs and their
derivatives have been reported since olden days and in the recent
years these compounds gained attention due to their biodegradable
properties and also to develop newer surfactants with higher bioac-
2
0–22
23–26
tivity.
These molecules find extensive use as surfactants,
1
7,27–29
antimicrobial agents,
fabric softeners.
antistatic agents, corrosion inhibitors,
Several reports showed the synthesis of nitro-
cides, hard-surface cleansers,7
–11
sanitizers/disinfectants,
12
30–32
13
14
bacterial biofilm eradicating agents and fungicides. The antimi-
crobial activity of QACs is primarily related to their cationic surfac-
tant properties. The main mode of antimicrobial action of QACs is
due to interaction with cell membranes, predominantly at the level
of the cytoplasmic membrane causing disruption and leakage of the
gen containing fatty esters like long chain aza, aziridine, azetidine
and heterocyclic (piperidine and pyridine) based fatty esters, but
3
,33
reports on their biological activity are limited.
It is important to
explore the possibilities of developing newer bio-based antimicro-
bials which can overcome the increased resistance of pathogenic
microbes and biofilms. Therefore, the present study is aimed to
synthesize fatty acid based novel amine esters and esterquats from
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.03.002
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0

S. R. Yasa et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1978–1982
1979
1
1-BUA, and to evaluate their antimicrobial and anti-biofilm
activities.
The novel esterquats (5a–h) were prepared via a new chemical
antimicrobial property indicating that the antimicrobial activity
2
8
is dependent on the alkyl chain lengths. For Gram-positive bacte-
ria, promising activity was observed with chain lengths of n =
12–14, while for Gram-negative bacteria the chain lengths of
synthetic approach starting from 11-BUA using different aliphatic
amines and methyl iodide as shown in Figure 1. The synthesized
intermediate amine esters (3a–h) and final esterquats (5a–h) were
characterized by spectroscopic studies. The synthesized com-
pounds were studied for their antimicrobial and biofilm inhibitory
activities in the following sections. Three classes of esterquats (5a–
h) were prepared and the synthetic route for each class involved
four steps as shown in Scheme 1. In the first step, 11-BUA (1)
was converted into methyl 11-bromoundecanoate (11-BUME) (2),
followed by the synthesis of methyl-11-(alkylamino) undecanoates
1
5
n = 14–16 showed better activity.
The results from the present study showed that the synthesized
esterquats with hexyl amine 5a and 5d showed good antimicrobial
activity on Gram-positive bacteria with MIC values ranging
ꢀ1
between 1.9 and 31.2 lg mL . Intermediate amine esters, i.e., sec-
ondary amine ester (3a), tertiary amine diester (3d) prepared using
hexyl amine showed good antimicrobial activity on Gram-positive
ꢀ1
bacteria with MIC values ranging between 3.9 and 62.5 lg mL ,
while the tertiary amine monoesters 3g–h (synthesized from dioc-
(
3a–c, secondary amine monoester and 3g–h, tertiary amine
tyl and dicyclohexyl amine, respectively) showed MIC values rang-
0
ꢀ1
monoester) and dimethyl-11,11 -(alkylazanediyl)diundecanoates
ing between 15.6 and 62.5
lg mL . However, in comparison
(
3d–f, tertiary amine diester) by the reaction of 11-BUME with dif-
between amine esters and esterquats, the antimicrobial activity
of esterquats was improved by quaternization of amine ester. We
observed that after the quaternization of the amine esters 3a–h,
the antimicrobial activity of esterquats 5a–h increased signifi-
cantly. Among all the synthesized esterquats, 5a, 5b, 5d, 5g and
5h displayed broad spectrum antimicrobial activity exhibiting
excellent inhibitory effects on Gram-positive bacterial strains and
less activity against Gram-negative bacterial strains.
ferent aliphatic amines. In the third step, the synthesized sec-
ondary amine monoesters (3a–c), tertiary amine mono (3g–h)
and diesters (3d–f) were converted into monoesterquats (4a–c
and 4g–h) and diesterquats (4d–f) by reacting with methyl iodide.
The resultant esterquats (4a–h) were converted to the final ester-
quats (5a–h) with chloride counter ion by passing through Amber-
ꢀ
34
lyst A-26 Cl ion exchange resin.
Amberlyst A-26 is a
ꢀ
macroporous, styrene divinylbenzene Cl anion exchanger, which
enables the halide switch. During the process of passing the ester-
quats through the resin, the iodide counter ion is swapped and
exchanged with the chloride counter ion. Further, all the synthe-
sized compounds were obtained in good yields in the range of
Among these five compounds, 5d was found to exhibit excellent
antimicrobial activity with MIC values ranging between 1.9 and
ꢀ1
7.8
lg mL . The compound 5d showed excellent antimicrobial
activity against Micrococcus luteus MTCC 2470 with the MIC value
ꢀ
1
of 1.9
lg mL . The compound 5d showed good inhibitory activity
8
5–96%.
Antimicrobial activity: The synthesized amine esters and ester-
on Staphylococcus aureus MTCC 96 and Staphylococcus aureus
ꢀ1
MLS16 MTCC 2940 strains with the value of 3.9
against Bacillus subtilis MTCC 121 the MIC value was 7.8
l
g mL
and
ꢀ
1
quats were screened for their antimicrobial activity against both
Gram-positive as well as Gram-negative bacterial strains along
with Candida albicans fungal strain.35 The results to this regard
are shown in Table 1 which indicate that some of the compounds
exhibited variable inhibitory effects with minimum inhibitory con-
lg mL .
Moreover, it also showed excellent antifungal activity on Candida
albicans MTCC 3017. The amine ester and esterquat prepared from
dicyclohexyl amine, 3h and 5h showed promising antimicrobial
ꢀ1
activity with MIC values ranging between 3.9 and 62.5 lg mL .
ꢀ1
centration (MIC) values ranging between 1.9 and 31.2
While, the compounds prepared from dodecyl/C12 carbon chain
3b, 3e and 5e) and octadecyl/C18 carbon chain (3c, 3f, 5c and 5f)
did not show any antimicrobial activity on the tested strains up
lg mL
.
The compounds prepared from dioctyl amine, 3g and 5g also
showed good antimicrobial activity with MIC values ranging
ꢀ1
(
between 3.9 and 62.5 lg mL . The antimicrobial screening results
indicate that the esterquats are more potent than intermediate
amine esters. Further, the minimum bactericidal concentration
(MBC) was also evaluated for the synthesized amine esters and
esterquats and the MBC values ranged between 3.9 and
ꢀ1
to the maximum tested concentration of >125 lg mL except for
the compound 5b. Earlier reports also suggest that the quaternized
compounds with alkyl chain length above C12 did not show any
O
O
Cl
Cl
N
O
N
n
n
O
O
O
N-alkyl, N,N-dimethyl monoesterquat,
n = 3 (5a), 6 (5b), 9 (5c)
N-alkyl, N-methyl diesterquat,
n = 3 (5d), 6 (5e), 9 (5f)
O
O
m
N
O
N
O
Cl
Cl
m
N,N-dioctyl, N-methyl monoesterquat,
m = 5 (5g)
N,N-dicyclohexyl, N-methyl monoesterquat
5h)
(
Figure 1. Molecular structures of the synthesized novel esterquats.

1
980
S. R. Yasa et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1978–1982
Br OH
1
O
i
Br
O
2
O
ii
a
iib
iic
O
H
N
R1
O
O
O
O
N
O
R
R
R
N
R
1
3
a-c
a-c
O
O
O
3g-h
iii
3d-f
O
iii
I
iii
N
R1
I
O
R1
N
O
4
I
O
O
O
4
g-h
iv
R N
O
iv
Cl
4d-f
O
N
O
R1
Cl
R
iv
R1
N
O
O
5a-c
O
5
g-h
Cl
R N
O
O
5d-f
O
R = CH3- CH
R = CH3- CH
R = CH3- CH
2
2
2
-(CH
-(CH
-(CH
2
)
3
-CH
-CH
2
2
-
-
=
3a, 3d, 4a, 4d, 5a and 5d
1
R
R
2 2 5 2
= CH3-CH -(CH ) -CH - = 3g, 4g, 5g
= Cyclohexyl group - = 3h, 4h, 5h
2
)
9
= 3b, 3e, 4b, 4e, 5b and 5e
= 3c, 3f, 4c, 4f, 5c and 5f
1
2
)
15-CH
2
-
Scheme 1. Reagents and conditions: (i) methanol, PTSA, reflux, 6 h, 99.5%; (ii
a
) R-NH
2
(1.1 equiv), K
2
CO
3
, 90–110 °C, 12 h, 85–90%; (ii
b
) R-NH
2
(0.55 equiv), K
2
CO
3
, 90–110 °C,
1
1
1
2 h, 88–91%; (ii
c
) R -NH-R (1.1 equiv), K
2
CO
3
, 90–110 °C, 12 h, 95–96% (iii) CH
3
I, K
2
CO , rt, 97–99%; (iv) Amberlyst A 26 chloride ion resin, 96–98%.
3
Table 1
Antimicrobial activity of synthesized amine esters and esterquats
Minimum inhibitory concentration (l )
g mL 1
ꢀ
Test compounds
aS. a.
aB. s.
aS. m.
aM. l.
bK. p.
bE. c.
bP. a.
cC. a.
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
a
b
c
d
e
f
3.9
7.8
3.9
15.6
>125
>125
31.2
>125
>125
31.2
15.6
3.9
7.8
>125
1.9
>125
>125
3.9
>125
>125
>125
>125
>125
>125
>125
>125
15.6
7.8
>125
>125
>125
>125
31.2
>125
0.9
>125
>125
>125
>125
>125
>125
>125
>125
31.2
15.6
>125
>125
>125
>125
>125
>125
0.9
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
0.9
15.6
>125
>125
>125
>125
>125
>125
>125
31.2
31.2
>125
7.8
>125
>125
15.6
15.6
—
>125
>125
31.2
>125
>125
15.6
31.2
3.9
15.6
>125
3.9
>125
>125
3.9
>125
>125
62.5
>125
>125
>125
62.5
7.8
15.6
>125
7.8
>125
>125
7.8
>125
>125
62.5
>125
>125
62.5
31.2
7.8
7.8
>125
3.9
>125
>125
7.8
g
h
3.9
0.9
—
7.8
0.9
—
3.9
0.9
—
7.8
0.9
—
Ciprofloxacin
Miconazole
—
—
—
7.8
MIC-minimum inhibition concentration and the values are mean of three determinations. a Gram-positive bacteria; b Gram-negative bacteria; c fungus; S. a. (Staphylococcus
aureus MTCC 96); B. s. (Bacillus subtilis MTCC 121); S. m. (Staphylococcus aureus MLS16 MTCC 2940); M. l. (Micrococcus luteus MTCC 2470); K. p. (Klebsiella planticola MTCC
5
30); E. c. (Escherichia coli MTCC 739); P. a. (Pseudomonas aeruginosa MTCC 2453); C. a. (Candida albicans MTCC 3017).
ꢀ
1
3
5
1.2
l
g mL which is twice the MIC value for the compounds 3a,
Antifungal activity: The synthesized compounds, i.e., amine ester
(3a) and esterquats (5a, 5b, 5d, 5g and 5h) showed good antifungal
activity against Candida albicans MTCC 3017 (Table 1), which
a, 5b, 5d, 5g and 5h. The results to this regard are shown in
Supplementary data Table S1.

S. R. Yasa et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1978–1982
1981
Table 2
screening results, it can be concluded that the compounds with
above C12 carbon chain length did not show any antifungal activity.
The selected compounds from the above study were further
tested for their minimum fungicidal concentration (MFC). The
compound 5d displayed a MFC value of 7.8 lg mL identical to
the standard Miconazole against Candida albicans MTCC 4748.
Antifungal activity of synthesized amine esters and esterquats
Fungal strain
Minimum inhibitory concentration (MIC, l
g mLꢀ1)
3a
5a
5b
5d
5h
5g
Miconazole
ꢀ
1
C. a. 3017
C. a. 183
C. a. 227
15.6
31.2
31.2
15.6
15.6
31.2
31.2
62.5
62.5
31.2
15.6
31.2
15.6
31.2
15.6
15.6
31.2
15.6
31.2
15.6
31.2
15.6
15.6
15.6
31.2
15.6
15.6
15.6
31.2
31.2
31.2
31.2
62.5
62.5
62.5
15.6
31.2
62.5
31.2
31.2
62.5
31.2
7.8
15.6
15.6
15.6
31.2
31.2
15.6
15.6
15.6
15.6
15.6
15.6
31.2
31.2
15.6
15.6
31.2
15.6
15.6
31.2
31.2
31.2
15.6
15.6
62.5
15.6
31.2
31.2
31.2
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
7.8
15.6
15.6
31.2
15.6
15.6
15.6
7.8
15.6
15.6
7.8
Moreover, the MFC results correlated well with the antifungal
C. a. 854
activity data and the MFC values ranged between 7.8 and
C. a. 1637
C. a. 3018
C. a. 3958
C. a. 4748
C. a. 7315
C. p. 1744
C. as. 1962
C. g. 3019
C. k. 3020
I.s. 4755
ꢀ1
6
2.5
l
g mL which is twice the MIC value for all the tested com-
pounds. The results to this regard are shown in Supplementary
data Table S2.
Anti-biofilm assay: Biofilms are complex communities of bacte-
ria that exist in a self-produced matrix of polysaccharides, proteins,
and extracellular DNA responsible for the growing resistance in
15.6
31.2
15.6
3
6
bacteria towards antibiotics. Several clinically significant patho-
gens producing biofilms are implicated in more than 80% of the
3
7
bacterial infections. Hence, the synthesized amine esters and
esterquats were studied for their biofilm inhibitory activity against
four bacterial strains, namely Staphylococcus aureus MTCC 96, Bacil-
lus subtilis MTCC 121, Staphylococcus aureus MLS16 MTCC 2940 and
Micrococcus luteus MTCC 2470. The results on the biofilm inhibitory
activity for the synthesized amine esters and esterquats shown in
Table 3 indicated that these compounds exhibited good to moder-
ate anti-biofilm activity against all the four tested strains. In partic-
ular, the compound 5d demonstrated promising anti-biofilm
MIC values are the mean of three estimations. C.a. 3017 (C. albicans MTCC 3017); C.
a. 183 (Candida albicans MTCC 183); C. a. 227 (C. albicans MTCC 227); C. a. 854 (C.
albicans MTCC 854); C. a. 1637 (C. albicans MTCC 1637); C. a. 3018 (C. albicans MTCC
3018); C. a. 3958 (C. albicans MTCC 3958); C. a. 4748 (C. albicans MTCC 4748); C. a.
7315 (C. albicans MTCC 7315); C. p. 1744 (Candida parapsilosis MTCC 1744); C. as.
1962 (Candida aaseri MTCC 1962); C. g. 3019 (Candida glabrata MTCC 3019); C. k.
3020 (Candida krusei MTCC 3020) and I. s. 4755 (Issatchenkia hanoiensis MTCC
4755).
ꢀ1
prompted us to further test these compounds against thirteen
more fungal strains and the results to this regard are tabulated in
Table 2. As evident from the results, all the six compounds showed
excellent to good antifungal activity with MIC values ranging
activity with IC50 values of 0.9, 1.9, 1.4 and 4.1 lg mL against Sta-
phylococcus aureus MLS16 MTCC 2940, Staphylococcus aureus MTCC
96, Micrococcus luteus MTCC 2470 and Bacillus subtilis MTCC 121,
respectively. Other compounds such as 3a (IC50 values ranged
ꢀ1
ꢀ1
ꢀ1
between 7.8 and 62.5
l
g mL . The compound 5d was found to
between 2.2 and 4.2
l
g mL ), 5a (2.1–4.8
l
g mL ), 5h (2.5–
ꢀ1
ꢀ1
be most effective among the all tested compounds which showed
4.4
l
g mL ) and 5g (2.0–4.8
l
g mL ) showed good biofilm inhi-
excellent activity identical to the standard antifungal drug
bitory activity on all the tested bacterial strains. It is important
to note that the ability to inhibit biofilm formation of these com-
pounds to a maximum extent is in coherence with the antimicro-
bial activity against the respective strain. Previous literature
reports also suggest that the QACs exhibited promising biofilm
ꢀ1
(
Miconazole) with a MIC value of 7.8 lg mL against C. albicans
MTCC 3017, C. albicans MTCC 4748 and Candida aaseri MTCC 1962.
Moreover, this compound also displayed good antifungal activ-
ity against Candida albicans MTCC 183, C. albicans MTCC 227, C.
albicans MTCC 1637, C. albicans MTCC 3018, C. albicans MTCC
3
8
inhibitory activity. Further, it is reported earlier that reactive
oxygen species (ROS) is an important inducer of apoptosis in bac-
teria which may cause oxidative damage to cellular compounds
3
958, C. albicans MTCC 7315, Candida parapsilosis MTCC 1744, Can-
dida glabrata MTCC 3019 and Issatchenkia hanoiensis MTCC 4755
ꢀ1
39
with the MIC value of 15.6
l
g mL and showed moderate activity
and lead to cellular dysfunction or cell death. Therefore, to eluci-
against C. albicans MTCC 854 and Candida krusei MTCC 3020 with
MIC value of 31.2 lg mL . The synthesized compounds from ami-
nes like dicyclohexyl amine (5h) and dioctyl amine (5g) showed
date whether oxidative stress is involved in the apoptotic cell
death, the intracellular ROS accumulation within the cells of
ꢀ1
0
0
mature biofilms was measured using the fluorescent probe 2 ,7 -
dichlorofluorescein-diacetate (DCFH-DA), a general ROS fluores-
cent probe. The DCFH-DA dye conversion mainly depends on the
metabolically active cells in the biofilm. DCFH-DA is deacetylated
in the cells where it can react quantitatively with intracellular
good to moderate antifungal activity with MIC values ranging
ꢀ1
between 15.6 and 31.2
lg mL , while the other compounds like
3
a, 5a (C carbon chain) and 5b (C12 carbon chain) also showed
6
good to moderate antifungal activity. Based on the antifungal
Table 3
Biofilm inhibition assay of synthesized compounds
Test compounds
IC50 values (
S.a
l
g mLꢀ1)
B.s
S.m
M.l
2.2 ± 0.24
3
3
3
3
5
5
5
5
5
a
d
g
h
a
b
d
g
h
2.6 ± 0.12
20.1 ± 0.52
14.5 ± 0.19
21.3 ± 0.27
2.5 ± 0.44
9.1 ± 0.33
1.9 ± 0.08
3.2 ± 0.26
2.5 ± 0.14
0.6 ± 0.07
4.2 ± 0.18
—
—
3.9 ± 0.16
—
—
20.8 ± 0.35
3.2 ± 0.25
22.2 ± 0.56
0.9 ± 0.11
4.8 ± 0.24
3.6 ± 0.34
0.4 ± 0.06
18.6 ± 0.32
17.2 ± 0.12
19.9 ± 0.62
2.1 ± 0.22
8.8 ± 0.44
1.5 ± 0.16
2.0 ± 0.16
2.5 ± 0.18
0.6 ± 0.08
—
4.8 ± 0.32
8.5 ± 0.26
4.1 ± 0.18
4.6 ± 0.22
4.4 ± 0.38
0.5 ± 0.06
Ciprofloxacin
IC50 (Half maximal inhibitory concentration) and the values are mean of three
determinations. S. a. (Staphylococcus aureus MTCC 96); B. s. (Bacillus subtilis MTCC
1
21); S. m. (Staphylococcus aureus MLS16 MTCC 2940); M. l. (Micrococcus luteus
Figure 2. Intracellular ROS accumulation in Staphylococcus aureus MLS16 MTCC
2940 of compound 5d.
MTCC 2470).

1
982
S. R. Yasa et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1978–1982
radicals (mainly H
2
O
2
) to get converted to its fluorescent product
References and notes
(
DCF), which is retained within the cells and thus provides an
1
.
Bergeron, R. J.; Feng, Y.; Weimar, W. R.; McManis, J. S.; Dimova, H.; Porter, C.;
Raisler, B.; Phanstiel, O. J. Med. Chem. 1997, 40, 1475.
Blaghbrough, I. S.; Carrington, S.; Geall, A. J. Pharm. Sci. 1997, 3, 223.
index of oxidation in the cell cytosol. The intracellular ROS accu-
mulation in the biofilms of Staphylococcus aureus MLS16 MTCC
2
2
.
940 was measured using DCFHDA dye, in which the dye conver-
3. Lie Ken Jie, M. S. F.; Syed-Rahmatullah, M. S. K. J. Am. Oil Chem. Soc. 1992, 69,
59.
4
4
0
3
sion depends on the metabolically active cells in the biofilm.
.
.
Las, C. D. J. Am. Oil Chem. Soc. 1984, 61, 184.
After treatment with esterquat (5d) and Ciprofloxacin, a significant
increase in the accumulation of ROS levels was observed in Staphy-
lococcus aureus MLS16 MTCC 2940 as shown in Figure 2.
5
Tezel, U.; Pavlostathis, S. G. Role of Quaternary Ammonium Compounds on
Antimicrobial Resistance in the Environment. In Antimicrobial Resistance in the
Environment; Keen, P. L., Montforts, M. H. M. M., Eds.; John Wiley & Sons, 2011;
pp 349–387.
When compared with untreated biofilms, the esterquat (5d)
treated biofilms showed increased levels of ROS accumulation.
The ROS measurements 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, suggesting that
the ROS accumulation is of intracellular origin. This accumulated
ROS may be responsible for the bactericidal activity. Recent studies
also indicate that under oxidative stress, the free radicals con-
tribute to arrest the cell growth or cause bacterial mediated cell
death by damaging the specific essential metabolic enzymes (cel-
lular respiratory chain), disrupting cellular membrane, and DNA
damage ultimately leading to cell lysis and death.41
In conclusion, we have synthesized new amine esters and ester-
quats by employing a simple and convenient synthetic approach
with good yields using different aliphatic amines and 11-bro-
moundecanoic acid. Synthesized compounds were evaluated for
antibacterial, antifungal and biofilm inhibitory activities. Some of
the synthesized amine esters and esterquats showed promising
to moderate antimicrobial activity. Further, some of the com-
pounds also displayed potent antifungal activity against fourteen
different Candida strains. Especially, the esterquat 5d was found
to exhibit excellent activity against Candida albicans MTCC 3017,
Candida albicans MTCC 4748 and Candida aaseri MTCC 1962 strains.
We observed that among the synthesized compounds, the antibac-
terial and antifungal activities decreased with increasing chain
length of amine esters and esterquats. The compounds prepared
using hexylamine, 3a, 5a and 5d showed antibacterial and antifun-
gal activities, while the compounds synthesized using dodecy-
lamine and octadecylamine did not show any antimicrobial
activity except the esterquat 5b. Furthermore, the compound 5d
6
.
.
Boethling, R. S. Environmental Aspects of Cationic Surfactants. In Cationic
Surfactants: Analytical and Biological Evaluations; Cross, J., Singer, E. J., Eds.;
Marcel Dekker: New York, USA, 1994; p 102.
Tiedink, J. Cationic Surfactants: Cationic Surfactants in Biocides In Handbook of
Applied Surface and Colloid Chemistry; Holmberg, K., Ed.; John Wiley & Sons:
West Sussex, England, 2001; Vol. 1, pp 318–321.
7
8. Thebault, P.; Taffin de Givenchy, E.; Levy, R.; Van den Berghe, Y.; Guittard, F.;
Geribaldi, S. Eur. J. Med. Chem. 2009, 44, 717.
9.
Kourai, H.; Yabuhara, T.; Shirai, A.; Maeda, T.; Nagamune, H. Eur. J. Med. Chem.
006, 41, 437.
10. Pernak, J.; Chawla, P. Eur. J. Med. Chem. 2003, 38, 1035.
2
11. Jennings, M. C.; Minbiole, K. P. C.; Wuest, W. M. ACS Infect. Dis. 2015, 1, 288.
12. Tezel, U.; Pavlostathis, S. G. Curr. Opin. Biotechnol. 2015, 33, 296.
13. Jennings, M. C.; Ator, L. E.; Paniak, T. J.; Minbiole, K. P.; Wuest, W. M.
ChemBioChem 2014, 15, 2211.
14. Oblak, E.; Piecuch, A.; Krasowska, A.; Luczynski, J. Microbiol. Res. 2013, 168, 630.
15. Gilbert, P.; Moore, L. E. J. Appl. Microbiol. 2005, 99, 703.
16. Ioannou, C. J.; Hanlon, G. W.; Denyer, S. P. Antimicrob. Agents Chemother. 2007,
51, 296.
17. Bodor, N.; Kaminski, J. J.; Selk, S. J. Med. Chem. 1980, 23, 469.
18. Haldar, J.; Kondaiah, P.; Bhattacharya, S. J. Med. Chem. 2005, 48, 3823.
19. Tyagi, R.; Tyagi, V. K.; Khanna, R. K. J. Oleo. Sci. 2006, 55, 337.
20. Farbenindustrie, A. G. A Process for the Preparation of Quaternary Ammonium
Compounds, Ger. Patent. 521035, 1931.
2
2
2
2
1. Kruger, G.; Boltersdorf, D.; Overkempe, K. In Esterquats in Novel Surfactants;
Holmberg, K., Ed.; M. Dekker: New York, 1998; pp 116–119.
2. Luczynski, J.; Fr a˛ ckowiak, R.; Wloch, A.; Kleszczynska, H.; Witek, S. Cell. Mol.
Biol. Lett. 2013, 18, 89.
3. Kim, Y. S.; Kim, H. W.; Lee, S. H.; Shin, K. S.; Hur, H. W.; Rhee, Y. H. Int. J. Biol.
Macromol. 2007, 41, 36.
4. Mohamed, A. S.; Mohamed, M. Z. J. Surfact. Deterg. 2010, 13, 159.
25. Han, B.; Geng, T.; Jiang, Y.; Ju, H. J. Surfact. Deterg. 2015, 18, 91.
2
2
2
2
6. Achouri, M. E.; Alehyen, S.; Assioui, A.; Chami, R.; Bensajjay, F.; PeRez, L.;
Infante, M. R. J. Surfact. Deterg. 2013, 16, 473.
7. Lindstedt, M.; Allenmark, S.; Thompson, R. A.; Edebo, L. Antimicrob. Agents
Chemother. 1949, 1990, 34.
8. Thorsteinsson, T.; Masson, M.; Kristinsson, K. G.; Hjalmarsdottir, M. A.;
Hilmarsson, H.; Loftsson, T. J. Med. Chem. 2003, 46, 4173.
9. Skrzypczakl, A.; Brycki, B.; Mirska, I.; Pernak, J. Eur. J. Med. Chem. 1997, 32, 661.
was identified as
investigation.
a
promising lead molecule for further
30. Benoit, H. Soap Cosmetics 2001.
31. Mishra, S.; Tyagi, V. K. J. Oleo. Sci. 2007, 56, 269.
32. Schramm, J. A. Liquid Detergents, Surfactant Science Series In In Lai, K.-Y., Ed.;
Marcel Dekker: New York, 1997; Vol. 67,.
3. Lie Ken Jie, M. S. F.; Pasha, M. K. J. Chem. Soc., Perkin Trans. I 1996, 12, 1331.
4. Srinivas, R.; Garu, A.; Moku, G.; Agawane, S. B.; Chaudhuri, A. Biomaterials 2012,
Acknowledgments
3
3
3
3, 6220.
One of the authors Y.S. Reddy gratefully acknowledges the
Department of Biotechnology (DBT), New Delhi for the financial
assistance under sponsored project and Director, CSIR-IICT for pro-
viding the facilities.
3
5. Amsterdam, D. Susceptibility Testing of Antimicrobials in Liquid Media. In
Antibiotics in Laboratory Medicine; Loman, V., Ed., 4th ed.; Williams and
Wilkins: Baltimore, MD, 1996; pp 52–111.
6. Olson, I. Eur. J. Clin. Microbiol. Infect. Dis. 2015, 34, 877.
7. Davies, D. Nat. Rev. Drug Disc. 2003, 2, 114.
8. Sandt, C.; Barbeau, J.; Gagnon, M. A.; Lafleur, M. J. Antimicrob. Chemother. 2007,
60, 1281.
9. Madeo, F.; Frohlich, E.; Ligr, M.; Grey, M.; Sigrist, S. J.; Wolf, D. H.; Frohlich, K. U.
J. Cell Biol. 1999, 145, 757.
0. Kumar, C. G.; Poornachandra, Y. Coll. Surf. B: Biointerf. 2015, 125, 110.
41. Dixon, S. J.; Stockwell, B. R. Nat. Chem. Biol. 2013, 10, 9.
3
3
3
Supplementary data
3
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.03.
4
0
02.