Synthesis, characterization, antimicrobial and anti-biofilm activity of a new class of 11-bromoundecanoic acid-based betaines

Article abstract of DOI:10.1007/s00044-017-1958-y

Novel betaines were synthesized from esterquats, which in turn were obtained from the reaction of 11-bromo undecanoic acid, different alkyl amines, and methyl iodide. The synthesized betaines were characterized by fourier transform infrared, proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and mass spectral analysis. These betaines were synthesized in four steps; in the first step, 11-bromo undecanoic acid was converted into methyl 11-bromoundecanoate followed by the synthesis of secondary amine monoester, and tertiary amine mono and diesters by the reaction of 11-bromoundecanoate with different aliphatic amines (hexyl, dodecyl, octadecyl, dioctyl, and dicyclohexyl amine). In the third step, the prepared secondary amine monoesters, tertiary amine mono, and diesters were converted into monoesterquats and diesterquats by reacting with methyl iodide. The resultant esterquats were converted into betaines by saponification reaction using LiOH.H₂O in water and tetrahydrofuran. The synthesized compounds (5a–h) were studied for their antimicrobial activity. Some of the compounds showed good to moderate antibacterial activity with minimum inhibitory concentration values ranging between 3.9–31.2 μg mL^?1 and antifungal activity with minimum inhibitory concentration values ranging between 7.8–62.4 μg mL^?1. Further, some of the betaines also showed good anti-biofilm activity with IC₅₀ values ranging between 2.1–25.3 μg mL^?1 on the tested pathogenic microbial and fungal strains.

Full text of DOI:10.1007/s00044-017-1958-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1958-y
ORIGINAL RESEARCH
Synthesis, characterization, antimicrobial and anti-biofilm activity
of a new class of 11-bromoundecanoic acid-based betaines
1 ●
2 ●
2 ●
Sathyam Reddy Yasa Y. Poornachandra C. Ganesh Kumar
1
Vijayalakshmi Penumarthy
Received: 27 March 2017 / Accepted: 10 June 2017
©
Springer Science+Business Media, LLC 2017
Abstract Novel betaines were synthesized from esterquats,
which in turn were obtained from the reaction of 11-bromo
undecanoic acid, different alkyl amines, and methyl iodide.
The synthesized betaines were characterized by fourier
transform infrared, proton nuclear magnetic resonance,
carbon-13 nuclear magnetic resonance, and mass spectral
analysis. These betaines were synthesized in four steps; in
the first step, 11-bromo undecanoic acid was converted into
methyl 11-bromoundecanoate followed by the synthesis of
secondary amine monoester, and tertiary amine mono and
diesters by the reaction of 11-bromoundecanoate with dif-
ferent aliphatic amines (hexyl, dodecyl, octadecyl, dioctyl,
and dicyclohexyl amine). In the third step, the prepared
secondary amine monoesters, tertiary amine mono, and
diesters were converted into monoesterquats and dies-
terquats by reacting with methyl iodide. The resultant
esterquats were converted into betaines by saponification
and antifungal activity with minimum inhibitory concentra-
−1
tion values ranging between 7.8–62.4 µg mL . Further,
some of the betaines also showed good anti-biofilm activity
−1
with IC50 values ranging between 2.1–25.3 µg mL on the
tested pathogenic microbial and fungal strains.
●
●
Keywords 11-Bromoundecanoic acid Esterquats
●
●
Betaines Antimicrobial activity Anti-biofilm activity
Introduction
Betaines are the quaternary ammonium-derived significant
zwitterionic compounds having diverse applications in
medicine, pharmacy, and biology due to their biocidal
properties (Baaman 1978; Lindstedt et al. 1990). The
betaines are extensively used as antimicrobial (Birnie et al.
2000; Peddie et al. 2003) and anti-biofilm agents in tooth-
paste formulations for the treatment of oral diseases to
control dental plaque without producing an esthetically
unacceptable discoloration of tooth surfaces since they have
zwitterionic structure with a non-exchangeable anion with
reaction using LiOH.H O in water and tetrahydrofuran. The
2
synthesized compounds (5a–h) were studied for their anti-
microbial activity. Some of the compounds showed good to
moderate antibacterial activity with minimum inhibitory
−
1
concentration values ranging between 3.9–31.2 µg mL
+
−
chemical structure of R R R N (CH ) COO (R =
1
2
3
2 n
1
Electronic supplementary material The online version of this article
doi:10.1007/s00044-017-1958-y) contains supplementary material,
which is available to authorized users.
chain length C12 to C , R , and R = methyl group, n = 7
18
2
3
(
to 15) (Robert et al. 1988; Kowalczyk 2008). Peddie and
coworkers reported the synthesis and antimicrobial activity
of aromatic and semi-aromatic betaines (Peddie et al. 2003).
Similarly, Cosquer and coworkers studied the antibacterial
activity of glycine betaines on different types of pathogenic
Gram-positive and Gram-negative bacterial strains, and
observed that some of the compounds showed good anti-
bacterial activity (Cosquer et al. 2004). Several authors also
reported the effective osmoprotectant property of amino
*
Vijayalakshmi Penumarthy
pvl@iict.res.in
1
2
Centre for Lipid Research, CSIR-Indian Institute of Chemical
Technology, Hyderabad, Telangana 500007, India
Medicinal Chemistry and Pharmacology Division, CSIR-Indian
Institute of Chemical Technology, Hyderabad, Telangana 500007,
India

Med Chem Res
acid based betaines like glycine and proline betaines for
bacteria (Abdel-Ghany et al. 1993; Amin et al. 1995).
Betaines show unique amphoteric surfactant properties
since they contain a hydrophobic carbon chain and find
applications mostly in the toiletries and personal care pro-
ducts niche areas (Kumar et al. 2007; Quan et al. 2012;
Domingo 1990; Zhou et al. 2016). Further, many of these
compounds display interesting physical properties, exhi-
biting phase transitions with ferroelectric, antiferroelectric,
and ferroelastic behavior as well as phases with commen-
surate and incommensurate superstructures (Schaack 1990).
Betaines play a significant role in various living systems as
osmoprotectors, osmoregulators and methyl transfer agents.
Complexes of these bases can be important as model sys-
tems due to the role of the hydrogen bonds in many bio-
logically important reactions (Godzisz et al. 2002). The
present study describes the synthesis of novel betaines by
employing 11-bromoundecanoic acid, various aliphatic
amines and methyl iodide in new synthetic approach. The
synthesized betaines were characterized and were further
studied for their antimicrobial and anti-biofilm activities.
recorded on Waters (Model: Q-STAR XL, Applied Bio-
systems, USA) Mass Spectrometer equipped with an Elec-
trospray Ionization source. HRMS data were recorded on a
Thermo Scientific Exactive Orbitrap mass spectrometer
(Germany) and are given in mass units (m/z).
Synthesis of methyl 11-(alkylamino) undecanoate (3a–c)
Initially, 11-bromoundecanoic acid (1) was converted into
11-bromoundecanoate (11-BUME) (2) by reacting with
methanol in presence of PTSA. Later, 11-BUME (1.0 eq)
and amine (hexyl, dodecyl, and octadecyl amine; 1.1 eq)
were dissolved in DMF solvent and stirred for 10 min fol-
lowed by the addition of anhydrous K CO (2 eqs to 11-
2
3
BUME), and the reaction was continued at 90‒110 °C for
12 h. The progress of the reaction was monitored by TLC
using CHCl and methanol (90:10 v/v) as a solvent system.
3
After completion of the reaction, the catalyst K CO was
2
3
filtered from the crude reaction product and extracted with
ethyl acetate followed by washing with water to remove the
DMF solvent. The crude reaction product was dried by
passing through anhydrous sodium sulfate and the ethyl
acetate solvent was removed under reduced pressure by
using rotary evaporator. Pure methyl 11-(alkylamino)
undecanoate was separated out from the dried crude reac-
tion product by column chromatography using silica gel
(60–120 mesh) with chloroform and methanol (98:2, v/v) as
eluent. Column chromatography was monitored by TLC
using chloroform and methanol (9:1, v/v) solvent system
and the product was identified by iodine vapor. The detailed
characterization of methyl 11-(alkylamino) undecanoates
(3a–c) has been reported by us previously (Yasa et al.
2016). The isolated yields were in the range of 85.0‒90.0%.
Materials and methods
Materials
The raw materials needed for the synthesis of betaines, such
as 11-bromoundecanoic acid, hexyl, dodecyl, octadecyl,
dioctyl, and dicyclohexyl amines were procured from
Sigma-Aldrich Chemicals (USA). Anhydrous potassium
carbonate (K CO ), p-toluenesulfonic acid (PTSA), dime-
2
3
thylformamide (DMF) and other solvents were obtained
from S.D. Fine Chemicals, Mumbai (India). All solvents
and chemicals were of reagent grade and were used directly
without further purification. Silica gel (60–120 mesh) for
column chromatography was bought from Acme Synthetic
Chemicals, Mumbai, India. Precoated thin-layer chromato-
graphy (TLC) plates were purchased from Merck, Darm-
stadt, Germany. All microbial strains were procured from
Microbial Type Culture Collection (MTCC) and Gene
Bank, CSIR-Institute of Microbial Technology, Chandi-
garh, India.
Synthesis of dimethyl 11,11′-(alkylazanediyl)
diundecanoate (3d–f)
11-BUME (1 eq) and amine (0.55 eq) were dissolved in
DMF solvent and stirred for 10 min followed by the addi-
tion of K CO , base catalyst (2 eqs to 11-BUME), and the
2
3
reaction continued at 90‒110 °C for 12 h. After completion
of the reaction, the catalyst K CO was filtered from crude
2
3
Analytical methods
reaction product and extracted with ethyl acetate followed
by washing with water to remove DMF solvent. Pure
dimethyl 11,11′-(alkylazanediyl)diundecanoate was sepa-
rated out from the crude product by column chromato-
graphy using silica gel (60–120 mesh) with chloroform and
methanol (99:1, v/v) as eluent. The detailed characterization
of dimethyl 11,11′-(alkylazanediyl) diundecanoates (3d–f)
has been reported by us previously (Yasa et al. 2016). The
isolated yields were in the range of 88.0‒91.5%.
IR spectra were recorded on a Perkin-Elmer (Model:
Spectrum BX) FT-IR spectrometer using CHCl and KBr.
All proton nuclear magnetic resonance ( H-NMR) spectra
were recorded on 300 MHz (Brucker) and 500 MHz (Var-
ian) spectrometers, respectively. All carbon-13 nuclear
3
1
1
3
magnetic resonance ( C-NMR) spectra were recorded on
25 MHz (Brucker) spectrometer. ESI-MS spectra were
1

Med Chem Res
Synthesis of methyl 11-(dialkylamino) undecanoate (3g–h)
solvent using 3.0% methanol in chloroform (v/v) as eluent.
A 3% solution of the above silica gel column purified car-
boxylic acid quaternary compound with iodide counterion
form in methanol was passed through a column of anion
exchange resin in hydroxide form (Dowex 1-X8) to get pure
betaine containing hydroxide counter ion. The betaines 5c
and 5f were recovered from the residue by recrystallization
using water, acetonitrile and aqueous acetone; whereas the
rest of the compounds were not recrystallized since they
were in liquid form. All the compounds 5a–h were char-
acterized by spectral techniques and the obtained yields
were in the range of 97.5‒99.0%.
1
1-BUME (1.0 eq) and dialkylamine (1.1 eq) were dis-
solved in DMF solvent and stirred for 10 min followed by
the addition of K CO (2 eqs to 11-BUME) at room tem-
2
3
perature and continued the reaction at 90‒110 °C for 12 h.
After completion of the reaction, the catalyst K CO was
2
3
filtered from crude reaction product and extracted with ethyl
acetate followed by washing with water to remove DMF
solvent. The crude reaction product was dried by passing
through anhydrous sodium sulfate and the ethyl acetate
solvent was removed under reduced pressure by using
rotary evaporator. The pure methyl 11-(dialkylamino)
undecanoate was separated out from the crude reaction
product by silica gel (60–120 mesh) column chromato-
graphy using chloroform and methanol (98:2, v/v) as eluent.
Column chromatography was monitored by TLC using
chloroform and methanol (9:1, v/v) solvent system and the
product was identified by iodine vapor. The detailed char-
acterization of methyl 11-(dialkylamino) undecanoates
Characterization of betaines (5a–h)
11-(hexyldimethylammonio) undecanoate (5a)
−
1
Light brown liquid, yield 98.0%; IR (Neat, cm ): 3424,
1
2925, 2855, 1727, 1463, 1184; H-NMR (CDCl , 500
3
+
MHz) δ 3.38–3.25 (4H, m, –H C–N –CH –), 3.11 (6H, s,
2
2
+
(
2
3g and 3h) was reported by us previously (Yasa et al.
016). Isolated yields were in the range of 95.0‒96.0%.
–N –(CH ) ), 2.24 (2H, t, J = 7.15 Hz, –H C–C=O),
3 2 2
1.79–1.64 (4H, m), 1.62–1.48 (2H, m), 1.43–1.22 (18H, m),
1
3
0
.91 (3 H, t, J = 6.60 Hz); C-NMR (CDCl , 125 MHz) δ
3
+
2 2
+
Synthesis of esterquats (4a–h)
174.73 (–C=O), 63.34 (–H C–N –CH –), 50.14 (−N –
(
CH ) ), 33.60 (–H C–C=O), 30.58, 28.64, 28.42, 25.67,
3 2 2
+
The synthesized secondary amine monoesters (3a–c), ter-
tiary amine monoesters (3g–h) and diesters (3d–f) were
converted into esterquats (4a–h) by reacting with methyl
iodide using the following reported procedure (Srinivas
et al. 2012). Amine ester (1 g) was dissolved in 2 mL
chloroform and 5 mL methyl iodide was added to the
solution followed by the addition of catalytic amount of
K CO . The reaction mixture was stirred at room tem-
25.33, 24.33, 21.82, 13.54 (–CH ); ESI-MS (m/z) (M + H)
314.44; HRMS-ESI (m/z) calculated for (C H NO ) , (M
+ H) is 314.3054, found at 314.3055.
3
+
1
9
40
2
+
11-(dodecyldimethylammonio) undecanoate (5b)
−
1
Light brown semi-solid, yield 98.0%; IR (Neat, cm ):
1
3423, 2923, 2851, 1718, 1467, 1180; H-NMR (CDCl , 500
2
3
3
+
perature for 12 h and the solvent and excess methyl iodide
were removed under reduced pressure by using rotary
evaporator. The crude product was purified on silica gel
MHz) δ 3.36–3.19 (4H, m, –H C–N –CH –), 3.09 (6H, s,
2
2
+
−N –(CH ) ), 2.21 (2H, t, J = 7.15 Hz, –H C–C=O),
3
2
2
1.78–1.62 (2H, m), 1.61–1.40 (4H, m), 1.39–1.14 (30H, m),
1
3
(
60‒120 mesh) column chromatography using 2.5–3.0%
0.87 (3H, t, J = 6.60 Hz); C-NMR (CDCl , 125 MHz) δ
3
+
+
methanol in chloroform (v/v) as eluent to afford the pure
174.63 (–C=O), 63.05 (–H C–N –CH –), 50.20 (−N –
2 2
esterquats (4a–h) in good yields (95.0‒98.5%).
(CH ) ), 37.21 (–H C–C=O), 31.40, 29.11, 28.81 X 2,
3 2 2
2
8.63, 28.56, 26.45, 25.83, 24.56, 22.19, 21.81, 13.98; ESI-
+
+
Synthesis of betaines (5a–h)
MS (m/z) (M + H) 398.31, (M + Na) 420.33; HRMS-ESI
+
+
(m/z) calculated for (C H NO ) , (M + H) is 398.3993,
25 52 2
The esterquat (4a–h) (1 g) was dissolved in 10 mL of tet-
found at 398.3999.
rahydrofuran (THF): H O (1:1, v/v) and added 3 eqs of
2
LiOH.H O. The reaction mixture was stirred at room tem-
11-(octadecyldimethylammonio) undecanoate (5c)
2
perature for 12 h. After completion of the reaction, the THF
solvent was removed under reduced pressure using rota
evaporator at 40 °C followed by neutralization using 1 N
HCl and the crude reaction mixture was extracted using
ethyl acetate and washed with water to remove acid traces.
The organic layer was dried over anhydrous sodium sulfate
and concentrated on rotary evaporator. The pure product
was separated by silica gel column chromatography using a
Light brown solid, yield 97.5%, melting point 158.2–158.4 °
−1
1
C; IR (KBr, cm ): 3422, 2919, 2850, 1711, 1468, 1181; H-
NMR (CDCl3, 500 MHz) 3.38–3.17 (4H, m,
δ
–
+
+
H C–N –CH –), 3.10 (6H, s, −N –(CH ) ), 2.23 (2H, t, J
2
2
3 2
= 7.15 Hz, –H C–C=O), 1.79–1.61(2H, m), 1.60–1.41 (4H,
2
1
3
m), 1.40–1.10 (30H, m), 0.87 (3H, t, J = 6.60 Hz); C-NMR
(CDCl , 125 MHz) δ 174.56 (–C=O), 63.02 (–H C–N –
+
3
2

Med Chem Res
+
CH –), 50.24 (−N –(CH ) ), 37.15 (–H C–C=O), 31.51,
–N–CH3), 2.23 (2H, t, J = 7.43 Hz, –H C–C=O),
2
3 2
2
2
2
2
9.15 X 2, 28.84 X 2, 28.63 X 2, 28.56, 26.49 X 2, 25.92,
1.79–1.62 (6H, m), 1.61–1.50 (2H, m), 1.47–1.18 (32H, m),
+
13
4.62, 22.27, 21.85, 13.99; ESI-MS (m/z) (M + H) 482.71,
0.89 (6H, t, J = 6.60 Hz); C-NMR (CDCl , 125 MHz) δ
3
+
+
(
(
M + Na)
504.66; HRMS-ESI (m/z) calculated for
174.78 (–C=O), 60.94 (–N (CH ) ), 47.84 (–N–CH ),
2
3
3
+
+
C H NO ) , (M + H) is 482.4932, found at 482.4913.
33.65 (–H C–C=O), 31.05, 28.64, 28.42, 25.73, 24.33,
3
1
64
2
2
+
2
Na)
(
1.97, 21.59, 13.66; ESI-MS (m/z) (M) 440.44, (M +
+
1
1,11′-(hexyl(methyl)ammonio)diundecanoate (5d)
462.39; HRMS-ESI (m/z) calculated for
+
+
C H NO ) , (M + H) is 440.4462, found at 440.4458.
2
8
58
2
−
1
Light brown gummy liquid, yield 99.0%; IR (Neat, cm ):
3
5
1
423, 2925, 2857, 1714, 1468, 1167; H-NMR (CDCl ,
11-(dicyclohexyl(methyl)ammonio)undecanoate (5h)
3
+
00 MHz) δ 3.27 (6H, br.t, J = 7.7 Hz, –N (CH ) ), 3.06
2
3
−
1
(
3H, s, –N–CH ), 2.24 (2H, t, J = 7.30 Hz, –H C–C=O),
Light brown liquid, yield 98.0%; IR (Neat, cm ): 3425,
3
2
1
1
0
1
3
2
.76–1.64 (4H, m), 1.63–1.49 (6H, m), 1.43–1.22 (30H, m),
2927, 2855, 1721, 1448, 1170; H-NMR (CDCl , 500
3
1
3
+
.88 (3H, t, J = 6.8 Hz); C-NMR (CDCl , 125 MHz) δ
MHz) δ 3.55 (2H, t, J = 11.2 Hz, –H C–N –CH –, dioctyl
3
2
2
+
74.83 (–C=O), 61.05 (–N (CH ) ), 47.85 (–N–CH ),
chain methylene protons adjacent to nitrogen), 3.31–3.17
2
3
3
+
3,68 (–H C–C=O), 30.62, 28.99, 28.67, 28.50,
(2H, m, N –(CH ), undecyl chain methylene protons
2
2
5,78, 25.46, 24.38, 21.87, 21.63, 13.56; ESI-MS (m/z)
adjacent to nitrogen), 2.99 (3H, s, –N–CH ), 2.22 (2H, t, J
3
+
(
(
M + 2H) 484.40; HRMS-ESI (m/z) calculated for
C H NO ) is 484.4360, found at 484.4368.
= 7.15 Hz, –H C–C=O), 2.18–2.06 (2H, m) 2.05–1.91
2
+
13
(4H, m), 1.80–1.51 (10H, m), 1.50–1.19 (20H, m); C-
NMR (CDCl , 125 MHz) δ 174.56 (–C=O), 69.95 (–N –
(CH)2), 56.82 (N –(CH )), 44.60 (–N–CH3), 33.63
2
9
58
4
+
3
+
1
1,11′-(dodecyl(methyl)ammonio)diundecanoate (5e)
2
(
–H C–C=O), 28.71, 28.61, 28.46, 28.31, 26.85, 26.60,
2
−
1
Light brown gammy liquid, yield 98.5%; IR (Neat, cm ):
26.28, 25.37, 25.20, 24.53, 24.34, 23.34; ESI-MS (m/z) (M
+ H) 380.38, (M + Na) 402.33; HRMS-ESI (m/z) cal-
culated for (C H NO ) , (M + H) is 380.3523, found at
380.3533.
1
+
+
3
5
–
1
0
1
3
2
421, 2926, 2855, 1710, 1461, 1164; H-NMR (CDCl ,
00 MHz) δ 3.42–3.30 (6H, m, –N (CH ) ), 3.22 (3H, s,
N–CH3), 2.33 (2H, t, J = 7.47 Hz, –H C–C=O),
.77–1.67 (4H, m), 1.66–1.53 (6H, m), 1.45–1.14 (42H, m),
.88 (3H, t, J = 6.8 Hz); C-NMR (CDCl , 125 MHz) δ
75.19 (–C=O), 61.15 (–N (CH ) ), 48.05 (–N–CH ),
3.83 (–H C–C=O), 31.43, 29.15, 28.95, 29.04, 28.84,
8.79, 28.62, 25.89, 24.50, 22.22, 21.76, 13.85; ESI-MS
3
+
+
+
2
3
24 46 2
2
1
3
Antimicrobial activity
3
+
2
3
3
Antimicrobial activity of the synthesized compounds (5a–h)
was determined using well diffusion method (Amsterdam
1996) against different pathogenic reference strains, such as
Micrococcus luteus MTCC 2470, Bacillus subtilis MTCC
2
+
(
(
m/z) (M + 2H) 568.69; HRMS-ESI (m/z) calculated for
+
C H NO ) is 568.5299, found at 568.5308.
3
5
70
4
121, Staphylococcus aureus MTCC 96, S. aureus MLS16
1
1,11′-(octadecyl(methyl)ammonio)diundecanoate (5f)
MTCC 2940, Klebsiella planticola MTCC 530, Escherichia
coli MTCC 739, Pseudomonas aeruginosa MTCC 2453
and different Candida strains such as Candida albicans
MTCC 183, C. albicans MTCC 227, C. albicans MTCC
854, C. albicans MTCC 1637, C. albicans MTCC 3017, C.
albicans MTCC 3018, C. albicans MTCC 3958, C. albi-
cans MTCC 4748, C. albicans MTCC 7315, C. para-
psilosis MTCC 1744, C. aaseri MTCC 1962, C. glabrata
MTCC 3019, C. krusei MTCC 3020, Issatchenkia
hanoiensis MTCC 4755 procured from the MTCC, CSIR-
Institute of Microbial Technology, Chandigarh, India. The
pathogenic reference strains were seeded on the surface of
the media Petri plates, containing Muller–Hinton agar with
Light brown solid, yield 99.40%, melting point 78.6–78.8 °
−1
C; IR (KBr, cm ): 3426, 2921, 2851, 1730, 1468, 1161;
1
H-NMR (CDCl3, 500 MHz)
δ
3.45–3.27 (6H, m,
+
-
N (CH ) ), 3.18 (3H, s, –N‒CH ), 2.31 (2H, t, J = 7.47
2
3
3
Hz, –H C–C=O), 1.80–1.65 (4H, m), 1.64–1.54 (6H, m),
1
2
1
3
.45–1.14 (54H, m), 0.88 (3H, t, J = 7.0 Hz); C-NMR
+
3 2 3
(
CDCl , 125 MHz) δ 174.60 (–C=O), 60.85 (–N (CH ) ),
4
2
7.73 (–N–CH ), 33.65 (–H C–C=O), 31.27, 29.03, 28.89,
3 2
8.79, 28.69, 28.48, 25.75, 24.36, 22.05, 21.56, 13.73; ESI-
+
MS (m/z) (M + 2H) 652.73; HRMS-ESI (m/z) calculated
for (C H NO ) is 652.6238, found at 652.6229.
+
4
1
82
4
0
.1 mL of earlier prepared microbial suspensions individu-
7
−1
1
1-(dioctyl(methyl)ammonio)undecanoate (5g)
ally containing 1.5 × 10 cfu mL (equal to 0.5 McFarland
standard). Wells of 6.0 mm diameter were prepared in the
media plates using a cork borer and the synthesized betaines
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.
Light brown liquid, yield 99.0%; IR (Neat, cm− ): 3428,
1
1
2
927, 2856, 1725, 1453, 1175; H-NMR (CDCl , 500
3
+
MHz) δ 3.37–3.21 (6H, m, –N (CH ) ), 3.07 (3H, s,
2
3

Med Chem Res
A standard antibiotic solution of ciprofloxacin (bacterial
strains) and miconazole (Candida strains) at a dose range of
C. albicans MTCC 4748 strain was cultured in 24-well
microtitre plates. After biofilm formation, the mature bio-
films were washed with 300 μL of 0.9% (w/v) NaCl, treated
with compound 5g and Miconazole (control) was run in
parallel and incubated for 24 h at 30 °C. Later, the biofilms
were incubated with 10 μM DCFH-DA dye, simultaneously
with the treated test sample. After 24-h incubation, the
fluorescence was measured on an Infinite M200 Pro
microtitre plate reader (Tecan, Switzerland) at excitation
and emission wavelengths of 485 and 535 nm, respectively.
The entire content in each well was removed and the cells
were separated from the supernatant by centrifugation at
1
25–0.9 μg perwell and the well-containing methanol
served as positive and negative controls, respectively. The
plates were incubated for 24 h at 37 °C for bacterial strains
and 30 °C for different Candida strains and the well con-
taining the least concentration showing the inhibition zone
was considered as the minimum inhibitory concentration
(
MIC). All experiments were carried out in duplicates and
the mean values are represented.
Anti-biofilm activity
5000×g for 10 min. The fluorescence of the supernatant and
The synthesized betaines (5a–h) were screened in sterile 96-
well polystyrene microtiter plates using the modified bio-
film inhibition assay against a panel of pathogenic bacterial
strains including S. aureus MTCC 96, S. aureus MLS16
MTCC 2940, B. subtilis MTCC 121, Micrococcus luteus
MTCC 2470, Klebsiella planticola MTCC 530 and
C. albicans MTCC 4748 which were cultured overnight in
tryptone soy broth (supplemented with 0.5% glucose)
that of the resuspended sessile cells in phosphate buffered
saline was measured separately to determine the generated
fluorescence in either intracellular or extracellular environ-
ment. The level of ROS accumulation was quantified in
triplicate and the values are indicated as mean ± S.D.
Results and discussion
Synthesis
(
Olson 2015). The test compounds of predetermined con-
−
1
centrations ranging from 0 to 125 μg mL were mixed with
the bacterial suspensions having an initial inoculum con-
5
−1
centration of 5 × 10 cfu mL . 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
Three types of betaines were prepared (Fig. 1) and the
synthetic route for each class involved four steps as shown
in Scheme 1. In the first step, 11-bromo undecanoic acid (1)
was converted into 11-BUME (2) by esterification reaction
in presence of methanol and PTSA, followed by the
synthesis of methyl-11-(alkylamino) undecanoates (3a–c,
secondary amine monoesters and 3g, h, tertiary amine
monoesters) and dimethyl-11, 11′-(alkylazanediyl) diunde-
canoates (3d–f, tertiary amine diesters) by the reaction of
11-BUME with different aliphatic amines in the presence of
K CO in DMF. In the third step, the synthesized secondary
2
3
amine monoesters (3a–c), tertiary amine mono (3g–h) and
diesters (3d–f) were quaternized by using methyl iodide in
the presence of K CO to monoesterquats (4a–c and 4g, h)
(
Dynex Technologies, Inc, Chantilly, VA, USA). Blank
wells were employed as a background check. The inhibition
data were interpreted from the dose-response curves,
where the 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.
2
3
and diesterquats (4d–f). The resultant esterquats (4a–h)
were converted to the final compounds, i.e., betaines (5a–h)
by saponification reaction with LiOH.H O in water: tetra-
2
hydrofuran (1:1, v/v).
All the synthesized compounds were characterized by
using different spectral techniques like fourier transform
1
13
infrared (FT-IR), H-NMR, C-NMR, and electrospray
ionization-high resolution mass spectrometry (ESI-HRMS).
Detection of intracellular reactive oxygen species (ROS)
accumulated in C. albicans MTCC 4748 biofilms
1
H-NMR spectra of compound 5a showed a multiplet at δ
3.38–3.25 ppm,which indicates the presence of four protons
The intracellular ROS accumulated in C. albicans MTCC
on methylene carbons adjacent to quaternary ammonium
+
4
748 sessile cells was detected using 2,7-dichloro-
group (−N –(CH ) ), a singlet at δ 3.11 ppm reveals the six
2
2
fluorescein diacetate (DCFH-DA), which is converted to
highly fluorescent dichlorofluorescein in the presence of
intracellular ROS (Madeo et al. 1999). Briefly, the
protons of two methyl groups were attached to quaternary
ammonium group and a triplet at δ 2.24 ppm shows two
carbon protons adjacent to carbonyl of carboxyl group. In

Med Chem Res
Fig. 1 Molecular structures of
the synthesized betaines (5a–h)
Scheme 1 Schematic representation for the synthesis of betaines (5a–h)
1
3
+
C-NMR spectra of compound 5a showed some important
(–H C–N –CH –), the peak at δ 50.14 ppm shows the two
2 2
+
characteristic peaks: δ 174.73 ppm indicates the carbonyl
carbon (–C=O), δ 63.34 ppm shows the methylene group
carbons adjacent to quaternary ammonium group
methyl carbons (−N –(CH ) ) and at δ 33.60 ppm indicates
the carbon adjacent to carbonyl functionality (–H C–C=O).
Mass spectral studies (ESI-HRMS) also showed that the
3 2
2

Med Chem Res
Table 1 Antimicrobial activity
of the synthesized betaines
−1
Test compounds
Minimum inhibitory concentration (µg mL )
a
a
a
a
b
b
b
c
(5a–h)
S.a
B.s
S.m
M.l
K.p
E.c
P.a
C.a
5
5
5
5
5
5
5
5
a
b
c
15.6
15.6
>125
>125
>125
>125
15.6
>125
0.9
31.2
15.6
>125
>125
>125
>125
15.6
>125
0.9
15.6
7.8
7.8
15.6
15.6
>125
>125
>125
>125
15.6
>125
0.9
>125
>125
>125
>125
>125
>125
>125
>125
0.9
>125
>125
>125
>125
>125
>125
>125
>125
0.9
31.2
15.6
>125
>125
>125
>125
7.8
7.8
>125
>125
>125
>125
7.8
>125
>125
>125
>125
3.9
d
e
f
g
h
>125
0.9
>125
0.9
>125
–
Ciprofloxacin
Miconazole
–
–
–
–
–
–
–
7.8
The minimum inhibition concentration 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 530); E. c. (Escherichia coli MTCC 739); P. a. (Pseudomonas aeruginosa MTCC 2453);
C. a. (Candida albicans MTCC 3017)
+
concentration of >125 μg mL−1, this could be due to more
hydrophobicity of monoalkyl methyl quaternary ammo-
nium dicarboxylates (5d–f), synthesized by the reaction of
two moles of 11-bromo methylundecanoate and primary
amine. The compound 5h prepared using dicyclohexyl
amine also showed no antimicrobial activity even up to the
base peak at 314.3054 (C H NO +H) corresponding to
1
9
39
2
the molecular weight of the compound which further con-
firmed the formation of desired product, i.e., 5a. In the
FT-IR spectra, the compound (5a) showed a strong
absorption band at 3424 cm− indicating the asymmetric
stretching mode of O–H of carboxyl group; the two strong
bands at 2925 and 2855 cm− show the alkyl chain (–C‒H)
1
1
−1
maximum tested concentration of >125 μg mL . The
−
1
stretching vibration and a strong band at 1727 cm indi-
cates the carbonyl functionality of carboxyl group.
compound 5h didn’t show any antimicrobial activity, this
could be due to more steric hindrance on quaternary
ammonium group by two cyclohexyl rings. Literature
revealed that the antimicrobial activity of quaternary
ammonium compounds is due to electrostatic interactions
between the positively charged quaternary ammonium
compounds and the negatively charged bacterial cellular
membrane, which is followed by permeation of the carbon
chain of quaternary ammonium compound into the intra-
membrane region, ultimately leading to leakage of cyto-
plasmic material and cellular lysis followed bacterial cell
death (Jennings et al. 2015).
Antimicrobial activity
The synthesized betaines (5a–h) were screened for their
antimicrobial activity against both Gram-positive and
Gram-negative bacterial strains as well as against C.
albicans, a fungal strain. It was observed that the majority
of compounds such as 5a, 5b and 5g showed good anti-
microbial activity in quaternary amino monocarboxylate
−
1
form with MIC values ranging between 3.9–31.2 μg mL
,
whereas the compound 5c prepared using long chain alkyl
group, i.e., octadecyl amine did not exhibit any activity on
the tested strains even up to the maximum tested con-
centration of >125 μg mL as shown in Table 1. Earlier
reports also suggested that the betaines with long alkyl
chains (above C14 carbon chain) did not show anti-
microbial activity, indicating that the antimicrobial activity
was dependent on the alkyl chain length (Birnie et al.
The results from the present study showed that the
betaines 5a and 5b in monocarboxylate form synthesized
using hexyl and dodecyl amines showed antimicrobial
activity on Gram-positive bacteria with MIC values ranging
−
1
−
1
between 7.8–62.4 μg mL , while the compound 5g syn-
thesized using dioctyl amine also showed good anti-
microbial activity on Gram-positive bacteria with MIC
−
1
values ranging between 3.9–15.6 μg mL . Whereas, the
intermediate compounds such as amine esters (3a, 3d, 3g,
and 3h) also showed moderate antimicrobial activity was
reported in our previous work (Yasa et al. 2016).
2
000). While the compounds 5d–f having dicarboxylate
ions in their structure did not exhibit antimicrobial activity
on the tested strains even up to the maximum tested

Med Chem Res
Anti-fungal activity
using hexyl and dodecyl amines, respectively, showed good
to moderate antifungal activity with MIC values ranging
−
1
The betaines 5a, 5b, and 5g showed good-to-moderate
antifungal activity against C. albicans MTCC 3017
between 15.6–62.5 μg mL . Based on the antifungal
screening results, it was concluded that the compounds
(5d–f) in quaternary ammonium dicarboxylate ion form,
and prepared from above C -alkyl amines did not show
(
Table 1), which prompted us to further test these com-
pounds against other fungal strains and the results to this
regard are tabulated in Table 2. As evident from the results,
the three compounds 5a, 5b, and 5g showed excellent-to-
good antifungal activity with MIC values ranging between
1
2
any antifungal activity even up to the maximum tested
−
1
concentration of >125 μg mL
.
−1
7
5
.8–62.5 μg mL on tested fungal strains. The compound
g was found to be most effective among the all tested
Anti-biofilm assay
compounds and exhibited excellent antifungal activity
identical to the standard Miconazole with MIC value of 7.8
Biofilms are complex communities of bacteria that exist in a
self-produced matrix of polysaccharides, proteins, and
extracellular DNA responsible for the growing resistance in
bacteria towards various antibiotics (Olson 2015). Several
clinically significant pathogens producing biofilms are
implicated in more than 80% of the bacterial infections
−1
μg mL on different fungal strains, except on some strains
like C. albicans MTCC 227, C. albicans MTCC 3018, C.
parapsilosis MTCC 1744 and C. glabrata MTCC 3019,
which showed good antifungal activity with MIC value of
−1
1
5.6 μg mL . The other compounds 5a and 5b synthesized
(Davies 2003). Therefore, the antimicrobial active com-
pounds 5a, 5b, and 5g were studied for their biofilm inhi-
bitory activity against five bacterial strains, namely S.
aureus MTCC 96, B. subtilis MTCC 121, S. aureus MLS16
MTCC 2940, Micrococcus luteus MTCC 2470 and Kleb-
siella planticola MTCC 530, and one fungal strain like
Candida albicans MTCC 4748.
Table 2 Antifungal activity of synthesized betaines (5a, 5b, and 5g)
Fungal strain
Minimum inhibitory
concentration (µg mL ) of test
−1
compounds
The results of the biofilm inhibitory activity for the
betaines, such as 5a, 5b, and 5g are shown in Table 3,
which suggests that these compounds exhibited good-to-
moderate anti-biofilm activity against all the six tested
strains. In particular, the compound 5g demonstrated good
anti-biofilm activity with IC values of 8.2, 10.1, 4.1, 2.1,
5
a
5b
5g
Miconazole
Candida albicans MTCC 183
C. albicans MTCC 227
C. albicans MTCC 854
C. albicans MTCC 1637
C. albicans MTCC 3018
C. albicans MTCC 3958
C. albicans MTCC 4748
C. albicans MTCC 7315
C. parapsilosis MTCC 1744
C. aaseri MTCC 1962
62.5 15.6 7.8 7.8
31.2 31.2 15.6 7.8
62.5 62.5 7.8 7.8
125
62.5 7.8 7.8
5
0
−
1
31.2 31.2 15.6 7.8
31.2 31.2 7.8 7.8
15.6 15.6 7.8 7.8
31.2 31.2 7.8 7.8
31.2 15.6 15.6 7.8
31.2 15.6 7.8 7.8
15.6 15.6 15.6 7.8
31.2 31.2 7.8 7.8
15.6 31.2 7.8 7.8
10.1, and 3.7 μg mL against S. aureus MTCC 96, B.
subtilis MTCC 121, S. aureus MLS16 MTCC 2940,
Micrococcus luteus MTCC 2470, Klebsiella planticola
MTCC 530 and C. albicans MTCC 4748, respectively.
Other compounds, such as 5a (IC values ranging between
5
0
−1
4
3
.2–25.3 μg mL ) and 5b (IC50 values ranging between
−1
.8–11.1 μg mL ) showed moderate biofilm inhibitory
C. glabrata MTCC 3019
C. krusei MTCC 3020
activity on all the tested strains. Particularly, the compound
5
g showed excellent biofilm inhibitory activity on C. albi-
Issatchenikia hanoiensis MTCC
−1
4
755
cans MTCC 4748 with IC50 value of 3.7 μg mL , while
the standard Miconazole value showed IC50 value of
Table 3 Antibiofilm activity of
synthesized betaines (5a, 5b,
and 5g)
IC50 values in (μg mL−1) of test compounds
Test compounds
5a
5b
5g
Ciprofloxacin Miconazole
Staphylococcus aureus MTCC 96 10.1 ± 0.36 9.8 ± 0.41 8.2 ± 0.25
0.7 ± 0.09
0.6 ± 0.09
0.7 ± 0.11
0.8 ± 0.09
0.6 ± 0.12
–
–
Bacillus subtilis MTCC 121
S. aureus MLS16 MTCC 2940
Micrococcus luteus MTCC 2470
25.3 ± 0.32 8.6 ± 0.18 10.1 ± 0.22
12.1 ± 0.16 3.8 ± 0.11 4.1 ± 0.21
4.2 ± 0.22 4.1 ± 0.18 2.1 ± 0.14
–
–
–
–
Klebsiella planticola MTCC 530 10.5 ± 0.16 11.1 ± 0.18 10.1 ± 0.22
Candida albicans MTCC 4748 11.1 ± 0.35 9.8 ± 0.36 3.7 ± 0.11
2.8 ± 0.12

Med Chem Res
(Halliwell and Aruoma 1991; Kobayashi et al. 2002).
Recent studies suggest that when yeast cells are subjected to
oxidative stress, these free radicals contribute to arrest in
cell growth or result in damage of the specific essential
metabolic enzymes (cellular respiratory chain), disrupting
cellular membrane, and DNA damage ultimately causing
cell lysis and death (Dixon and Stockwell 2013).
Conclusion
In conclusion, a new type of betaine (5a–h) was synthesized
using a simple and convenient synthetic approach with good
yields by employing various aliphatic amines and
Fig. 2 Intracellular ROS accumulation in Candida albicans MTCC
4
748 of compound 5g
11-bromoundecanoic acid. The synthesized betaines (5a–h)
were evaluated for antibacterial, antifungal and anti-biofilm
activities. Some of the synthesized betaines showed good-
tomoderate antimicrobial activity. Further, the several
compounds also displayed excellent-to-good antifungal
activity against different Candida strains. Particularly, the
compound 5g was found to exhibit excellent antifungal
activity against C. albicans MTCC 183, C. albicans MTCC
−
1
2
.8 μg mL . It is important to note that the ability to inhibit
biofilm formation of these compounds to a maximum extent
which is in coherence with the antimicrobial activity against
the respective strain.
Detection of intracellular ROS in mature Candida
biofilms
854, C. albicans MTCC 1637, C. albicans MTCC 3958, C.
albicans MTCC 4748, C. albicans MTCC 7315, C. aaseri
MTCC 1962, C. krusei MTCC 3020 and I. hanoiensis
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). In order to elucidate whether
oxidative stress is involved in the apoptotic cell death, the
intracellular ROS accumulation within the sessile cells of
C. albicans MTCC 4748 mature biofilms was measured
using 2,7-dichlorofluorescein diacetate dye, in which the
dye conversion depends on the state of the metabolically
active cells in the biofilm (Kumar and Poornachandra
−
1
MTCC strains with 7.8 μg mL . Further, it was observed
that among the synthesized betaines the antimicrobial
activity of the compounds prepared using above C -alkyl
1
2
amine, and the compounds 5d–f with dicarboxylate ion
form did not show any activity. The betaines 5a, 5b, and 5g
showed anti-biofilm activity. Among the synthesized
betaines, the compound 5g displayed promising anti-biofilm
activity against C. albicans MTCC 4748 with IC value of
5
0
−1
3
.7 μg mL , while the standard Miconazole showed IC
5
0
−1
2
015). After treatment of C. albicans MTCC 4748 cells
value of 2.8 μg mL . Moreover, the compound 5g was
identified as a promising lead molecule for further
investigation.
with different concentrations of the test compound 5g, a
significantly increased level of intracellular ROS accumu-
lation was observed in the tested C. albicans MTCC
−
1
4
748 strain. At a concentration of 1, 2, and 4 μg mL , the
Acknowledgements One of the authors Sathyam Reddy gratefully
acknowledges the Department of Biotechnology (DBT), New Delhi
for the financial assistance under sponsored project and the Director,
CSIR-IICT for providing the facilities.
compound 5g-treated biofilms showed increased levels of
intracellular ROS accumulation, which was closer to the
standard drug Miconazole. The ROS measurements were
also carried out separately in both sessile cells and in the
supernatant (data not shown). The ROS-induced increase in
fluorescence was observed only for the sessile cells, sug-
gesting that the ROS accumulation was of intracellular
origin (Fig. 2). This accumulated ROS may also be
responsible for the fungicidal activity. Oxygen-containing
free radical molecules and their precursors formed in bio-
logical systems are collectively termed as ROS, comprising
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