Membrane Active Small Molecules Show Selective Broad Spectrum Antibacterial Activity with No Detectable Resistance and Eradicate Biofilms

Article abstract of DOI:10.1021/acs.jmedchem.5b00443

Treating bacterial biofilms with conventional antibiotics is limited due to ineffectiveness of the drugs and higher propensity to develop bacterial resistance. Development of new classes of antibacterial therapeutics with alternative mechanisms of action has become imperative. Herein, we report the design, synthesis, and biological evaluations of novel membrane-active small molecules featuring two positive charges, four nonpeptidic amide groups, and variable hydrophobic/hydrophilic (amphiphilic) character. The biocides synthesized via a facile methodology not only displayed good antibacterial activity against wild-type bacteria but also showed high activity against various drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactam-resistant Klebsiella pneumoniae. Further, these biocides not only inhibited the formation of biofilms but also disrupted the established S. aureus and E. coli biofilms. The membrane-active biocides hindered the propensity to develop bacterial resistance. Moreover, the biocides showed negligible toxicity against mammalian cells and thus bear potential to be used as therapeutic agents.

Full text of DOI:10.1021/acs.jmedchem.5b00443

Article
pubs.acs.org/jmc
Membrane Active Small Molecules Show Selective Broad Spectrum
Antibacterial Activity with No Detectable Resistance and Eradicate
Biofilms
Jiaul Hoque, Mohini M. Konai, Spandhana Gonuguntla, Goutham B. Manjunath, Sandip Samaddar,
Venkateswarlu Yarlagadda, and Jayanta Haldar*
Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific
Research, Jakkur, Bengaluru 560064, India
*
S Supporting Information
ABSTRACT: Treating bacterial biofilms with conventional
antibiotics is limited due to ineffectiveness of the drugs and
higher propensity to develop bacterial resistance. Development
of new classes of antibacterial therapeutics with alternative
mechanisms of action has become imperative. Herein, we
report the design, synthesis, and biological evaluations of novel
membrane-active small molecules featuring two positive
charges, four nonpeptidic amide groups, and variable hydro-
phobic/hydrophilic (amphiphilic) character. The biocides
synthesized via a facile methodology not only displayed good
antibacterial activity against wild-type bacteria but also showed high activity against various drug-resistant bacteria such as
methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactam-resistant
Klebsiella pneumoniae. Further, these biocides not only inhibited the formation of biofilms but also disrupted the established S.
aureus and E. coli biofilms. The membrane-active biocides hindered the propensity to develop bacterial resistance. Moreover, the
biocides showed negligible toxicity against mammalian cells and thus bear potential to be used as therapeutic agents.
15−17
INTRODUCTION
nosocomial infections in humans.
Therefore, searching for
■
antibacterial agents that are not only highly active and broadly
antibacterial but also able to inhibit and/or disperse bacterial
biofilms and stall bacterial resistance are very essential.
Antimicrobial peptides (AMPs) and lipopeptides are
Coupled with the inexorable proliferation of drug-resistant
microorganisms, the antimicrobial drugs getting approved by
the Food and Drug Administration (FDA) are declining
1
steadily. This poses a grim scenario for the generation to come
18,19
Nature’s very own design of membrane-active agents.
when most antimicrobials may prove ineffective, taking human
medicine to the preantibiotic era. Antibiotics, which in general
target the cellular processes of bacteria, are rendered ineffective
due to point mutations, production of drug-inactivating
enzymes, or highly effective efflux pumps developed by
Cationic AMPs and lipopeptides, composed of positively
charged amino acids, hydrophobic moieties from hydrophobic
amino acids or fatty acids, and peptide (or amide) bonds, are
known to interact with the bacterial cell membrane in a
20,21
2
−4
nonspecific manner.
Consequently, the propensity to
bacteria.
Another major threat to human health is the
5
develop bacterial resistance against these agents is low. Further,
these are also shown to have the potency to destroy preformed
formation of bacterial biofilms. Pathogens living in biotic
biofilms induce persistent chronic infections and significantly
elevate bacterial resistance to antibiotics and the host immune
system as a result of the diffusion barrier due to extracellular
matrix (EPS), slow metabolism, genetic mutation, persister
cells, and so on.
bacteria in a biofilm state are 10−1000-times more resistant to
host immune responses and conventional antibiotic treat-
2
2,23
bacterial biofilms.
Thus, they appear to be ideal
antibacterial agents to supplement or to replace the existing
antibiotic arsenal. However, low in vivo potency, high cost of
manufacture, low stability in plasma, and low selectivity toward
mammalian cells limit the large-scale use of these natural
peptides as clinical antibacterial agents. Non-natural peptidomi-
metic approaches that mimic AMPs or lipopeptides to improve
plasma stability and selectivity may circumvent the problems
associated with the natural peptides. To this end, various
synthetic mimics of these agents were developed such as α-
6
−8
Compared with the planktonic cells,
9
−11
ment.
5% of microbial infections in humans, for example,
endocarditis, periodontitis, and chronic lung infections in cystic
More importantly, bacterial biofilms account for over
7
11−14
fibrosis (CF) patients are some of the prominent diseases.
Furthermore, bacterial biofilms on abiotic surfaces such as
implantable medical devices (e.g., urinary catheters, cardiovas-
cular stents, etc.) play a significant role in surface-associated
Received: March 19, 2015
©
XXXX American Chemical Society
A
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Scheme 1. Synthesis of Lipophilic Cationic Small Molecular Biocides Bearing Non-peptidic Amide Groups
peptoids, β-peptides, oligoureas, oligoacyl lysines, aryl-amide
foldamers, antimicrobial polymers, alkylated peptoids, etc.
lipophilic alkyl chains pending from quaternary ammonium
groups were varied. Further, to fine-tune the structure−activity
relationship of the compounds even more, lipophilic moieties
(methylene spacer) were also varied between the amine groups
of the diaminoalkane frameworks. To synthesize the small
molecular compounds, first various intermediates (1a−1d, 2a−
2d, and 3a−3d) were prepared. Intermediates 1a−1d were
synthesized following a previously reported protocol with slight
24−33
However, the rational design of these peptidomimitics often
turns out to be complicated due to the involvement of a variety
of molecular frameworks and functional groups in order to tune
34
their structure−activity relationship and selectivity. Further-
more, synthetic complexity associated with such molecules even
limits their large-scale usage.
37
Recently, we have developed selective antibacterial small
modifications. N-Alkyl amines were reacted with bromoacetyl
bromide in the presence of aqueous K CO to give N-alkyl-2-
molecular peptidomimitics in a facile and cost-effective way
2
3
3
5,36
based on natural and unnatural amino acids.
In this report,
bromoethanamides (1a-1d, Scheme 1). The reaction was
carried out at 5 °C for 30 min (initial addition time of highly
reactive bromoacetyl bromide) and then at room temperature
for about 4 h to give 100% yield. Intermediates 2a−2d were
synthesized with quantitative yields by the reaction of 1a−1d
with N,N-dimethylamine at room temperature for 12 h and
subsequent deprotonation with aqueous KOH. Another set of
intermediates (3a−3d) was prepared following a protocol
similar to the preparation of 1a−1d. To this end,
diaminoalkanes were reacted with bromoacetyl bromide to
yield various reactive diamides quantitatively. Finally, the amine
intermediates (2a−2d) were reacted individually with the
diamides (3a−3d) to give various cationic small molecular
compounds (4−19, Scheme 1). The products were purified and
isolated by precipitation to give more than 99% yield. Different
lipophilic alkyl moieties (R = −C H , −C H , −C H , and
we have taken an initiative to develop highly antibacterial and
nontoxic small molecules by introducing nonpeptidic amide
bonds (devoid of amino acids), positive charges through
quaternary ammonium groups, and hydrophobic character
through a lipophilic alkyl chain using commercially available
diaminoalkanes as a framework. Antibacterial activity against
various bacteria and toxicity against different mammalian cells
were evaluated. Also, a structure−activity relationship (SAR)
was established in order to find out the effect of molecular
architecture on antibacterial activity in different media.
Membrane active mechanism of action, plasma stability studies,
activity in the presence of complex mammalian fluids, and
ability of these compounds to withstand bacterial resistance
were evaluated. Further, the potential to inhibit biofilms and to
destroy preformed biofilms was also evaluated in order to find
the utility of these small molecules to be developed as potential
antibacterial agents.
6
13
8
17
12 25
−
C H ) and lipophilic spacer moieties (m = 2, 3, 4, and 6)
were used. Thus, a library of 16 cationic small molecules was
16 33
obtained. All the final compounds were characterized by FTIR,
H NMR, C NMR, HRMS, and elemental analyses (Figures
RESULTS AND DISCUSSION
1
13
■
Synthesis and Characterization. We have designed small
molecules bearing two positive charges, two lipophilic moieties,
and four nonpeptidic amide bonds by using diaminoalkanes,
commercially available cheap starting materials, as a framework.
The molecules were synthesized only in three steps (Scheme
S1−S16, Table S5, Supporting Information). Three important
features of these compounds are variable hydrophobic character
not only from pendent lipophilic alkyl chains but also from the
lipophilic spacer chains in their design, inclusion of nonpeptidic
amide bonds instead of peptide bonds formed by the
conventional amino acids in AMPs or lipopeptides, and
1). In order to assess the importance of amphiphilic balance,
B
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Figure 1. Minimum inhibitory concentrations (MICs) of cationic small molecules against (A) S. aureus and (B) E. coli in different media. Upward
arrow indicates higher MIC values than the values shown.
Table 1. Antibacterial Activity (MIC) and Hemolytic Activity (HC ) of Small Molecules
5
0
MIC (μg/mL)
a
drug-sensitive bacteria
drug-resistant bacteria
VRE
biocides
S. aureus
E. coli
P. aeruginosa
MRSA
K. pneumoniae
HC50 (μg/mL)
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
16
16
15
4
62
32
375
187
93
32
24
24
8
32
24
16
7.8
12
6
187
125
93
440
420
400
338
385
330
255
132
46
32
24
48
93
4
24
32
4
93
4
12
32
4
48
0
2
4
24
2
4
24
1
2
3
4
5
6
7
8
9
0.9
3.5
2.8
3.4
4
2
16
1
1
16
32
367
367
375
375
>1000
>1000
>1000
>1000
6
6
375
375
375
375
>1000
>1000
>1000
>1000
24
3.9
3.9
3.9
94
94
94
125
54
0.64
4
44
15.6
31
4
43
4
41
94
94
125
125
20
0.63
>1000
>1000
>1000
>1000
0.4
187
187
187
250
>199
750
80
52
38
36
colistin
0.4
1.2
>250
vancomycin
b
b
b
>1000
a
b
MRSA = Methicillin-resistant S. aureus, VRE = vancomycin-resistant E. faecium, K. pneumonia = β-lactam-resistant Klebsiella pneumoniae. Not
determined.
permanent positive charges from quaternary ammonium groups
instead of soft charges in AMPs or lipopeptides. In order to
compare the hydrophobic character of the amphiphilic
molecules, retention time was measured by reverse phase
high performance liquid chromatography (HPLC) for all the
compounds (Figures S17−S20, Supporting Information). As
expected with the increase in pending alkyl chain length,
retention time increased greatly, whereas with the increase in
spacer chain length, retention time increased slightly thus
indicating the fine-tuning of the amphiphilic character of the
compounds.
Antibacterial Activity. To evaluate the antibacterial
efficacy, these molecules were challenged against a wide
spectrum of drug-sensitive bacteria such as S. aureus, E. coli,
and P. aeruginosa and drug-resistant bacteria such as methicillin-
resistant S. aureus (MRSA), vancomycin-resistant E. faecium
S. aureus (NRSA), and colistin-resistant E. coli (CREC). The
antibacterial efficacy was determined in suitable nutrient media
and expressed as minimum inhibitory concentration (MIC),
that is, the minimum concentration of the molecules required
to inhibit the growth of the bacteria (Figure 1, Table 1, and
Tables S1−S4, Supporting Information). A glycopetide anti-
biotic vancomycin and a lipopetide antibiotic colistin were also
used in this study to compare the results. In general, all the
compounds except 16−19 (with −C H lipophilic chain and
m = 2−6) showed good activity against all the bacteria tested in
growth media. Moreover, the cationic small molecules were
found to be more active toward Gram-positive bacteria than the
Gram-negative bacteria. For example, the range of MIC values
for the small molecules were 1−125 μg/mL against Gram-
positive S. aureus whereas the range of MIC values was 4−1000
μg/mL for Gram-negative E. coli. However, the antibacterial
activity of the cationic small molecules was found to be quite
16
33
(VRE), β-lactam-resistant K. pneumoniae, norfloxacin-resistant
C
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different depending on the type of media used. For example,
compound 18 (with −C H lipophilic chain and m = 4) was
μg/mL, respectively. These biocides were also found to be
active against β-lactam-resistant K. pneumoniae. The MIC values
of compounds 10 and 11 were 24 and 16 μg/mL, respectively,
against this Gram-negative drug-resistant bacterium. The
cationic molecules were also found to be active against
norfloxacin-resistant S. aureus (NRSA) and colistin-resistant
E. coli (CREC). Norfloxacin and colistin, the two most
common drugs used to treat the Gram-positive S. aureus and
Gram-negative E. coli infections, are becoming ineffective due
1
6
33
found be active in minimum essential medium (MIC = 3.9−7.8
μg/mL) compared with very little or no activity in nutrient/LB
media (MIC = 125−1000 μg/mL) against the same bacteria.
Effect of Lipophilic Alkyl Chain Length on Antibacterial
Activity (in Nutrient Media). Among the various sets of
compounds (4−7 with −C H lipophilic chain and m = 2−6;
6
13
8
−
−
−11 with −C H lipophilic chain and m = 2−6; 12−15 with
8
17
39,40
C H lipophilic chain and m = 2−6 and 16−19 with
to the development of bacterial resistance.
Interestingly,
12
25
C H lipophilic chain and m = 2−6), 8−11 were found to
compounds 10 and 11 displayed MIC values of 4 and 2 μg/mL,
respectively, against both NRSA and CREC (Table S2,
Supporting Information).
16
33
be most active against both Gram-positive S. aureus and Gram-
negative E. coli in the growth media (Table 1). For example, the
range of MIC values for compounds 8−11 was 1−4 μg/mL
against S. aureus and 2−16 μg/mL against E. coli, whereas the
MIC values for compounds 4−7, 12−15, and 16−19 were 4−
Bactericidal Activity (in Growth Media). In order to find
out whether these biocides are bacteriostatic or bactericidal or
both, aliquots from the wells that appeared to have less or no
turbidity in MIC experiments were plated on suitable agar
plates and minimum bactericidal concentrations (MBC) were
determined as the concentration where no bacterial colony was
observed. Against S. aureus, the most active compounds 10 and
11 displayed MBC values of 4 and 2 μg/mL, respectively. The
MBC values of biocides 10 and 11 against E. coli were 8 and 4
μg/mL, respectively (Table S3, Supporting Information).
Overall, the excellent bactericidal activity demonstrated that
these small molecular biocides might be used as potential
antibacterial agents.
16 μg/mL, 3−4 μg/mL, and 94−125 μg/mL against S. aureus
and 24−62 μg/mL, 16−32 μg/mL, and >1000 μg/mL against
E. coli, respectively. The above results further suggested that an
optimum lipophilic alkyl chain length is required for the
maximum activity. To understand the role of the lipophilic alkyl
chain more closely, the MIC values of the compounds of
different alkyl chains (from −C H to −C H ) and a
6
13
16 33
particular spacer chain (m = 6) were compared. Compounds 7
(
(
R = −C H ), 11 (R = −C H ), 15 (R = −C H ), and 19
6
13
8
17
12 25
R = −C H ) showed MIC values of 4, 0.9, 4, and 125 μg/mL
16
33
against S. aureus and 24, 2, 31, and >1000 μg/mL against E. coli,
respectively (Figure S21A, Supporting Information). Thus,
these compounds showed a parabolic pattern with the variation
lipophilic chain lengths. Interestingly, these compounds showed
activity against P. aeruginosa, a difficult-to-treat Gram-negative
bacterium that causes many nosocomial infections (hospital-
acquired infections) and is known to show resistance to almost
all the clinically approved drugs. The MIC values displayed by
the most active compounds 10 and 11 were 24 and 16 μg/mL,
respectively.
Role of Medium on Antibacterial Activity. Since the growth
media used in determining MIC constitute anionic and
hydrophobic peptides, interaction of the cationic small
molecules with the growth media might play a major role in
determining the antibacterial efficacy. In order to understand
the effect of media, antibacterial efficacy of the cationic small
molecules was determined in minimum essential medium
(MEM), a relatively less complex medium that constitutes
nonessential amino acids, sodium pyruvate, lipoic acid, vitamin
B , biotin, and ascorbic acid (Gibco, ref. 41061-029, without
38
12
Effect of Lipophilic Spacer Chain Length on Antibacterial
Activity (in Growth Media). In order to fine-tune the
antibacterial activity of these small molecules further,
amphiphilic balance was also varied by varying the lipophilic
spacer chain length by one or two methylene units (Table 1).
With the increase in lipophilic spacer length while keeping the
optimum alkyl chain length constant (R = −C H ),
phenol red). Interestingly, the cationic molecules with higher
lipophilic chain length (less or not active in growth media)
were found to be active in MEM. For example, compound 18
(with −C H lipophilic chain and m = 4) showed MIC values
16
33
of 2.0 μg/mL against S. aureus and 7.8 μg/mL against E. coli in
MEM, whereas the MIC values were 125 μg/mL against S.
aureus in nutrient medium and >1000 μg/mL against E. coli in
LB medium, respectively (Figure 1, Table S3, Supporting
Information). On the other hand, antibacterial activity of the
small molecules with lower lipophilic chain length was found to
be somewhat lower in MEM compared with the growth media.
For example, compound 10 (with −C H lipophilic chain and
8
17
antibacterial efficacy within the most active set of compounds
8−11) was found to increase, for example, MIC values of 8 (m
2), 9 (m = 3), 10 (m = 4), and 11 (m = 6) were 4, 4, 2, and
.9 μg/mL against S. aureus and 24, 12, 4, and 2 μg/mL against
(
=
0
E. coli, respectively (Figure S21B, Supporting Information).
Among all the cationic small molecules, compounds 10 and 11
were found to be most active. However, irrespective of spacer
chain length, compounds with −C H and −C H lipophilic
8
17
m = 4) showed MIC values of 1.0 μg/mL each against S. aureus
and E. coli in MEM, whereas the MIC values of 10 were 2 μg/
mL against S. aureus and 4 μg/mL against E. coli in growth
medium (Figure 1 and Table S2, Supporting Information).
Thus, for the cationic small molecules, the nature of the
medium and hence the interaction of medium with these
molecules played a vital role in determining the antibacterial
efficacy. This could be possibly due to the presence of mostly
negatively charged and highly hydrophobic peptide residues
present in peptone (an additive in nutrient media) or tryptone
(an additive in LB), which resulted in strong interaction with
the cationic and hydrophobic small molecules (16−19) in a
6
13
16 33
alkyl chains were found to be less active or not active against
both Gram-positive and Gram-negative bacteria in the growth
media used.
Antibacterial Activity against Drug-Resistant Bacteria (in
Growth Media). Not only active against drug-sensitive bacteria,
these small molecules showed broad-spectrum activity against
various drug-resistant bacteria (Table 1). Like the drug-
sensitive bacteria, the cationic molecules showed similar trends
in their activity against drug-resistant bacteria. The most active
compounds 10 and 11 displayed MIC values of 2 and 1 μg/mL,
respectively, against MRSA. The activity of 10 and 11 against
VRE was also excellent as they exhibited MIC values of 4 and 1
41
way that affected the antibacterial activity.
Antibacterial Activity in Mammalian Fluids. One of the
major disadvantages of the natural antibacterial peptides is the
D
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Figure 2. Biofilm disruption by compound 10: (A, B) cell viability in biofilms of S. aureus and E. coli, respectively, obtained by plating and counting
the viable bacteria after treating with different concentrations of the compound 10; (C, D) images of the treated and nontreated biofilms of S. aureus
and E. coli, respectively after staining with crystal violet. Star represents <50 CFU/mL.
loss of antibacterial efficacy due to protease degradation, which
fluids like serum, plasma, and blood, thus implying its potential
as an antibacterial agent. The 2−4-fold increase of MBC value
could be possibly due to negatively charged proteins and
macromolecules in human serum, plasma, or blood that tightly
bind to the cationic molecule, thereby deactivating it toward
in turn results in decreased antibacterial activity in the blood
3
3
plasma. In order to determine the stability of the nonpeptidic
amide containing biocides under plasma conditions, first these
compounds were treated in 50% human plasma for different
periods of time and subsequently antibacterial efficacy of the
plasma-treated biocides was evaluated. The biocide used was 10
against S. aureus. Interestingly compound 10, after treatment
with 50% plasma for 0, 3 and 6 h did not reveal any significant
loss in activity. The MIC values of 10 were found to be 2 μg/
mL each after 0 and 3 h treatment and 3 μg/mL after 6 h
treatment, respectively in 50% plasma (Figure S22A,
Supporting Information). Notably, MSI-78, a naturally
occurring antimicrobial peptide, is known to show 2-fold
increase in MIC in the presence of 50% human serum itself
4
1,43
membrane disruption of bacteria.
Antibacterial Kinetics. Since these compounds are
bactericidal in nature, the rate of bactericidal action was
investigated toward both S. aureus and E. coli using compound
10 at two different concentrations (MIC and 6 × MIC). The
compound showed rapid bactericidal activity against both types
of bacteria. It killed S. aureus (∼5 log reduction) at 120 min at 6
× MIC, whereas it showed bacteriostatic effect at MIC. On the
other hand, the compound killed E. coli (∼5 log reduction) at
240 min at 6 × MIC and remained bacteriostatic at MIC
(Figure S23, Supporting Information). Interestingly, when
antibacterial kinetics was performed in HEPES/glucose buffer
(5 mM HEPES/5 mM glucose = 1:1), compound 10 killed
both S. aureus and E. coli instantly (Figure S23, Supporting
Information). Thus, the rate of bactericidal activity was also
found to be dependent on the nature of medium.
42
than in the absence of serum (100% media). The above result
thus indicated that the compound 10 did not lose antibacterial
efficacy upon pretreatment in plasma.
Another serious concern of the antibacterial agents is the loss
of antibacterial activity in the presence of complex mammalian
4
3
fluids. Hence the antibacterial efficacy of these compounds
was directly investigated in various complex mammalian fluid
systems (50% serum, 50% plasma, and 50% blood) against
MRSA, a drug-resistant Gram-positive bacterium that causes
many infections in humans. Antibacterial activity was
investigated by determining the minimum bactericidal concen-
tration (MBC) of the compound 10 in 50% serum, 50%
plasma, and 50% blood supplemented with 50% minimum
Antibiofilm Activity. Bacteria within biofilms are inher-
ently insensitive to antiseptics, microbicides, and antimicrobial
peptides or defensive cells derived from the host. Further, they
are 10−1000-fold more resistant to conventional antibiotic
11
treatment. More importantly, biofilms account for more than
80% of microbial infections in humans and thus pose a
5
−8
considerable impediment to antimicrobial therapy.
Both
5
essential media (MEM) containing 10 CFU/mL of MRSA.
Gram-positive S. aureus and Gram-negative E. coli form biofilms
and are known to cause many infection in humans. S. aureus
biofilms are known cause dental caries, musculoskeletal
infections, penile prostheses, and various other nosocomial
infections from sutures, catheters, contact lenses, etc., whereas
E. coli biofilms cause binary tract infections, urinary tract
The compound was found to be active in 50% serum, 50%
plasma, and 50% blood. The MBC values of 10 were 4 μg/mL
in 50% serum and 8 μg/mL each in 50% plasma and 50%
blood, respectively (Figure S22B, Supporting Information).
The above results indicated that the compound was able to
retain its antibacterial activity even in very complex mammalian
5
cystitis, bacterial prostatitis, etc. Hence we decided to evaluate
E
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Figure 3. Mechanism of antibacterial action of the cationic small molecular biocides at 40 μg/mL: (A, B) membrane depolarization against S. aureus
and E. coli, respectively, using diSC3(5) dye as fluorescence probe and (C, D) cytoplasmic membrane permeabilization against S. aureus and E. coli,
respectively, using propidium iodide (PI) as fluorescence probe.
the ability of these compounds to inhibit both S. aureus and E.
coli biofilm formation. Further, the ability of these molecules to
disperse established S. aureus and E. coli biofilms was also
investigated.
the cell viabilities in biofims decreased to 9.77, 8.45, and 6.9
log10 CFU/mL at 2, 4, and 16 μg/mL and to 0 at 32 μg/mL,
whereas cell viability in nontreated biofilm increased to 13.6
log10 CFU/mL. Thus, the EC50 (the concentration of
compound that reduces 50% bacterial titer of a preformed
biofilm) value of the biocide was 16.8 μg/mL. More
importantly, the biocide at the highest tested concentration
(32 μg/mL) showed zero cell viability in 24 h S. aureus biofilm
indicating complete eradication of established biofilm (Figure
2A). Matured E. coli biofilm (developed for 72 h) having an
initial count of 22.5 log10 CFU/mL of bacteria was similarly
treated at four different concentrations (4, 8, 32, and 64 μg/mL
respectively). After the treatment, the cell viabilities in biofims
for the different concentrations decreased to 21.1, 20.5, 13.1,
and 11.4 log CFU/mL at 4, 8, 32, and 64 μg/mL respectively
Biofilm Inibition. The ability of the cationic biocides to
inhibit the formation of bacterial biofilm was investigated with
one of the most potent biocides, 10, against both S. aureus and
E. coli. Compound 10 was found to be an effective inhibitor of
both S. aureus and E. coli biofim formation. The IC50 (the
concentration of the compounds that inhibits 50% biofilm
4
4
development ) values of 10 against S. aureus and E. coli biofilm
formation were found to be 2 and 4 μg/mL, respectively
(Figure S24, Supporting Information). Moreover, the com-
pound at the above concentrations was found to be
nonbactericidal. Thus, the ability to inhibit the biofilm
formation by these biocides makes them promising antibiofilm
agents.
Biofilm Disruption. Any antibacterial agent having the
potency not only to inhibit bacterial biofilm formation but
also to disperse or eradicate established biofilms would be an
ideal agent to tackle infections caused by bacteria. In order to
evaluate the efficiency of this class of compounds to eradicate
preformed biofilms, one of the best compounds, 10, was used
against established S. aureus and E. coli biofilms. Matured S.
aureus biofilm (developed for 24 h) having an initial count of
10
(Figure 2B). Thus, the EC value compound 10 was 63.4 μg/
50
mL. Moreover, the compound was able to reduce viable
bacteria even in 72 h grown matured E. coli biofilm. The above
results thus indicated the ability of the biocide to disperse the
biofilms and hence to kill the microorganism present in the
biofilm. The disruption of bacterial biofilms at different
concentrations was also evident from the crystal violet staining
of the treated biofilms (Figure 2C,D for S. aureus and E. coli,
respectively).
Mechanism of Action. To confirm that these cationic
small molecular biocides act by disrupting the bacterial cell
membrane integrity, the detailed molecular mechanism of
12.8 log10 CFU/mL of bacteria was treated at four different
concentrations (2, 4, 16, and 32 μg/mL). After the treatment,
F
DOI: 10.1021/acs.jmedchem.5b00443
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Journal of Medicinal Chemistry
Article
Figure 4. Propensity of development of bacterial resistance against compound 10: (A) for S. aureus where norfloxacin was used as control; (B) for E.
coli where colistin was used as control.
action was investigated using spectroscopic methods against
both Gram-positive S. aureus and Gram-negative E. coli. Also in
order to assess the structure−activity relationship of the
cationic biocides, membrane depolarization, intracellular
potassium ion leakage, and both inner-membrane and outer-
membrane permeabilization experiments were performed with
all the compounds.
Outer Membrane (OM) Permeabilization. Hydrophobic
dye N-phenyl naphthylamine (NPN) is generally excluded from
the OM of Gram-negative bacteria. When the OM is ruptured
or damaged, NPN partitions into the perturbed OM, exhibiting
35
an increase in fluorescence. Upon addition of the cationic
biocides, E. coli suspensions in the presence of NPN showed
increased fluorescence. Thus, the above result indicated that E.
coli outer membrane was damaged by the cationic biocides.
Moreover, all the biocides showed outer membrane permeabi-
lization (Figure S26, Supporting Information). Surprisingly,
biocides 16−19 (m = 2−6 and R = −C H ) also showed OM
Cytoplasmic Membrane Depolarization. To find out
whether the biocides act by depolarizing the bacterial cell
membrane and to establish the effect of molecular architecture
on membrane depolarization, fluorescence spectroscopy was
used with the membrane-potential sensitive dye 3,3′-
16
33
permeabilization at 40 μg/mL (MIC values in LB broth of
these biocides were ≥1000 μg/mL). Since the MIC experiment
was performed in LB broth, which is composed of negatively
charged and hydrophobic peptides, the interaction of these
cationic and hydrophobic molecules inhibited interaction with
bacteria in such medium as explained earlier. Thus, compounds
16−19 being less or not active in LB medium showed
membrane permeabilization in buffer solution. In order to
confirm further, antibacterial activity (MBC) of compounds 11
and 18 having lower and higher lipophilic chain lengths,
respectively, was determined in both buffer and LB broth at
different time intervals (1, 2, 4, and 6 h). Notably, compound
18 showed activity in buffer solution (Table S4, Supporting
Information).
4
5,46
dipropylthiadicarbocyanine iodide (diSC 5).
In general,
3
due to the potential gradient, the dye is taken up by the bacteria
and accumulation in the membrane led to a decrease in
fluorescence intensity due to self-quenching. However, when
bacteria were treated with the biocides, an increase in
fluorescence intensity was observed due to the displacement
of diSC 5 into the solution against both S. aureus and E. coli.
3
Thus, the above fact indicated that the biocides dissipated the
membrane potential of both types of bacteria. Further, different
biocides showed different extent of dissipation of membrane
potential. Biocides 8−11 (m = 2−6 and R = −C H ) and 12−
8
17
1
5 (m = 2−6 and R = −C H ) showed the maximum
1
2
25
membrane depolarization, whereas biocides 4−7 (m = 2−6 and
R = −C H ) and 16−19 (m = 2−6 and R = −C H )
Inner Membrane (IM) Permeabilization. The ability of
biocides to permeabilize the cytoplasmic membrane of bacteria
was studied using propidium iodide (PI) dye. PI enters inside
bacteria only through compromised membranes and fluoresces
6
13
16 33
exhibited less or negligible membrane depolarization against
both S. aureus and E. coli (Figure 3A,B). The above results
further indicated that the biocides with −C H and −C H
8
17
12 25
36
lipophilic moiety are more active in disrupting the potential
upon binding to the cellular DNA. Upon treatment with the
biocides, an enhancement in the fluorescence intensity was
observed in S. aureus and E. coli. Thus, the biocides were
efficient in permeabilizing the cytoplasmic membranes of both
Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria.
All the biocides were found to show the membrane
permeabilization against both bacteria. Biocides 8−11 and
12−15 showed the maximum membrane permeabilization,
whereas biocides 4−7 and 16−19 exhibited less or negligible
membrane permeabilization against S. aureus (Figure 3C),
which is well correlated with their MIC values. Against E. coli,
all the biocides showed membrane permeabilization at the
tested concentrations. However, biocides 16−19 along with the
active molecules 8−11 and 12−15 also showed cytoplasmic
permeabilization (Figure 3D) probably due to similar reason as
than the biocides with −C H and −C H , which are well
6
13
16 33
correlated with their MIC values.
+
Intracellular K Ion Leakage. Upon disruption of membrane
potential, the intracellular potassium ion is released for both
47
Gram-positive and Gram-negative bacteria. Thus, the leakage
+
of K ions was studied using potassium ion sensitive dye PBFI-
AM against both S. aureus and E. coli by fluorescence
spectroscopy. PBFI-AM is known to show an increase in
+
+
fluorescence after binding to K ions if K ions leak out from
the cells. However, no increase in fluorescence intensity was
observed thus indicating that none of the biocides caused
+
significant leakage of K ions against both types of bacteria,
+
whereas valinomycin, used as positive control, showed K ion
leakage (Figure S25, Supporting Information).
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Figure 5. Microscopy images of HEK 293 cells: (A) cells without any treatment (control); (B) cells treated with compound 10 at 2 μg/mL; (C)
cells treated with 10 at 24 μg/mL, and (D) cells treated with triton-X (1 vol %). Scale bar 20 μm.
explained earlier. In summary, all the above studies indicated
that these cationic small molecules indeed interacted with the
negatively charged bacterial cell membrane and disrupted the
membrane integrity.
Hemolytic Activity. The ability of the small molecular
biocides to lyse human red blood cells (hRBC) was expressed
as HC50 (μg/mL), that is, the concentration of the biocides at
which 50% of RBC gets lysed (Figure S27, Supporting
Information). In general, hemolytic activity was found to
increase with the increase in liphophilic chain length pending
from quaternary nitrogen (Table 1). For example, the HC50
values of compounds 4 (m = 2 and R = −C H ), 8 (m = 2 and
Propensity To Induce Bacterial Resistance. The
development of bacterial resistance against most of the
clinically approved drugs is one of the major problems of
1
−3
current times.
Thus, it would be of importance to evaluate
6
13
the potential emergence of bacterial resistance against these
biocides. The ability of these biocides to suppress the
development of resistance was evaluated by taking one of the
most active biocides (10) against both Gram-positive S. aureus
R = −C H ), 12 (m = 2 and R = −C H ) and 16 (m = 2 and
8
17
12 25
R = −C H ) were 440, 385, 66, and 52 μg/mL, respectively.
16
33
On the other hand, with the increase in lipophilic spacer chain
length, the hemolytic activity was found to increase but to a
lesser extent. For example, the HC values of compounds 4 (m
47,48
and Gram-negative E. coli.
Norfloxacin, an antibiotic
50
commonly used to treat the Gram-positive infections, was
used as a positive control for S. aureus, whereas colistin, a
lipopeptide antibiotic active against Gram-negative bacteria, was
used in the case of E. coli. These antibacterial agents were
repeatedly challenged against bacteria at their sub-MIC values
to allow bacteria to develop resistance. Interestingly, no change
in the MIC of 10 was observed against both S. aureus and E. coli
even after 20 passages, whereas around 810-fold and 248-fold
increase in MIC was observed in case of norfloxacin and
colistin, respectively (Figure 4). The above results thus
indicated that bacteria find it difficult to develop resistance
against this type of biocide within the experimental time period.
Cytotoxicity against Mammalian Cells. One of the
major concerns in the development of clinically useful
antibacterial agents for biomedical applications is their
selectivity toward mammalian cells over bacterial cells. In
order to evaluate the cytotoxic effect, hemolytic activity against
human erythrocytes as well as cytotoxicity against human
embryonic kidney (HEK) cells and human cervical cancer
= 2 and R = −C H ), 5 (m = 3 and R = −C H ), 6 (m = 4
6
13
6
13
and R = −C H ), and 7 (m = 6 and R = −C H ) were 440,
6
13
6
13
420, 400, and 338 μg/mL, respectively. However, compound
10, one of the most potent biocides, showed HC value of 255
50
μg/mL thus giving selectivity (S = HC /MIC) of 128 and 64
50
against S. aureus and E. coli respectively (Table 1). Compound
10 showed no hemolytic activity at 4 μg/mL and 1−2%
hemolysis at 40 μg/mL (at MIC and 10 × MIC values for E.
coli and most of the drug-resistant bacteria). The above results
thus indicated that these compounds are selectively active
toward bacteria.
Cytotoxicity. To strengthen the selectivity of the cationic
biocides, cytotoxicity study was also performed with HEK 293
and HeLa cells by optical microscopy. Cells were treated with
the biocides 10 and 11 at lowest and highest MIC values (2 and
24 μg/mL for 10 and 1 and 16 μg/mL for 11), and the treated
cell lines were imaged to visualize the morphology (Figure 5,
Figures S28−S30, Supporting Information). The biocide
treated cells were found to have spindle shape with intact
morphology at both concentrations and were similar to the
4
9,50
derived HeLa cells were performed.
H
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Journal of Medicinal Chemistry
Article
untreated cell lines (Figure 5A−C), whereas cells treated with
triton-X were found to have completely irregular shape and
damaged morphology (Figure 5D). These results thus indicated
that these biocides are indeed nontoxic toward mammalian cells
at their MIC values.
funnel, and the aqueous layer was subjected to repeated washes with
DCM (2 × 50 mL). All the organic solutions were combined and
washed with water repeatedly (3 × 100 mL). The final organic layer
was then passed through anhydrous Na SO . The DCM layer was
2
4
collected, and the solvent was evaporated to obtain colorless liquids or
1
solids with 100% yield. The products were characterized by FT-IR, H
NMR, and 13C NMR spectroscopy.
−
1
CONCLUSION
2-Bromo-N-hexylethanamide (1a). FTIR: 3250 cm (amide N−
■
−1
H str.), 2928 cm (−CH − assym. str.), 2850 (−CH − sym. str.),
2
2
In conclusion, novel membrane-active small molecules have
been developed via simple yet quantitative methodology. The
compounds displayed excellent antibacterial activity against
various wild-type and drug-resistant bacteria (both Gram-
positive and Gram-negative). Variation of amphiphilic nature of
the compounds drastically improved their antibacterial activity
as well as selectivity. Additionally, the small molecules were
found to be stable under plasma conditions and showed
excellent activity in various complex mammalian fluids such as
serum, plasma, and blood. Further, these compounds not only
inhibited the formation of biofilm but also dispersed the
established bacterial biofilms. These molecules acted on
bacteria rapidly by disrupting the membrane and did not
allow bacteria to develop resistance. The compounds showed
negligible toxicity against mammalian cells. Thus, the
membrane-active small molecules developed herein could be
used as therapeutic agents to treat bacterial infections caused by
multidrug-resistant bacteria in both plankotonic and biofilm
state.
−1
−1
1
680 cm (Amide I, CO str.), 1560 cm (Amide II, N−H ben.),
−1
1
1
470 cm (−CH − scissor). H NMR (400 MHz, CDCl ): δ 0.859
2
3
(
t, terminal −CH , 3H), 1.309 (m, −(CH ) −, 6H), 1.530 (q,
3
2 3
−
CH (CH ) −, 2H), 3.248 (m, −CONHCH −, 2H), 3.861 (s,
2
2
3
2
1
3
−COCH Br, 2H), 6.581 (br s, amide −NHCO, 1H). C NMR (100
2
MHz, CDCl ): δ 14.190, 22.770, 26.912, 29.711, 31.994, 40.408,
3
165.589.
−
1
2
-Bromo-N-octylethanamide (1b). FTIR: 3252 cm (amide N−H
−1
str.), 2929 cm (−CH −assym. str.), 2851 (−CH − sym. str.), 1680
2
2
−
1
−1
cm (Amide I, CO str.), 1556 cm (Amide II, N−H ben.), 1469
−
1
1
cm (−CH − scissor). H NMR (400 MHz, CDCl ): δ 0.877 (t,
2
3
terminal −CH , 3H), 1.291 (m, −(CH ) −, 10H), 1.535 (q,
3
2 5
−
−
CH (CH ) −, 2H), 3.252 (t, −CONHCH −, 2H), 3.865 (s,
2
2
5
2
13
COCH Br, 2H), 6.565 (br s, amide −NHCO, 1H). C NMR
2
(100 MHz, CDCl ): δ 14.188, 22.698, 26.904, 29.411, 29,589, 29.998,
3
31.989, 40.402, 165.578.
−
1
2-Bromo-N-dodecylethanamide (1c). FT-IR: 3251 cm (amide
−1
N−H str.), 2929 cm (−CH −assym. str.), 2851 (−CH − sym. str.),
2
2
−
1
−1
1
1
(
685 cm (Amide I, CO str.), 1558 cm (Amide II, N−H ben.),
−
1
1
470 cm (−CH − scissor). H NMR (400 MHz, CDCl ): δ 0.858
2
3
t, terminal −CH , 3H), 1.289 (m, −(CH ) −, 18H), 1.533 (q,
3
2 9
−
CH (CH ) −, 2H), 3.278 (t, −CONHCH −, 2H), 3.881 (s,
2
2
9
2
13
EXPERIMENTAL SECTION
Materials and Instrumentation. 1-Aminohexane, 1-aminooctane,
-aminododecane, 1-aminohexadecane, bromoacetyl bromide, 1,2-
−
COCH Br, 2H), 6.475 (br s, amide −NHCO, 2H). C NMR
■
1
2
(
100 MHz, CDCl ): δ 14.192, 22.765, 26.901, 29.321, 29.420, 29.585,
3
2
9.643, 29.705, 31.992, 40.401, 165.587.
-Bromo-N-hexadecylethanamide (1d). FT-IR: 3250 cm (amide
−1
2
diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, 1,6-diamino-
hexane, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
−1
N−H str.), 2927 cm (−CH − assym. str.), 2849 (−CH − sym. str.),
2
2
−
1
−1
1
1
679 cm (Amide I, CO str.), 1562 cm (Amide II, N−H ben.),
(
HEPES), and α-D(+)-glucose were purchased from Sigma-Aldrich
−
1
1
468 cm (−CH − scissor). H NMR (400 MHz, CDCl ): δ 0.878
and used as received. Anhydrous potassium carbonate (K CO ),
2
3
2
3
(
−
−
t, terminal −CH , 3H), 1.300 (m, −(CH ) −, 26H), 1.547 (q,
anhydrous sodium sulfate (Na SO ), phosphorus pentaoxide (P O ),
3
2 13
2
4
2
5
CH (CH ) −, 2H), 3.279 (t, −CONHCH −, 2H), 3.883 (s,
calcium hydride (CaH ), N,N-dimethylamine solution (40%),
2
2
13
2
2
13
COCH Br, 2H), 6.575 (br s, amide −NHCO, 2H). C NMR
dichloromethane (DCM), chloroform, ethanol, acetonitrile, diethyl
ether, acetone, and molecular sieves (4 Å) were purchased from SD
Fine, India, and were of analytical grade. Acetonitrile, DCM, and
chloroform were dried over P O , and ethanol was dried over CaH
2
2
(100 MHz, CDCl ): δ 14.193, 22.769, 26.903, 29.334, 29.413, 29.578,
3
2
9.636, 29.718, 29.818, 32.095, 40.413, 165.567.
General Procedure for the Synthesis of 2-(N,N-Dimethyl)-N′-
2
5
alkylethanamide (2a−2d). N-Alkyl-1-bromoethanamides 1a−1d
(40 mmol) were dissolved in dry chloroform (40 mL) in a screw-
and stored over molecular sieves. Nuclear magnetic resonance (NMR)
1
spectra were recorded using Bruker AMX-400 (400 MHz for H NMR
and 100 MHz for 13C NMR) spectrometer. The chemical shift (δ)
top pressure tube. Dry NHMe gas was added to the solution of N-
2
alkyl-1-bromoethanamide in chloroform at 0 °C until the volume of
the resulting solution was roughly doubled (80 mL). Then the reaction
mixture was stirred for 24 h at 80 °C overnight. After the reaction, the
pressure tube was cooled, the reaction mixture was transferred to a
round-bottom (RB) flask, and the final volume of the reaction mixture
was brought to 150 mL by adding chloroform. The unreacted gas was
removed carefully by heating slowly until wet litmus paper no longer
turns blue on exposure to the emerging vapors. The solution was
washed with 2 M KOH solution (3 × 100 mL) in order to
deprotonate the products. Finally, the organic layer was passed
values are reported in parts per million (ppm). Mass spectra were
recorded on a 6538-UHD Accurate mass Q-TOF LC-MS high
resolution mass spectrometer (HRMS). Infrared (IR) spectra were
recorded by Bruker IFS66 V/s spectrometer on NaCl crystal for liquid
compounds and KBr pellet for solid compounds. Elemental analyses
were performed by a Thermo Finnigan FLASH EA 1112 CHNS
analyzer. For optical density (OD) measurement, Tecan Infinite Pro
series M200 microplate reader was employed. Staphylococcus aureus
(
MTCC 737) and Escherichia coli (MTCC 443) were purchased from
MTCC (Chandigarh, India). Pseudomonas aeruginosa (ATCC 424),
methicillin-resistant Staphylococcus aureus (ATCC 33591), vancomy-
cin-resistant Enterococcus faecium (ATCC 51559), and β-lactam-
resistant Klebsiella pneumoniae (ATCC 700603) were obtained from
ATCC (Rockville, MD, USA).
through anhydrous Na SO and was dried to give yellow colored
2
4
liquids with 100% yield. The products were characterized by FT-IR,
1
13
H NMR, and C NMR spectroscopy.
2-(N,N-Dimethyl)-N′-hexylethanamide (2a). FT-IR (KBr): 3230
−
1
−1
−1
−1
General Procedure for the Synthesis of N-Alkyl-2-bromoe-
thanamides (1a−1d). 1-Aminoalkanes (60 mmol) were dissolved in
DCM (100 mL), and a solution of K CO (12.4 g, 90 mmol) in water
cm (amide N−H str.), 2926 cm (−CH − assym. str.), 2852 cm
2
−
1
(−CH − sym. str.), 1680 cm (Amide I, CO str.), 1560 cm
2
−1
1
(Amide II, N−H ben.), 1470 cm (−CH − scissor). H NMR (400
2
3
2
(
100 mL) was added to it. The binary mixture was then cooled to 5 °C
MHz, CDCl ): δ 0.879 (t, terminal −CH , 3H), 1.239 (m,
3
3
in a cold incubator connected to a chiller. Bromoacetyl bromide (18.2
g, 90 mmol) was dissolved in DCM (75 mL) and was added to the
mixture dropwise for about 30 min. After the completion of addition,
the reaction mixture was stirred at room temperature for about 4 h.
After the reaction, the organic layer was separated using a separating
CH (CH ) −, 6H), 1.547 (q, CH (CH ) CH −, 2H), 2.286 (s,
3
2
3
3
2
3
2
(CH ) N−, 6H), 2.883 (s, (CH ) NCH CO−, 2H), 3.224 (q,
3
2
3
2
2
−CH NHCO−, 2H), 7.137 (br s, amide −NH, 1H).
2
2-(N,N-Dimethyl)-N′-octylethanamide (2b). FT-IR (KBr): 3232
−1
−1
−1
cm (amide N−H str.), 2936 cm (−CH − assym. str.), 2855 cm
2
I
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Journal of Medicinal Chemistry
Article
−
1
−1
(
(
−CH − sym. str.), 1678 cm (Amide I, CO str.), 1560 cm
N,N′-(Hexane-1,6-diyl) Bis(2-bromoethanamide) (3d). ATR FT-
2
−1
1
−1
−1
Amide II, N−H ben.), 1470 cm (−CH − scissor). H NMR (400
IR (Solid): 3295 cm (amide N−H str.), 2937 cm (−CH − assym.
2
2
−
1
−1
MHz, CDCl ): δ 0.831 (t, terminal −CH , 3H), 1.237 (m,
str.), 2858 cm (−CH − sym. str.), 1639 cm (Amide I, CO str.),
3
3
2
−
1
−1
1
CH (CH ) −, 10H), 1.489 (q, CH (CH ) CH −, 2H), 2.236 (s,
1540 cm (Amide II, N−H ben.), 1483 cm (−CH
2
− scissor). H
3
2
5
3
2
5
2
(
−
CH ) N−, 6H), 2.884 (s, (CH ) NCH CO−, 2H), 3.274 (q,
NMR (400 MHz, CDCl
4H), 1.539 (m, −CH
−CH CH (CH CH CH
(CH NHCOCH −, 4H), 6.548 (br s, amide −CON-
H(CH NHCO−, 2H). C NMR (100 MHz, CDCl
3
): δ 1.369 (m, −CH
CH (CH CH CH
−, 4H), 3.877 (s, −CH
2 2 2 2 2 2
CH (CH ) CH CH −,
3
2
3
2
2
CH NHCO−, 2H), 7.139 (br s, amide −NH, 1H).
2
2
2
)
2
2
2
−, 4H), 3.293 (q,
CONH-
2
2
-(N,N-Dimethyl)-N′-dodecylethanamide (2c). FT-IR (KBr): 3229
2
2
2
)
2
2
2
2
−
1
−1
−1
−1
cm (amide N−H str.), 2934 cm (−CH − assym. str.), 2856 cm
2
)
6
)
2
2
−1
13
(
(
−CH − sym. str.), 1680 cm (Amide I, CO str.), 1559 cm
2
6
): δ 26.268,
3
2
−1
1
Amide II, N−H ben.), 1471 cm (−CH − scissor). H NMR (400
29.264, 29.485, 40.034, 165.531.
2
MHz, CDCl ): δ 0.869 (t, terminal −CH , 3H), 1.247 (m,
General Procedure for the Synthesis of Cationic Small
3
3
Molecules (4−19). To the individual solutions of 3a−3d (4 mmol)
CH (CH ) −, 18H), 1.489 (q, CH (CH ) CH −, 2H), 2.229 (s,
3
2
9
3
2
9
2
in organic solvents (EtOH for 3a, CHCl for 3b, and MeCN for 3c
(
−
CH ) N−, 6H), 2.926 (s, (CH ) NCH CO−, 2H), 3.281 (q,
3
3
2
3
2
2
and 3d, 50 mL) were added 2a−2d (12 mmol) separately, and the
reaction mixtures were stirred at 85 °C for about 24 h. After the
reaction, the mixtures were cooled to room temperature and
transferred to a RB flask. Then the volume of the reaction mixtures
was reduced to 1/10th its original volume by rotary evaporator.
Finally, the products were precipitated with excess diethyl ether/
acetone (150 mL). Either the organic solvent was decanted off and the
precipitate was washed three times with diethyl ether, or the
precipitates were filtered through sintered glass funnel and washed
repeatedly with diethyl ether and vacuum-dried to give more than 99%
CH NHCO−, 2H), 7.136 (br s, amide −NH, 1H).
2
2
-(N,N-Dimethyl)-N′-hexadecylethanamide (2d). FT-IR (KBr):
−1 −1
3
228 cm (amide N−H str.), 2935 cm (−CH − assym. str.), 2859
2
−1
−1
−1
cm (−CH − sym. str.), 1681 cm (Amide I, CO str.), 1559 cm
2
−1
1
(
Amide II, N−H ben.), 1470 cm (−CH − scissor). H NMR (400
2
MHz, CDCl ): δ 0.871 (t, terminal −CH , 3H), 1.239 (m,
3
3
CH (CH ) −, 26H), 1.529 (q, CH (CH ) CH −, 2H), 2.268 (s,
3
2
13
3
2
13
2
(
−
CH ) N−, 6H), 2.934 (s, (CH ) NCH CO−, 2H), 3.265 (q,
3
2
3
2
2
CH NHCO−, 2H), 7.120 (br s, amide −NH, 1H).
2
General Procedure for the Synthesis of Alkane Dibromoe-
1
yield. All the final products (4−19) were characterized by FT-IR, H
thanamide (3a−3d). Diaminoalkanes (50 mmol) were dissolved in
NMR, 13C NMR, HRMS, and elemental analysis. The purity of the
compounds checked by reverse phase high performance liquid
chromatography (HPLC) using 0.1% trifluoroacetic acid (TFA) in
water/acetonitrile (0−100%) as mobile phase was more than 95%.
N,N′-(Ethane-1,2-diethanamide) Bis(N,N-dimethyl-(N′-hexyletha-
chloroform (100 mL), and a solution of K CO (20.7 g, 150 mmol) in
2
3
water (100 mL) was added to it. The binary mixture was then cooled
to 5 °C in a cold incubator. Bromoacetyl bromide (30.4 g, 150 mmol)
was dissolved in chloroform (100 mL) and was added to the mixture
dropwise for about 30 min. After the completion of addition, the
reaction mixture was stirred at room temperature for 12 h. After the
reaction, the insoluble solid was filtered through a sintered glass funnel
and washed repeatedly with water. Finally, the precipitate was dried in
a vacuum oven at 60 °C to obtain white colored products (1st
collection). Also, the organic layer from the reaction mixture after the
filtration was separated using a separating funnel, and the aqueous
layer was subjected to repeated washes with chloroform (2 × 50 mL).
All the organic solutions were then combined and washed with water
−1
namide) ammonium bromide) (4). FTIR (Solid): 3208 cm (amide
−
1
−1
N−H str.), 2909 cm (−CH − assym. str.), 2860 cm (−CH −
2
2
−1
−1
sym. str.), 1675 cm (amide I, CO str.), 1555 cm (amide II, N−
−1
1
H ben.), 1468 cm (−CH − scissor). H NMR (400 MHz, CDCl ):
2
3
δ 0.875 (t, terminal −CH , 6H), 1.259 (m, −CH (CH ) CH −, 12H),
3
3
2
3
2
1
. 581 (m, CH (CH ) CH CH −, 4H), 3. 231 (m,
3
2
3
2
2
CH (CH ) CH CH −, 4H), 3.439 (m, −NHCH CH NH−, 4H),
3
2
3
2
2
2
2
+
3
. 611 (s, −C H N (CH ) CH −, 12H), 4. 630 (s,
2
3
2
2
+
−
CH N (CH ) CH −, 4H), 8.219 (br s, CH (CH ) NHCO−, 2H),
2
3
2
2
3
2 5
(
3 × 75 mL). The final organic layer was passed through anhydrous
Na SO . The organic solvent was evaporated to obtain white products
2nd collection). The products were characterized by FT-IR, H
13
8
1
6
.684 (br s, −CONHCH −, 2H). C NMR (100 MHz, CDCl ): δ
2
3
2
4
4.219, 22.777, 27.281, 29.225, 29.415, 38.941, 40.409, 52.829, 64.182,
1
(
6.226, 162.664, 162.880. HRMS (ESI) m/z calculated for
NMR, and 13C NMR spectroscopy. The products obtained in both
ways (from precipitate and solution) were the same, and their
combined weight gave quantitative (100%) yield of 3a−3d.
+
2+
C H N O Br [M − Br] and [M − 2Br] : 593.3571, 595.3571,
2
6
54
6
4
2
and 257.2194. Found: 593.3394, 595.3379, and 257.2112.
N,N′-(Propane-1,3-diethanamide) Bis(N,N-dimethyl-(N′-hexyle-
N,N′-(Ethane-1,2-diyl) Bis(2-bromoethanamide) (3a). FT-IR
−1
thanamide) ammonium bromide) (5). FTIR (Solid): 3212 cm
−1
−1
(
Solid): 3294 cm (amide N−H str.), 2938 cm (−CH − assym.
−1
−1
−1
2
(
(
amide N−H str.), 2928 cm (−CH − assym. str.), 2860 cm
−1
−1
2
str.), 2860 cm (−CH − sym. str.), 1645 cm (Amide I, CO str.),
−1
2
−CH − sym. str.), 1685 cm (amide I, CO str.), 1565 cm
−1
−1
1
2
1
542 cm (Amide II, N−H ben.), 1480 cm (−CH − scissor). H
−1
1
2
(amide II, N−H ben.), 1465 cm (−CH − scissor). H NMR (400
2
NMR (400 MHz, CDCl ): δ 3.290 (m, −NH(CH ) NH−, 4H), 3.873
3
2
2
MHz, CDCl ): δ 0.862 (t, terminal −CH , 6H), 1.269 (m,
3
3
(
−
2
s, −CH CONH(CH ) NHCOCH −, 4H), 6.551 (br s, amide
2
2
2
2
−CH (CH ) CH −, 12H), 1.615 (m, CH (CH ) CH CH −, 4H),
3
2
3
2
3
2
3
2
2
13
CONH(CH ) NHCO−, 2H). C NMR (100 MHz, CDCl ): δ
2
2
3
1.851 (m, −NHCH CH CH NH−, 2H), 3.231 (m,
2
2
2
9.495, 40.134, 165.531.
N,N′-(Propane-1,3-diyl) Bis(2-bromoethanamide) (3b). FT-IR
CH (CH ) CH CH −, 4H), 3.381 (m, −NHCH CH CH NH−,
3
2
3
2
2
2
2
2
+
4
H), 3.580 (s, −CH N (CH ) CH −, 12H), 4.614 (s,
2
3
2
2
−1
−1
(
Solid): 3295 cm (amide N−H str.), 2942 cm (−CH − assym.
+
2
−CH N (CH ) CH −, 4H), 8.208 (br s, CH (CH ) NHCO−, 2H),
2
3
2
2
3
2 5
−1
−1
str.), 2863 cm (−CH − sym. str.), 1645 cm (Amide I, CO str.),
13
2
8.665 (br s, −CONHCH −, 2H). C NMR (100 MHz, CDCl ): δ
2
3
−1
−1
1
1
544 cm (Amide II, N−H ben.), 1485 cm (−CH − scissor). H
2
14.209, 22.707, 26.138 27.278, 29.224, 29.409, 38.978, 40.412, 52.838,
NMR (400 MHz, CDCl ): δ 1.542 (m, −CH CH CH −, 4H), 3.291
3
2
2
2
64.194, 66.234, 162.647, 162.856. HRMS (ESI) m/z calculated for
(
(
m, −NHCH CH CH NH−, 4H), 3.876 (s, −CH CONH-
+
2+
2
2
2
2
C H N O Br [M − Br] and [M − 2Br] : 607.3726, 609.3726, and
2
7
56
6
4
2
CH ) NHCOCH −, 4H), 6.589 (br s, amide −CON-
2
3
2
264.2272. Found: 607.2185, 609.3509, and 264.2185.
13
H(CH ) NHCO−, 2H). C NMR (100 MHz, CDCl ): δ 29.239,
2
3
3
N,N′-(Butane-1,4-diethanamide) Bis(N,N-dimethyl-(N′-hexyle-
−
1
2
9.482, 40.194, 165.538.
N,N′-(Butane-1,4-diyl) Bis(2-bromoethanamide) (3c). FT-IR
thanamide) ammonium bromide) (6). FTIR (Solid): 3210 cm
−1
−1
−1
(amide N−H str.), 2920 cm (−CH − assym. str.), 2860 cm
2
−1
−1
−1
(
Solid): 3294 cm (amide N−H str.), 2939 cm (−CH − assym.
(−CH − sym. str.), 1678 cm (amide I, CO str.), 1557 cm
2
2
−1
−1
−1
1
str.), 2859 cm (−CH − sym. str.), 1642 cm (Amide I, CO str.),
(amide II, N−H ben.), 1470 cm (−CH − scissor). H NMR (400
2
2
−1
−1
1
1
545 cm (Amide II, N−H ben.), 1480 cm (−CH − scissor). H
MHz, CDCl ): δ 0.872 (t, terminal −CH , 6H), 1.254 (m,
2
3
3
NMR (400 MHz, CDCl ): δ 1.541 (m, −CH (CH ) CH −, 4H),
−CH (CH ) CH −, 12H), 1.534 (m, CH (CH ) CH CH −, 4H),
3
2
2
2
2
3
2
3
2
3
2
3
2
2
3
(
.293 (q, −CH (CH ) CH −, 4H), 3.874 (s, −CH CONH-
1.578 (m, −NHCH (CH ) CH NH−, 4H), 3.220 (m,
2
2
2
2
2
2
2
2
2
CH ) NHCOCH −, 4H), 6.554 (br s, amide −CON-
CH (CH ) CH CH −, 4H), 3.321 (m, −NHCH (CH ) CH NH−,
2
4
2
3
2
3
2
2
2
2
2
2
13
+
H(CH ) NHCO−, 2H). C NMR (100 MHz, CDCl ): δ 29.254,
4H), 3.558 (s, −CH N (CH ) CH −, 12H), 4.610 (s,
2
4
3
2
3
2
2
+
2
9.479, 40.294, 165.540.
−CH N (CH ) CH −, 4H), 8.205 (br s, CH (CH ) NHCO−, 2H),
2
3
2
2
3
2 7
J
DOI: 10.1021/acs.jmedchem.5b00443
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
3
8
1
5
.447 (br s, −CONHCH −, 2H). C NMR (100 MHz, CDCl ): δ
14.221, 22.760, 26.108, 27.144, 27.173, 29.308, 29.336, 31.160, 40.056,
2
3
4.221, 22.760, 26.108, 27.144, 27.173, 29.308, 29.336, 31.160, 40.056,
2.788, 64.447, 65.532, 162.773, 162.943. HRMS (ESI) m/z calculated
for C H N O Br [M − Br] and [M − 2Br] : 677.4510, 679.4510,
and 299.2663. Found: 677.4306, 679.4290, and 299.2618.
52.788, 64.447, 65.532, 162.773, 162.943. HRMS (ESI) m/z calculated
+
2+
for C H N O Br [M − Br] and [M − 2Br] : 677.4510, 679.4510,
32
66
6
4
2
+
2+
and 299.2663. Found: 677.4306, 679.4290, and 299.2618. Elemental
analysis: C 50.76, H 8.79, N 11.11 (calculated); C 50.64, H 8.77, N
11.06 (found).
28
58
6
4
2
N,N′-(Hexane-1,6-diethanamide) Bis(N,N-dimethyl-(N′-hexyle-
−1
thanamide) ammonium bromide) (7). FTIR (Solid): 3207 cm
N,N′-(Hexane-1,6-diethanamide) Bis(N,N-dimethyl-(N′-octyle-
−
1
−1
−1
−1
(
(
(
amide N−H str.), 2914 cm (−CH − assym. str.), 2855 cm
thanamide) ammonium bromide) (11). FTIR (Solid): 3207 cm
2
−
1
−1
−1
−1
−CH − sym. str.), 1675 cm (amide I, CO str.), 1558 cm
(amide N−H str.), 2914 cm (−CH − assym. str.), 2855 cm
2
2
−1
1
−1
amide II, N−H ben.), 1470 cm (−CH − scissor). H NMR (400
(−CH − sym. str.), 1675 cm (amide I, CO str.), 1558 cm
2
2
−1
1
MHz, CDCl ): δ 0.875 (t, terminal −CH , 6H), 1.262 (m,
(amide II, N−H ben.), 1470 cm (−CH − scissor). H NMR (400
3
3
2
−
−
C H 3 ( C H 2
)
C H 2 − ,
2 0 H ) ,
1 . 4 1 4
( m ,
MHz, CDCl ): δ 0.875 (t, terminal −CH , 6H), 1.262 (m,
5
3
3
NHCH CH (CH ) CH CH NH−, 4H), 1.554−1.571 (m, −
− C H ( C H 2
)
C H 2 − ,
2 0 H ) ,
1 . 4 1 4
( m ,
2
2
2
2
2
2
3
5
CH (CH ) CH CH − and −NHCH CH (CH ) CH CH NH−,
−NHCH CH (CH ) CH CH NH−, 4H), 1.554−1.571 (m, −
3
2
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
−
1
H ), 3. 241 −3 . 3 16 (m , C H ( C H ) C H C H − a n d
CH (CH ) CH CH − and −NHCH CH (CH ) CH CH NH−,
3
2
5
2
2
3
2
5
2
2
2
2
2
2
2
2
+
NHCH (CH ) CH NH−, 8H), 3.586 (s, −CH N (CH ) CH −,
8 H ) , 3 . 2 4 1 −3 . 3 1 6 ( m , C H ( CH ) C H CH − a n d
2
2
4
2
2
3
2
2
3
2
5
2
2
+
+
2H), 4.641 (s, −CH N (CH ) CH −, 4H), 8.229 (br s,
−NHCH (CH ) CH NH−, 8H), 3.586 (s, −CH N (CH ) CH −,
2
3
2
2
2
2
4
2
2
3
2
2
13
+
CH (CH ) NHCO−, 2H), 8.440 (br s, −CONHCH −, 2H).
C
12H), 4.641 (s, −CH N (CH ) CH −, 4H), 8.229 (br s,
3
2
7
2
2
3
2
2
13
NMR (100 MHz, CDCl ): δ 14.206, 22.743, 26.198, 27.129, 28.308,
CH (CH ) NHCO−, 2H), 8.440 (br s, −CONHCH −, 2H).
C
3
3
2
7
2
2
9.156, 29.297, 29.320, 39.313, 40.040, 52.621, 64.782, 65.243,
NMR (100 MHz, CDCl ): δ 14.206, 22.743, 26.198, 27.129, 28.308,
3
1
62.765, 162.862. HRMS (ESI) m/z calculated for C H N O Br
29.156, 29.297, 29.320, 39.313, 40.040, 52.621, 64.782, 65.243,
30
62
6
4
2
+
2+
[
M − Br] and [M − 2Br] : 649.4197, 651.4197, and 285.2507.
162.765, 162.862. HRMS (ESI) m/z calculated for C H N O Br
34
70
6
4
2
+
2+
Found: 649.4109, 651.4098, and 285.2493.
[M − Br] and [M − 2Br] : 705.4824, 707.4824, and 313.2820.
Found: 705.4618, 707.4602, and 313.2761. Elemental analysis: C
52.01, H 8.99, N 10.71 (calculated); C 51.95, H 8.84, N 10.36
(found).
N,N′-(Ethane-1,2-diethanamide) Bis(N,N-dimethyl-(N′-octyletha-
−1
namide) ammonium bromide) (8). FTIR (Solid): 3215 cm (amide
−1
−1
N−H str.), 2918 cm (−CH − assym. str.), 2855 cm (−CH −
2
2
−1
−1
sym. str.), 1678 cm (amide I, CO str.), 1560 cm (amide II, N−
N,N′-(Ethane-1,2-diethanamide) Bis(N,N-dimethyl-(N′-dodecyle-
−1
1
−1
H ben.), 1465 cm (−CH − scissor). H NMR (400 MHz, CDCl ):
thanamide) ammonium bromide) (12). FTIR (Solid): 3212 cm
2
3
−1
−1
−1
δ 0.866 (t, terminal −CH , 6H), 1.266 (m, −CH (CH ) CH −, 20H),
(amide N−H str.), 2920 cm (−CH − assym. str.), 2860 cm
3
3
2
5
2
2
−1
1
. 564 (m, CH (CH ) CH CH −, 4H), 3. 229 (m,
(−CH − sym. str.), 1682 cm (amide I, CO str.), 1565 cm
3
2
5
2
2
2
−1
1
CH (CH ) CH CH −, 4H), 3.459 (m, −NHCH CH NH−, 4H),
3
(amide II, N−H ben.), 1468 cm (−CH − scissor). H NMR (400
3
2
5
2
2
2
2
2
+
. 605 (s, −CH N ( CH ) CH −, 12H) , 4 . 6 2 9 ( s ,
CH N (CH ) CH −, 4H), 8.234 (br s, CH (CH ) NHCO−, 2H),
MHz, CDCl ): δ 0.886 (t, terminal −CH , 6H), 1.259 (m,
2
3
2
2
3
3
+
−
−CH (CH ) CH −, 36H), 1.589 (m, CH (CH ) CH CH −, 4H),
2
3
2
2
3
2
7
3
2
9
2
3
2
9
2
2
1
3
8
1
5
.734 (br s, −CONHCH −, 2H). C NMR (100 MHz, CDCl ): δ
3.219 (m, CH (CH ) CH CH −, 4H), 3.459 (m, −NHCH CH NH−,
2
3
3
2
9
2
2
2
2
+
4.228, 22.767, 27.163, 29.180, 29.313, 29.338, 31.939, 38.822, 40.403,
4H), 3.612 (s, −CH N (CH ) CH −, 12H), 4.627 (s,
2
3
2
2
+
2.829, 64.122, 66.238, 162.765, 163.380. HRMS (ESI) m/z calculated
−CH N (CH ) CH −, 4H), 8.229 (br s, CH (CH ) NHCO−,
2
3
2
2
3
2 11
+
2+
13
for C H N O Br [M − Br] and [M − 2Br] : 649.4197, 651.4197,
and 285.2507. Found: 649.4109, 651.4098, and 285.2493. Elemental
analysis: C 49.43, H 8.58, N 11.54 (calculated); C 49.23, H 8.60, N
2H), 8.824 (br s, −CONHCH −, 2H). C NMR (100 MHz,
30
62
6
4
2
2
CDCl ): δ 14.198, 22.801, 27.098, 29.280, 29.320, 29.431, 29.615,
3
29.718, 32.401, 38.816, 40.415, 52.909, 64.221, 66.285, 162.749,
+
1
1.29 (found).
163.258. HRMS (ESI) m/z calculated for C H N O Br [M − Br]
38
78
6
4
2
2+
N,N′-(Propnae-1,3-diethanamide) Bis(N,N-dimethyl-(N′-octyle-
and [M − 2Br] : 761.5450, 763.5450, and 341.3133. Found:
761.5241, 763.5229, and 341.3037. Elemental analysis: C 54.26, H
9.35, N 9.99 (calculated); C 54.16, H 9.41, N 9.91 (found).
N,N′-(Propane-1,3-diethanamide) Bis(N,N-dimethyl-(N′-dodecy-
−
1
thanamide) ammonium bromide) (9). FTIR (Solid): 3210 cm
−
1
−1
−1
(
(
(
amide N−H str.), 2925 cm (−CH − assym. str.), 2860 cm
2
−
1
−CH − sym. str.), 1680 cm (amide I, CO str.), 1565 cm
2
−1
1
−1
amide II, N−H ben.), 1470 cm (−CH − scissor). H NMR (400
lethanamide) ammonium bromide) (13). FTIR (Solid): 3214 cm
2
−
1
−1
−1
MHz, CDCl ): δ 0.860 (t, terminal −CH , 6H), 1.264 (m,
−
1
(amide N−H str.), 2929 cm (−CH − assym. str.), 2862 cm
3
3
2
−
1
CH (CH ) CH −, 20H), 1.555 (m, CH (CH ) CH CH −, 4H),
(−CH − sym. str.), 1681 cm (amide I, CO str.), 1562 cm
3
2
5
2
3
2
5
2
2
2
−1
1
.848 (m, −NHCH CH CH NH−, 2H), 3.241 (m,
(amide II, N−H ben.), 1468 cm (−CH − scissor). H NMR (400
2
2
2
2
CH (CH ) CH CH −, 4H), 3.377 (m, −NHCH CH CH NH−,
4
MHz, CDCl ): δ 0.877 (t, terminal −CH , 6H), 1.264 (m,
3
2
5
2
2
2
2
2
3
3
+
H), 3.583 (s, −CH N (CH ) CH −, 12H), 4.666 (s,
CH N (CH ) CH −, 4H), 8.178 (br s, CH (CH ) NHCO−, 2H),
−CH (CH ) CH −, 36H), 1.521 (m, CH (CH ) CH CH −, 4H),
2
3
2
2
3
2
9
2
3
2
9
2
2
+
−
1.839 (m, −NHCH CH CH NH−, 2H), 3.250 (m,
2
3
2
2
3
2
7
2
2
2
1
3
8
1
5
.601 (br s, −CONHCH −, 2H). C NMR (100 MHz, CDCl ): δ
CH (CH ) CH CH −, 4H), 3.379 (m, −NHCH CH CH NH−,
2
3
3
2
9
2
2
2
2
2
+
4.201, 22.790, 26.128, 27.134, 27.189, 29.412, 29.289, 31.260, 40.456,
4H), 3.579 (s, −CH N (CH ) CH −, 12H), 4.689 (s,
2
3
2
2
+
2.898, 64.401, 65.516, 162.768, 162.848. HRMS (ESI) m/z calculated
−CH N (CH ) CH −, 4H), 8.210 (br s, CH (CH ) NHCO−,
2
3
2
2
3
2 11
+
2+
13
for C H N O Br [M − Br] and [M − 2Br] : 663.4354, 665.4354,
and 292.2585. Found: 663.4164, 665.4133, and 292.2564. Elemental
analysis: C 50.11, H 8.69, N 11.32 (calculated); C 50.01, H 8.60, N
2H), 8.612 (br s, −CONHCH −, 2H). C NMR (100 MHz,
31
64
6
4
2
2
CDCl ): δ 14.218, 22.789, 27.112, 29.310, 29.318, 29.428, 29.595,
3
29.710, 32.412, 38.821, 40.421, 52.913, 64.230, 66.290, 162.752,
+
1
1.24 (found).
163.308. HRMS (ESI) m/z calculated for C H N O Br [M − Br]
39
80
6
4
2
2+
N,N′-(Butane-1,4-diethanamide) Bis(N,N-dimethyl-(N′-octyletha-
and [M − 2Br] : 775.1874, 777.1874, and 348.1345. Found:
775.1396, 777.1381, and 348.1123. Elemental analysis: C 54.79, H
9.43, N 9.90 (calculated); C 54.72, H 9.51, N 9.86 (found).
N,N′-(Butane-1,4-diethanamide) Bis(N,N-dimethyl-(N′-dodecyle-
−
1
namide) ammonium bromide) (10). FTIR (Solid): 3205 cm
−
1
−1
−1
(
(
(
amide N−H str.), 2915 cm (−CH − assym. str.), 2852 cm
2
−
1
−CH − sym. str.), 1674 cm (amide I, CO str.), 1560 cm
2
−1
1
−1
amide II, N−H ben.), 1468 cm (−CH − scissor). H NMR (400
thanamide) ammonium bromide) (14). FTIR (Solid): 3210 cm
2
−
1
−1
−1
MHz, CDCl ): δ 0.864 (t, terminal −CH , 6H), 1.262 (m,
−
1
(amide N−H str.), 2912 cm (−CH − assym. str.), 2855 cm
3
3
2
−
1
CH (CH ) CH −, 20H), 1.544 (m, CH (CH ) CH CH −, 4H),
(−CH − sym. str.), 1675 cm (amide I, CO str.), 1563 cm
3
2
5
2
3
2
5
2
2
2
−1
1
.580 (m, −NHCH (CH ) CH NH−, 4H), 3.213 (m,
(amide II, N−H ben.), 1470 cm (−CH − scissor). H NMR (400
2
2
2
2
2
CH (CH ) CH CH −, 4H), 3.316 (m, −NHCH (CH ) CH NH−,
4
MHz, CDCl ): δ 0.878 (t, terminal −CH , 6H), 1.257 (m,
3
2
5
2
2
2
2
2
2
3
3
+
H), 3.558 (s, −CH N (CH ) CH −, 12H), 4.610 (s,
CH N (CH ) CH −, 4H), 8.205 (br s, CH (CH ) NHCO−, 2H),
−CH (CH ) CH −, 36H), 1.539 (m, CH (CH ) CH CH −, 4H),
2
3
2
2
3
2
9
2
3
2
9
2
2
+
−
1.578 (m, −NHCH (CH ) CH NH−, 4H), 3.218 (m,
2
3
2
2
3
2
7
2
2
2
2
1
3
8
.447 (br s, −CONHCH −, 2H). C NMR (100 MHz, CDCl ): δ
CH (CH ) CH CH −, 4H), 3.320 (m, −NHCH (CH ) CH NH−,
2
3
3
2
9
2
2
2
2
2
2
K
DOI: 10.1021/acs.jmedchem.5b00443
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
+
4
−
2
H), 3.560 (s, −CH N (CH ) CH −, 12H), 4.612 (s,
−CH (CH ) CH −, 52H), 1.537 (m, CH (CH ) CH CH −, 4H),
2
3
2
2
3
2
13
2
3
2
13
2
2
+
CH N (CH ) CH −, 4H), 8.218 (br s, CH (CH ) NHCO−,
1.581 (m, −NHCH (CH ) CH NH−, 4H), 3.220 (m,
2
3
2
2
3
2
11
2
2
2
2
13
H), 8.451 (br s, −CONHCH −, 2H). C NMR (100 MHz,
CH (CH ) CH CH −, 4H), 3.324 (m, −NHCH (CH ) CH NH−,
2
3
2
13
2
2
2
2
2
2
+
CDCl ): δ 14.220, 22.791, 27.110, 29.312, 29.321, 29.425, 29.598,
4H), 3.556 (s, −CH N (CH ) CH −, 12H), 4.607 (s,
3
2
3
2
2
+
2
1
9.707, 32.417, 38.821, 40.418, 52.924, 64.232, 66.288, 162.756,
−CH N (CH ) CH −, 4H), 8.211 (br s, CH (CH ) NHCO−,
2
3
2
2
3
2 15
+
13
63.310. HRMS (ESI) m/z calculated for C H N O Br [M − Br]
2H), 8.454 (br s, −CONHCH −, 2H). C NMR (100 MHz,
40
82
6
4
2
2
2+
and [M − 2Br] : 789.763, 791.5763, and 355.329. Found: 789.5557,
CDCl ): δ 14.217, 22.788, 23.867, 27.151, 29.318, 29.328, 29.441,
3
7
91.5545, and 355.3197. Elemental analysis: C 55.29, H 9.52, N 9.67
29.542, 29.708, 32.418, 38.820, 40.420, 52.918, 64.228, 66.290,
(
calculated); C 55.25, H 9.49, N 9.62 (found).
N,N′-(Hexane-1,6-diethanamide) Bis(N,N-dimethyl-(N′-dodecyle-
162.753, 163.322. HRMS (ESI) m/z calculated for C48H N O Br
98 6 4 2
+
2+
[M − Br] and [M − 2Br] : 901.7016, 903.7016, and 411.3916.
Found: 901.6819, 903.6814, and 411.3865. Elemental analysis: C
58.74, H 10.07, N 8.57 (calculated); C 58.68, H 10.12, N 8.50
(found).
−
1
thanamide) ammonium bromide) (15). FTIR (Solid): 3210 cm
−
1
−1
−1
(
(
(
amide N−H str.), 2918 cm (−CH − assym. str.), 2858 cm
2
−
1
−CH − sym. str.), 1676 cm (amide I, CO str.), 1560 cm
2
−1
1
amide II, N−H ben.), 1465 cm (−CH − scissor). H NMR (400
N,N′-(Hexane-1,6-diethanamide) Bis(N,N-dimethyl-(N′-hexade-
2
MHz, CDCl ): δ 0.879 (t, terminal −CH , 6H), 1.258 (m,
cylethanamide) ammonium bromide) (19). FTIR (Solid): 3208
3
3
−
1
−1
−1
−1
−
−
C H 3 ( C H 2
)
C H 2 − ,
3 6 H ) ,
1 . 4 2 1
( m ,
cm (amide N−H str.), 2920 cm (−CH
2
− assym. str.), 2860 cm
9
−1
NHCH CH (CH ) CH CH NH−, 4H), 1.552−1.574 (m, −
(−CH
(amide II, N−H ben.), 1467 cm (−CH
MHz, CDCl ): δ 0.889 (t, terminal −CH
− C H ( C H − , 5 2 H ) ,
−NHCH CH
CH (CH − and −NHCH
(CH
NH−, 8H), 3.591 (s, −CH
2
− sym. str.), 1675 cm (amide I, CO str.), 1563 cm
2
2
2
2
2
2
−1
1
CH (CH ) CH CH − and −NHCH CH (CH ) CH CH NH−,
2
− scissor). H NMR (400
, 6H), 1.254 (m,
1 . 4 2 9 ( m ,
3
2
9
2
2
2
2
2
2
2
2
8
−
1
H ), 3. 240 −3 . 3 18 (m , C H ( C H ) C H C H − a n d
3
3
3
2
9
2
2
+
NHCH (CH ) CH NH−, 8H), 3.588 (s, −CH N (CH ) CH −,
3
) 1 3 C H
2 2
2
2
4
2
2
3
2
2
+
2H), 4.642 (s, −CH N (CH ) CH −, 4H), 8.227 (br s,
2
2
CH
2
(CH
2
)
2
CH
2
2
NH−, 4H), 1.550−1.575 (m, −
2
3
2
2
1
3
CH (CH ) NHCO−, 2H), 8.438 (br s, −CONHCH −, 2H).
NMR (100 MHz, CDCl ): δ 14.218, 22.786, 27.108, 29.318, 29.341,
2
1
−
8
9
C
3
)
13CH
2
CH
2
2
CH
2
(CH
2
)
2
CH
2
CH
2
NH−,
and
) CH −,
3
2
11
2
8H), 3. 241−3. 320 (m, CH
−NHCH (CH CH
12H), 4.640 (s, −CH
CH (CH 15NHCO−, 2H), 8.451 (br s, −CONHCH
NMR (100 MHz, CDCl ): δ 14.220, 22.784, 23.860, 27.150, 29.314,
)
1 3 CH
CH
−
3
3
2
2
2
+
9.435, 29.599, 29.714, 32.423, 38.828, 40.419, 52.928, 64.241, 66.294,
2
)
2
4
2
2
N (CH
N (CH ) CH −, 4H), 8.225 (br s,
2 3 2 2
3 2 2
+
62.757, 163.312. HRMS (ESI) m/z calculated for C H N O Br [M
4
2
86
6
4
2
+
2+
13
Br] and [M − 2Br] : 817.6076, 819.6076, and 369.3446. Found:
3
)
2
−, 2H).
2
C
17.5849, 819.5837, and 369.3339. Elemental analysis: C 56.22, H
.67, N 9.37 (calculated); C 56.20, H 9.69, N 9.29 (found).
N,N′-(Ethane-1,2-diethanamide) Bis(N,N-dimethyl-(N′-hexadecy-
3
29.324, 29.440, 29.540, 29.712, 32.422, 38.814, 40.425, 52.925, 64.230,
66.294, 162.758, 163.323. HRMS (ESI) m/z calculated for
−1
+
2+
lethanamide) ammonium bromide) (16). FTIR (Solid): 3208 cm
C H N O Br [M − Br] and [M − 2Br] : 929.7329, 931.7329,
50
102
6
4
2
−
1
−1
−1
(
(
(
amide N−H str.), 2925 cm (−CH − assym. str.), 2862 cm
and 425.4073. Found: 929.7130, 931.7121, and 425.4075. Elemental
analysis: C 59.48, H 10.19, N 8.33 (calculated); C 59.38, H 10.12, N
8.27 (found).
2
−
1
−CH − sym. str.), 1680 cm (amide I, CO str.), 1560 cm
2
−1
1
amide II, N−H ben.), 1467 cm (−CH − scissor). H NMR (400
2
MHz, CDCl ): δ 0.880 (t, terminal −CH , 6H), 1.249 (m,
−
3
−
4
CH (CH ) NHCO−, 2H), 8.828 (br s, −CONHCH −, 2H).
NMR (100 MHz, CDCl ): δ 14.212, 22.745, 27.112, 29.321, 29.347,
2
1
−
8
9
Microorganisms and Culture Conditions. The antibacterial
activity of all the small molecular biocides was evaluated against both
Gram-positive bacteria (S. aureus, MRSA, VRE, and NRSA) and Gram-
negative bacteria (E. coli, P. aeruginosa, K. pneumonia, and CREC). S.
aureus, P. aeruginosa, MRSA, K. pneumonia, and NRSA were cultured
in nutrient broth (5.0 g of peptone, 1.0 g of beef extract, 2.0 g of yeast
extract, and 5.0 g of NaCl in 1000 mL of sterile distilled water), while
E. coli was grown in Luria−Bertani broth (5.0 g of yeast extract, 10.0 g
of tryptone, and 10.0 g of NaCl in 1000 mL of sterile distilled water).
For VRE brain−heart infusion broth (5.0 g of beef heart infusion form,
3
3
CH (CH ) CH −, 52H), 1.580 (m, CH (CH ) CH CH −, 4H),
3
2
13
2
3
2
13
2
2
.217 (m, CH (CH ) CH CH −, 4H), 3.458 (m,
3
2
1 3
2
2
+
NHCH CH NH−, 4H), 3.618 (s, −CH N (CH ) CH −, 12H),
2
2
2
3
2
2
+
.628 (s, −CH N (CH ) CH −, 4H), 8.224 (br s,
2
3
2
2
13
C
3
2
15
2
3
9.429, 29.578, 29.726, 32.420, 38.824, 40.422, 52.931, 64.244, 66.289,
62.754, 163.307. HRMS (ESI) m/z calculated for C H N O Br [M
4
6
94
6
4
2
+
2+
Br] and [M − 2Br] : 873.6703, 875.6703, and 397.6760. Found:
73.6499, 875.6489, and 397.3687. Elemental analysis: C 57.95, H
.94, N 8.82 (calculated); C 57.89, H 9.98, N 8.78 (found).
N,N′-(Propane-1,3-diethanamide) Bis(N,N-dimethyl-(N′-hexade-
12.5 g of calf brain infusion form, 2.5 g of Na HPO , 2.0 g of D-
2 4
glucose, 10 g of peptone, and 5.0 g of NaCl in 1000 mL of sterile
distilled water) was used as growth medium. For solid media, 2.5%
agar was used along with the above respective medium. The bacterial
freeze-dried stock samples in 30% glycerol were stored at −80 °C.
About 5 μL of these stock solutions was added to 3 mL of the
respective broth medium, and the cultures were grown for 6 h at 37 °C
prior to the antibacterial experiments.
cylethanamide) ammonium bromide) (17). FTIR (Solid): 3215
−
1
−1
−1
−1
cm (amide N−H str.), 2930 cm (−CH − assym. str.), 2860 cm
2
−1
(
(
−CH − sym. str.), 1682 cm (amide I, CO str.), 1565 cm
2
−1
1
amide II, N−H ben.), 1465 cm (−CH − scissor). H NMR (400
2
MHz, CDCl ): δ 0.881 (t, terminal −CH , 6H), 1.260 (m,
−
1
CH (CH ) CH CH −, 4H), 3.368 (m, −NHCH CH CH NH−,
4
−
2
CDCl ): δ 14.220, 22.789, 23.878, 27.142, 29.321, 29.334, 29.438,
2
1
3
3
CH (CH ) CH −, 52H), 1.531 (m, CH (CH ) CH CH −, 4H),
Antibacterial Assay. Antibacterial efficacy of all synthesized
3
2
13
2
3
2
13
2
2
.842 (m, −NHCH CH CH NH−, 2H), 3.252 (m,
compounds (4−19) was assayed by the microdilution broth method as
2
2
2
5
1
described in CLSI guidelines. The 6 h grown culture as described in
3
2
13
2
2
2
2
2
+
9
H), 3.584 (s, −CH N (CH ) CH −, 12H), 4.682 (s,
Microorganism and Culture Conditions gives about 10 CFU/mL of
2
3
2
2
+
CH N (CH ) CH −, 4H), 8.206 (br s, CH (CH ) NHCO−,
bacteria determined by the spread plating method. The bacterial
cultures were diluted to give approximately 105 CFU/mL in the
respective media or buffer and used for determining antibacterial
efficacy. All the final compounds except 16−19 were water-soluble at
room temperature. Compounds 16−19 were solubilized in water by
heating. Stock solutions were prepared by serial dilution of the
compounds using sterilized Milli-Q water. The aqueous solutions of
serial dilutions (50 μL) were added to the wells of a 96 well plate
2
3
2
2
3
2 15
13
H), 8.624 (br s, −CONHCH −, 2H). C NMR (100 MHz,
2
3
9.584, 29.714, 32.425, 38.829, 40.427, 52.920, 64.238, 66.297,
62.757, 163.314. HRMS (ESI) m/z calculated for C H N O Br
47
96
6
4
2
+
2+
[
M − Br] and [M − 2Br] : 887.6859, 889.6859, and 404.3838.
Found: 887.6662, 889.6652, and 404.3786. Elemental analysis: C
8.35, H 10.01, N 8.69 (calculated); C 58.27, H 10.11, N 8.58
5
(
5
found).
followed by the addition of about 150 μL of bacterial suspension (10
N,N′-(Butane-1,4-diethanamide) Bis(N,N-dimethyl-(N′-hexadecy-
CFU/mL). The plates were then incubated at 37 °C for 24 h in shaker
incubator. The optical density (OD) of the bacterial suspension was
recorded using a TECAN (Infinite series, M200 pro) plate reader at
600 nm. Each concentration had triplicate values, the whole
experiment was repeated at least twice, and the antibacterial efficacy
was determined by taking the average of triplicate OD values for each
−1
lethanamide) ammonium bromide) (18). FTIR (Solid): 3205 cm
−
1
−1
−1
(
(
(
amide N−H str.), 2910 cm (−CH − assym. str.), 2850 cm
2
−
1
−CH − sym. str.), 1674 cm (amide I, CO str.), 1560 cm
2
−1
1
amide II, N−H ben.), 1464 cm (−CH − scissor). H NMR (400
2
MHz, CDCl ): δ 0.869 (t, terminal −CH , 6H), 1.251 (m,
3
3
L
DOI: 10.1021/acs.jmedchem.5b00443
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
concentration and plotting it against concentration. The data was then
subjected to sigmoidal fitting. From the curve, the antibacterial activity
was determined as the point where the OD value was similar to that of
control having no bacteria and expressed as minimum inhibitory
concentration (MIC). A glycopeptide antibiotic vancomycin and a
lipopeptide colistin were used in this study to compare the
antibacterial efficacy.
of sterile water as control. The plates were then incubated under
stationary conditions for 24 h. After incubation, the medium was
removed and washed a single time with 1× PBS. Then 0.1% crystal
violet (CV) solution (100 μL) was added into the wells and allowed to
incubate for 30 min. Crystal violet solution was then discarded, and
the plates were washed with 1× PBS. The residual was solubilized with
200 μL of 95% ethanol solution and diluted 10-fold. The OD at 540
nm was then recorded using a plate reader. Biofilm inhibition was
quantified by considering 100% biofilm formation in the case of
nontreated control.
Antibacterial Activity in the Presence of Human Plasma.
Bacteria (S. aureus) was grown similarly as mentioned earlier and
5
finally diluted in the respective media to give 10 CFU/mL. Fresh
human blood donated by a healthy donor was centrifuged at 3500 rpm
for 5 min. The plasma, which was separated from the blood cells, was
carefully collected. The test compound 10 was dissolved in sterile
water at a concentration of 2000 μg/mL. This was further diluted 2-
fold into the plasma so that the final concentration of compound 10
was 1000 μg/mL in 50% plasma. Three such test samples were
preincubated in 50% plasma for 0, 3, and 6 h, respectively, at 37 °C.
Then 50 μL of the above solutions was added to wells of a 96-well
Biofilm Disruption Assay. Bacteria (S. aureus and E. coli) (6 h
grown, midlog phase) were suspended to 10 CFU/mL into suitable
5
broths (nutrient media supplemented with 1% glucose and NaCl for S.
aureus and M9 media supplemented with 0.02% casamino acid and
0.5% glycerol for E. coli). The well plates containing 100 μL of these
suspensions were then incubated under stationary conditions (for
about 24 h for S. aureus and 72 h for E. coli). After incubation, the
medium was removed and washed with 1× PBS single time.
Compound 10 (100 μL at various concentrations) was then added
to the wells containing established bacterial biofilms and allowed to
incubate for 24 h. A control was made where 100 μL of medium was
added instead of test compound. After 24 h, medium was discarded
and planktonic cells were removed by washing with 1× PBS. Then 100
μL of trypsin−EDTA solution was added to the treated biofilm to
dissolve. Cell suspension of biofilms was then assessed by plating serial
10-fold dilutions on suitable agar plates. After 24 h of incubation,
bacterial colonies were counted, and cell viability was expressed as
log (CFU/mL) along with the control. For visualizing the disruption
5
plate, and 150 μL of the bacterial suspension (10 CFU/mL) was
added to wells containing the test samples in 50% plasma. The plate
was then incubated for 24 h at 37 °C, and antibacterial efficacy of the
test compound was determined as described above.
Antibacterial Activity in Complex Mammalian Fluids. Blood
sodium heparin as anticoagulant) was donated by healthy human
(
donors. Plasma was isolated by centrifugation of the blood at 3500
rpm for 5 min as mentioned above. Serum was obtained by using SST
II Advance serum tube (BD vacutainer) (ref. 367956) containing
human blood and then centrifuging the blood at 3500 rpm for 5 min.
Methicillin-resistant S. aureus (MRSA) was grown similarly as above in
10
of biofilm, 100 μL of 0.1% of crystal violet (CV) was added into the
wells and incubated for 10 min. Crystal violet solution was then
discarded, and the plates were washed once with 1× PBS. Finally,
stained wells were imaged using a digital camera.
9
nutrient broth for 6 h to give ∼10 CFU/mL. Finally, MRSA was
diluted in minimum essential medium (MEM) and mixed with the
5
mammalian systems individually to give 10 CFU/mL in 50% serum,
5
0% plasma, and 50% blood supplemented with 50% MEM. The test
Cytoplasmic Membrane Depolarization Assay. The 6 h grown
bacteria (midlog phase) were harvested (3500 rpm, 5 min), washed in
a mixture of 5 mM glucose and 5 mM HEPES buffer (pH = 7.2) (1:1),
and resuspended in a mixture of 5 mM HEPES buffer, 5 mM glucose,
compound 10 was dissolved in sterile water at a concentration of 4000
μg/mL. This was further diluted 2-fold, and then 50 μL of the solution
was added to wells of a 96-well plate. Bacterial suspension (150 μL,
5
8
1
0 CFU/mL) in 50% serum, 50% plasma, and 50% blood was added
and 100 mM KCl solution (1:1:1) to give ∼10 CFU/mL. Bacterial
individually to the wells containing the test solutions. The plates were
then incubated for 24 h at 37 °C, and antibacterial efficacy of the test
compound was determined by plating the bacterial suspension (20 μL)
directly from the wells onto a nutrient agar plate. The agar plates were
incubated at 37 °C for 24 h, and colonies were observed to determine
the minimum bactericidal concentration (MBC) (the minimum
concentration at which the compound killed all the bacterial cells).
Bactericidal Time-Kill Assay. The rate of bactericidal activity, that
is, the rate at which the compounds killed bacteria was evaluated by
performing time-kill kinetics. Briefly, bacteria (S. aureus and E. coli)
were grown in suitable growth medium at 37 °C for 6 h and diluted in
respective media. The compound 10 was added to the bacterial
suspension (150 μL) was added to wells of a 96-well plate (black plate,
clear bottom with lid). Then 8 μM of 3,3′-dipropylthiadicarbocyanine
iodide (diSC 5) (50 μL) was added to the wells containing bacterial
3
suspension and preincubated for 30 min for S. aureus and 60 min for E.
coli (additional 50 μL of 200 μM EDTA was also added in the case of
E. coli). After the incubation, fluorescence was measured for about 8
min at 2 min intervals at an excitation wavelength of 622 nm (slit
width = 10 nm) and emission wavelength of 670 nm (slit width = 5
nm). Then, the bacterial suspensions were transferred to another well-
plate containing 10 μL of 840 μg/mL biocides, and fluorescence
intensity was monitored similarly for another 12 min. A control
experiment was performed by treating the preincubated bacterial and
dye solution only with Milli-Q water (10 μL).
5
solution (S. aureus of approximately 1.8 × 10 CFU/mL) at
+
concentrations of MIC and 6 × MIC in a 96-well plate. The plate
was then incubated at 37 °C. At different time intervals (0, 30, 60, 90,
1
Intracellular K Ion Leakage Assay. The 6 h grown bacteria
were harvested (3500 rpm, 5 min), washed, and resuspended in a
8
20, 240, and 360 min), 20 μL of aliquots from the solution were
mixture of 10 mM HEPES buffer and 0.5% glucose (1:1) to give ∼10
taken out and serially diluted (10-fold serial dilution) in 0.9% saline.
Then 20 μL of the dilutions was plated on respective agar plates and
incubated at 37 °C for 24 h. The bacterial colonies were counted, and
results are represented in logarithmic scale, that is, log (CFU/mL) vs
CFU/mL. Then 150 μL of the bacterial suspension was added into the
wells of a 96-well plate (black plate, clear bottom with lid). Then 4 μM
of PBFI-AM dye (50 μL) was added to the wells containing bacterial
suspension and preincubated for 30 min for S. aureus and 60 min for E.
coli. After the incubation, fluorescence was measured for about 8 min
at 2 min intervals at an excitation wavelength of 346 nm (slit width =
10 nm) and emission wavelength of 505 nm (slit width = 5 nm). Then,
the bacterial suspensions were transferred to another well-plate
containing 10 μL of 840 μg/mL biocides, and fluorescence intensity
was monitored similarly for another 12 min. Two control experiments
were performed by treating the preincubated bacterial and dye
solution only with Milli-Q water (10 μL) and valinomycin (10 μM),
respectively, as negative and positive controls.
10
time (in min). A similar experiment was performed with buffer (5 mM
HEPES/5 mM glucose = 1:1) with compounds 10 and 18 at 23.6 μg/
5
5
mL against S. aureus (5.53 × 10 CFU/mL) and E. coli (5.03 × 10
CFU/mL), respectively, at 0, 10, 20, 30, 40, 50, and 60 min time
intervals.
Biofilm Inhibition Assay. Midlog phase bacteria (S. aureus and E.
5
coli, 6 h grown culture) were diluted to a concentration of 10 CFU/
mL in suitable broths supplemented with 1% glucose and NaCl.
Compound 10 was serially diluted (2-fold), and 25 μL of these serial
dilutions was added to the wells of a 96-well plate. Then 75 μL of
Outer Membrane Permeabilization Assay. Midlog phase E. coli
cells were harvested (3500 rpm, 4 °C, 5 min), washed, and
resuspended in a mixture of 5 mM glucose/5 mM HEPES buffer
5
bacterial suspension (∼10 CFU/mL) was added into the wells
containing biocide solutions. A similar experiment was performed by
5
8
taking 75 μL of the bacterial suspension (∼10 CFU/mL) and 25 μL
(1:1) at pH 7.2 to give ∼10 CFU/mL. Bacterial suspension (150 μL)
M
DOI: 10.1021/acs.jmedchem.5b00443
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
was transferred into the wells of a 96-well plate (black plate, clear
bottom with lid). Then 10 μM NPN dye (50 μL) was added to the
wells containing bacterial suspension and preincubated for 60 min.
After the incubation, fluorescence was measured for about 8 min at 2
min intervals at excitation wavelength of 350 nm (slit width = 10 nm)
and emission wavelength of 420 nm (slit width = 5 nm). Then, the
bacterial suspensions were transferred to another well-plate containing
FT, Fourier transform; HRMS, high-resolution mass spectrom-
etry; HEK, human embryonic kidney; IC , half-maximum
inhibitory concentration; IR, infrared; m, multiplet (spectral);
50
m/z, mass-to-charge ratio; Me, methyl; MIC, minimal
+
inhibitory concentration; M , parent molecular ion; MRSA,
methicillin-resistant Staphylococcus aureus; OD, optical density;
RBC, red blood cell; rt, room temperature; s, singlet (spectral);
t, triplet (spectral)
1
0 μL of 840 μg/mL biocides, and fluorescence intensity was
monitored similarly for another 12 min. A control experiment was
performed by treating the preincubated bacterial and dye solution only
with Milli-Q water (10 μL).
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and E. coli, midlog phase) were harvested (3500 rpm, 5 min), washed,
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wells of a 96-well plate (black plate, clear bottom with lid). Then 10
μM propidium iodide (PI, 50 μL) was added to the wells containing
bacterial suspension and preincubated for 30 min for S. aureus and 60
min for E. coli. After the incubation, fluorescence was measured for
about 8 min at 2 min intervals at excitation wavelength of 535 nm (slit
width = 10 nm) and emission wavelength of 617 nm (slit width = 5
nm). Then, the bacterial suspensions were transferred to another well-
plate containing 10 μL of 840 μg/mL biocides, and fluorescence
intensity was monitored similarly for another 12 min. A control
experiment was performed by treating the preincubated bacterial and
dye solution only with Milli-Q water (50 μL).
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AUTHOR INFORMATION
Corresponding Author
E-mail: jayanta@jncasr.ac.in. Phone: 080-2208-2565.
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*
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ACKNOWLEDGMENTS
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We thank Prof. C. N. R. Rao, FRS (JNCASR), for his constant
support and encouragement. J. Hoque thanks JNCASR for
senior research fellowship (SRF). The work was supported by
Ramanujan Fellowship (SR/S2/RJN-43/2009 for J. Haldar)
from Department of Science and Technology, Government of
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ABBREVIATIONS USED
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