Antibacterial and Antibiofilm Activity of Cationic Small Molecules with Spatial Positioning of Hydrophobicity: An in Vitro and in Vivo Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.6b01435

More than 80% of the bacterial infections are associated with biofilm formation. To combat infections, amphiphilic small molecules have been developed as promising antibiofilm agents. However, cytotoxicity of such molecules still remains a major problem. Herein we demonstrate a concept in which antibacterial versus cytotoxic activities of cationic small molecules are tuned by spatial positioning of hydrophobic moieties while keeping positive charges constant. Compared to the molecules with more pendent hydrophobicity from positive centers (MIC = 1-4 μg/mL and HC₅₀ = 60-65 μg/mL), molecules with more confined hydrophobicity between two centers show similar antibacterial activity but significantly less toxicity toward human erythrocytes (MIC = 1-4 μg/mL and HC₅₀ = 805-1242 μg/mL). Notably, the optimized molecule is shown to be nontoxic toward human cells (HEK 293) at a concentration at which it eradicates established bacterial biofilms. The molecule is also shown to eradicate preformed bacterial biofilm in vivo in a murine model of superficial skin infection.

Full text of DOI:10.1021/acs.jmedchem.6b01435

Subscriber access provided by University of Otago Library
Article
Antibacterial and Antibiofilm Activity of Cationic Small Molecules with
Spatial Positioning of Hydrophobicity: An In Vitro and In Vivo Evaluation
Jiaul Hoque, Mohini Mohan Konai, Shanola Smitha Sequeira, Sandip Samaddar, and Jayanta Haldar
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01435 • Publication Date (Web): 04 Nov 2016
Downloaded from http://pubs.acs.org on November 6, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Antibacterial and Antibiofilm Activity of Cationic Small
Molecules with Spatial Positioning of Hydrophobicity: An In
Vitro and In Vivo Evaluation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jiaul Hoque, Mohini M. Konai, Shanola S. Sequeira, Sandip Samaddar and Jayanta Haldar*
Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal
Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, India
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 46
1
2
3
4
5
6
7
8
9
ABSTRACT. More than 80% of the bacterial infections are associated with biofilm
formation. To combat infections, amphiphilic small molecules have been developed as
promising antibiofilm agents. However, cytotoxicity of such molecules still remains a major
problem. Herein we demonstrate a concept in which antibacterial versus cytotoxic activities
of cationic small molecules is tuned by spatial positioning of hydrophobic moieties while
keeping positive charges constant. Compared to the molecules with more pendent
hydrophobicity from positive centres (MIC = 1ꢀ4 ꢀg/mL and HC50 = 60ꢀ65 ꢀg/mL),
molecules with more confined hydrophobicity between two centres show similar antibacterial
activity but significantly less toxicity towards human erythrocytes (MIC = 1ꢀ4 ꢀg/mL and
HC50 = 805ꢀ1242 ꢀg/mL). Notably, the optimized molecule is shown to be nonꢀtoxic towards
human cells (HEK 293) at concentration it eradicates established bacterial biofilms. The
molecule is also shown to eradicate preformed bacterial biofilm inꢀvivo in murine model of
superficial skin infection.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 46
Journal of Medicinal Chemistry
1
2
3
4
INTRODUCTION
5
6
7
8
It is estimated that more than 80% of bacterial infections are accompanied by the biofilm
formation.1 Biofilms that form on biotic surfaces, e.g., mammalian tissue, are associated with
a vast number of bacterial infections such as chronic lung infections in cystic fibrosis
patients, periodontitis, endocarditis, skin infections, etc.2,3 Moreover, bacterial biofilms play
significant role in many nosocomial infections due to colonization on abiotic surfaces such as
catheters and other medical devices.4,5 In order to prevent infections associated with bacterial
biofilms, various synthetic antibiofilm agents capable of inhibiting biofilm formation have
been developed.6ꢀ13 Nonetheless, from a clinical standpoint, infections caused by already
established biofilms pose significant threat to human health as biofilms are inherently
resistant to host immune system and conventional therapeutic antibiotics.14ꢀ16 It is therefore
necessary to develop molecules that are capable of eradicating preformed bacterial biofilms
while sparing the mammalian cells.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As the biofilms are covered by anionic exopolymeric substances (EPS) composed of
mostly polysaccharide, proteins and extracellular negatively charged DNA, cationic small
molecules are known to interact with the biofilm matrix thereby disrupts the matrix.17ꢀ22
Further, small molecules have also been shown to destroy the matrixꢀembedded bacteria
which are difficult to kill otherwise thereby leading to eradication of established biofilm.23ꢀ27
Interestingly, some of the molecules were shown to have the abilities of both inhibiting
bacterial biofilm formation and eradicating established biofilms.28ꢀ31 However, identification
of small molecules that eradicate biofilm and kill encased bacteria without affecting the
mammalian cell viability still remains a major challenge. In general antibacterial activity
versus cytotoxicity of amphiphilic small molecules has been tuned by varying length of the
hydrophobic chain, the number of positive charges and the hydrophobic/hydrophilic balance
of the molecules.32ꢀ42 Importantly, the role of positive charges and thereby tuning the
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 46
1
2
3
4
5
6
7
8
9
amphiphilic balance were effectively shown to improve the antibacterial activity while
minimising the toxicity of cationic small molecules.43ꢀ46 It has also been observed that the
choice of scaffold and hence spacing of positive charges improves the selectivity of the
molecules.43ꢀ46 However, how the activity and toxicity of an amphiphilic small molecule
varies as a function of spatial positioning of the hydrophobic moieties while keeping the
positive charges unchanged remains largely unexplored. For instance, how the activity and
toxicity of a small molecule bearing two positive charges differ when the hydrophobic
moieties are more confined between two positive centres as opposed to being pendent from
the positive centres. It is well known that optimum amphiphilicity is an essential criterion to
achieve antibacterial activity; however increase in hydrophobic character for improving
antibacterial activity is also known to increase cytotoxicity. Herein we hypothesized that
cytotoxicity of the molecules could further be tuned by spatial positioning of hydrophobic
moiety in molecules with optimum amphiphilicity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Interestingly, spatial controlling of hydrophobicity was shown to significantly affect
the toxicity of cationic small molecules with negligible changes in antibacterial activity.
Confining hydrophobic moiety more between two positive centres of a small molecule
remarkably reduced its toxicity towards mammalian cells compared to a molecule with less
confined hydrophobicity (Figure 1). In a model lipidꢀsmall molecule interaction studies, it
was observed that molecules with more confined hydrophobicity interacted selectively with
negatively charged lipid membrane of bacteria whereas molecules with more pendent
hydrophobicity interacted with both negatively charged lipid membrane of bacteria and
zwitterionic lipid membrane of mammalian cells. Moreover, the optimised molecule showed
excellent efficacy in eradicating established bacterial biofilms inꢀvitro. While the lack of
ability to eradicate established bacterial biofilms under complex inꢀvivo conditions has been a
major setback for antibiofilm agents to be translated into clinics, we also show that the
4
ACS Paragon Plus Environment

Page 5 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
optimized molecule is capable of annihilating preformed methicillinꢀresistant Staphylococcus
aureus (MRSA) biofilms in murine model. The above facts therefore indicated that the study
might hold promise in designing and developing safe and potent biofilm disruptors.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS AND DISCUSSION
Design and Synthesis of Small Molecules. To establish the above facts, we synthesized two
series of cationic small molecules (1aꢀ3a and 1bꢀ3b) that differ only in spatial positioning of
hydrophobicity (1a (n = 2; R = −C10H21), 1b (n = 4; R = −C10H21), 2a (n = 6; R = −C8H17), 2b
(n = 8; R = −C8H17), 3a (n = 10; R = −C6H13) and 3b (n = 12; R = −C6H21)) (Figure 1). In
each series the number of methylene groups was gradually increased between the positive
centers of the molecules with the gradual decrease in the number of methylene groups in
pendent alkyl chains (Figure 1a to 1b to 1c). Two series of molecules with a total of 22 and
24 carbon atoms in their pendent and confined alkyl chains were synthesized (the number
includes only the carbon atoms present in the alkyl chains except the common positive
charges and amide bonds) (Figure 1). Molecules 1a and 1b possess the maximum pendent
hydrophobicity (Figure 1a), 2a and 2b possess middling pendent hydrophobicity with
moderately confined hydrophobic chain (Figure 1b) whereas 3a and 3b possess the least
pendent hydrophobicity with mostly confined hydrophobic chain (Figure 1c). We deliberately
avoided using very low pendent alkyl chain, as the molecules with such chains did not yield
reasonable antibacterial activity. All the small molecules were found to be soluble in water.
However, solubility of the molecules with more confined hydrophobicity (3a and 3b) was
less compared to the molecules with pendent or moderately confined hydrophobicity (1a, 1b,
2a and -2b).
The molecules were synthesized in a simple three step method (Scheme 1).32 First,
N,N,N′,N′ꢀtetramethylꢀα,ωꢀdiaminoalkanes were synthesized from α,ωꢀdibromoalkanes by
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 46
1
2
3
4
5
6
7
8
9
reacting the bromoalkanes with N,Nꢀdimethylamine. The length of the hydrocarbon chain in
α,ωꢀdibromoalkanes was varied accordingly to obtain molecules with more confined or more
pendent hydrophobicity (Scheme 1). Second, Nꢀalkylꢀ1ꢀbromoethanamide with different
prefixed chain lengths was synthesized by reacting alkylamines with bromoacetyl bromide.
Finally, N,N,N′,N′ꢀtetramethylꢀα,ωꢀdiaminoalkanes were reacted with Nꢀalkylꢀ1ꢀ
bromoethanamide to obtain the cationic small molecules (Scheme 1). The molecules were
characterized by FTꢀIR, 1H NMR, 13C NMR and high resolution mass spectrometry (HRMS).
These molecules bear structural resemblance with antimicrobial peptides (AMPs) that are
known to interact with the bacterial cell membrane in a nonꢀspecific manner which make
them less susceptible towards microbial resistance development. However, while the usage of
AMPs is limited due to their proteolytic susceptibility, high production cost and high toxicity,
easily synthesizable, nonꢀtoxic cationic small molecules with nonꢀpeptidic amide bonds and
spatially tuned hydrophobicity would be helpful. Recently other than expected electrostatic
and hydrophobic interactions, amphiphilic antimicrobials with amide groups have been
shown to interact with bacterial cell membrane via hydrogen bonding thereby leading to
enhanced activity.47 In addition to the spatial control of hydrophobicity, we also studied the
role of increasing hydrophobicity on biological activity profile of the small molecules. A total
of 12 more cationic small molecules were synthesized by varying confined alkyl chain length
while keeping the pendent chain length constant (1c-1f; 2c-2f and 3c-3f; Scheme 1). Thus a
total of 18 compounds were synthesized and studied (1c (n = 6; R = −C10H21), 1d (n = 8; R =
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
−C10H21), 1e (n = 10; R = −C10H21) and 1f (n = 12; R = −C10H21); 2c (n = 2; R = −C8H17), 2d
(n = 4; R = −C8H17), 2e (n = 10; R = −C8H17) and 2f (n = 12; R = −C8H17), and 3c (n = 2; R =
−C6H13), 3d (n = 4; R = −C6H13), 3e (n = 6; R = −C6H13), 3f (n = 8; R = −C6H13). The details
of characterization and spectral data were provided in the experimental section and in the
Supporting Information (Figure S1ꢀS18, Supporting Information). The purity of the
6
ACS Paragon Plus Environment

Page 7 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
molecules was tested by HPLC and was found to be more than 95% (Figure S19, Supporting
Information).
7
8
9
Antibacterial and Hemolytic Activities. Each compound was first tested for its antibacterial
activity against Gramꢀpositive S. aureus and Gramꢀnegative E. coli. The activity was
represented in terms of minimum inhibitory concentration (MIC) and defined as the
minimum concentration required to inhibit bacterial growth.48 Notably MIC of the molecules
within a series (1a-3a or 1b-3b) were found to be similar thus indicating that the spatial
positioning of hydrophobicity did not alter their antibacterial efficacy (Figure 2). For
example, MIC values of 1b, 2b and 3b were found to be 1ꢀ2 ꢀg/mL against S. aureus and 2
ꢀg/mL each against E. coli respectively (Figure 2a). However, when tested against human
erythrocytes, hemolytic activity (represented as HC50, concentration at which 50% hemolysis
occurs) of the molecules within a series was found to vary remarkably. For example, HC50
values of 1b, 2b and 3b were found to be 60 ꢀg/mL, 212 ꢀg/mL and 805 ꢀg/mL respectively.
The above results thus indicated that the molecule with confined hydrophobicity (3b) was
more selective towards bacteria over human erythrocytes than the molecules with pendent
hydrophobicity (1b and 2b). To compare the potency of the molecules, selectivity (S =
HC50/MIC) which is one of the measures of therapeutic efficacy was calculated. Selectivity of
the molecules 1b, 2b and 3b were 30, 182 and 805 against S. aureus and 30, 106 and 402
against E. coli respectively (Figure 2b). Thus the above results indicated that the 3b with
more confined hydrophobicity are most selective. Similar trends of antibacterial and
hemolytic activities were observed for the other series (series 1). MIC values of 1a, 2a and 3a
were found to be 2ꢀ4 ꢀg/mL against S. aureus and 4ꢀ6 ꢀg/mL against E. coli respectively
whereas HC50 values were found to be 65 ꢀg/mL, 300 ꢀg/mL and 1242 ꢀg/mL respectively
(Figure 2a). Consecutively, selectivity of 1a, 2a and 3a were 32, 150 and 310 towards S.
aureus and 16, 50 and 207 towards E. coli respectively (Figure 2b). The above results thus
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 46
1
2
3
4
5
6
7
8
9
demonstrated the versatility of the concept. Notably, when tested against methicillinꢀresistant
S. aureus (MRSA), similar results were observed against MRSA for both the series (Table 1).
The optimised molecule 3b showed MIC value of 2 ꢀg/mL against MRSA thereby making it
a promising antibacterial agent.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Interestingly, when the hydrophobic character hence the overall amphiphilic balance
of the molecules was varied mainly by varying the confined alkyl chain while keeping the
pendent alkyl chain constant (1a-1f or 2a-2f or 3a-3f); both antibacterial and hemolytic
activities were found to vary differently depending on the length of both pendent and
confined alkyl chains. For example, when the length of confined chain was increased for the
molecules with −C10H21 pendent chain, antibacterial activity and hemolytic toxicity of the
molecules were found to vary nonꢀsignificantly except molecule 1f (Table 1). For example,
MIC values of molecules 1a-1e were 2ꢀ4 ꢀg/mL against S. aureus and 2ꢀ8 ꢀg/mL against E.
coli respectively. However, HC50 values of molecules were found to be 50ꢀ65 ꢀg/mL against
hRBC (Table 1). The low activity of 6a (MIC values were 31 ꢀg/mL against S. aureus and
500 ꢀg/mL against E. coli respectively) could be due to its very high hydrophobic character
which might lead to aggregation of the molecules in tested media. On the other hand, when
length of the confined chain was increased for the molecules with −C8H17 pendent chain,
antibacterial activity was found to vary slightly while toxicity towards human erythrocytes
varied greatly (Table 1). For example, MIC values of molecules 2a-2f were 1ꢀ2 ꢀg/mL
against S. aureus and 1ꢀ8 ꢀg/mL against E. coli respectively. Whereas HC50 values of these
molecules were found to be 50ꢀ350 ꢀg/mL against hRBC (Table 1). Notably, when the length
of confined chain was increased for the molecules with −C6H13 pendent chain, antibacterial
activity was found to vary significantly while toxicity towards human erythrocytes remained
almost unchanged (Table 1). For example, MIC values of molecules 3a-3f were 1ꢀ125 ꢀg/mL
8
ACS Paragon Plus Environment

Page 9 of 46
Journal of Medicinal Chemistry
1
2
3
4
against S. aureus and 2ꢀ250 ꢀg/mL against E. coli respectively. Whereas HC50 values of the
5
6
7
8
molecules were found to be close to or greater than 1000 ꢀg/mL against hRBC (Table 1). The
results are therefore in good agreement with the earlier reports.29 In summary, above facts
indicated that confining hydrophobicity in molecules with optimized amphiphilic balance is
important to obtain active yet nonꢀtoxic compounds.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To strengthen the nonꢀcytotoxic behaviour of the molecules with confined
hydrophobicity, human embryonic kidney (HEK 293) cells were treated with 1b, 2b and 3b,
all at 32 ꢀg/mL and were imaged by fluorescence microscopy using membrane permeable
green fluorescent dye calceinꢀAM and membrane impermeable red fluorescent dye propidium
iodide (PI).20 While the untreated cells showed complete green fluorescence (Figure 3a), cells
treated with molecule 1b showed complete red fluorescence at the tested concentration
indicative of cell death (Figure 3b). Cells treated with molecule 2b displayed partially green
fluorescence thereby indicating the presence of some live cells (Figure 3c). Interestingly,
cells treated with molecules 3b showed complete green fluorescence at the same
concentration thus indicating the survival of kidney cells (Figure 3d). It should be mentioned
that the molecules showed similar activity against HEK cells when treated at a lower
concentration (at 16 ꢀg/mL) and at a higher concentration (at 64 ꢀg/mL). When the cells
were treated at 16 ꢀg/mL, 3b treated cells showed complete green fluorescence with higher
cell density in contrast to the cells treated with 1b and 2b which also showed green
fluorescence but with lesser cell density (Figure S20, Supporting Information). When the
cells treated at 64 ꢀg/mL, 3b treated cells still showed mostly green fluorescence in sharp
contrast to the cells treated with 1b and 2b at the same concentration which showed
completely red fluorescence (Figure S21, Supporting Information). The above facts thereby
indicated the lower cytotoxic nature of 3b compared to molecules with less confined
hydrophobicity (1b and 2b).
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 46
1
2
3
4
5
6
7
8
9
Effect on Membrane Fluidity and Membrane Permeabilization. We speculated that the
molecules with less confined hydrophobicity would interact more strongly with both anionic
bacterial cell membrane as well as zwitterionic mammalian cell membrane because of more
flexible pendent alkyl chain.49 Hence we studied the interaction of these molecules with both
bacterial and mammalian cell membranes using model lipid bilayers. Liposomes were made
using anionic DPPG and zwitterionic DPPE (DPPG:DPPE = 88:12) mimicking the bacterial
cell membrane, and zwitterionic DPPC mimicking the red blood cell membrane with a
hydrophobic dye Laurdan (6ꢀDodecanoylꢀ2ꢀdimethylaminoꢀnaphthalene).47 In phospholipid
liposomes, the dye is known to get located closer to the hydrated aqueous surface of
liposomes and sense freely rotating water molecules. Upon interaction with the cationic
lipophilic molecules, disorder of lipid molecules in the membrane allows penetration of more
water molecules into the bilayer leading to more hydration of the membrane surface. The
extent of hydrataion is commonly quantified by determining the general polarization (GP =
(I440−I490)/(I440+I490) where I is the fluorescence intensity at 440 nm and 490 nm for an
excitation at 350 nm. In general, lower the GP, higher is the membrane disorder.50,51
Interestingly, GP for both 1b and 3b treated liposomes was found to be lower compared to
the untreated DPPG:DPPE liposome at all three concentrations tested (25 ꢀg/mL, 50 ꢀg/mL
and 100 ꢀg/mL) (Figure 4a). These results suggested that both 1b and 3b were effective in
disordering anionic DPPG:DPPE bilayer to the similar extent. However, against DPPC
liposomes, 1b showed large reduction in GP than 3b when compared to the untreated bilayer
(Figure 4b). These results thus indicated that the molecule with less pendent hydrophobicity
interacted to a much lesser extent with the zwitterionic mammalian cell membrane.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To confirm that the cationic small molecules act by disrupting the bacterial cell
membrane integrity, molecular mechanism of action was studied using spectroscopic
methods against both Gramꢀpositive and Gramꢀnegative bacteria. To establish whether the
10
ACS Paragon Plus Environment

Page 11 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
molecules act by depolarizing the cell membrane of bacteria, 3, 3'ꢀ
dipropylthiadicarbocyanine iodide (DiSC35), a membraneꢀpotential sensitive dye, was used.52
In general, the dye is taken up by the bacteria due to the potential gradient and accumulation
in the membrane leads to a decrease in its fluorescence intensity because of selfꢀquenching.
When the membraneꢀpotential is lost, the dye would be displaced into the solution thus would
lead to an enhancement in fluorescence intensity. Notably, upon treating bacteria with the
molecules, fluorescence intensity of DiSC35 was found to increase for both S. aureus and E.
coli (Figure 5a and 5b). The above fact therefore indicated that the molecules dissipated
membraneꢀpotential of both Gramꢀpositive and Gramꢀnegative bacteria. The ability of the
small molecules to permeabilize the cell membrane of bacteria was further studied using a
cell membrane impermeable dye propidium iodide (PI).53 Once the cellꢀmembrane of bacteria
is compromised, PI is known to enter inside bacteria and fluoresce upon intercalating to the
cellular DNA. Notably, when bacteria were treated with the molecules, fluorescence intensity
of PI was found to increase against both S. aureus and E. coli (Figure 5c and 5d). Thus the
molecules were also shown to be efficient in permeabilizing the cell membranes of bacteria.
In summary, the above studies indicated that the cationic small molecules indeed interacted
with the negatively charged cell membrane of bacteria and disrupted the membrane integrity.
Biofilm Disrupting Ability. Next we tested the ability of the optimized molecule to eradicate
established bacterial biofilms. S. aureus and E. coli biofilms were treated with 3b (at 32
ꢀg/mL) and analyzed for cell counting as well as observed by confocal laser scanning
microscopy (CLSM).54 Molecule 3b was found to reduce 9.2 log bacterial burden against S.
aureus biofilm and 7.1 log against E. coli biofilm respectively thus showing the ability to
destroy biofilmꢀencased bacteria. The biofilm disrupting property of the molecule was further
established by CLSM via staining with green fluorescent dye SYTO 9. In the untreated
samples, thick and matured biofilms of thickness ∼14ꢀ18 ꢁm were seen (Figure 6a and 6c)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 46
1
2
3
4
5
6
7
8
9
while treated samples showed scattered mono layered bacteria of only 2ꢀ3 ꢁm thickness
against both S. aureus and E. coli respectively (Figure 6b and 6d). These results further
demonstrated the ability of the molecules to eradicate establish bacterial biofilms at a
concentration at which it did not cause any cytotoxic effect. We also determined the
minimum biofilm eradication concentration (MBEC) for the cationic molecules. MBEC
values of 1b and 3b were found to be 31.2ꢀ62.5 ꢀg/mL against S. aureus and 62.5ꢀ125 ꢀg/mL
against E. coli respectively (Table S1). The above results thus indicated that these molecules
are capable of annihilating established biofilms completely. To evaluate the potential of these
small molecules as antibiofilm agents, MBEC values were also compared with some common
antiseptics (BACꢀ14 and DDACꢀ10) and cationic amphiphiles (DTAB and CTAB). Notably,
the molecules showed similar or even better MBEC values compared to those of commercial
quaternary compounds (Table S1).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In-vivo Toxicity and Antibiofilm Activity. Inꢀvivo toxicity of the cationic small molecules
still remains a major concern for the molecules to be used in healthcare applications.55 Thus
we briefly evaluated the toxic effect of the optimized molecule (3b) by determining the lethal
dose (LD50) upon topical application in mice. LD50 values for both 1b and 3b were found to
be more than 200 mg/kg in an acute dermal toxicity study (OECD 425). However, no visible
inflammation or abnormal skin reaction was observed for 3b at 200 mg/kg. On the other
hand, 1b showed high onꢀset toxicity as darkening of the skin and relatively much lesser fur
appearance were observed after 14 days of postꢀtreatment. Another important limitation of
the antimicrobials is their relatively poor potency under inꢀvivo conditions. In general, it has
been observed that small molecules lack the ability to eliminate preformed bacterial biofilms
inꢀvivo, which has largely limited their preꢀclinical entry.55 Herein we studied inꢀvivo efficacy
of the most potent molecule to eradicate preformed biofilm of MRSAꢀone of the leading
biofilm forming and the most common bacteria that causes many nosocomial infections.
12
ACS Paragon Plus Environment

Page 13 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Importantly, the optimised molecule showed remarkable biofilm eradicating ability under inꢀ
vivo conditions. In a murine model of superficial skin infection and biofilm formation, a
wound was first induced on the back of mice (dorsal midline) and then infected with
MRSA.56 The biofilms formed after 24 h of infection were then treated by 3b topically (40
mg/kg once a day) (Figure 7a) for four days. After 18 h of the application of last dosage, mice
were sacrificed and skin tissues were collected. Notably, 3b treated skin tissue samples
showed 5.3 log (> 99.999%) reduction of MRSA compared to the untreated tissue samples (p
value <0.0001). In contrast, fusidic acid and silver nanoparticle containing commercial
ointment (ReliHeal) which are used in clinics to treat skin infections, were found to reduce
MRSA count by 2.2 and 2.3 log reduction at 40 mg/kg and 200 mg/kg respectively (Figure
7b) (p values were 0.0183 and 0.0025 respectively). The fact that the molecule 3b was much
more effective in comparison to the approved drug fusidic acid emphasizes on the clinical
potential of the molecule. The ability of the small molecule in eradicating matured biofilm
under inꢀvivo conditions was further established by imaging skin tissue samples with
scanning electron microscopy. Tissue samples after 24 h of infection showed the presence of
a thick dense MRSA layer thereby indicated the formation and persistence of biofilm (Figure
7c). The treated skin tissue sample, on the other hand, showed negligible number of bacteria
on the tissue surface (Figure 7d). These result thus indicated the ability of the optimised
molecule to eradicate bacterial biofilms under inꢀvivo conditions.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
In summary, we demonstrated a concept of developing nonꢀtoxic small molecular biofilm
disruptors by spatial positioning of the hydrophobicity. Molecules with more pendent
hydrophobicity showed higher membrane disrupting ability, as indicated by their high
antibacterial as well as cytotoxic activities. Molecules, on the other hand, with more confined
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 46
1
2
3
4
5
6
7
8
9
hydrophobicity showed higher antibacterial but significantly lower cytotoxic activities. Using
model lipid bilayers and spectroscopic methods, the cationic molecules were shown to
depolarize and permeabilize the bacterial cell membrane thereby leading to cell inactivation.
Importantly, the optimized molecule was found to be nonꢀtoxic towards mammalian cells at a
concentration at which it eradicated established bacterial biofilms. Moreover, the molecule
was also found to eradicate preformed bacterial biofilm under more complex inꢀvivo
conditions without causing substantial skin toxicity. The results furnished herein thus
emphasize the potential of this class of molecules to be useful in guiding and developing
future antibacterial and antibiofilm small molecules with improved biocompatibility.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
1. Materials and Instrumentation. 1ꢀAminohexane, 1ꢀaminooctane, 1ꢀaminodecane,
bromoacetyl bromide, N,N,N′,N′ꢀtetramethylꢀ1,2ꢀdiaminoethane, N,N,N′,N′ꢀtetramethylꢀ1,4ꢀ
diaminobutane, 1,6ꢀdibromohexane, 1,8ꢀdibromooctane, 1,10ꢀdibromodecane and 1,12ꢀ
dibromododecane were purchased from SigmaꢀAldrich and used as received. Anhydrous
potassium carbonate (K2CO3), anhydrous sodium sulphate (Na2SO4), phosphorous pentaoxide
(P2O5), dichloromethane, chloroform, acetonitrile and anhydrous diethylether were purchased
from SD Fine, India and were of analytical grade. Dichloromethane, chloroform and
acetonitrile were dried over P2O5 and stored over activated molecular sieves (4 Å). Nuclear
1
magnetic resonance (NMR) spectra were recorded using Bruker AMXꢀ400 (400 MHz for H
NMR and 100 MHz for 13C NMR) spectrometer in suitable deuterated solvents. High
resolution mass spectra were recorded on a 6538ꢀUHD Accurate mass QꢀTOF LCꢀMS high
resolution massꢀspectrometer (HRMS). Fourier transform infrared (FTꢀIR) spectra were
recorded on an attenuated total reflectance (ATR) FTꢀIR spectrometer using diamond crystal
as ATR crystal. The purity (>95%) of the small molecules was determined by HPLC. For
14
ACS Paragon Plus Environment

Page 15 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
optical density and fluorescence measurements, Tecan Infinite Pro series M200 microplate
reader was used. Staphylococcus aureus (MTCC 737) and Escherichia coli (MTCC 443)
were obtained from MTCC (Chandigarh, India). Methicillinꢀresistant Staphylococcus aureus
(MRSA) (ATCC 33591), were purchased from ATCC (Rockville, MD, USA). Bacterial
growth media and agar were supplied by HIMEDIA, India. Studies on human subjects such
as human red blood cells (hRBC) and human embryo kidney cells (HEK 293) were
performed according to the guidelines approved by Institutional BioꢀSafety Committee
(IBSC) at Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR). Studies on
animals were performed in accordance with protocols approved by the Institutional Animal
Ethics Committee (IAEC) at the Jawaharlal Nehru Centre for Advanced Scientific Research
(JNCASR).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2. Synthesis of Small Molecules
2. 1. General Synthesis of N,N,N′,N′ꢀTetramethylꢀα,ωꢀdiaminoalkanes. N,N,N′,N′ꢀ
tetramethylꢀα,ωꢀdiaminoalkanes were synthesized with the slight modifications following an
earlier report.32 Briefly, NHMe2 gas was collected into dry CHCl3 (40 mL) in a screwꢀtop
pressure tube set at 0 °C until the volume of the resulting solution became 1.5 fold (~60 mL).
Dibromoalkane (10 g) was then added through syringe and stirred for 24 h at room
temperature. After cooling, the reaction mixture was transferred into a round bottomed flask
quantitatively. Excess gas was removed by heating the reaction mixture slowly. Finally, the
mixture was diluted with CHCl3 followed by washing with NaOH (2 M, 100 mL × 2)
solution. Organic layer was then collected and passed through anhydrous Na2SO4. Finally,
the organic solution was dried and light yellow gummy liquid was obtained as product with
quantitative yield.
1
N,N,N′,N′ꢀTetramethylꢀ1,6ꢀdiaminohexane: H NMR (400 MHz, CDCl3): δ 1.288ꢀ1.342 (m,
−CH2CH2CH2NMe2, 4H), 1.421ꢀ1.476 (m, −CH2CH2CH2NMe2, 4H), 2.204ꢀ2.255 (br. m,
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 46
1
2
3
4
5
−CH2CH2N(CH3)2 and −CH2CH2CH2NMe2, 16H); 13C NMR (100 MHz, CDCl3): δ 27.636,
27.879, 45.656, 60.030.
6
7
8
9
N,N,N′,N′ꢀTetramethylꢀ1,8ꢀdiaminooctane: 1H NMR (400 MHz, CDCl3): δ 1.223ꢀ1.314 (br.
m, −(CH2)2CH2CH2NMe2, 8H), 1.398ꢀ1.451 (m, −(CH2)2CH2CH2NMe2, 4H), 2.179ꢀ2.228
(br. m, −CH2CH2N(CH3)2 and −(CH2)2CH2CH2NMe2, 16H); 13C NMR (100 MHz, CDCl3): δ
27.615, 27.877, 29.685, 45.638, 60.092.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N,N,N′,N′ꢀTetramethylꢀ1,10ꢀdiaminodecane: 1H NMR (400 MHz, CDCl3): δ 1.257 (br. m,
−(CH2)3CH2CH2NMe2, 12H), 1.409ꢀ1.444 (m, −(CH2)3CH2CH2NMe2, 4H), 2.192ꢀ2.242 (br.
m, −CH2CH2N(CH3)2 and −(CH2)3CH2CH2NMe2, 16H); 13C NMR (100 MHz, CDCl3): δ
27.671, 27.908, 29.703, 29.753, 45.643, 60.119.
N,N,N′,N′ꢀTetramethylꢀ1,12ꢀdiaminododecane: 1H NMR (400 MHz, CDCl3): δ 1.246ꢀ1.262
(br. m, −(CH2)4CH2CH2NMe2, 16H), 1.410ꢀ1.445 (m, −(CH2)4CH2CH2NMe2, 4H), 2.190ꢀ
2.231 (br. m, −CH2CH2N(CH3)2 and −(CH2)4CH2CH2NMe2 and, 16H); 13C NMR (100 MHz,
CDCl3): δ 27.682, 27.922, 29.753, 29.775, 45.652, 60.130.
2.2. Synthesis of NꢀAlkylꢀ1ꢀbromoethanamide. 1ꢀAminoalkanes (60 mmol) was dissolved in
CH2Cl2 (200 mL). K2CO3 (90 mmol) was dissolved in water (150 mL) and the aqueous
solution was then added to the organic solution. The two phase solution was cooled to 4ꢀ5 °C.
Bromoacetyl bromide (90 mmol) in CH2Cl2 (50 mL) was added drop wise to the solution
while maintaining temperature at 4ꢀ5 °C for about 30 min. The reaction mixture was then set
at room temperature and stirred for about 4 h. After the reaction organic layer was collected
and the aqueous solution was washed with CH2Cl2 (3 × 50 mL). Organic solution was then
washed with water (3 × 100 mL), passed over the anhydrous Na2SO4 and concentrated to
give product with quantitative yields.
NꢀHexylꢀ1ꢀbromoethanamide: FTꢀIR: 3255 cmꢀ1 (amide NH str.), 2935 cmꢀ1 (CH2 assym.
str.), 2852 (CH2 sym. str.), 1678 cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.),
16
ACS Paragon Plus Environment

Page 17 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
1468 cmꢀ1 (CH2 scissor); 1H NMR: (400 MHz, CDCl3): δ 0.880 (t, terminal –CH3, 3H),
1.325 (m, −CH2(CH2)3CH3, 6H), 1.539 (m, −CH2(CH2)3CH3, 2H), 3.277 (m, −CONHCH2−,
6
7
8
9
2H), 3.884 (s, −COCH2Br, 2H), 6.468 (br. s, amide –NHCO, 1H); 13C NMR (100 MHz,
CDCl3): δ 14.111, 22.643, 26.601, 29.357, 29.534, 31.531, 40.411, 165.335.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NꢀOctylꢀ1ꢀbromoethanamide: FTꢀIR: 3258 cmꢀ1 (amide NH str.), 2943 cmꢀ1 (CH2 assym.
str.), 2857 (CH2 sym. str.), 1671 cmꢀ1 (amide I, C=O str.), 1561 cmꢀ1 (amide II, NH ben.),
1460 cmꢀ1 (CH2 scissor); 1H NMR: (400 MHz, CDCl3): δ 0.882 (t, terminal –CH3, 3H),
1.290 (m, −CH2(CH2)5CH3, 10H), 1.521 (m, −CH2(CH2)5CH3, 2H), 3.275 (m, −CONHCH2−,
2H), 3.884 (s, −COCH2Br, 2H), 6.462 (br. s, amide –NHCO, 1H); 13C NMR (100 MHz,
CDCl3): δ 14.214, 22.765, 26.947, 29.293, 29.325, 29.401, 29.548, 31.901, 40.425, 165.326.
NꢀDecylꢀ1ꢀbromoethanamide: FTꢀIR: 3254 cmꢀ1 (amide NH str.), 2928 cmꢀ1 (CH2 assym.
str.), 2855 (CH2 sym. str.), 1678 cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.),
1472 cmꢀ1 (CH2 scissor). 1H NMR: (400 MHz, CDCl3): δ 0.880 (t, terminal –CH3, 3H),
1.263ꢀ1.304 (m, −CH2(CH2)7CH3, 14H), 1.539 (m, −CH2(CH2)7CH3, 2H), 3.290 (m,
−CONHCH2−, 2H), 3.884 (s, −COCH2Br, 2H), 6.463 (br. s, amide –NHCO, 1H); 13C NMR
(100 MHz, CDCl3): δ 14.247, 22.815, 26.953, 29.368, 29.405, 29.425, 29.559, 29.639,
29.652, 32.021, 40.443, 165.316.
2.3. General Synthesis of Cationic Small Molecules. N,N,N′,N′ꢀTetramethylꢀα,ωꢀ
diaminoalkanes (5 mmol) was reacted with Nꢀalkylꢀ1ꢀbromoethanamide (15 mmol) in dry
°
CHCl3 (40 mL) in a screwꢀtop pressure tube at 85 C for about 24 h. After the reaction, the
solvent was evaporated and the product was precipitated using excess diethylether. Finally
the product was either filtered or washed or excess solvent was decanted and washed multiple
times with diethylether. The precipitate was then dried overnight in vacuum oven at 55 ˚C to
give 1a-1f, 2a-2f and 3a-3f quantitatively.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 46
1
2
3
4
5
1a: FTꢀIR: 3258 cmꢀ1 (NH str.), 2920 cmꢀ1 (CH2 assym. str.), 2848 cmꢀ1 (CH2 sym. str.),
1
1685 cmꢀ1 (amide I, C=O str.), 1550 cmꢀ1 (amide II, NH ben.), 1472 cmꢀ1 (CH2 scissor); H
6
7
8
9
NMR (400 MHz, CDCl3): δ 0.84 (t, terminal –CH3, 6H), 1.26 (m, −CH2(CH2)7CH3, 28H),
1.52 (m, −CH2(CH2)7CH3, 4H), 3.21 (m, −CONHCH2−, 4H), 3.61 (s, −(CH3)2N+−, 12H),
4.68 (br. m, −CH2N+Me2−, 4H), 4.81 (s, −NHCOCH2−, 4H), 8.34 (br. s, amide –NH, 2H); 13C
NMR (100 MHz, CDCl3): δ 14.1, 22.7, 27.1, 29.1, 29.3, 29.4, 29.6, 31.9, 40.1, 57.9, 63.0,
162.1; HRMS: calculated m/z 591.4207, 593.4188 [M–Br−]+, 256.2509 [M–2Br−]2+; observed
m/z 591.4212, 593.4198 [M–Br−]+, 256.2529 [M–2Br−]2+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1b: FTꢀIR: 3261 cmꢀ1 (NH str.), 2928 cmꢀ1 (CH2 assym. str.), 2851 cmꢀ1 (CH2 sym. str.),
1
1682 cmꢀ1 (amide I, C=O str.), 1554 cmꢀ1 (amide II, NH ben.), 1476 cmꢀ1 (CH2 scissor); H
NMR (400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.29 (br. m, −CH2(CH2)7CH3, 28H),
1.55 (m, −CH2(CH2)7CH3, 4H), 2.21 (br. m, −CH2CH2N+Me2−, 4H), 3.27 (m, −CONHCH2−,
4H), 3.45 (s, −(CH3)2N+−, 12H), 3.89 (br. m, −CH2CH2N+Me2−, 4H), 4.44 (s, −NHCOCH2−,
4H), 8.56 (br. s, amide –NH, 2H); 13C NMR (100 MHz, CDCl3): δ 14.2, 20.4, 22.7, 27.2,
29.2, 29.4, 29.4, 29.7, 31.9, 40.1, 63.1, 65.5, 162.6; HRMS: calculated m/z 619.4520,
621.4520 [M–Br−]+, 270.2665 [M–2Br−]2+; observed m/z 619.4525, 621.4521 [M–Br−]+,
270.2683 [M–2Br−]2+.
1c: FTꢀIR: 3255 cmꢀ1 (NH str.), 2925 cmꢀ1 (CH2 assym. str.), 2852 cmꢀ1 (CH2 sym. str.), 1680
1
cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1470 cmꢀ1 (CH2 scissor); H NMR
(400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.24ꢀ1.29 (m, −CH2(CH2)7CH3, 28H),
1.53ꢀ1.58 (m, −CH2(CH2)7CH3 and −(CH2)2CH2CH2N+Me2, 8H), 1.94 (br. m,
−CH2CH2N+Me2, 4H), 3.25 (m, −CONHCH2−, 4H), 3.44 (s, −(CH3)2N+−, 12H), 3.75 (br. m,
−CH2N+Me2−, 4H), 4.57 (s, −NHCOCH2−, 4H), 8.61 (br. s, amide –NH, 2H); 13C NMR (100
MHz, CDCl3): δ 14.2, 21.7, 22.7, 24.4, 27.2, 29.1, 29.4, 29.4, 29.6, 32.1, 40.1, 52.4, 62.9,
18
ACS Paragon Plus Environment

Page 19 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
66.9, 162.9; HRMS: calculated m/z 647.4833, 649.4815, [M–Br−]+, 284.2822 [M–2Br−]2+;
observed m/z 647.4830, 649.4813, [M–Br−]+, 284.2827 [M–2Br−]2+.
6
7
8
1d: FTꢀIR: 3257 cmꢀ1 (NH str.), 2925 cmꢀ1 (CH2 assym. str.), 2849 cmꢀ1 (CH2 sym. str.),
9
1
1682 cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1482 cmꢀ1 (CH2 scissor); H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NMR (400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.25ꢀ1.48 (m, −CH2(CH2)7CH3,
28H), 1.53ꢀ1.61 (m, −(CH2)4CH2CH2N+Me2, 8H), 1.96 (br. m, −CH2CH2N+Me2, 4H) 3.23
(m, −CONHCH2−, 4H), 3.42 (s, −(CH3)2N+−, 12H), 3.72 (br. m, −(Me)2 NCH2−, 4H), 4.55
+
(s, −NHCOCH2−, 4H), 8.74 (br. s, amide –NH, 2H); 13C NMR (100 MHz, CDCl3): δ 14.1,
21.7, 22.8, 24.5, 27.2, 29.2, 29.4, 29.5, 29.6, 32.1, 40.2, 52.5, 62.9, 66.9, 162.7; HRMS:
calculated m/z 675.5148, 677.5130 [M–Br−]+, 298.2978 [M–2Br−]2+; observed m/z 675.5146,
677.5133 [M–Br−]+, 298.3002 [M–2Br−]2+.
1e: FTꢀIR: 3260 cmꢀ1 (NH str.), 2920 cmꢀ1 (CH2 assym. str.), 2849 cmꢀ1 (CH2 sym. str.), 1682
1
cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1477 cmꢀ1 (CH2 scissor); H NMR
(400 MHz, CDCl3): δ 0.88 (t, terminal –CH3, 6H), 1.27ꢀ1.35 (m, −CH2(CH2)7CH3 and
−CH2(CH2)4CH2CH2N+Me2, 32H), 1.43 (m, −CH2(CH2)4CH2CH2N+Me2, 8H), 1.58 (m,
−CH2(CH2)7CH3, 4H), 1.87 (br. m, −CH2CH2N+Me2, 4H), 3.26 (m, −CONHCH2−, 4H), 3.39
(s, −(CH3)2N+−, 12H), 3.70 (br. m, −CH2N+Me2−, 4H), 4.57 (s, −NHCOCH2−, 4H), 8.84 (br.
13
s, amide –NH, 2H); C NMR (100 MHz, CDCl3): δ 14.2, 22.5, 22.8, 25.7, 27.2, 28.1, 28.2,
29.1, 29.3, 29.7, 32.1, 40.1, 52.1, 63.4, 66.9, 162.8; HRMS: calculated m/z 703.5459,
705.5450 [M–Br−]+, 312.3135 [M–2Br−]2+; observed m/z 703.5467, 705.5454 [M–Br−]+,
312.3177 [M–2Br−]2+.
1f: FTꢀIR: 3267 cmꢀ1 (NH str.), 2931 cmꢀ1 (CH2 assym. str.), 2854 cmꢀ1 (CH2 sym. str.), 1688
1
cmꢀ1 (amide I, C=O str.), 1550 cmꢀ1 (amide II, NH ben.), 1480 cmꢀ1 (CH2 scissor); H NMR
(400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.25ꢀ1.30 (m, −CH2(CH2)5CH3 and
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 46
1
2
3
4
5
−(CH2)4(CH2)4CH2CH2N+Me2, 36H), 1.39 (m, −(CH2)4(CH2)4CH2CH2N+Me2, 8H), 1.56ꢀ1.61
(m, −CH2(CH2)5CH3, 4H), 1.85 (br. m, −CH2CH2N+Me2, 4H), 3.25 (m, −CONHCH2−, 4H),
6
7
8
+
3.37 (s, −(CH3)2N+−, 12H), 3.64 (br. m, −(Me)2 NCH2−, 4H), 4.58 (s, −NHCOCH2−, 4H),
9
8.88 (br. s, amide –NH, 2H); 13C NMR (100 MHz, CDCl3): δ 14.2, 22.4, 22.8, 25.7, 27.2,
28.1, 28.3, 29.2, 29.4, 29.7, 32.2, 41.1, 52.6, 63.5, 66.9, 162.7; HRMS: calculated m/z
731.5772, 733.5759 [M–Br−]+, 326.3291 [M–2Br−]2+; observed m/z 731.5776, 733.5761 [M–
Br−]+, 326.3290 [M–2Br−]2+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2a: FTꢀIR: 3260 cmꢀ1 (NH str.), 2922 cmꢀ1 (CH2 assym. str.), 2850 cmꢀ1 (CH2 sym. str.),
1
1680 cmꢀ1 (amide I, C=O str.), 1554 cmꢀ1 (amide II, NH ben.), 1480 cmꢀ1 (CH2 scissor); H
NMR (400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.26ꢀ1.29 (m, −CH2(CH2)5CH3,
20H), 1.56ꢀ1.62 (m, −CH2(CH2)5CH3 and −(CH2)2CH2CH2N+Me2, 8H), 2.12 (br. m,
−CH2CH2N+Me2, 4H) 3.21 (m, −CONHCH2−, 4H), 3.61 (s, −(CH3)2N+−, 12H), 4.68 (br. m,
+
−(Me)2 NCH2−, 4H), 4.81 (s, −NHCOCH2−, 4H), 8.69 (br. s, amide –NH, 2H); 13C NMR
(100 MHz, CDCl3): δ 14.1, 21.9, 22.7, 24.7, 27.1, 29.1, 29.2, 29.3, 31.8, 39.9, 62.9, 66.2,
162.9; HRMS: calculated m/z 591.4207, 593.4188 [M–Br−]+, 256.2509 [M–2Br−]2+; observed
m/z 591.4214, 593.4202 [M–Br−]+, 256.2522 [M–2Br−]2+.
2b: FTꢀIR: 3259 cmꢀ1 (NH str.), 2927 cmꢀ1 (CH2 assym. str.), 2854 cmꢀ1 (CH2 sym. str.),
1
1685 cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1479 cmꢀ1 (CH2 scissor); H
NMR (400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.25ꢀ1.28 (br. m, −CH2(CH2)5CH3,
20H), 1.44 (m, −(CH2)4CH2CH2N+Me2, 8H), 1.58 (m, −CH2(CH2)5CH3, 4H), 1.95 (br. m,
−CH2CH2N+Me2, 4H) 3.24 (m, −CONHCH2−, 4H), 3.40 (s, −(CH3)2N+−, 12H), 3.71 (br. m,
+
−(Me)2 NCH2−, 4H), 4.55 (s, −NHCOCH2−, 4H), 8.79 (br. s, amide –NH, 2H); 13C NMR
(100 MHz, CDCl3): δ 14.1, 22.3, 22.6, 25.5, 27.1, 27.7, 29.1, 29.2, 29.2, 31.8, 39.8, 52.2,
20
ACS Paragon Plus Environment

Page 21 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
63.1, 66.2, 162.9; HRMS: calculated m/z 619.4520, 621.4520 [M–Br−]+, 270.2665 [M–
2Br−]2+; observed m/z 619.4522, 621.4511 [M–Br−]+, 270.2679 [M–2Br−]2+.
6
7
8
2c: FTꢀIR: 3265 cmꢀ1 (NH str.), 2927 cmꢀ1 (CH2 assym. str.), 2860 cmꢀ1 (CH2 sym. str.), 1675
9
1
cmꢀ1 (amide I, C=O str.), 1550 cmꢀ1 (amide II, NH ben.), 1474 cmꢀ1 (CH2 scissor); H NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(400 MHz, CDCl3): δ 0.88 (t, terminal –CH3, 6H), 1.26ꢀ1.31 (br. m, −CH2(CH2)5CH3, 20H),
1.57 (m, −CH2(CH2)5CH3, 4H), 3.23 (m, −CONHCH2−, 4H), 3.62 (s, −(CH3)2N+−, 12H),
+
4.68 (br. m, −(Me)2 NCH2−, 4H), 4.81 (s, −NHCOCH2−, 4H), 8.37 (br. s, amide –NH, 2H);
13C NMR (100 MHz, CDCl3): δ 14.2, 22.7, 27.1, 29.1, 29.3, 29.4, 31.9, 40.2, 53.1, 58.1, 63.1,
162.2; HRMS: calculated m/z 535.3581, 537.3569 [M–Br−]+, 228.2196 [M–2Br−]2+; observed
m/z 535.3592, 537.3578 [M–Br−]+, 228.2203 [M–2Br−]2+.
2d: FTꢀIR: 3269 cmꢀ1 (NH str.), 2933 cmꢀ1 (CH2 assym. str.), 2855 cmꢀ1 (CH2 sym. str.),
1
1685 cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1480 cmꢀ1 (CH2 scissor); H
NMR (400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.26ꢀ1.34 (br. m, −CH2(CH2)5CH3,
20H), 1.44 (m, −(CH2)4CH2CH2N+Me2, 8H), 1.58 (m, −CH2(CH2)5CH3, 4H), 2.15 (br. m,
−CH2CH2N+Me2, 4H) 3.23 (m, −CONHCH2−, 4H), 3.46 (s, −(CH3)2N+−, 12H), 3.92 (br. m,
+
−(Me)2 NCH2−, 4H), 4.47 (s, −NHCOCH2−, 4H), 8.52 (br. s, amide –NH, 2H); 13C NMR
(100 MHz, CDCl3): δ 14.2, 20.4, 22.7, 27.2, 29.1, 29.3, 29.3, 31.9, 40.1, 52.3, 63.1, 65.7,
162.6; HRMS: calculated m/z 563.3894, 565.3878 [M–Br−]+, 242.2352 [M–2Br−]2+; observed
m/z 563.3895, 565.3881 [M–Br−]+, 242.2356 [M–2Br−]2+.
2e: FTꢀIR: 3260 cmꢀ1 (NH str.), 2935 cmꢀ1 (CH2 assym. str.), 2855 cmꢀ1 (CH2 sym. str.), 1685
1
cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1480 cmꢀ1 (CH2 scissor); H NMR
(400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.26ꢀ1.35 (br. m, −CH2(CH2)5CH3 and
−(CH2)4CH2CH2CH2N+Me2, 24H), 1.43 (m, −(CH2)4CH2CH2CH2N+Me2, 8H), 1.54 (m,
−CH2(CH2)5CH3, 4H), 1.87 (br. m, −CH2CH2N+Me2, 4H) 3.25 (m, −CONHCH2−, 4H), 3.39
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 46
1
2
3
4
+
(s, −(CH3)2N+−, 12H), 3.70 (br. m, −(Me)2 NCH2−, 4H), 4.57 (s, −NHCOCH2−, 4H), 8.84
(br. s, amide –NH, 2H); 13C NMR (100 MHz, CDCl3): δ 14.1, 22.7, 25.7, 27.1, 28.1, 28.2,
29.1, 29.3, 31.9, 40.1, 52.1, 63.4, 66.9, 162.8; HRMS: calculated m/z 647.4833, 649.4815,
[M–Br−]+, 284.2822 [M–2Br−]2+; observed m/z 647.4835, 649.4819, [M–Br−]+, 284.2877 [M–
2Br−]2+.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2f: FTꢀIR: 3263 cmꢀ1 (NH str.), 2935 cmꢀ1 (CH2 assym. str.), 2860 cmꢀ1 (CH2 sym. str.), 1682
1
cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1480 cmꢀ1 (CH2 scissor); H NMR
(400 MHz, CDCl3): δ 0.86 (t, terminal –CH3, 6H), 1.25ꢀ1.29 (br. m, −CH2(CH2)5CH3 and
−(CH2)8CH2CH2N+Me2, 36H), 1.57 (m, −CH2(CH2)3CH3, 4H), 1.82 (br. m, −CH2CH2N+Me2,
+
4H) 3.26 (m, −CONHCH2−, 4H), 3.38 (s, −(CH3)2N+−, 12H), 3.66 (br. m, −(Me)2 NCH2−,
4H), 4.57 (s, −NHCOCH2−, 4H), 8.81 (br. s, amide –NH, 2H); 13C NMR (100 MHz, CDCl3):
δ 14.1, 22.7, 22.9, 26.1, 27.2, 28.7, 28.8, 29.1, 29.3, 29.5, 29.6, 31.9, 39.9, 52.1, 63.4, 66.3,
162.8; HRMS: calculated m/z 675.5148, 677.5130 [M–Br−]+, 298.2978 [M–2Br−]2+; observed
m/z 675.5148, 677.5133 [M–Br−]+, 298.3002 [M–2Br−]2+.
3a: FTꢀIR: 3257 cmꢀ1 (NH str.), 2925 cmꢀ1 (CH2 assym. str.), 2850 cmꢀ1 (CH2 sym. str.),
1
1679 cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1480 cmꢀ1 (CH2 scissor); H
NMR (400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.28ꢀ1.43 (m, −CH2(CH2)3CH3, and
−(CH2)6CH2CH2N+Me2, 24H), 1.59 (m, −CH2(CH2)3CH3 4H), 1.87 (br. m, −CH2CH2N+Me2,
+
4H) 3.25 (m, −CONHCH2−, 4H), 3.39 (s, −(CH3)2N+−, 12H), 3.68 (br. m, −(Me)2 NCH2−,
4H), 4.56 (s, −NHCOCH2−, 4H), 8.82 (br. s, amide –NH, 2H); 13C NMR (100 MHz, CDCl3):
δ 14.1, 22.6, 22.6, 25.8, 26.7, 28.2, 28.4, 29.1, 31.4, 39.942, 52.1, 63.323, 66.4, 162.8;
HRMS: calculated m/z 591.4207, 593.4188 [M–Br−]+, 256.2509 [M–2Br−]2+; observed m/z
591.4206, 593.4192 [M–Br−]+, 256.2524 [M–2Br−]2+.
22
ACS Paragon Plus Environment

Page 23 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
3b: FTꢀIR: 3260 cmꢀ1 (NH str.), 2928 cmꢀ1 (CH2 assym. str.), 2855 cmꢀ1 (CH2 sym. str.),
1
1680 cmꢀ1 (amide I, C=O str.), 1555 cmꢀ1 (amide II, NH ben.), 1480 cmꢀ1 (CH2 scissor); H
6
7
8
9
NMR (400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.28ꢀ1.39 (br. m, −CH2(CH2)3CH3
and −(CH2)8CH2CH2N+Me2, 28H), 1.56 (m, −CH2(CH2)3CH3, 4H), 1.83 (br. m,
−CH2CH2N+Me2, 4H) 3.27 (m, −CONHCH2−, 4H), 3.37 (s, −(CH3)2N+−, 12H), 3.65 (br. m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
−(Me)2 NCH2−, 4H), 4.58 (s, −NHCOCH2−, 4H), 8.89 (br. s, amide –NH, 2H); 13C NMR
(100 MHz, CDCl3): δ 14.1, 22.6, 22.6, 22.9, 26.1, 26.5, 26.8, 28.6, 28.7, 28.7, 29.1, 29.3,
31.5, 40.1, 49.8, 52.1, 63.4, 162.8; HRMS: calculated m/z 619.4520, 621.4520 [M–Br−]+,
270.2665 [M–2Br−]2+; observed m/z 619.4527, 621.4516 [M–Br−]+, 270.2687 [M–2Br−]2+.
3c: FTꢀIR: 3263 cmꢀ1 (NH str.), 2925 cmꢀ1 (CH2 assym. str.), 2852 cmꢀ1 (CH2 sym. str.), 1688
1
cmꢀ1 (amide I, C=O str.), 1562 cmꢀ1 (amide II, NH ben.), 1481 cmꢀ1 (CH2 scissor); H NMR
(400 MHz, CDCl3): δ 0.88 (t, terminal –CH3, 6H), 1.26ꢀ1.35 (m, −CH2(CH2)3CH3, 12H), 1.57
(m, −CH2(CH2)3CH3 4H), 3.24 (m, −CONHCH2−, 4H), 3.62 (s, −(CH3)2N+−, 12H), 4.67 (br.
+
m, −(Me)2 NCH2−, 4H), 4.79 (s, −NHCOCH2−, 4H), 8.81 (br. s, amide –NH, 2H); 13C NMR
(100 MHz, CDCl3): δ 14.3, 22.6, 26.8, 29.1, 31.5, 40.2, 53.1, 63.2, 162.2; HRMS: calculated
m/z 479.2955, 481.2940 [M–Br−]+, 200.1883 [M–2Br−]2+; observed m/z 479.2954, 481.2938
[M–Br−]+, 200.1886 [M–2Br−]2+.
3d: FTꢀIR: 3260 cmꢀ1 (NH str.), 2928 cmꢀ1 (CH2 assym. str.), 2860 cmꢀ1 (CH2 sym. str.),
1
1681 cmꢀ1 (amide I, C=O str.), 1556 cmꢀ1 (amide II, NH ben.), 1484 cmꢀ1 (CH2 scissor); H
NMR (400 MHz, CDCl3): δ 0.89 (t, terminal –CH3, 6H), 1.26ꢀ1.29 (br. m, −CH2(CH2)3CH3
12H), 1.54 (m, −CH2(CH2)3CH3, 4H), 2.14 (br. m, −CH2CH2N+Me2, 4H) 3.25 (m,
+
−CONHCH2−, 4H), 3.47 (s, −(CH3)2N+−, 12H), 3.91 (br. m, −(Me)2 NCH2−, 4H), 4.46 (s,
−NHCOCH2−, 4H), 8.51 (br. s, amide –NH, 2H); 13C NMR (100 MHz, CDCl3): δ 14.1, 20.4,
22.6, 26.7, 29.1, 31.4, 40.1, 52.2, 63.1, 65.5, 162.6; HRMS: calculated m/z 507.3268,
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 46
1
2
3
4
5
509.3291 [M–Br−]+, 214.2039 [M–2Br−]2+; observed m/z 507.3250, 509.3285 [M–Br−]+,
214.2063 [M–2Br−]2+.
6
7
8
3e: FTꢀIR: 3261 cmꢀ1 (NH str.), 2925 cmꢀ1 (CH2 assym. str.), 2850 cmꢀ1 (CH2 sym. str.), 1680
9
1
cmꢀ1 (amide I, C=O str.), 1560 cmꢀ1 (amide II, NH ben.), 1482 cmꢀ1 (CH2 scissor); H NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.26ꢀ1.37 (m, −CH2(CH2)3CH3, 12H), 1.55
(m, −CH2(CH2)3CH3 and −(CH2)2CH2CH2N+Me2, 8H), 2.1 (br. m, −CH2CH2N+Me2, 4H)
3.25 (m, −CONHCH2−, 4H), 3.44 (s, −(CH3)2N+−, 12H), 3.74 (br. m, −(Me)2 NCH2−, 4H),
+
4.58 (s, −NHCOCH2−, 4H), 8.61 (br. s, amide –NH, 2H); 13C NMR (100 MHz, CDCl3): δ
14.1, 21.8, 22.6, 24.5, 26.7, 29.1, 31.4, 39.9, 52.3, 62.8, 66.8, 162.8; HRMS: calculated m/z
535.3581, 537.3569 [M–Br−]+, 228.2196 [M–2Br−]2+; observed m/z 535.3593, 537.3576 [M–
Br−]+, 228.2207 [M–2Br−]2+.
3f: FTꢀIR: 3260 cmꢀ1 (NH str.), 2930 cmꢀ1 (CH2 assym. str.), 2860 cmꢀ1 (CH2 sym. str.), 1685
1
cmꢀ1 (amide I, C=O str.), 1552 cmꢀ1 (amide II, NH ben.), 1480 cmꢀ1 (CH2 scissor); H NMR
(400 MHz, CDCl3): δ 0.87 (t, terminal –CH3, 6H), 1.28ꢀ1.35 (br. m, −CH2(CH2)3CH3 12H),
1.48 (m, −CH2(CH2)4CH2−, 8H), 1.59 (m, −CH2(CH2)3CH3, 4H), 1.95 (br. m,
−CH2CH2N+Me2, 4H) 3.26 (m, −CONHCH2−, 4H), 3.41 (s, −(CH3)2N+−, 12H), 3.70 (br. m,
+
−(Me)2 NCH2−, 4H), 4.55 (s, −NHCOCH2−, 4H), 8.76 (br. s, amide –NH, 2H); 13C NMR
(100 MHz, CDCl3): δ 15.9, 24.5, 24.6, 27.8, 28.3, 30.4, 30.5, 33.2, 42.1, 54.9, 65.3, 67.8,
162.8; HRMS: calculated m/z 563.3894, 565.3878 [M–Br−]+, 242.2352 [M–2Br−]2+;
observed m/z 563.3900, 565.3885 [M–Br−]+, 242.2359 [M–2Br−]2+.
3. In-vitro Antibacterial Assay
3.1. Minimum Inhibitory Concentration. The 6 h grown culture (∼109 CFU/mL of bacteria)
were diluted to give ∼105 CFU/mL in respective media. Solutions of the small molecules
were prepared in water and serial dilution was performed for all the compounds (2ꢀfold serial
24
ACS Paragon Plus Environment

Page 25 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
dilution). These dilutions (50 ꢁL) were then added to the wells of 96ꢀwell plate followed by
addition of 150 ꢁL of previously made bacterial suspension (∼105 CFU/mL). Two controls,
one with only water and bacterial suspension and the other with compound solution with only
media, were made. The plates were then incubated for 24 h at 37 °C. After incubation, optical
density (OD) of the bacterial suspension was recorded using Plate Reader at 600 nm
(TECAN, Infinite series, M200 pro). Each concentration was added in triplicate and all the
experiments were repeated at least twice. Finally, antibacterial efficacy, expressed as
minimum inhibitory concentration (MIC), was determined by considering the concentration
at which the OD values were found to be similar to that of only media.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3.2. Biofilm Disruption Assay. Glass cover slips were sterilised by dipping them in ethanol
and then drying in flame. The sterilised cover slips were placed in the wells of a 6ꢀwell plate.
Bacteria (6 h grown) were then diluted to ∼105 CFU/mL in nutrient medium supplemented
with 1% glucose and 1% NaCl for S. aureus and M9 medium supplemented with 0.5%
glycerol and 0.02% casamino acid for E. coli respectively and were added to the wells
containing cover slips (2 mL). The well plate was then incubated under stationary conditions
at 37 ˚C for about 24 h for S. aureus and 72 h for E. coli respectively. After incubation,
medium was removed and planktonic bacteria were carefully washed once with PBS (pH =
7.4). Cover slips containing biofilms were then placed into the wells of another 6ꢀwell plate
and 2 mL of test compound 3b dissolved in the above respective media (at 32 ꢀg/mL) was
added to the wells containing the cover slips with already established bacterial biofilm. The
plate was then allowed to incubate under stationary conditions at 37 ˚C for 24 h. Only 2 mL
of the respective media was used as negative control. After 24 h, medium was discarded and
cover slips were washed with PBS. Then trypsinꢀEDTA solution (100 µL) was added to the
wells containing treated and nonꢀtreated cover slips to dissolve the biofilm matrix. Bacterial
count was performed by serially diluting the suspension (10ꢀfold dilution) and then plating on
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 46
1
2
3
4
5
6
7
8
9
nutrient agar plates. Bacterial colonies grown on the plate after 24 h were counted and
viability of the cell within the matrix was expressed as log10 (CFU/mL). For imaging via
CLSM, cover slips were stained with green fluorescent dye SYTO 9 (5 µM, 10 ꢀL) and
imaged using a confocal laserꢀscanning microscopy (Zeiss 510 Meta Confocal Microscope).
3.3. Minimum Biofilm Eradication Concentration (MBEC) Assay. The minimum biofilm
eradication concentration for the cationic small molecules was determined following an
earlier report.18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3.4. Membrane Hydration Assay. Lipids (0.5 mM DPPG:DPPE (88:12) or 0.5 mM of DPPC)
and Laurdan dye (5 ꢁM) were taken in glass vials in chloroform. Thin films were made under
constant flow of dry argon gas. Films were then dried under vacuum. Finally, lipid films were
hydrated with PBS (pH = 7.4) overnight. Hydrated films of lipids were then processed for 10
freeze thaw cycles from 70 °C to 4 °C with alternate vortexing. The solutions were then
sonicated at 70 °C for 15 min to get unilamellar vesicles from multilamellar vesicles that
form during vortexing. Laurdan dye embedded liposome (2 mL) (lipid: dye = 100:1) was
taken in a fluorescence cuvette along with the compound solutions in PBS (pH = 7.4) (lipid:
compound = 7.4:1). Fluorescence emission was then measured at 440 nm and 490 nm for an
excitation wavelength at 350 nm. The measurements were performed using Water Peltier
system attached PerkinElmer LSꢀ55 Luminescence Spectrometer at 37 °C. Membrane
hydration and hence changes in polarity are studied by shifts in the emission spectrum of
Laurdan dye and are quantified by calculating the generalized polarization (GP).
GP was calculated by using the following equation.
GP = (I440 ꢀ I490)/(I440 + I490) where I440 and I490 represent fluorescent emission intensity of the
dye at 440 nm and 490 nm respectively for the excitation at 350 nm.
4. In-vitro Toxicity Assay
26
ACS Paragon Plus Environment

Page 27 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4.1. Hemolytic Assay. Studies on human subjects were performed according to the guidelines
approved by Institutional BioꢀSafety Committee (IBSC) at Jawaharlal Nehru Centre for
Advanced Scientific Research (JNCASR). Blood was donated by a healthy human donor and
red blood cells (RBCs) were isolated from the heparinised blood. The cells were then washed
twice and finally resuspended in PBS (5 vol%). Cell suspension (150 ꢁL) was then added to
solutions of serially diluted small molecules taken in wells of a 96ꢀwell plate (50 ꢁL). Two
controls were prepared, one without the compounds (only 50 ꢀL water) and the other with 0.1
vol% solution of Triton Xꢀ100 (TX, 50 ꢁL). The plate was then incubated at 37 °C for 1 h.
Next, cells were centrifuged at 3500 rpm for 5 minutes. Supernatant from the wells (∼100 ꢁL)
was then transferred to a new 96ꢀwell plate and absorbance of the supernatant was recorded
at 540 nm. Percentage of hemolysis was calculated as (A–Ao)/(Atotal–Ao)×100, where A is the
absorbance for the test samples, Ao is the absorbance for the wells contained only water and
RBC suspension , and Atotal the absorbance of fully lysed cells (wells with TX), all at 540 nm.
4.2. Cytotoxicity Assay (Fluorescence Microscopy). Human embryo kidney (HEK 293) cells
were seeded onto the wells of a tissue culture treated 96ꢀwell plate (∼104 cells/well). The
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
seeded cellswere then treated with the small molecules at 32 ꢀg/mL. 0.1% TritonꢀX was used
as positive control whereas untreated cells were used as negative controls. Both the treated
and untreated cells were then washed once with PBS and stained with calcein AM (2 ꢁM,
Fluka) and propidium iodide (PI, 4.5 ꢁM) (SigmaꢀAldrich) for 15 min at 37 °C under 5%
CO2ꢀ95% atmosphere (50 ꢀL of 1:1 calcein AM:PI). Finally, the cells were washed with PBS
to remove the excess dyes and images were captured with a 20X objective in Leica DM2500
fluorescence microscope using. A bandꢀpass filter for calcein AM at 500ꢀ550 nm and a longꢀ
pass filter for PI at 590ꢀ800 nm were used while imaging.
5. In-vivo Ttoxicity. Inꢀvivo toxicity was determined according to the OECD guidelines
(OECD 425) and following an earlier report.21
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 46
1
2
3
4
5
6
7
8
9
6. In-vivo Activity. BALB/c mice (female, 6 to 8 weeks old, 18ꢀ22g) were used for the
experiments. First, the mice were rendered neutropenic (∼100 neutrophils/mL) by injecting
two doses of cyclophosphamide intraperitoneally (i.p.) (150 mg/kg first dose and 100 mg/kg
after 3 days of the first dose). After 24 h of the second dose, mice were anesthetized by
ketamineꢀxylazine (40 mg/kg ketamine and 2 mg/kg) mixture i.p.) The back of each mouse
was clipped and then shaved using a razor. While shaving, a wound (reddening and glistening
of the skin without bleeding) was introduced on the dorsal midline of each mouse. To the
wound site, methicillinꢀresistant S. aureus (MRSA) was added drop wise (∼109 cells/mL, 20
ꢀL) and allowed to dry to ensure that the bacteria remained within the shaved area. Mice (n =
5 in each group) were left for 24 h of the infection to allow to form the biofilm. Next, mice
were treated with 3b (40 mg/kg), fusidic acid (40 mg/kg), and ReliHealꢀSilver ointment
(Carboxymethylcellulose IP 3.5% w/w, colloidal silver nanoparticle 50 ppm, Reliance Life
Advances, India) (200 mg/kg) at the site of infection. The compound solution was carefully
added and spread on the entire wound surface to avoid any loss of solution. The dosages were
continued for four days (per day one dose). One group of mice (n = 5) were left untreated and
used as a control. Mice were sacrificed 18 h after the last dose using isofluorane and the
infected skin was collected, homogenized, diluted and plated for cell counting. The bacterial
count was finally expressed as log CFU/g of the tissue collected and expressed as mean ±
standard error of mean. For imaging the skin tissue by SEM, some portion of skin tissue
samples were fixed in formalin for 24 h, dried subsequently using 30, 50, 70, 90 and 100%
ethanol. The skin tissue samples were then adhered on silicon wafers; sputter coated with
gold and imaged by Quanta 3D FEG, FEI field emission scanning electron microscope.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ANCILLARY INFORMATION
Supporting Information
28
ACS Paragon Plus Environment

Page 29 of 46
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
1H NMR and high resolution mass spectra, figures showing fluorescence microscopy of
mammalian cells, table showing antibiofilm activity. The Supporting Information is available
free of charge on the ACS Publications website.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Acknowledgements
We thank Prof. C. N. R. Rao, FRS for constant support and guidance. J. Hoque thanks
JNCASR for senior research fellowship (SRF).
Abbreviation Used:
FTꢀIR = Fourier Transform Infrared Spectroscopy
NMR = Nuclear Magnetic Resonance
HRMS = High Resolution Mass Spectrometry
SEM = Scanning Electron Microscopy
HPLC = High Performance Liquid Chromatography
PBS = Phosphateꢀbuffered Saline
MIC = Minimum Inhibitory Concentration
MBEC = Minimum Biofilm Eradication Concentration
MRSA = Methicillinꢀresistant Staphylococcus aureus
CFU = Colony Forming Unit
PI = Propidium Iodide
HEK = Human Embryonic Kidney
TX = Triton X
HC50 = 50% Hemolytic Concentration
LD50 = 50% Lethal Dose
Author Information
*Corresponding author: jayanta@jncasr.ac.in; Tel: +91ꢀ080ꢀ2208ꢀ2565
Notes
The authors declare no competing financial interests.
29
ACS Paragon Plus Environment