Click inspired synthesis of: P-tert -butyl calix[4]arene tethered benzotriazolyl dendrimers and their evaluation as anti-bacterial and anti-biofilm agents

Article abstract of DOI:10.1039/d0nj02591g

Calixarenes with their three-dimensional structural architecture and ease of functionalization at the upper and/or lower rim are well-known for their plethora of applications in chemical, physical, biological and other disciplines. Herein, CuAAC click ins

Full text of DOI:10.1039/d0nj02591g

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2020, DOI: 10.1039/D0NJ02591G.
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ARTICLE
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Click inspired synthesis of p-tert-butyl calix[4]arene
tethered benzotriazolyl dendrimers and their evaluation as
anti-bacterial and anti-biofilm agents
Cite this: DOI: 10.1039/x0xx00000x
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Anand K. Agrahari,a Ashish K. Singh,b Anoop S. Singh,a Mala Singh,a Pathik Maji,c
Shivangi Yadav,b Sanchayita Rajkhowa,d Pradyot Prakash,b* and Vinod K. Tiwari*a
Received 00th January 2012,
Accepted 00th January 2012
Calixarenes with their three dimensional structural architecture and suitable functionalization at upper and/or lower
rim are well-known for their plethora of application in chemical, physical, biological and other deciplines. Herein, we
have devised the CuAAC click inspired p-tert-butylcalix[4]arene tethered benzotriazolyl dendrimers for example, N-
1, N-2 type 2 fold compound 6 and 7 ‘G(0)’ and 6 fold compound 16 and 17 ‘G(1)’ generation benzotriazolyl
dendrimer and also evaluated for their therapeutic potential both in vitro and in vivo. These developed calix[4]arene
tethered benzotriazolyl dendrimers have been characterized by standard spectroscopic analysis including NMR (1H
and 13C), MS, and SEC datas. Among all the developed benzotriazolyl dendrimer, the compound 7 was identified as
efficacious one which providing potential anti-bacterial and anti-biofilm activities against drug-resistant and slime
producing organisms without imparting cytotoxicity to the eukaryotic systems.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
conjugates, such as four α-l-C-fucosyl units to the calix[4]arene
platform developed by Grazia et al., have displayed anti-biofilm
potential against the pathogenic Pseudomonas aeruginosa.22
Authors showed concentration-dependent anti-biofilm activity of
these calixarene conjugates at 200μM concentration and the
percentage of biofilm inhibition was found to be 73% against the
PAO1 strain of Pseudomonas aeruginosa.22 Likewise, anti-bacterial
Calixarenes, with their distinctive three dimensional architecture
and easy functionalization at upper and/or lower rim are attractive
building blocks for well-ordered self-assembly and thus well
explored as suitable scaffolds for various purposes in organic
synthesis.1-4 In addition, calixarenes form stable complexes with
biomolecules which were quite useful in host-guest chemistry,5
molecular recognition particularly in chiral recognition,6
chemosensor and biosensor technology,7 catalysis, biotechnology,8
platonic micelles,9 gene delivery,10 etc. In addition to their
fascinating physical and chemical behavior, calix[4]arene systems
have been further identified as imperative structural characteristics
that are preferably explored like promising molecular scaffold for
the development of new chemical entities (NCE's) as anti-fungal,11
and anti-biofilm activities of functionalized
amphiphillic
tetraalkoxycalix[4]arenes which was dependence on the alkyl chain
length on the lower rim of calixarene has also been well
documented.23 Recently, a number of 1,4-disubstituted 1,2,3-
triazoles displayed a wide range of pharmacological activities.24
Fascinatingly, similar hybrid molecules also displayed potent in vitro
and in vivo anti-bacterial activities against both drug-sensitive and
drug-resistant pathogens.25 However, there are only few reports
available about the anti-biofilm activity of 1,4-disubstituted
triazoles or their conjugates.26,27 The glamour feature of this
skeleton can be further perceived in the recent report on triazole
appended water soluble curcumin, which fostered apoptosis like
phenomenon among multi drug resistant pathogens like Klebsiella
pneumoniae along with interesting anti-biofilm activity.28
Surprisingly, similar anti-biofilm activity is still not well documented
for dendrimers comprising with benzotriazole residue, which we
investigated herein. Benzotriazoles, being a non-toxic, cheap and
stable moiety have been widely explored as an attractive synthetic
auxiliary in organic synthesis for an easy access of diverse range of
biologically relevant molecules.29-31 Few simple benzotriazole
derivatives such as vorozole, 4,5,6,7-tetrabromobenzotriazole
(TBB), etc showed broad bioactivity spectrum, where water
solubility and effective pharmacological activity with minimum side
anti-viral,12 anti-HIV,13 anti-cancer,14 anti-tumour activity.15
A
number of calixarenes and their conjugates are known to display
potent anti-bacterial activities.16-18 Furthermore, this scaffold
displayed a high-affinity ligand of cholera toxin19 and bacterial
lectin.20 For their successful medical implications, the toxicity of
NCE's is obviously a key feature; interestingly calixarene and their
conjugates were in general neither have immune responses nor the
toxicity.21 In addition, some calixarenes based multivalent
aDepartment of Chemistry, Centre of Advanced Study, Institute of
Science, Banaras Hindu University, Varanasi-221005, India.
Bacterial Biofilm & Drug Resistance Research Laboratory, Department of
Microbiology, Institute of Medical Sciences, Banaras Hindu University,
Varanasi-221005, India.*E-mail: Pradyot_micro@bhu.ac.in
c Department of Chemistry, Guru Ghasidas University, Bilaspur-495009, India
dDepartment of Chemistry, Jorhat Institute of Science & Technology, Jorhat-
785010, Assam, India.
b
*E-mail: Tiwari_chem@yahoo.co.in; Vinod.Tiwari@bhu.ac.in
This journal is © The Royal Society of Chemistry 2013
New J. Chem., 2020, 00, 1-3 | 1

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effects can formulate this skeleton as an appropriate NCE's.32-34 and 5, respectively.42 The resulted azide functionalized alkylated
High applicability in medicinal field due to its larger conjugated benzotriazole 4 on CuAAC reaction with bis-propargyloxy-p-tert-
system, better π-π stacking interaction, and it’s three nitrogen butylcalix[4]arene core unit 1 in presence of CuSO4 5H2O/sodium
.
atoms easily form the non-covalent interaction such as hydrogen ascorbate as standard click condition in THF/water (1:1) at room
bond, ion-dipole, coordination bond and Van der Waals with the temperature gave 91% yield of G(0) generation benzotriazolyl
biocatalyst which facillitate a broad spectrum of biological action.35
dendrimer 6. Similar CuAAC reaction of azide functionalized
The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC, commonly benzotriazole derivative 5 under similar standard click condition
known as Click chemistry) resulted to regioselective formation of furnished the desired G(0) generation benzotriazolyl dendrimer 7 in
9
1,4-disubstituted triazoles with
a great ease have diverse 88% yield (Scheme 2).
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applications,36 where since last 19 years, this reliable and modular
protocol was extensively explored in order to achieve variety of
dendrimers.4,24,37 The Click driven triazole motif has been
acknowledged as non-classical amide isostere that mimic the amide
linkage in biological system and this way offered multiple biological
applications.38-40 Therefore, we endeavour to amalgamate the
synthetic advantages and biological potency of benzotriazoles and
calixarenes through click coupling of these two moieties to
assemble
a new class of p-tert-butyl-calix[4]arene tethered
benzotrizolyl dendrimers. We, herein wish to report the CuAAC click
inspired synthesis of novel benzotriazolyl dendrimer with their
possible biological in vitro as well as in vivo investigations.
Scheme 2 Synthesis of azide functionalized dendrons 4 and 5, and
dendrimer 6, 7.
Disappearance of the azide peaks in IR and appearance of triazolyl
Results and Discussion
1
peaks at δ 8.02 and 7.97 ppm in H NMR, strengthen the tethering
Chemistry
of two scaffolds and confirm for the successful synthesis of G(0)
generation dendrimers (6 and 7), which was further supported by
its HRMS analysis.
Furthermore, to synthesize the first generation dendrimers G(1), we
followed the procedure according to the (Scheme 3), where free
amine functionality of tris-hydroxyaminomethane 8 was protected
The p-tert-butylcalix[4]arene tethered benzotrizolyl dendrimers 6,
7, 16 and 17 were derived from bis-propargyloxy-p-tert-
butylcalix[4]arene. Our synthesis commence with the generation of
bis-propargyloxy-p-tert-butylcalix[4]arene core unit 1 which was
obtained in 57% yield from p-tert-butylcalix[4]arene by reacting it
with propargyl bromide in anhydrous acetone in presence of K2CO3
as base. The core unit 1 was isolated in pure form by column
chromatography (SiO2) and characterized by standard spectral data
(1HNMR, 13CNMR and IR), which were closely matched to the
standard available literature sample.41
as N-BOC
propargylation
9
using di-tert-butyl dicarbonate, followed by
to furnish N-(tert-butyloxycarbonyl) tris
[(propargyloxy)methyl]aminomethane 10.43 Moreover, removal of
BOC group followed by subsequent addition of chloroacetylchloride
using N,N-diisopropylethylamine (DIPEA), resulted the 2-
chloroacetamide-tris[(propargyloxy)methyl] aminomethane 11.
OH
OH
O
HO
HO
di-tert-butyl dicarbonate,
BuOH/MeOH,
rt,18 h
HO
HO
NH2
O
N
H
Br
K2CO3,
9
8
Anhyd. Acetone
KOH,0oC-rt
Br
OH
O
O
OH
OH
HO
OH
OH
O
O
1
O
O
TFA, Dry DCM,0oC
Cl
O
O
Scheme 1 Synthesis of bis-propargylated calixarane core unit 1.
N
H
O
N
Chloroacetyl chloride,
DIPEA, Dry DCM
0 oC-rt
H
O
O
10
11
To synthesize the azide functionalized dendritic benzotriazolyl
architecture, first of all benzotriazole was alkylated using 1,4
dibromobutane in presence of NaH in anhydrous THF to furnish
respective 1-(4-bromobutyl)-1H-benzo[d][1,2,3]triazole 2 (33%) and
2-(4-bromobutyl)-2H-benzo[d][1,2,3]triazole 3 (18%), which were
separated in pure form by column chromatography (SiO2).
Compound 2 and 3, separately on treatment with sodium azide in
DMF via routine nucleophilic substitution reaction method
furnished azide functionalized alkylated benzotriazole derivatives 4
Scheme 3 Synthesis of 2-chloroacetamide-tris[(propargyloxy)methyl]
aminomethane (11).
The compound 11 was coupled with 1-(4-azidobutyl)-1H-
benzo[d][1,2,3]triazole
4
and
2-(4-azidobutyl)-2H-
benzo[d][1,2,3]triazole 5 scaffolds by utilizing modular copper(I)-
catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) tool, to
obtain the chlorine functionalized dendrons
12 and 13,
2 | New J. Chem., 2020, 00, pp-pp
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respectively. Next, the developed dendrons were elucidated by H (SEC) data has displayed the monodispersity of the synthesized 16
NMR spectra, the triazolyl peak for 12 and 13 at δ 8.0 and 7.56 ppm and 17 benzotriazolyl dendrimers (Figure 2).
confirmed the formation of said dendrons, furthermore, their
synthesis was supported by 13C NMR. As it is evident from the
experimental results chlorine is a good leaving group in comparison
to the benzotriazole.44 Therefore, chloro group of dendrons 12 and
13 were substituted by azide group using NaN3 in DMF to furnish
desired azide functionalized dendrons 14 and 15, respectively
(Scheme 4).
O
N
OH
OH
O
N
O
N
OH
O
N
OH
N
N
N
N
N
N
N
N
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N
N
N
N
N
N
N
N
N
N
O
O
N
N
O
Cl
7
6
N
H
O
11
N
N N
N
N N
CuAAC
N
N
N
N
CuSO4.5H2O
NaAsc, THF/H2O
CuSO4.5H2O
NaAsc, THF/H2O
N
N
OH
O
OH
O
4
5
N
N
N
N
N
N
N
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N
N
N
N
O
HN
NH
O
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O
O
N
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O
O
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O
O
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O
O
N
N
O
O
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N
N
O
O
Cl
N
N
Cl
N
N
N
N
N
N
N
N
N
N
N
H
O
N
N
N
O
H
N
N
N
12
NaN3, DMF
13
NaN3, DMF
N
N
N
N
N
N
N
N
N
N
N
N
N
N
16
N
N
N
N
N
O
N
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O
N
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N
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N
N
N
N
N
N
O
N
O
O
OH
O
O
OH
O
N3
N3
N
N
N
N
O
H
N
N
N
N
N
O
H
N
N
N
N
N
O
N
N
14
HN
15
N
NH
N
O
N
N
N
O
O
1
1
N
Calix derivative
2nd Click
(CuAAC)
Calix derivative
2nd Click
(CuAAC)
N
N
N
N
O
N
O
O
O
N
N
N
N
N
N
N
N
N
First Generation (G1)
17
First Generation (G1)
16
N
N
N
N
Dendrimer
Dendrimer
Scheme 4 Schematic representation for the synthesis of first
generation dendron 14, 15 and dendrimer 16 and 17.
N
N
N
N
N
N
N
N
N
N
N
N
The peak attribute for azide group appeared at 2103.96 and
2106.89 cm-1 in IR spectrum confirmed for the successful generation
of azide functionality. In continuation, core 1 was clicked separately
with dendritic architecture 14 and 15 via CuAAC conjugation to
furnish the targeted benzotriazolyl first generation dendrimers 16
and 17, respectively. Appearance of triazolyl peaks at 8.03 and 8.07
ppm in 1H NMR spectrum of dendrimer 16 and 17, respectively,
depicts the formation of new regioselective 1,4-disubstituted
triazole. The structure was further supported by its 13C NMR
spectrum. Besides this, the complete vanishing of azide peak in the
IR spectra of compound 16 and 17 have also inferred the
unequivocal formation of desired dendrimers. The structure of
developed dendrimer was further elucidated by MALDI- TOF and
ESI-MS analysis. In contrast to this, size exclusion chromatography
17
Fig. 1 Structure of developed benzotriazolyl dendrimers 6, 7, 16, and 17.
Fig. 2 SEC diagram of benzotriazolyl dendrimers 16 and 17.
New J Chem., 2020, 00, pp-pp | 3
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Anti-biofilm Assays:
Additionally, the electronic UV-Visible absorbance and emission
studies of the developed benzotriazolyl dendrimer 6, 7, 16, and 17
was performed in acetonitrile at 10-3 M concentration to record
their absorbance and emission spectra (Figure 3).
Tissue culture plate assay for the elucidation of anti-biofilm
potential of the synthesized compounds:
The synthesized compounds exhibited dose-dependent biofilm
inhibition against all the tested bacterial isolates. Interestingly, at
the concentration 8 nM, compound 6 was found to inhibit the
MSSA biofilm by only 14.4%, however at the same concentration,
significant inhibition of 41.76%, was observed in E. Coli biofilms
(Table 2). All the synthesized compounds exhibited poor anti-
biofilm activities against the pre-formed biofilms. Nonetheless, the
compound 7, was found significantly effective in inhibiting the
biofilms of all the test pathogens (Table 2, Figure 4).
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Fig. 3 Electronic absorption spectra (10-3 M, in CH3CN) and emission
spectra of developed benzotriazolyl dendrimers 1, 6, 16, and 17.
Biological evaluations
Minimum inhibitory and minimum bactericidal concentration (MIC
and MBC) determination:
Antimicrobial activity of the synthesized compounds (6, 16, 7, 17,
and 1) along with ciprofloxacin against the aforementioned bacteria
was evaluated by determining MIC by broth micro-dilution assay.
The compound 6 inhibited the growth of Methicillin-resistant
Staphylococcus aureus (MRSA) clinical isolate at
a minimal
Fig. 4 (1) Biofilm reduction plot of compounds 1, 6, 7, 16, and 17
against the five high priority multi-drug resistant isolates at 8 nm
concentration signifies reduction of biofilm biomass. (2) Biofilm
degradative effects of synthesized compounds over MRSA biofilms.
Lanes 1-6 represent the untreated, compounds 6, 16, 7, 17, and 1
treated groups. (3) Biofilm inhibitory effects of synthesized
compounds over MSSA biofilms. Lanes 1-6 represent the untreated,
compound 6, 16, 17, 7, and compound 1 treated groups.
concentration of 26 nM, while at the concentration 12 nM, the
compound 16 completely inhibited the MRSA growth. Similarly, the
MIC of compound 7 was found to be 3.46 nM against the
mentioned MRSA isolate. Despite of reduction in log10CFU/mL
count compared to the control, ciprofloxacin was found almost
ineffective in the tested range. These results depict the strong and
immediate bactericidal activity of all the synthesized compounds (6,
16, 7, 17, and 1). The MIC and MBC of the synthesized compounds
(6, 16, 7, 17, and 1) against the tested pathogens are listed in Table
1.
Table 1. Minimum inhibitory concentration of the synthesized compounds and Ciprofloxacin
4 | New J. Chem., 2020, 00, pp-pp
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Comp
Inhibition
Indices
Staphylococ
cus aureus
(ATCC
Staphylococcus
aureus
(MRSA, 1024/
2019)
Klebsiella
pneumoniae
(ATCC 700603)
Klebsiella
pneumoniae
(2859/2019)
Pseudomonas
aeruginosa
(ATCC 25619)
Pseudomonas
aeruginosa
(3047/2019)
Escherichia coli
(ATCC 25922)
Escherichia
coli
(2157/2019)
29213)
6
16
MIC (nM)
MBC (nM)
MIC (nM)
MBC (nM)
MIC (nM)
MBC (nM)
MIC (nM)
MBC (nM)
MIC (nM)
MBC (nM)
MIC (nM)
13.00
27.00
6.00
26.00
52.00
12.00
24.00
3.46
13.00
27.00
6.00
26.00
52.00
12.00
24.00
13.84
41.52
60.16
96.24
197.40
1579.20
13.00
27.00
6.00
39.00
104.00
24.00
13.00
27.00
6.00
13.00
52.00
12.00
24.00
3.46
12.00
1.73
12.00
6.92
12.00
6.92
48.00
12.00
1.73
9
7
27.68
10
11
12
13
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15
16
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13.84
7.52
6.92
27.60
15.04
48.12
49.34
394.80
27.68
15.04
48.12
49.34
789.60
110.72
120.32
192.48
394.80
3158.40
6.92
13.84
15.04
48.12
12.34
197.40
17
7.52
7.52
24.06
3.08
12.03
12.34
197.40
12.03
3.08
1
98.70
24.67
Ciprofl
oxacin MBC (nM)
0.75
1.51
12.07
193.15
1.50
3.02
48.29
96.67
1.50
3.01
193.15
386.31
0.75
1.51
12.07
24.14
Table 2. Comparison of biofilm inhibitory and degradative activity of the synthesized compounds (at concentration 8µM) based on
percent reduction in biofilm biomass.
Bacterial strains
MSSA29213
Reduction in biofilm biomass
14.39±0.54
20.97±0.61
25.17±0.72
41.76±0.84
23.41±0.45
6
0.61±0.47
26.08±0.52
25.82±0.43
52.59±0.36
23.64±0.88
16
86.24±0.37
90.90±0.64
87.43±0.71
89.47±0.39
82.73±0.57
7
16.55±0.61
22.11±0.83
25.35±0.38
46.45±0.62
23.33±0.34
17
7.73±0.73
15.96±0.91
21.37±0.42
37.34±0.78
17.45±0.82
1
MRSA1024/2019
K. pneumonia2859/2019
E.coli 2157/2019
Biofilm inhibitory effects
P. aeruginosa 3047/2019
Compounds
MSSA29213
10.25±1.62
15.84±0.96
12.24±0.93
28.24±1.49
4.76±2.89
15.22±1.27
19.33±2.37
9.02±2.44
38.13±2.38
6.35±3.65
69.18±1.94
73.74±2.68
65.13±2.72
75.21±1.62
63.54±2.89
9.51±2.03
14.32±1.81
8.34±1.95
28.31±1.53
17.25±2.33
4.24±1.36
8.55±0.79
5.42±2.67
14.71±2.66
3.25±1.88
MRSA1024/2019
K. pneumonia2859/2019
E.coli 2157/2019
Biofilm degradation effects
P. aeruginosa 3047/2019
Fluorescent Confocal Microscopic evaluation of anti-biofilm rarefication of bacterial populations in panels A’, B’, and C’ of Figure
potential: 5, respectively. As we move down from panel A to C, selective
The effects of the compound 7 on biofilms of high slime producing uptake of the compound 7 by the bacteria can be conjectured as
multi-drug resistant clinical isolate of Escherichia coli was the panels depict the rarefication of the bacteria down the lane.
investigated by confocal microscopy. Seventy-two hours old biofilm Further, the differential interference contrast (DIC) imaging (Lanes
of the said isolate was then challenged with the MBIC concentration A’ to D’) also supports the confocal observations. In addition, this
of compound 7 (0.05 nM) and then, imaged by staining with the dye can be further understood by the loss of bacteria in the matrix in
propidium iodide. Interestingly, the matured biofilm of the said the 2.5D confocal micrographs as depicted in A’’-C’’.
bacterium showed an intense PI-staining, indicating the prevalence
of dead cells after exposure to the compound 7 (Figure 5A-D).
After administering the minimal concentration of 0.05 nM and
incubation of 180 mins, we observed only a few bacteria down the
lanes indicating the complete inhibition of biofilm. This
disintegration is accompanied by the dispersal (flow) of the biofilm
debris. The results further show an obvious disintegration of the
biofilm matrix (Figure 5A-C, A’-C’). This was evident by the observed
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Fig. 5 The Confocal Microscopic validation of time-dependent
effects of the compound 7 (0.05 nM) on biofilm of MDR Escherichia
coli using propidium iodide. (A - D) Post treatment time dependent
evaluation of E. coli biofilm every hour. Panel a represents the
effect of molecule after 1 hrs while panels b and c represent the
effects after 2, and 3 hrs of treatment. Panel D is the dead control
bacteria after treatment with meropenem. Intermediary yellow
colour shows some interaction between the compound 7 and the
dye. (A’ -D’) Post treatment differential interference contrast (DIC)
micrographs of E. coli biofilm. Panel A represents the effect of drug
after 1 hrs while panels B and C represent the effects after 2, and 3
hrs of treatment. Panel D is the dead control bacteria after
treatment with meropenem. Meropenem kills the bacteria but
poses no effect over the biofilm matrix. (A’’-C’’) Layer wise 2.5D
depiction of progressive loss of bacterial presence also the matrix
components after percolation of drug to the bottom regions as well.
Fig. 6 Time dependent flow cytometry analysis of Escherichia coli
with ROS-sensitive dye, DCFDA for ROS production analysis in the
groups treated with compound 7 at different concentrations and
ciprofloxacin. (a) Histogram of untreated log phase Escherichia coli
cells labelled with DCFDA. (b) Histogram of compound 7 (1/4 MIC
concentration) treated Escherichia coli cells showing the increase in
cell population (33.5%) that took up DCFDA showing ROS
generation. (c) Histogram of compound 7 treated (1/2 MIC)
Escherichia coli cells showing the increase in cell population (37.6%)
that had taken up DCFDA, showing the fair increase in ROS
generation. (d) Histogram of the compound 7 treated (MIC
concentration) Escherichia coli cells. The panel demonstrates the
increase in cell population (66.7%) that had taken up DCFDA. Note
the bimodal population distribution. (e) Histogram of the
ciprofloxacin treated Escherichia coli cells. The panel demonstrates
the increase in cell population (61.1%) that had taken up DCFDA.
We observed PI to be localized in the biofilm matrix throughout.
Therefore we tried to further explore the percolation of drug to the
bottom of the matrix. As can be seen in the panels A’’-C,’’ there is
progressive loss of bacterial presence and the panel C’’ clearly
reveals that upon administration of compound 7, there is not only
the loss of bacterial population but also the matrix components.
This indicates that the compound 7, which might be simultaneously
interacting with surface associated biofilm-forming proteins/sugars
(leading to biofilm disintegration) and the intracellular components.
The verity that propidium iodide binds to the nucleic acid of the
The results obtained are in consonance with the recent report,
which suggests that the insertion of hydrophobic moiety in the
membrane fosters negative curvature in the bilayer that
permeabilizes the cells and eventually result in the cell death.45
In vitro evaluation of cytotoxicity:
Sulforhodamine B assay:
We assess the in vitro cell viability of HCT116 cells in presence of
lysed cells, we, therefore, deduced that the compound 7 is all the synthesized compounds using SRB assay (Figure 7).
triggering the cascade(s) of events that is ultimately leading to the
bacterial lysis and biofilm inhibition (Figure 5).
Incubation of HCT116 cells with compound 7 did not influence
cell viability in the tested range (2-1024 µg/mL) which can be
inferred from the fact that upon exposure to 2 µg/mL of
compound 7, cell viability was around 99% which remained
91.07% (almost unchanged) when exposed to 1024 µg/mL.
Unlike compound 7, the MoS2 QDs affected the cell viability in a
concentration-dependent manner. As seen, clearly in figure 7,
after 72 hrs, the viability remained unaltered compared to the
MoS2 treated cells indicating its improved biocompatibility.
Reactive oxygen species generation:
The test bacteria were exposed to the different concentrations of
compound 7 and ciprofloxacin, and assessed for ROS generation by
examining with 2′, 7′-dichlorfluorescein-diacetate (DCFH-DA) dye
using flow cytometry. We observed dose dependent ROS
generation profile of the compound 7 as well as the ciprofloxacin
treated cells (Figure 6). At the MIC concentration, compound 7
treated cells exhibited more right shifting (66.7%) compared to
ciprofloxacin treated cells (61.1%) indicating more pronounced ROS
generation and subsequent killing (Figure 6).
LDH Assay:
Although benzotriazole based moieties have promising
activities, however, a detailed biocompatibility evaluation is
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lacking in the literature. Therefore, we further evaluated the compound 7 (16, 32, 64, 128, 256, 512, and 1024 µg/mL)
biocompatibility of our compound 7 using HCT116 cells after showed almost no LDH activity. However, we noted dose
exposure for 72 hrs by phase contrast, and LDH-assay. Images dependent cell leakage in the MoS2 QDs treated groups. At the
reveal unaltered proliferation over the time with more concentration of 1024 µg/mL, around 72% cytotoxicity was
pronounced polygonal morphology that suggests restoration of noted in the MoS2 QDs treated cells while on the same
HCT116 functionality (Figure 7). Table 3 sums up the said concentration no leakage was noted in compound 7 treated
enzyme activity as mean absorbances in different groups and cells (Table 3). This indicates biocompatibility of compound 7,
percent toxicity imparted by the positive control and different which was in consonance with the phase contrast micrographs
concentrations of the compound 7 after 48 hrs exposure of the (Figure 7a-h, Table 3).
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HCT116 cells. At every concentration, HCT116 cells treated with
Table 3. Lactate Dehydrogenase Cytotoxicity Assay against HCT116 Cells
Medium
Absorbance
(λmax 500)
Cell + Medium
Absorbance
(λmax 500)
Cell + 2% Triton X
100
Absorbance (λmax
500)
Concentration
(µg/mL)
Compound 7
Treated Cells
Absorbance (λmax
500)
MoS2QDs
Treated Cells
Absorbance (λmax 500)
%
Cytotoxicity by
MoS2QDs
32
64
1.39±0.16
2.44±0.22
2.75±0.22
3.10±0.14
3.52±0.25
3.96±0.29
4.35±0.29
0.71
6.12
18.10
38.03
54.01
71.78
1.47±0.13
1.58±0.11
1.69±0.13
1.78±0.10
1.89±0.13
1.12±0.28
1.30±0.30
4.21±0.26
128
256
512
1024
Fig. 7a-h Evaluation of cytotoxicity over HCT116 cells post-treatment. (a) Untreated HCT116 cells on day 1; (b) Untreated HCT116 cells after 48 hrs;
(c) HCT116 cells treated with compound 6; (d) HCT116 cells treated with compound 16; (e) HCT116 cells treated with compound 7; (f) HCT116 cells
treated with compound 17; (g) HCT116 cells treated with compound 1; (h) HCT116 cells treated with Imatinib.
aminotransferase (ALT/GPT), and aspartate aminotransferase (AST/
GOT) in compound 7 treated groups vis a vis MoS2 QDs treated
groups with respect to the untreated control group. Interestingly,
no significant increase in the ALT/ GPT and AST/ GOT activities in
compound 7 treated group III was observed, however, the MoS2
QDs treated group II had shown the marked increase in ALT and AST
activities (ESI Table S1). Similarly, we documented significantly
increased concentration of serum urea and creatinine (p≤0.05) in
group II whereas in compound 7 treated group III no significant
change was found. The activity of SOD and catalase in blood serum
were found decreased markedly (p≤0.05) in group II as compared to
In vivo evaluation of cytotoxicity:
Biochemical evaluation of serum:
Toxicity testing of the compound 7 has been done on rats and
different cellular parameters were assessed in blood serum as listed
in Supplementary information 1. No significant change in serum
glucose concentration was found among different groups. However,
a significant reduction in the level of Hb in MoS2 QDs treated group
(group II) was noted. Unlike group II, no significant changes were
observed in Hb concentration among compound 7 treated test
group III and it was found comparable to the untreated group I
(See, ESI Table S1). We further evaluated the levels of alanine
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the untreated control group I. We observed insignificant changes in the morphological changes. A figure 7 summarizes the histological
the activity of SOD and catalase in compound 7 exposed group III status of each organ (liver and kidney) in respective groups.
(See, ESI Table S1).
As expected, the histopathological sections of the untreated control
group I reveal the normal structure and compact arrangement of
hepatocytes (Figure 8). Same as the untreated group I, no obvious
hepatic damage was manifested in compound 7 treated group III.
However, many obvious morphological alterations in the liver
tissues of the MoS2 QDs treated group II was noted such as
degeneration of hepatocytes, mild necrosis, partial damage of the
central vein, hydropic degeneration, and hepatocyte vacuolations
(Figure 8B) as reported in case of toxicity evaluations of carbon-
nano tubes.49 Interestingly, the histological status of the compound
7 treated group III was almost the same as that of untreated control
group I (Figure 8C).
Likewise, the histopathological study of kidney sections showed no
remarkable alteration(s) in the compound 7 treated group III as
compared to the control group I (Figure 8D, F). However, the
sections of the kidney of MoS2 QDs treated groups showed mild
nephrotoxicity, such as swelling of the glomerulus and decreased
bowman's space (Figure 8E).
Oxidative stress analysis:
To evaluate the toxicity imparted by compound 7 and MoS2 QDs we
evaluated the anti-oxidative indicators in the vital organs like liver,
and kidney. The protein content and the enzyme activities results
have been summed up in Supplementary information (Table S2,
and S3). The lipid peroxidation level, measured by the
malondialdehyde assay (Table S3), clearly reveals an insignificant
increase in compound 7 treated group III compared to the
untreated control. However, a significant increase in the lipid
peroxidation level was noted in MoS2 QDs treated positive control
group (p < 0.05). The superoxide dismutase (SOD) and the catalase
activities (CAT) have shown a significant decrease in MoS2 treated
group II (p˂0.05) while the compound 7 treated group III has shown
significant decrease in SOD and CAT activities as compared to the
control group (p>0.05) (See, Table S3).
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The results obtained clearly show that even administering such a
higher dose of compound 7, no significant reduction in the levels of
SOD, CAT activities were manifested (See SI file). While its
counterpart, MoS2 QDs, triggered excessive production of reactive
oxygen species (ROS) as evident from the significant decrease in the
activities of the said enzymes (See, Table S3).
A plethora of literature documents the decrease in SOD activity due
to excessive superoxide radical formation and H2O2 accumulation.46
Liver is primarily involved in xenobiotic metabolism, which helps
detoxification, therefore, it is always on high risk with regard to the
reactive oxygen species (free radical) attack, which may lead to lipid
peroxidation.47 Numerous other studies also support lipid
peroxidation via free radical generation due to nanoparticles
induced toxicity.48 Our results are also in accordance with another
earlier report that MoS2 increased lipid peroxidation in rats. Lipids
are more predisposed macromolecules to oxidative stress and our
results showed that the level of lipid peroxides, significantly
increased due to MoS2 QDs exposure indicating tissue damage. In
this study, compound 7 was observed to significantly reduce the LPs
level by scavenging free radicals and inhibiting the propagating
chain reaction of LPs.
Fig. 8 Histopathological evaluation (HE staining, 10×) of vital organs
(Liver, and Kidney) of Charles Foster strain rats both untreated and
treated with highest dose of compound 7, and MoS2 QDs. A, B, and
C are the liver sections of the untreated control group I, MoS2
treated group II, and Compound 7 treated group III. , D, E, and F are
the kidney sections of untreated group I, MoS2 QDs treated group II,
and Compound 7 treated group III. (1) Undamaged central vein of
group I. (2) Compact and healthy hepatocytes of group I. (3)
Damaged and dilated central vein of group II. (4) Degenerated,
necrotized, vacuolated, and diffused hepatocytes of group II. (5)
Undamaged central vein of group III. (6) Compact and healthy
hepatocytes of group III. (7) Healthy glomerulus and Bowman’s
space of group I. (8) Healthy and undistorted loops of group I. (9)
Swollen glomerulus and dilated Bowman’s space of group II. (10)
Necrotized glomerulus and Bowman’s space of group II. (11)
Distorted loops of group II. (12) Healthy glomerulus and Bowman’s
space of group III. (13) Healthy and undistorted loops of group III.
The results obtained from our study showed that upon oral
administration of MoS2 QDs, we noted obvious signs of hepato and
nephro-toxicity such as vacuolations and degeneration of
hepatocytes, necrosis, damage to the central vein, hydropic
MoS2 QDs administration resulted in an increase in the level of free
radicals, which fostered cellular damage, and this observation could
be insinuated by the low levels of free radical scavenging enzymes
such as catalase and superoxide dismutase, the first line of cellular
defense against the oxidative stress. Conversely, compound 7 had
not affected the pool of the said enzymes compared to the
untreated control group I, which establishes its compatibility to the
biological system.
Histopathological investigations of toxicity:
We further performed the histopathological studies of liver, and
kidney to access the compound 7 mediated toxicity by monitoring
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degeneration, and swelling of glomerulus and increase in Bowman’s physical and spectroscopic data closely matched with the standard
space diameter. Interestingly, no evidence of hepato and nephro sample (see, supporting information file).
toxicity was observed after exposure to the compound 7.
Synthesis of benzotriazolyl dendritic architecture, 1-(4-bromobutyl)-
5
1H-benzo[d][1,2,3]triazole
(2)
and
2-(4-bromobutyl)-2H-
6
7
8
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benzo[d][1,2,3]triazole (3) was followed by the literature procedure
and physical and spectroscopic data closely matched with the with
the standard reported literature24 (see, supporting information file).
Conclusions
In this study, we demonstrated
a facile protocol for the
development of benzotriazole based novel theranostic hybrid, as a
new chemical entities (NCE's) for the possible treatment of hyper
virulent Klebsiella pneumoniae infections. The developed molecules
were characterized by standard spectroscopic analysis, such as
NMR (1H and 13C), UV-Vis, IR, ESI-Ms, HRMS, and GPC. Moreover,
there was an excellent chemo-sensitizing effect of the synthesized
compounds. In addition, we have explored its mode of action using
fluorescent probes for studying its reactive oxygen species
generation (ROSG). Furthermore, we also demonstrated the
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Procedure for the synthesis of compound 4 and 5.
To the compound 2 or 3 (1.0 g, 3.93 mmol) in anhydrous DMF (5
mL) in 50 mL R.B. flask, sodium azide (383 mg, 5.9 mmol, 1.5 equiv.)
was added to the mixture and stirred for 24 h at 40oC. After the
completion of reaction, reaction mixture was evaporated under
high vacuum to afford crude residue, which was extracted with
ethyl acetate (2 x 50 mL), washed with the water (2 x 20 mL).
Organic layers were collected and evaporated under rota-vapor to
afford the crude mass and after column chromatography (SiO2), the
individual compounds 4 and 5 were isolated in pure form.
biocompatibility of compound
7 both in vitro and in vivo.
Distribution and organ-specific accumulation of compound 7 was
also investigated after oral administration into rats to ensure its
biocompatibility. In all, the current study depicts the synthesis and
application of G(0) and G(1) generation p-tert-butyl-calixarene
tethered benzotriazole dendrimer. Considered together, the
compound 7 represents NCE's, providing potential antibacterial and
anti-biofilm activities against drug-resistant and slime producing
organisms without imparting cytotoxicity to the mammalian cell
1-(4-azidobutyl)-1H-benzo[d][1,2,3]triazole (4). Yellow viscous,
yield: 714 mg (84%); Rf = 0.4 (30% ethyl acetate/n-hexane); IR:
3417.8, 2929.4, 2857.5, 2098.9, 1615.3, 1455.2, 1268.4, and 747.4
cm-1; 1H NMR (500 MHz, CDCl3): δ 8.04 (d, J = 7.5 Hz, 1H), 7.52-7.46
(m, 2H), 7.37-7.34 (m, 1H), 4.67-4.64 (m, 2H), 3.32-3.39 (m, 2H),
2.12-2.06 (m, 2H), 1.63-1.58 (m, 2H); 13C NMR (125 MHz, CDCl3): δ
145.9, 132.7, 132.7, 123.8, 120.0, 109.0, 50.6, 47.4, 26.7 and 26.0
ppm.
line. However,
a thorough study is needed for further
improvements in downstream processing techniques for scalable
production, with further understanding of in vivo trafficking of
these hybrid compounds.
2-(4-azidobutyl)-2H-benzo[d][1,2,3]triazole (5). Yellow oil, yield:
731 mg (86%); Rf = 0.4 (20% ethyl acetate/n-hexane); IR: 3061.8,
2928.8, 2857.3, 2096.8, 1567.0, and 1448.4 cm-1; 1H NMR (500 MHz,
CDCl3): δ 7.86-7.83 (m, 2H), 7.36-7.33 (m, 2H), 4.75-4.72 (m, 2H),
3.29-3.26 (m, 2H), 2.20-2.14 (m, 2H), 1.61-1.57 (m, 2H); 13C NMR
(125 MHz, CDCl3): δ 144.0, 126.0, 117.7, 55.5, 50.4, 26.8 and 25.6
ppm.
Experimental
General
All the chemicals and solvents were used of pure grade. All the
reactions were carried out under inert atmosphere except CuAAC
click reactions. Thin-layer chromatography (TLC) was performed on
60 F254 Silica gel pre-coated Aluminium plates and seen under UV
lamp Violet lamp (λ max = 254 nm) or iodine vapour. Column
chromatography (SiO2) was performed on silica gel (230-400 mesh,
Merck) by using distilled n-hexane, ethyl acetate and commercially
available dichloromethane and methanol. Solvents were
evaporated under rota-vapour. Mass spectra were recorded by
electron spray ionization mass spectrometry (ESI-MS), HRMS,
MALDI-TOF MS. IR Spectra were recorded as Nujol mulls in KBr
pellets and in ATR. 1H and 13C NMR were taken at 500 MHz (1H) and
125 MHz (13C), respectively. Chemical shifts were given in ppm
downfield from internal TMS; J values in Hz.
General procedure for the synthesis of benzotriazolyl dendrimers
6 and 7.
Core 1 (100 mg, 0.137 mmol) was dissolved in THF/water (1:1, 30
mL with ratio (1:1), followed by addition of 1-(4-azidobutyl)-1H-
benzo[d][1,2,3]triazole (87 mg, 0.34 mmol) or 2-(4-azidobutyl)-2H-
.
benzo [d] [1,2,3]triazole (87 mg, 0.34 mmol) after that CuSO4 5H2O
(20 mg, 0.08 mmol) and sodium ascorbate (16 mg, 0.08 mmol) were
added and reaction mixture was stirred for overnight. After
completion of the reaction, it was passed through celite.
Subsequent work-up and extraction was done using ethyl acetate
and water. Organic layers were collected, dried over anhydrous
sodium sulfate, filtered and concentrated under high vacuum to
obtain crude residue, which was further purified by column
chromatography (SiO2) to afford the targeted product 6 or 7.
Core-5,11,17,23-tetra-p-tert-butyl-25,27-dihydroxy-
dipropargylcalix[4]arene (1) was synthesized in 57% yield from p-
tert-butylcalix[4]arene by following the literature procedure23 and
26,28-
Physical data of compound 6. Off white solid, yield: 145 mg (91%);
Rf = 0.3 (80% ethyl acetate/n-hexane); m. p. = 98-101 °C; IR: 3438.0,
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2956.5, 2924.6, 2854.0, and 1484.4 cm-1; 1H NMR (500 MHz, CDCl3): 2.44-2.43 (m, 3H); 13C NMR (125 MHz, CDCl3): δ 165.8, 79.2, 74.7,
δ 8.01 (d, J = 8.0 Hz, 2H), 7.94 (s, 2H), 7.50 (s, 2H), 7.42 (s, 2H), 7.34- 68.0, 59.5, 58.5, and 42.7 ppm.
7.32 (m, 4H), 7.03 (s, 4H), 6.82 (s, 4H), 5.10 (s, 4H), 4.66-4.63 (m,
4H), 4.35 (t, J = 6.5 Hz, 4H), 4.24 (d, J = 13.5 Hz, 4H), 3.28 (d, J = 13.5
Hz, 4H), 2.04-2.03 (m, 4H), 1.95-1.89 (m, 4H), 1.28 (s, 18H), 0.98 (s,
18H); 13C NMR (125 MHz, CDCl3): δ 150.2, 149.6, 147.4, 145.8,
144.0, 141.9, 132.8, 132.5, 127.6, 127.3, 125.7, 125.1, 123.9, 123.5,
119.9, 109.2, 69.7, 49.4, 47.1, 33.9, 33.8, 31.7, 31.6, 30.9, 29.6, 27.4
and 26.4 ppm. m/z (HRMS) for [C70H85N12O4]+ =1157.6811 (M+H)+,
observed = 1157.6833.
Procedure for the synthesis of benzotriazolyl dendritic structures
12 and 13.
1-(4-azidobutyl)-1H-benzo[d][1,2,3]triazole 4 (242 mg, 1.12 mmol,
3.5 equiv.) or 2-(4-azidobutyl)-2H-benzo[d][1,2,3]triazole 5 (242 mg,
1.12 mmol, 3.5 equiv.) was taken in a mixture of THF and water (1:1
ratio). 2-Chloroacetamide-tris [(propargyloxy)methyl] amino
methane 11 (100 mg, 0.32 mmol, 1.0 equiv) was added to the
reaction mixture followed by addition of CuSO4.5H2O (72 mg, 0.29
mmol, 0.3 equiv. per propargyl) and sodium ascorbate (57 mg, 0.29
mmol). The resulting suspension was stirred for 12h at 40˚C. After
the completion of the reaction (monitored by TLC), suspension was
passed through the cellite to remove the metal and the filtrate was
extracted with ethyl acetate (3 x 50 mL). Organic layers were
collected and passed over the anhydrous Na2SO4, and evaporated
under rota-vapor to obtain the crude residue which was further
refined by column chromatography (SiO2) to afford the desired
dendritic structure 12 and 13, respectively. Further, obtained
product was completely characterized by NMR.
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Physical data of compound (7). Off white solid, yield: 140 mg (88%);
Rf = 0.3 (60% ethyl acetate/n-hexane); m.p. = 110-115°C; IR: 3437.8,
1
2957.3, 2867.2, and 1484.6 cm-1; H NMR (500 MHz, CDCl3): δ 7.97
(s, 2H), 7.81-7.80 (m, 4H), 7.42 (s, 2H), 7.34-7.32 (m, 4H), 7.04 (s,
4H), 6.84 (s, 4H), 5.14 (s, 4H), 4.77-4.74 (m, 4H), 4.37-4.34 (m, 4H),
4.24 (d, J = 13.5 Hz, 4H), 3.30 (d, J = 13.5 Hz, 4H), 2.17-2.11 (m, 4H),
1.95-1.89 (m, 4H), 1.27 (s, 18H), 0.99 (s, 18H); 13C NMR (125 MHz,
CDCl3): δ 150.2, 149.6, 147.4, 144.3, 144.1, 141.8, 132.6, 127.5,
126.2, 125.7, 125.1, 123.4, 117.9, 69.8, 59.5, 49.5, 33.9, 33.8, 31.8,
31.6, 30.9, 29.6, 27.3 and 26.7 ppm. m/z (HRMS) for
[C70H84N12O4Na]+ =1179.6630 (M+Na)+, observed=1179.6615
Physical data of benzotriazolyl dendritic structure (12).
Off white solid, yield: 249 mg (81%); Rf
= 0.35 (5%
methanol/dichloromethane); 1H NMR (500 MHz, CDCl3): δ 8.00-7.98
(m, 3H), 7.52 (s, 3H), 7.49-7.41 (m, 6H), 7.32 (t, J = 7.5 Hz, 3H), 6.89
(s, 1H), 4.64-4.61 (m, 6H), 4.52 (s, 6H), 4.36-4.34 (m, 6H), 3.87 (s,
2H), 3.74 (s, 6H), 2.02-1.90 (m, 12H); 13C NMR (125 MHz, CDCl3): δ
165.6, 145.8, 144.7, 132.7, 127.3, 123.9, 122.5, 119.8, 109.1, 68.4,
64.6, 59.8, 49.2, 47.0, 42.9, 27.1 and 26.2 ppm.
N-(tert-Butyloxycarbonyl) tris(hydroxymethyl)aminomethane (9)
was synthesized by following the literature procedure and physical
and spectroscopic data closely matched with the with the standard
reported literature.25
N-(tert-Butyloxycarbonyl)tris[(propargyloxy)methyl]amino methane
(10) was synthesized by following the literature procedure and
physical and spectroscopic data were closely matched with the
standard known one.25
Physical data of benzotriazolyl dendritic structure (13).
1
White solid, yield: 231 mg (75%); Rf = 0.4 (5% methanol/DCM); H
NMR (500 MHz, CDCl3): δ 7.84-7.81 (m, 6H), 7.56 (s, 3H), 7.36-7.34
(m, 6H), 6.94 (s, 1H), 4.77-4.74 (m, 6H), 4.57 (s, 6H), 4.39-4.36 (m,
6H), 3.91 (s, 2H), 3.79 (s, 6H), 2.16-2.10 (m, 6H), 1.97-1.91 (m, 6H);
13C NMR (125 MHz, CDCl3): δ 165.5, 144.4, 144.0, 126.1, 122.4,
117.6, 68.3, 64.4, 59.7, 55.2, 49.1, 42.7, 26.9 and 26.4 ppm.
2-Chloroacetamide-tris[(propargyloxy)methyl]aminomethane
(11).43
Compound 10 (1.0 g, 3.21 mmol) was dissolved in anhydrous
dichloromethane (15 mL) and cooled at 0oC, drop-wise addition of
trifluroacetic acid (4 mL, 52.20 mmol) and stirred for 2 h, next,
subjected to co-evaporation with toluene (3 x 5.0 mL). Resulting
residue was dissolved in dichloromethane (5 mL) and cooled at 0oC.
DIPEA (1.2 mL, 7.43 mmol) was added to the reaction mixture
followed by addition of chloroacetyl chloride (590 µL, 7.43 mmol)
solution in DCM (10 mL). Reaction was then transferred at room
temperature and stirred for the 15 h under argon atmosphere.
Trifluroacetic acid was co-evaporated with toluene followed by
quenching with the addition of DIPEA. The resulting reaction
mixture was washed with 0.5 M hydrochloric acid and water (3 x 10
mL), organic layers were collected, dried over anhydrous Na2SO4
and evaporated under rota-vapor to gave the crude mass which was
subjected to purification by column chromatography (SiO2) to
afford compound 11. Yellow oil, Yield: 566 mg (61%); Rf = 0.4 (30%
ethyl acetate/n-hexane); MS: m/z = 334 (M+Na); 1H NMR (500 MHz,
CDCl3): δ 6.80 (s, 1H), 4.13-4.12 (m, 6H), 3.94 (s, 2H), 3.81 (s, 6H),
Synthesis of azide functionalized dendrons 14 and 15.
Chlorine functionalized dendrons 12 (200 mg, 0.21 mmol) or 13
(200 mg, 0.21 mmol) was dissolved in anhydrous DMF followed by
addition of sodium azide (20 mg, 0.31 mmol). The solution was
stirred for 24 h at 60˚C, after completion of the reaction (monitored
by TLC), crushed ice were added to the reaction mixture and
resulting precipitate was filtered off by Buchner funnel and washed
with water to obtain the crude product which was subjected to
column chromatography (SiO2) using the gradient of methanol/
dichloromethane afforded the compound 14 and 15, respectively.
Physical data of benzotriazolyl dendritic structure (14).
White solid, yield: 161 mg (80%); Rf
= 0.3 (5% methanol/
dichloromethane); IR: 2952.4, 2917.7, 2849.3, 2103.9, 1681.6, and
1454.0 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.99-7.97 (m, 3H), 7.52 (s,
3H),7.48-7.47 (m, 3H), 7.44-7.41 (m, 3H), 7.31 (t, J = 7.5 Hz, 3H),
6.71 (s, 1H), 4.62 (t, J = 6.5 Hz, 6H), 4.51 (s, 6H), 4.36-4.33 (m, 6H),
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3.78 (s, 2H), 3.73 (s, 6H), 2.01-1.89 (m, 12H); 13C NMR (125 MHz, 6H), 7.33-7.29 (m, 12H), 7.24 (br s, 2H), 6.99 (s, 4H), 6.78 (s, 4H),
CDCl3): δ 166.6, 145.7, 144.7, 132.7, 127.3, 123.9, 122.5, 119.8, 5.09 (s, 4H), 5.06 (s, 4H), 4.72-4.69 (m, 12H), 4.49 (s, 12H), 4.34-4.31
109.1, 68.5, 64.5, 59.7, 52.4, 49.1, 47.0, 27.1 and 26.2 ppm.
(m, 12H), 4.23 (d, J = 12.0 Hz, 4H), 3.74 (s, 12H), 3.25 (d, J = 13.0 Hz,
4H), 2.09-2.05 (m, 12H), 1.92-1.88 (m, 12H), 1.23 (s, 18H), 0.93 (s,
18H); 13C NMR (125 MHz, CDCl3): δ 165.2, 150.2, 149.5, 147.2,
144.5, 144.1, 141.7, 132.4, 127.6, 126.2, 125.6, 125.3, 125.0, 122.9,
117.8, 69.6, 68.5, 64.5, 60.2, 55.4, 52.8, 49.3, 33.8, 33.7, 31.5, 30.8,
27.0 and 26.6 ppm. Calculated m/z (ESI-MS) for [C140H169N44O12]+ =
2659.3995 (M+H)+, observed= 2659.4147.
Physical data of benzotriazolyl dendritic structure (15):
Light yellow solid, yield: 157 mg (78%); Rf
= 0.3 (5%
methanol/dichloromethane); IR: 3397.8, 2923.8, 2871.9, 2106.8,
1682.6, and 1446.8 cm-1; 1H NMR (500 MHz, CDCl3): δ 7.83-7.81 (m,
6H), 7.51 (s, 3H), 7.37-7.33 (m, 6H), 6.65 (s, 1H), 4.76-4.73 (m, 6H),
4.56 (s, 6H), 4.38-4.35 (m, 6H), 3.79 (s, 2H), 3.76 (s, 6H), 2.14-2.10
(m, 6H), 1.97-1.92 (m, 6H); 13C NMR (125 MHz, CDCl3): δ 165.5,
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144.7, 144.2, 126.3, 122.4, 117.8, 68.6, 64.6, 59.8, 55.4, 52.6, 49.3, Biological evaluation:
27.1 and 26.7 ppm.
The developed compounds 1, 6, 7, 16, and 17 were screened for
their activities against the drug-resistant bacterial isolates.
Subsequently, we investigated the mode of action of compounds 6
and 7 (better activities against bacteria) against the MDR bacterial
isolates. Besides, a detailed biocompatibility profile of compound 7
was investigated both in vitro and in vivo. For in vitro analysis, we
employed cell proliferation assay using sulforhodamine B,
membrane integrity analysis by assaying lactate dehydrogenase
while in vivo biocompatibility was tested in Charles foster strain rats
by investigating their haematological/serological profiling, lipid
peroxidation, superoxide dismutase activity, and catalase activity
with subsequent validation by histopathological screenings.
Synthesis of N1-linked benzotriazolyl dendrimers (16).
Core 1 (32 mg, 44 µmol) was added in THF/water having ratio (1:1),
followed by addition of dendritic azide 14 (107 mg, 0.111 mmol, 2.5
equiv) and then after addition of CuSO4.5H2O (7.0 mg, 28.0 µmol,
0.3 equiv. per propargyl) and sodium ascorbate (6.0 mg, 30.2 µmol,
equiv. per propargyl). Reaction suspension was stirred for 24 h at 40
˚C, which was filtered through celite cake. Filtrate was extracted
with ethyl acetate (3 x 50 mL) and organic layers were collected and
dried over anhydrous Na2SO4 followed by evaporation under rota-
vapor. Crude mass thus obtained was subjected to purification by
column chromatography (SiO2) to afford the compound 16 as
yellow viscous. Yield: 106 mg (73%); Rf = 0.4 (10% methanol/DCM);
IR: 3429.3, 2958.6, 2925.5, 2854.9, 1731.4, and 1463.4 cm-1; 1H
NMR (500 MHz, CDCl3): δ 8.03 (s, 2H), 7.98 (d, J = 8.5 Hz, 6H), 7.60
(s, 6H), 7.50-7.48 (m, 6H), 7.43-7.40 (m, 6H), 7.34 (s, 2H), 7.32-7.29
(m, 6H), 7.14 (s, 2H), 7.00 (s, 4H), 6.76 (s, 4H), 5.05-5.03 (m, 8H),
4.62-4.59 (m, 12H), 4.51-4.47 (m, 12H), 4.34-4.32 (m, 12H), 4.24-
4.21 (m, 4H), 3.73 (s, 12H), 3.24 (d, J = 12.5 Hz, 4H), 1.99-1.88 (m,
24H), 1.24 (s, 18H), 0.93 (s, 18H); 13C NMR (125 MHz, CDCl3): δ
165.2, 150.2, 149.6, 147.3, 145.8, 144.7, 143.9, 141.7, 139.2, 132.8,
132.3, 126.6, 127.3, 125.6, 125.1, 123.9, 123.0, 119.8,114.0, 109.3,
68.5, 64.5, 60.3, 49.2, 47.1, 33.9, 33.7, 31.6, 30.9, 29.6, 27.2 and
26.3 ppm. Calculated m/z (MALDI TOF-MS) for [C140H168N44O12Na]+
=2681.3815 (M+Na)+, observed=2679.8118.
Anti-Bacterial Investigations:
Bacterial strains and culture conditions
We initially incorporated select control bacterial strains namely
Staphylococcus aureus (ATCC 29213), Klebsiella pneumoniae (ATCC
700603), Escherichia coli (ATCC 25922), and Pseudomonas
aeruginosa (ATCC 25619). In addition, we utilized multi-drug
resistant hypervirulent clinical isolate of Klebsiella pneumoniae [hv
KP, Lab code: 2859/2019], Methicillin-resistant Staphylococcus
aureus (MRSA, lab code: 1024/2019), Escherichia coli (lab code:
2157/2019), and Pseudomonas aeruginosa (Lab code: 3047/2019)
were investigated in this study. The bacterial identification was
performed using conventional bacteriological techniques, such as
colony morphology, gram-staining, and different biochemical tests
described elsewhere.50 We defined multi-drug resistance in the
present study as absolute resistance against at least five different
classes of drugs. Antibiotic susceptibility testing of the isolates was
performed by modified Kirby–Bauer method in accordance with the
Clinical and Laboratory Standards Institute guidelines 2019. We
used 15 antibiotic discs namely Ampicillin (10 µg), Cefuroxime (30
µg), cefoxitin (30 µg), Cephalexin (30 µg), Amoxicillin/clavulanate
(20/10 µg), Levofloxacin (5 µg), Ciprofloxacin (5 µg), Amikacin (30
µg), Gentamicin (120 µg), Cefepime (30 µg), Co-trimoxazole
(23.75/1.25 µg), Piperacillin & tazobactam (100/10 µg), Ertapenem
(10 µg), Meropenem (10 µg),and Imipenem (10 µg).
Synthesis of N2-linked benzotriazolyl dendrimers (17):
Core 1 (40 mg, 55 µmol) was added in THF/water (1:1 ratio),
followed by addition of dendritic azide 15 (133 mg, 0.14 mmol, 2.5
equiv) after that incorporation of CuSO4.5H2O (9.0 mg, 36.0 µmol,
0.3 equiv per propargyl) and sodium ascorbate (7.0 mg, 35.3 µmol,
0.6 equiv. per propargyl). Further, the reaction suspension was
stirred for 24 h at 40 0C, which was filtered through celite cake.
Filtrate was extracted with ethyl acetate (3 x 50 mL) and organic
layers was dried over anhydrous Na2SO4 followed by evaporation
under rota-vapor to produce the crude mass. Moreover,
purification of crude residue was performed by column
chromatography (SiO2) which afforded the pure compound 17 as off
white solid.
Minimum Inhibitory Concentration (MIC) determination
The minimum inhibitory concentration of the synthesized
Yield: 104 mg (71%); Rf = 0.6 (10% methanol/DCM); m.p. = 90-92°C;
IR: 3411.7, 2954.8, 2925.1, 2868.4, 1692.8, and 1483.4 cm-1; 1H
NMR (500 MHz, CDCl3): δ 8.07 (s, 2H), 7.79-7.76 (m, 12H), 7.59 (s,
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compounds was determined against the previously mentioned dependent treatment with MIC concentration of compound 7. Prior
isolates by the broth microdilution method as described earlier.51
to staining; chambered slide was gently tapped to remove the
Briefly, the said bacteria were grown in 5 mL BHI broth aerobically leftover broth with subsequent washing with PBS (pH 7.5).
for 18 hrs. Bacterial culture was then diluted to 1:1000 in fresh BHI Biofilm was fixed using 4% (v/v) paraformaldehyde for 30 mins. The
broth. The stock solutions (1024 µg/mL) of the synthesized syto9 and PI were reconstituted with DMSO and the stock solution
compounds (1, 6, 7, 16, and 17 initially dissolved in DMSO) were of 1mg/mL was prepared and stored frozen in aliquots of 100 µl.
prepared and diluted two-folds ranging from 0.5 to 512 µg/mL in For use, stock solutions were diluted with phosphate buffer to the
sterile BHI broth microtiter wells. Wells were inoculated with 100 µl concentration of 10 µg/mL. Ten microliter samples of these staining
of standardized cell suspension (105 CFU/mL) and incubated at 37°C solutions were applied directly to the top of the biofilms.
for next 18 hrs along with the drug suspension made. The MIC was The Zeiss LSM 510 inverted confocal laser-scanning microscope
the minimal concentration of the test compound(s) at which no (Carl Zeiss, Jena, Germany) was used to detect the green and red
observable growth was perceived. Positive controls were devoid of fluorescence from the dyes.
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any test compound but with ciprofloxacin (reference bactericidal
drug), while the sterile broth was used as negative control and all
the experiments were performed in triplicate (Table 1).
2′, 7′-dichlorfluorescein-diacetate (DCFH-DA) analysis for ROS
production in bacteria using flow cytometry:
We monitored endogenous reactive oxygen species (ROS)
production in test bacteria after the exposure of MIC concentration
of compound 7 for 48 hrs using flow cytometry using 2′, 7′-
dichlorofluorescein-diacetate (DCFH-DA) dye as described
previously.55 After 48 hrs of exposure, we harvested the bacterial
cells by centrifuging them at 3,000 X for 20 mins. We washed the
bacterial pellet thrice with phosphate-buffered saline (PBS, pH~7.2)
and re-suspended the cells in PBS to adjust the cell density to 106
/mL by. Subsequently, the resuspended cells were incubated for 30
mins with 5μM DCFH-DA followed by analysis of ROS production on
a BD Accuri C6 Flow cytometer. The BD Accuri C6 software was used
for all analyses. The assay was performed at a low sample flow rate
(14-μl min-1). A total of 10, 000, 00 events was taken into account
for each sample.
Anti-biofilm Activity Determination:
Tissue Culture Plate Assay (TCP)
The antibiofilm assay was performed in 96-well tissue culture plate
as described previously with minor modifications.52 Briefly,
overnight cultures of both control and clinical isolates of MSSA, and
MRSA were diluted 1:100 in brain heart infusion (BHI) broth
(HiMedia laboratories, India) supplemented with glucose (4%) at pH
6.6, whereas, control and clinical isolates of Escherichia coli,
Klebsiella pneumoniae, and Pseudomonas aeruginosa were grown
in Luria Bertani broth (HiMedia laboratories, India). Total 190μl of
each diluted bacterial suspension (OD 0.2 at λmax600nm) was
~
dispensed into flat-bottom polystyrene 96-well tissue culture plate
(Nunc, Denmark) and 10μl of synthesized compounds (6, 16, 7, 17,
and 1) at concentration 8µM was added and incubated at 37°C
without shaking for 24 h. Wells without compounds were set up as
positive controls. After incubation, biofilm was quantitated by
crystal violet (CV) assay (Merck, Germany) as described earlier.53
In vitro evaluation of cytotoxicity:
The freshly prepared compounds (6, 16, 7, 17, and 1) along with
MoS2 QDs were further investigated for their comparative toxicity
profiles. Prior to the experiment, all the synthesized compounds,
and MoS2QDs were dispersed by ultra-sonication for about 15 min
as described elsewhere.54
Cell culture:
The assays were performed in triplicate, and the results expressed
as mean OD570 ± the standard error of the mean (SEM) (Table 2).
The reduction in biofilm biomass compared to the control biofilms
incubated without CurQDs (Positive control), was calculated
according to the proposed formula.
The HCT116 cells were grown in Dulbecco’s modified Eagle medium
augmented with 10% fetal bovine serum, 100 U/mL penicillin, and
100 mg/mL streptomycin in a 5% CO2 atmosphere at 37 °C in a CO2
incubator. The cells were exposed to the compounds 6, 16, 7, 17,
and 1 for 24 hrs. The stock solution (2048 µg/mL) was prepared and
stored in small aliquots at 4°C and diluted two folds in a different
dose ranging from 2 to 1024 µg/mL in growth medium.
Fluorescent Confocal Microscopy for determination of anti-biofilm
properties
For fluorescent microscopic analysis of the effects of the
synthesized compounds, we grew E. Coli biofilm in chambered
slides as described previously.54 Briefly, the isolate was grown in BHI
broth overnight and then diluted 1:100 in fresh BHI broth such that
absorbance was adjusted to 0.2 at λmax 600nm. Twenty-microliters
of this suspension was then dispensed into 8-well flat-bottom
chambered slide containing 480 µl of BHI broth. The biofilm was
statically grown for 72 hrs. This was followed by concentration
Cell proliferation assay:
We performed cell proliferation assay using the sulforhodamine B
(SRB) described elsewhere.56 We seeded the exponential phase
HCT116 cells at a density of 5 × 103 cells per well into the 96-well
plates and allowed to adhere overnight. The cells were further
incubated for next 48 hrs with a range of concentrations of
compounds 6, 16, 7, 17, and 1 along with MoS2 quantum dots (2-
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1024 µg/mL). The 50% (w/v) TCA was used for fixation while 0.4% were fed only with Mili Q water. The group II rats were designated
(w/v) sulforhodamine B (SRB) for 30 min was used for staining as the positive control and were fed with 1024 µg/mL MoS2 QDs
followed by washing with 1% (v/v) acetic acid.
and the third group (Group III) was set as the test group fed with
compound 7 (512 µg/mL).
Lactate dehydrogenase assay:
Initially we made preliminary observations like changes in body
weight, activity, and physical appearance for the first 24 hours. We
euthanized rats on the 15th day and vital organs like liver, kidney,
and spleen were aseptically excised. For histological studies, part of
these organs was stored in 20% formalin. In addition, we prepared
10% tissue homogenate (w/v) in phosphate buffer (pH 7.4)
containing 0.1 mM EDTA to harvest the supernatant. We executed
biochemical estimations of alanine aminotransferase (ALT),
The comparative cytotoxicity of the synthesized compounds (6, 16,
7, 17, and 1) were evaluated over HCT116 cells by measuring
lactate dehydrogenase (LDH) activity as described earlier with
minor modifications.57 As a positive control, we used MoS2 QDs
treated cells. The synthesized compounds were evaluated in
concentration range 16, 32, 64, 128, 256, 512, 1024 µg/mL. The 12
hrs treated cells were spun at 500X g for 5 mins to harvest the
growth medium. The obtained broth and the LDH reagent were
mixed in 2:1 ratio and incubated for 30 mins followed by the
absorbance reading at 500 nm. For control, we used 2% Triton X
treated cells (Table 3). The extent of percentage (%) cytotoxicity
was calculated as follows:
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aspartate
aminotransferase
(AST),
glucose,
alanine
aminotransferase, aspartate aminotransferase, creatinine, and urea
from the obtained supernatant. In addition, from the tissues, we
also estimated lipid peroxidation levels, SOD, and catalase activities
(For detailed method, see the ESI-page S3).
Histopathological investigation
We washed the excised tissues with ice-cold normal saline (0.9%
NaCl) and 20mM EDTA and then fixed immediately in 20% formalin
for next 72hrs. After that, the tissues were stored in 70% ethanol.
The tissue specimens (liver, and kidney) were embedded in paraffin
and diced into 0.5 μm thick blocks. The slices were mounted and
stained with haematoxylin and eosin (H&E) for histopathological
inspection under an optical microscope. Minimum five slides per
sample were prepared and subjected to histopathological
evaluations.
In vivo evaluation of cytotoxicity:
Animal:
We further evaluated the toxicity of compound 7 on Charles Foster
strain male rats. Ethical guidelines were strictly followed for care
and use of rats, which were approved by the Institutional Animal
Ethics Committee (IAEC), Institute of Medical Sciences, Banaras
Hindu University, India (Ethical committee letter
#
No
Statistical Analysis:
2017/CAEC/720). We procured eighteen Charles Foster strain rats
(male, 100–120 g) from central animal house, Banaras Hindu
University and maintained at 25°C under a standard regimen of 12
hours: 12-hour light-dark cycle. Prior to the start of the experiment,
the animals were acclimatized to the experimental environment for
one week
We performed all the experiments in triplicate and reported the
results as the average of these independent experiments with the
corresponding standard deviations (SD).Statistical significance was
analyzed by one-way analysis of variance (ANOVA) applying
Dunnett’s post hoc test, Mann Whitney U test, Student’s t-test
(two-tailed, unequal variance), Two-way analysis of variance
(ANOVA) for multiple comparisons followed by Newman-keul post
hoc analysis as and when required using GraphPad Prism version
7.0 (GraphPad Software, Inc., La Jolla, CA, USA). The P-value<0.05
was considered statistically significant.
In vivo evaluation of cytotoxicity:
Animal:
We further evaluated the toxicity of compound 7 on Charles Foster
strain male rats. Ethical guidelines were strictly followed for care
and use of rats, which were approved by the Institutional Animal
Ethics Committee (IAEC), Institute of Medical Sciences, Banaras
Acknowledgements
VKT gratefully acknowledge Science and Engineering Research
Board (SERB), New Delhi for the funding (Grant No.:
EMR/2016/001123), CISC-BHU for providing spectroscopic studies
and Prof. B. Ray for SEC analysis. PP acknowledge DST-PURSE grant
sanctioned to microbiology Department, IMS, BHU. Authors
sincerely acknowledge the financial support from CSIR, New Delhi
for the contingency grant to AKA as SRF [09/013/(0671)/2017-EMR-
I], UGC, New Delhi for AKS as SRF [2061430918 vide 22/06/2014(i)
EU-V], and SY as SRF [1121630870 vide 18/12/2016(ii)EU-V] to CSIR,
New Delhi.
Hindu University, India (Ethical committee letter
#
No
2017/CAEC/720). We procured eighteen Charles Foster strain rats
(male, 100–120 g) from central animal house, Banaras Hindu
University and maintained at 25°C under a standard regimen of 12
hours: 12-hour light-dark cycle. Prior to the start of the experiment,
the animals were acclimatized to the experimental environment for
one week.
We randomly divided the rats into three groups such that each
group contains six rats. Group I rats served as negative control and
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18 H. M. Dibama, I. Clarot, S. Fontanay, A. D. Salem, M. Mourer, C.
Finance, R. E. Duval, and J. B. Regnouf-de-Vains, Bioorg. Med.
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Conflicts of interest
There are no conflicts to declare.
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