S1
N-Phosphorylated Derivatives of 5-Nitroindazole as Antimicrobial and
Antioxidant Agents and Docking Study against DNA GyraseA
SK Thaslim Basha1, Devineni Subba Rao1, Golla Madhava1, Shaik Thahir Basha2, Mundla
Nagalakshmi Devamma3, Saddala Madhu Sudhana4, Asupatri Usha Rani4, Chamarthi
Naga Raju1*
1
Department of Chemistry, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh,
India.
2
Department of Virology, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India.
3
4
Department of Botany, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India.
Department of Zoology, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India.
E-mail: rajuchamarthi10@gmail.com
Supplemental Materials
Materials and Methods
Chemicals and reagents were purchased from sigma-aldrich and merck and used without
further purification. The solvents were distilled according to the standard procedures before being
used. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 for the investigation
of progress of the reaction and employed the column chromatography using 100-200 silica gel
mesh for purification of the products. Guna melting point apparatus was used to determine melting
points and are uncorrected. Bruker ALPHA interferometer instrument was used for infrared
spectroscopy. 1H, 13C and 31P NMR were recorded on Bruker 500 MHz instrument using DMSOd6 solution and operating at 500 MHz, 125 MHz and 161.9 MHz respectively. TMS was used as
internal standard for recording of 1H and
13
C spectra, and 85% H3PO4 for
31
P NMR spectra as
S2
external standard. Mass spectra were recorded on ESI-MS in positive mode and the elemental
analysis was performed in FLASH EA Thermo Finnigan 1112 instrument.
Biological
Antibacterial activity
The newly synthesized compounds 7a-l were screened for their antibacterial activity against
two Gram-positive bacteria viz Bacillus subtilis (MTCC-441) and Staphylococcus aureus (MTCC737) and two Gram-negative bacteria viz Escherichia coli (MTCC-443) and Pseudomonas
aeruginosa (MTCC-741) by agar well diffusion method.25 Dimethylsulphoxide (DMSO) was used
as diluent to prepare 100 µg/mL concentrations of test sample and the antibiotic Streptomycin was
used as standard. 20 mL of Muller Hinton Agar medium was poured in each petri plate. After
solidification of the medium, 24 h old bacterial culture containing approximately 105-106 colony
forming units (CFU) per mL was spread on the surface of the medium and 5-8 mm wells were
created on the surface of the culture plates with sterile metallic borer and 100 µg/mL concentration
of 1 mL of test samples were loaded in each well and incubated at 37 oC for 24 h. The inhibition
of the test pathogens by the synthesized compounds around the wells was measured to investigate
antibacterial (zone of inhibition) activity of the samples under the study. The experiments were
conducted in triplicate and average tabulated as final result. The antibacterial screening data was
summarized in Table S 1 and activity of the title products were compared with the standard drug
Streptomycin.
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Table S 1 In vitro antibacterial activity of the synthesized compounds 7a-l.
Zone of Inhibition* (mm)
Compd.
Bacillus
subtilis
Staphylococcus
aureus
Escherichia
coli
Pseudomonas
aeruginosa
(MTCC-441)
(MTCC-737)
(MTCC-443)
(MTCC-741)
7a
19 ± 0.85
18 ± 0.45
18 ± 0.55
12 ± 0.65
7b
17 ± 0.74
19 ± 0.90
19 ± 0.44
11 ± 0.35
7c
19 ± 0.45
18 ± 0.52
15 ± 0.61
11 ± 0.65
7d
15 ± 0.47
19 ± 0.42
15 ± 0.39
12 ± 0.35
7e
12 ± 0.39
17 ± 0.74
13 ± 0.64
15 ± 0.52
7f
15 ± 0.55
15 ± 0.64
10 ± 0.68
13 ± 0.45
7g
19 ± 0.35
18 ± 0.55
18 ± 0.59
11 ± 0.64
7h
18 ± 0.62
18 ± 0.32
17 ± 0.43
14 ± 0.29
7i
19 ± 0.52
17 ± 0.68
18 ±0.72
12 ± 0.79
7j
18 ± 0.25
18 ± 0.36
19 ± 0.49
16 ± 0.47
7k
16 ± 0.34
19 ± 0.86
14 ± 0.66
13 ± 0.39
7l
15 ± 0.79
17 ± 0.54
16 ± 0.72
12 ± 0.50
Standard
25
26
25
27
Standard: Streptomycin
*Zone of inhibition at 100 µg/mL
# mean of triplicaton
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Antifungal activity
The antifungal activity of the target compounds 7a-l were evaluated against three soil borne
fungi viz Aspergillus niger, Aspergillus flavus and Sclerotium rolfsii,and one foliar pathogen
Colletotrichum gloeosporioides adopting poisoned food technique.26 Test compounds were
dissolved in dimethylsulphoxide (DMSO) and adjusted to 150µg/mL concentration. 100 mL of
PDA (Potato dextrose Agar) medium and required amount test sample were mixed thoroughly and
poured into petri dishes. After solidification 8 mm diameter of fungal discs were inoculated in the
center of the petri plates and incubated at 28±2 oC for 1 week. Growth and zone of inhibition were
measured after recording full growth of the pathogen in control plate. PDA medium without test
sample was used as negative control and nystatin was used as standard drug. Percentage of
inhibition of mycelia was calculated using following equation. Three replications were maintained
in each treatment and the average value was taken as final results are shown in Figure S 2.
I
Where
C T
X 100
C
I= Percentage of inhibition of mycelia growth.
C= Radial growth of mycelium over control.
T= Radial growth of mycelium on treatment.
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80
Aspergillus niger
Aspergillus flavus
Sclerotium rolfii
Colletotrichum gloeosporioides
70
% of Inhibition
60
50
40
30
20
10
0
Compounds
Figure S 1 In vitro antifungal activity of N-phosphorylated derivatives of 5-nitroindazole.
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DPPH radical scavenging activity
A simple 2, 2- diphenyl 1-picrylhydrazyl (DPPH) radical assay27 was employed to investigate
the radical scavenging activity the synthesized compounds 7a-l. Ascorbic acid is a natural
antioxidant, used as positive control for comparing the antioxidant activity of the title compounds.
DPPH (4 mg) was dissolved in 100 mL of methanol to obtain0.004% concentration of DPPH
solution. Title compounds concentrations, 50 and 100 µg/mL were prepared by adding aliquot
amount of methanol.1 mL of these test solutions were added to 4 mL of DPPH methanol solution
containing in a set of test tubes and then incubated for 30 min in darkwith occasional shaking.
Absorbance of the incubated test samples were measured at 517 nm using UV-Visible
spectrophotometer. The percentage of DPPH radical scavenging activity of the title compounds
were calculated by following equation. The experiments were evaluated intriplicate and mean
values were summarized in Table 3.
% 𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏𝒐𝒇𝑫𝑷𝑷𝑯 =
(𝑨𝒃𝒔𝒄𝒐𝒏𝒕𝒓𝒐𝒍 − 𝑨𝒃𝒔𝒔𝒂𝒎𝒑𝒍𝒆 )
× 𝟏𝟎𝟎
𝑨𝒃𝒔𝒄𝒐𝒏𝒕𝒓𝒐𝒍
Where ‘Abscontrol’ represents the absorbance of the DPPH solution and ‘Abssample’ represents the
absorbance of the sample and DPPH solution.
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Hydrogen peroxide scavenging activity
H2O2 antioxidant activity of the title compounds 7a-l was determined according to the method
of Ruch et al.28 with small modifications. A solution of hydrogen peroxide (40 mM) was prepared
in phosphate buffer (pH 7.4). Different concentrations (50 and 100 µg/mL) of test samples were
prepared in phosphate buffer. The prepared test samples (3.4 mL) were added to H2O2 solution
(0.6 mL, 40 mM) containing in a set of test tubes and incubated for 30 min in dark with occasional
shaking. The absorbance of the test samples were recorded at 230 nm using a spectrophotometer.
The percentage of scavenging activity was calculated by the following equation. Natural
antioxidant like ascorbic acid was used as a standard. The experiments were carried out in triplicate
and mean values were reported as final results.
% 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛𝑜𝑓H2 O2 =
(𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒 )
× 100
𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙
Where ‘Abscontrol’ represents the absorbance of the standard H2O2 solution and ‘Abssample’
represents the absorbance of the sample and the standard H2O2 solution.
S8
Table S 2 In vitro antioxidant activity of the synthesized compounds 7a-l by DPPH method and
Hydrogen peroxide method.
DPPH method
H2O2 method
Compd.
50 µg/mL
100 µg/mL
50 µg/mL
100 µg/mL
7a
51.85 ± 0.47
61.72 ± 0.57
47.62 ± 0.25
59.12 ± 0.17
7b
34.56 ± 0.58
45.97 ± 0.61
39.48 ± 0.41
50.97 ± 0.39
7c
43.20 ± 0.55
59.55 ± 0.49
50.14 ± 0.54
67.64 ± 0.48
7d
67.77 ± 0.12
71.59 ± 0.27
60.31 ± 0.67
77.34 ± 0.51
7e
56.61 ± 0.29
68.23 ± 0.36
52.87 ± 0.12
60.48 ± 0.32
7f
59.25 ± 0.41
69.45 ± 0.40
49.70 ± 036
58.60 ± 0.47
7g
64.07 ± 0.69
70.07 ± 0.31
53.18 ± 0.29
63.89 ± 0.31
7h
52.43 ± 0.62
68.11 ± 0.42
37.91 ± 0.71
43.27 ± 0.92
7i
45.78 ± 0.28
57.38 ± 0.68
41.28 ± 0.49
54.18 ± 0.32
7j
67.84 ± 0.37
74.24 ± 0.33
69.05 ± 0.37
79.90 ± 0.40
7k
51.59 ± 0.39
63.82 ± 0.19
57.64 ± 0.33
52.47 ± 0.39
7l
39.58 ± 0.22
46.21 ± 0.45
55.19 ± 0.25
69.56 ± 0.59
Standard
79.45
82.15
80.51
85.40
Blank
--
--
--
--
Standard-Ascorbic acid; Blank- Methanol; -- indicates No radical scavenging activity
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Table S 3 Molecular docking studies of synthesized compounds 7a-l against DNA GyraseA
enzyme (E. coli).
Interactions
Entry
Compd
Protein------Ligand
1
7a
2
7b
3
7c
4
7d
5
7e
6
7f
7
7g
8
7h
ILE264CN-------OH
PRO95CO--------ON
LYS298CO--------NH
ARG518ON------ON
GLN267CN-------ON
TYR266CO------ ON
TYR266CO-------NC
TYR266CO------OH
GLN267CN-------OP
HIS45CN------NO
HIS45CN-----OH
THR88CO------OH
ARG91CN-----OH
ARG91CN-----NO
TRP59CO------HO
ARG126CN------ON
ARG126CN------ OH
ARG126CN------ON
ILE130CN-------OP
ILE112CN------ OP
SER97CO-------OH
TYR266CO------OH
TYR266CO------NC
TYR266CO------ON
GLN267CN------ON
GLN94CN------OP
ILE112CN-------OH
VAL268CO------ OH
Binding
energies
(kcal/mol)
Bond length
(Ǻ)
Bond angle
-7.6
3.41
119.36
3.45
3.47
3.39
3.27
3.02
3.03
3.02
2.82
3.44
3.26
2.93
3.05
3.39
2.70
3.08
3.03
3.43
2.83
112.83
125.78
116.60
146.75
131.32
136.06
153.22
148.96
136.84
87.11
136.33
131.91
121.85T
130.68
111.61
85.45
120.53
116.72
2.83
116.76
2.89
3.14
3.13
3.11
2.93
2.89
3.03
2.92
108.60
140.03
123.30
211.07
138.63
135.84
127.55
105.04
-7.9
-7.7
-7.8
-7.6
-7.9
-7.7
-7.6
S 10
9
7i
10
7j
11
7k
12
7l
13.
5nitroindazol
14.
Standard
Standard-Streptomycin
ILE112CN-------OP
THR219CO-----OH
THR219CO-----ON
HIS262CO-----ON
ILE264CN---- OH
ILE264CN---- OH
LYS298CO-----OH
HIS45CN-------ON
TYR266CO-------NH
TYR266CO-------OP
GLN267CN------- OP
ARG517CN------OH
ARG517CN------NC
ARG517CN------ON
GLN94CO------OP
SER97CO------- OP
SER97CO-------OC
ALN117CN-------ON
GLY40CO-----ON
ASN165CN---NO
ASN165CN---ON
ASN165CN---ON
ASN169CN---ON
LYS42CN------OC
SER97CO-----NC
SER97CO-----OH
ILE112CO-----NC
ASN169CO---OH
SER172CN----OC
SER172CO----OC
TYR266CO----OC
TYR266CO----NC
GLN267CN---OC
-7.5
-9.2
-8.3
-6.9
-6.7
-6.3
3.09
2.93
3.32
3.55
3.06
3.16
3.00
3.36
3.10
3.12
3.05
2.87
3.36
3.17
3.34
3.15
3.15
3.00
3.25
3.47
2.99
3.21
3.26
2.81
3.11
2.95
2.97
3.04
3.06
3.40
3.07
2.88
2.84
110.67
92.94
103.43
123.95
114.86
117.78
84.26
116.54
154.76
104.34
113.19
132.94
113.46
92.59
160.63
140.54
108.69
115.74
117.95
117.92
106.68
118.30
92.72
156.95
88.06
110.64
104.99
148.78
98.61
160.82
108.46
116.63
133.14
S 11
Figure S 2 Crystal structure of DNA Gyrase A (GyrA).
Concentration (µg/mL)
100
90
80
70
60
50
40
30
20
10
0
89.47
88.2
IC50 values
57.87
52.6
46.21
46.99
45.68
18.53 21.43 22.17 19.7
18.21
17.3
Title Compounds
Figure S 3 Half inhibitory concentration of synthesized compounds by DPPH method.
S 12
7a
7l
7b
7c
7d
7e
7f
7g
7h
7i
S 13
Figure S 4 Graphical representation of interaction of 7a-l compounds with active site of
DNA gyraseA (E.coli).
Structural activity relationships studies of 7j and 7f
Table S 3 Molecular properties calculated by Molinspiration online server.
Compd.
miLogp
TPSA
natoms
MW
nON
nOHNH
nviolations
nrotb Volume
7j
4.008
109.323
34.0
498.86
10
0
0
6
404.04
7f
3.21
96.431
29.0
435.80
9
0
0
5
353.35
Table S 4 Bioactivity calculated by Molinspiration online server.
Compd.
GPCR
Ion channel
Kinase
modulator
inhibitor
Nuclear
receptor
ligand
Protease
Enzyme
inhibitor
inhibitor
7j
0.07
-0.02
0.26
-0.04
0.09
0.12
7f
0.04
-0.06
0.22
-0.01
0.11
0.08
Table S 5 Toxicity predicted by Lazar Toxicity Predictions tool.
Compd. Mutagenic
Carcinogenic
LD50
Dose
mmol
7j
0.038
0.0109
0.134
0.11
7f
0.042
0.0113
0.146
0.11
S 14
Figure S 5 1H NMR of 4-Chlorophenyl 5-nitro-1H-indazol-1-yl(4-(pyridin-2-yl)piperazin-1-yl)phosphinate (7j).
S 15
Figure S 6 13C NMR of 4-Chlorophenyl 5-nitro-1H-indazol-1-yl(4-(pyridin-2-yl)piperazin-1-yl)phosphinate (7j).
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Figure S 7 31P NMR of 4-Chlorophenyl 5-nitro-1H-indazol-1-yl(4-(pyridin-2-yl)piperazin-1-yl)phosphinate (7j).
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