Novel pyranopyrazole derivatives comprising a benzoxazole core as antimicrobial inhibitors: Design, synthesis, microbial resistance and machine aided results

Article abstract of DOI:10.1016/j.bioorg.2020.103908

From a medical point of view lot of existing antibiotics became unusable because microbial gained strong antibiotic resistance. The combination of two compounds in one core may lead to kill such type of pathogens. Herein, we developed pyranopyrazole derivatives comprising benzoxazole moiety by green approach strategy and studied their antimicrobial performance on four bacteria and two fungi. As a result, most of the compounds delivered reliable toxicity to kill the pathogens. In those, 6a exhibited considerable activity against the microbial pathogens. Moreover, compounds 6d, 6l, and 6n showed prominent antibacterial activity. In addition, molecular docking studies of docked compounds revealed the strong bonding interaction with DNA-Gyrase and were docked into the intercalation location of DNA of the DNA-gyrase complex. The molecule bounded to the DNA stabilized by the H bonds, hydrophobic interactions, and π-π interaction. In addition, the linked 5-chlorobenazoxazole structure stabilized by the DT-8 and DG2009 of the F chain with pi-pi interactions. From the computer-aided results, it was observed that compound 6a demonstrated maximum docking score ?10.0 kcal/mole towards DNA-gyrase. Overall, this investigation suggested that these biologically active compounds can be utilized as leads for preclinical studies with the goal of developing newer antimicrobial drugs.

Full text of DOI:10.1016/j.bioorg.2020.103908

BioorganicChemistry100(2020)103908
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel pyranopyrazole derivatives comprising a benzoxazole core as
antimicrobial inhibitors: Design, synthesis, microbial resistance and
machine aided results
Guda Mallikarjuna Reddya,b, Avula Krishna Kumaric, Vemulapati Hanuman Reddyc,
⁎
Jarem Raul Garciab,
a Department of Chemistry, State University of Ponta Grossa, Ponta Grossa, Parana State, Brazil
b Ural Federal University, Chemical Engineering Institute, Yekaterinburg 620002, Russia
c Natural Product Chemistry, Indian Institute of Chemical Technology, Tarnaka 500007, Hyderabad, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
From a medical point of view lot of existing antibiotics became unusable because microbial gained strong an-
tibiotic resistance. The combination of two compounds in one core may lead to kill such type of pathogens.
Herein, we developed pyranopyrazole derivatives comprising benzoxazole moiety by green approach strategy
and studied their antimicrobial performance on four bacteria and two fungi. As a result, most of the compounds
delivered reliable toxicity to kill the pathogens. In those, 6a exhibited considerable activity against the microbial
pathogens. Moreover, compounds 6d, 6l, and 6n showed prominent antibacterial activity. In addition, mole-
cular docking studies of docked compounds revealed the strong bonding interaction with DNA-Gyrase and were
docked into the intercalation location of DNA of the DNA-gyrase complex. The molecule bounded to the DNA
stabilized by the H bonds, hydrophobic interactions, and π-π interaction. In addition, the linked 5-chlor-
obenazoxazole structure stabilized by the DT-8 and DG2009 of the F chain with pi-pi interactions. From the
computer-aided results, it was observed that compound 6a demonstrated maximum docking score −10.0 kcal/
mole towards DNA-gyrase. Overall, this investigation suggested that these biologically active compounds can be
utilized as leads for preclinical studies with the goal of developing newer antimicrobial drugs.
Synthesis
Antimicrobial
Docking studies
Binding energy
1. Introduction
encourage the investigation and progress of innovative antimicrobials
to benefit tackle worldwide antimicrobial resistance. [6]. In this con-
Seventy years ago, before the introduction of antibiotics as drugs,
microbial infections denoted a key reason of death. Unluckily, anti-
biotic resistance has grown-up and is nowadays spreading rapidly in-
spite of introduction of novel active antimicrobial medicines into the
market, possibly leading to a health emergency in the nearby future [1].
Nowadays, a lot of infections happen to cause by microorganisms that
are unaffected to most of the antibacterial drugs presently available [2].
It is expected that 70% of clinically isolated microbes are opposed to
the frequently recommended antibiotics including methicillin along
with additional b-lactam microbial resistant, like oxacillin and ampi-
cillin [3,4]. Interestingly, vancomycin antibiotic drug was resisted by
some microbial strains like Enterococcus faecium and Staphylococcus
aureus [5]. The WHO (World Health Organization) recently circulated a
list of antimicrobial-resistant ‘priority pathogens’ that involve a parti-
cular risk to community health. The vital goal of WHO is to guide and
text, the development of innovative biologically active combinations
under an environmentally friendly path is one of the most important
and successful strategy. Consequently, eco-catalyst, nontoxic reagents,
experimental simplicity, cost minimization and less hazardous solvent
medium are the greenest synthetic key points [7,8]. Scientific works
have also contributed to the growing pollutions and therefore harming
nature. Keeping the above understandings in mind, the current work bis
heterocyclic composites and their synthesis has been taken up for the
development of antimicrobials. A lot of compounds are known to con-
tain azole structural moieties with antimicrobial activity such as Ke-
toconazole, Fluconazole, Voriconazole, Itraconazole, Posaconazole and
more [9–12]. Due to the increasing resistance power of microbes, these
drugs become inefficient. There are some more synthetic heterocyclic
compounds which contain two nitrogen possess significant anti-
microbial activities. Among them, pyranopyrazoles and benzoxazoles
⁎ Corresponding author.
E-mail address: nagareddy.organic@gmail.com (J.R. Garcia).
https://doi.org/10.1016/j.bioorg.2020.103908
Received 20 January 2020; Received in revised form 20 March 2020; Accepted 30 April 2020
Availableonline06May2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

G.M. Reddy, et al.
BioorganicChemistry100(2020)103908
Scheme 1. Synthesis of pyranopyrazole linked benzoxazole derivatives.
Table 1
The in-vitro toxic values of prepared moieties 6(a–n) on bacteria.
Samples
Zone of inhibition (mm)
Gram-positive
Gram-negative
S. aureus (ATCC-19433)
B. subtilis (ATCC-6633)
25 μg/well 50 μg/well
P. vulgaris (ATCC-29213)
E. coli (ATCC-8739)
25 μg/well
50 μg/well
25 μg/well
50 μg/well
25 μg/well
50 μg/well
6a
6b
6c
6d
6e
6f
32
16
22
30
19
23
03
05
15
12
25
27
09
28
34
–
2
1
1
3
2
3
1
1
3
2
1
2
2
1
2
35
18
23
33
22
25
04
08
16
14
26
29
11
30
37
–
1
3
1
2
1
3
3
2
2
1
1
1
2
1
1
29
11
15
26
13
18
02
02
08
05
19
22
03
25
30
–
2
1
1
3
2
2
1
1
2
3
1
1
1
1
1
33
12
17
28
14
20
02
04
10
07
21
26
04
27
35
–
1
2
3
1
2
2
3
3
1
3
3
1
1
2
2
25
10
15
24
13
16
02
04
09
06
19
21
05
22
28
–
2
2
1
3
1
2
1
2
1
2
3
1
1
1
1
31
13
18
29
15
21
01
05
12
10
24
26
08
28
32
–
1
3
1
2
1
1
1
1
2
3
2
2
1
1
1
22
08
10
19
09
11
01
03
07
05
12
15
05
17
24
–
1
2
1
1
1
2
1
2
2
1
1
1
1
3
2
25
11
14
23
13
16
03
04
10
09
18
19
08
21
27
–
1
3
1
2
1
2
2
3
2
1
1
2
2
1
2
6g
6h
6i
6j
6k
6l
6m
6n
Ref
*
Ref = Ciprofloxacin; * = Control(DMSO).
are medicinally important moieties.
nitrogen elements are pyranopyrazoles. Pyranopyrazole and their de-
rivatives revealed many biological properties such as anti-inflammatory
[21,22], analgesic & antimicrobial [23,24] antibacterial [25] and an-
ticancer [26]. A combination of the above two biological significance
heterocycle structures in a single moiety delivers to the materialization
of a fresh two heterocycle contained system which is prospective to
exhibit novel biological activities by a synergistic influence. With our
familiar knowledge in the field of bis heterocycles, not much work
Benzoheterocycles like benzoxazoles are prominent heterocycles.
Because in the medicinal chemistry point of view these compounds
exhibited different types of medicinal properties [13–15]. Moreover,
almost ten efficient drugs in the top two hundred medical products are
benzoxazole related products [16]. Benzoxazole composites are key
pharmacophore with no toxicities, which have delivered a diversity of
medicinal activities [17–20]. Besides, the other compounds having two
2

G.M. Reddy, et al.
BioorganicChemistry100(2020)103908
Table 2
(2:1) in the volume of 5 mL was added followed by the addition of
green montmorillonite K10 catalyst (half percentage by mass virtual to
5a). The total set up kept at 60–70°temperature for 5–7 hrs. After TLC
check, the reaction mixture filtered through funnel which contained
cotton, the product contained filtrate solvent evaporated using rotary
evaporate. The outcome product solid 6a recrystallized with the help of
methanol. The catalyst contained cotton dipped into ethyl acetate sol-
vent. Consequently, catalyst settle down the bottom, decanted the sol-
vent and the catalyst dried in oven at 50°. The recovered clay reused for
further reactions. Similarly, the other targets 6(b–n) were synthesized.
The in-vitro toxic values of prepared moieties 6(a–n) on fungi.
Samples
Zone of inhibition (mm)
A. flavus (MTCC-1884)
A. niger (MTCC-1881)
25 μg/well 50 μg/well
25 μg/well
50 μg/well
6a
6b
6c
6d
6e
6f
35
15
19
33
18
22
04
06
14
11
24
27
09
31
39
–
1
2
1
3
1
2
1
3
2
1
1
1
3
3
1
41
19
24
38
21
26
08
11
16
15
29
32
13
35
43
–
3
1
3
3
2
1
3
2
2
3
1
2
3
2
2
28
11
15
25
13
17
02
04
10
08
18
21
06
22
31
–
3
1
1
2
1
3
1
1
1
1
3
2
2
3
1
32
14
18
30
16
20
03
05
12
09
23
25
08
26
36
–
2
1
1
1
2
1
1
2
2
2
3
2
3
1
1
2.1.3. 4-(o-Tolyl)-3-(((5-chlorobenzo[d]oxazol-2-yl)thio)methyl)-6-
amino-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (6a)
6g
6h
6i
Light Yellow Powder; Yield 91%; m.p. 175–179 °C; IR (KBr) (cm−1):
3437, 3342 (NH2), 2242 (C^N), 1668 (C]N) 1641 (C]C); 1H NMR
(500 MHz, CDCl3): δ 2.29 (s, 3H, CH3), 3.61 (s, 2H, CH2), 4.51 (s, 1H,
CH), 7.34–7.70 (m, 7H, Ar-H), 8.56 (br, 2H, NH2) ppm; 13C NMR
(75 MHz, CDCl3): δ 167.2 (Amine Attached C), 161.3 (N]C-S), 155.4
(OeCeNH), 150.9, 142.6, 135.2, 133.1, 132.2, 130.5, 129.6, 127.2,
126.9, 126.2, 124.5, 123.2, 122.5, 120.1 (Aromatic carbon), 109.3
(CN), 56.5(C-CN), 33.5 (CH), 29.2 (CH2), 16.9 (CH3). ppm; HRMS: m/z
calcd for C22H17ClN5O2S (M + H)+ 450.0791; Found 450.0789.
6j
6k
6l
6m
6n
Ref
*
Ref = Ketoconazole, * = Control (DMSO).
reported in which benzoxazole and pyranopyrazole structures in one
moiety. Thus, the current work describes the green synthesis of ben-
zoxazole fused pyranopyrazoles and their biological evaluation. Fur-
thermore, Molecular docking is also discussed.
2.1.4. 4-(2-Chlorophenyl)-3-(((5-chlorobenzo[d]oxazol-2-yl)thio)
methyl)-6-amino-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (6b)
Light Yellow Powder; Yield 89%; m.p. 215–217 °C; IR (KBr) (cm−1):
3442, 3339 (NH2), 2241 (C^N), 1660 (C]N) 1641 (C]C); 1H NMR
(500 MHz, CDCl3): δ 3.62 (s, 2H, CH2), 4.58 (s, 1H, CH), 7.30–7.85 (m,
7H, Ar-H), 8.66 (br, 2H, NH2) ppm; 13C NMR (75 MHz, CDCl3): 169.0
(Amine Attached C), 161.5 (N]C-S), 156.3 (OeCeNH), 150.2 , 144.3,
135.4, 133.4, 131.6, 130.2, 128.4, 126.3, 125.9, 125.3, 124.5, 123.2,
122.1, 120.2 (Aromatic carbon), 110.3 (CN), 56.0 (C-CN), 36.1 (CH),
31.3 (CH2). ppm; HRMS: m/z calcd for C21H14Cl2N5O2S (M + H)+
470.0245; Found 470.0241
2. Experimental
2.1. Chemistry
All the initial compounds, reagent and solvents were acquired and
commercially available. Melting points verified by micro melting point
device and were incorrect. Tetramethylsilane (TMS) as an internal
standard (δ ═0) for 1H NMR, and for 13C NMR, CDCl3 (δ ═ 77.27) was
used as internal standard. For 1H NMR: 500 MHz and for 13C NMR:
75 MHz used. Carbon NMR spectra were obtained with complete proton
decoupling. Low-resolution MS and HRMS data were obtained using ESI
ionization.
2.1.5. 4-(3-hydroxyphenyl)-3-(((5-chlorobenzo[d]oxazol-2-yl)thio)
methyl)-6-amino −1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (6c)
Yellow Powder; Yield 92%; m.p. 186–188 °C; IR (KBr) (cm−1):
3445, 3331 (NH2), 2230 (C^N), 1674 (C]N) 1641 (C]C); 1H NMR
(500 MHz, CDCl3): δ 3.69 (s, 2H, CH2), 4.59 (s, 1H, CH), 7.29–7.84 (m,
7H, Ar-H), 8.49 (br, 2H, NH2) ppm; 13C NMR (75 MHz, CDCl3): δ 168.3
(Amine Attached C), 161.2 (N]C-S), 155.7 (OeCeNH), 151.6, 141.8,
135.4, 134.1, 133.2, 132.1, 130.9, 129.5, 128.3, 126.2, 125.2, 123.2,
121.5, 120.4 (Aromatic carbon), 109.5 (CN), 56.2 (C-CN), 35.5 (CH),
30.3 (CH2). ppm; HRMS: m/z calcd for C21H15ClN5O3S (M + H)+
452.0584; Found 452.0580.
2.1.1. Neat reaction procedure for synthetic routes of 6a, 6b, 6c and 6d
The four starting reagents, 5-chlorobenzo[d]oxazole-2-thiol (1a,
one mmol), ethyl 4-chloro-3-oxobutanoate (2, one mmol), hydrazine
(3, one mmol), malononitrile (4, one mmol) and 2-methylbenzaldehyde
(5a, one mmol) were taken in a round bottom flask and proceeded the
reaction at 60°under inert atmosphere for 5–7 hrs. After the completion
of reaction checked by TLC, the product 6a was isolated by using
chromatography technique and ethyl acetate-hexane solvent mixture as
eluent. The parallel procedure was followed to prepare 6(b–d).
2.1.6. 4-Phenyl-3-(((5-chlorobenzo[d]oxazol-2-yl)thio)methyl)-6-Amino-
1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (6d)
Light Yellow Powder; Yield 90%; m.p. 206–208 °C; IR (KBr) (cm−1):
3442, 3330 (NH2), 2235 (C^N), 1669 (C]N) 1635 (C]C); 1H NMR
(500 MHz, CDCl3): 3.69 (s, 2H, CH2), 4.50 (s, 1H, CH), 7.19–7.84 (m,
8H, Ar-H), 8.54 (br, 2H, NH2) ppm; 13C NMR (75 MHz, CDCl3): δ 168.3
2.1.2. Solvent and catalyst used preparation procedure of targets 6(a–n)
To all starting compounds of product 6a, solvent water–ethanol
Table 3
The MIC(MBC/MFC) of compounds 6d, 6f and 6g.
Samples
MIC(MBC/MFC)
S. aureus
B. subtilis
P. vulgaris
E. coli
A. flavus
A. niger
6a
12.5 (25)
25 (50)
25 (50)
6.25
25 (50)
50 (200)
25 (200)
6.25
50 (200)
25 (100)
100 (> 200)
12.5
100 (> 200)
50(200)
100 (> 200)
12.5
25 (> 200)
25 (50)
50 (100)
–
100 (> 200)
50 (> 200)
25 (100)
–
6d
6n
Ref1
Ref2
–
–
–
–
6.25
12.5
Ref1 = Ciprofloxacin; Ref2 = Ketoconazole.
3

G.M. Reddy, et al.
BioorganicChemistry100(2020)103908
Table 4
Protein ligand interaction energies of ligands and active residues (chain in parenthesis) with DNA-gyrase calculated using binding analysis.
Molecule
Binding affinity (in kcal/
Residues
mol)
6a
6d
6f
−10.0
−9.8
−8.6
−8.4
−8.5
−8.9
−9.1
−9.8
Thy-8(F), Gua-2009(F), Gua-2010 (F), Cyt-2011(E), Cyt-2012(E), Ade-2013(E), Ser-84(A), Arg-122(C), Asp-437(B), Arg-458(B), Gly-
459 (B), Asn-476(B), Glu-477(B)
Thy-8(F), Gua-2009(F), Gua-2010 (F), Cyt-2011(E), Cyt-2012(E), Ade-2013(E), Ser-84(A), Arg-122(C), Asp-437(B), Arg-458(B), Gly-
459 (B), Asn-476(B), Glu-477(B)
Thy-8(F), Gua-2009(F), Cyt-2012(E), Ade-2013(E), Thy-2014(E), Arg-122(C), Arg-458(B), Gly-459 (B), Lys-460(B), Ile-461(B), Leu-
462(B), Asn-475(B), Asn-476(B), Glu-477(B)
6g
6h
6k
6l
Thy-8(F), Gua-2009(F), Gua-2010 (F), Cyt-2012(E), Ade-2013(E), Ser-84(A), Arg-122(C), Asp-437(B), Arg-458(B), Gly-459 (B), Asn-
475(B), Asn-476(B), Glu-477(B)
Thy-8(F), Gua-2009(F), Gua-2010 (F), Cyt-2011(F), Cyt-2012(E), Ade-2013(E), Ser-84(A), Arg-122(C), Asp-437(B), Arg-458(B), Asn-
476(B)
Thy-8(F), Gua-2009(F), Gua-2010 (F), Cyt-2011(E), Cyt-2012(E), Ade-2013(E), Ser-84(A), Arg-122(C), Asp-437(B), Arg-458(B), Gly-
459 (B)
Thy-8(F), Gua-2009(F), Gua-2010 (F), Cyt-2011(F), Cyt-2012(E), Ade-2013(E), Arg-122(C), Asp-437(B), Arg-458(B), Gly-459 (B),
Asn-476(B), Glu-477(B)
6n
Thy-8(F), Gua-2009(F), Gua-2010 (F), Cyt-2011(F), Cyt-2012(E), Ade-2013(E), Arg-122(C), Asp-437(B), Arg-458(B), Gly-459 (B),
Asn-476(B), Glu-477(B)
6f
6k
6l
6n
Fig. 1. Molecules 6f, 6k, 6l and 6n were docked in the active site of the DNA-gyrase cleavage complex of S. aureus (PDB_ID:5CDQ). Inhibitor molecules shown in
white color stick style and the DNA side chains and amino acid side chains are shown in line style.
(Amine Attached C), 162.5 (N]C-S), 155.3 (OeCeNH), 150.3, 144.1,
138.2, 136.1, 132.2, 130.9, 126.4, 123.2, 122.5, 121.3, 120.5, 118.4
(Aromatic carbon), 110.2 (CN), 57.3 (C-CN), 35.4 (CH), 28.2 (CH2).
ppm; HRMS: m/z calcd for C21H15ClN5O2S (M + H)+ 436.0635; Found
436.0632.
2.3. Molecular docking study
Molecular docking studies of molecules into the crystal structures of
DNA-gyrase cleavage complex of S. aureus (Gram-positive bacteria)
with PDB_ID:5CDQ were carried out using Autodock Vina software
[28,29], open source molecular docking software. We have generated a
grid box with desired parameters around the active site of DNA-gyrase
cleavage complex of S. Aureus (PDB_ID:5CDQ) [30] as centre
:x = 40.124, y = -46.732, z = 64.933 and grid box size: x = 22,
y = 36, z = 26. We generated 20 conformations in each docking output
by using advanced Genetic algorithm method in Vina. Protein/DNA
complex and molecule input preparations and docking output analysis
were carried out using MGLTools-1.5.6 software.
2.2. Antimicorbial assay
By using four bacterias and two fungi pathogens, microbial toxicity
results were screened according to the method sited there in [27]. For
this, Ciprofloxacin and Ketoconazole were used as references and in 25
and 50 μg/well concentrations were used.
4

G.M. Reddy, et al.
BioorganicChemistry100(2020)103908
Molecule-6a
Molecule-6d
Molecule-6g
Molecule-6h
Fig. 2. Molecules 6a, 6d, 6g and 6h were docked in the active site of the DNA-gyrase cleavage complex of S. aureus (PDB_ID:5CDQ). Inhibitor molecules shown in
white color stick style and the DNA side chains and amino acid side chains are shown in line style.
3. Results and discussion
conditions, the rest of the products 6(e–n) were prepared by this
method, resulted in a high yield of the products. Interestingly, the spur
K10 catalyst was recycled up to five times and used for other reactions.
This was the additional advantage of this eco-friendly method over
other methods having similar type of molecules [31,32]. Moreover,
synthetic method under environmental favor conditions is the most
valuable research method over other. In addition, no harmful catalysts
were used in this method. Compare to other reported methods, using of
eco-friendly solvent media and quantitative yield of the product are the
most advantageous tools that are existed in this method
3.1. Chemistry
The simple and green synthetic method for the preparation of pyr-
anopyrazole attached benzoxazole and their derivatives were depicted
in scheme 1. In addition mechanism of product formation was depicted
in scheme 2. Foremost, few compounds 6a, 6b, 6c, and 6d were pre-
pared in different eco-friendly methods to rationalize the best method.
After, other targets 6(e-n) were prepared via an effective technique. In
this fashion, all starting compounds of product 6a were mixed and kept
the reaction without solvent and catalytic agent. As a result, 6a was
formed, but only 32% yield was obtained along with byproduct (7a)
(Scheme 3). This byproduct was formed due to the side reaction be-
tween aldehyde and hydrazine. The same situation existed in the case of
targets 6b, 6c and 6d, their outcome yields in this method were 28%,
34%, and 30%, correspondingly. In addition, side products (7(b-d))
also formed. The side product formation in this method may lead to low
yield of the product. The other cause of the trace profits may be due to
deficiency of solvent or reagent or both. Further, to obtain higher yields
of the product along with sustaining the ecologically benign condition,
we decided to use the montmorillonite K10 catalyst and solvent. When
K 10 catalyst and a green solvent such as a mixture of water–ethanol
(2:1) were added to the flask which already contained total preliminary
compounds of product 6a and proceeded reaction again. Sub-
sequently, 6a was formed with a 91% yield. In the identical technique,
the amalgams 6b, 6c, and 6d resulted 89, 92 and 90% of yields. That
means outstanding results were observed in this method. Moreover,
absent of byproduct formation was observed in this second method. The
reason may be due to the presence of a solvent that will help to mix the
reactants clearly and the spur which promotes all initial compounds to
target, as a result, yields come quantitatively. Based on the optimized
3.2. Compounds toxic effect on microbial
To detect any toxicity behavior of above synthetic molecules on
microbes the available four bacteria and two fungi (Table 1) were
chosen. The antimicrobial screening of all synthesized conjugates
against four bacteria are tabulated in Table 1. We were delighted to
perceive that from the bacterial point of view all compounds 6(a–n)
delivered reliable toxicity nature. In those, some compounds exhibited
excellent antibacterial nature on four bacteria, few compounds dis-
played moderate antibacterial performance and few compounds be-
haved mild antibacterial potential towards four bacteria. Captivatingly,
all 6(a–n) products exhibited excellent antibacterial nature against
Gram-positive bacteria only. Also, against S. aureus, these compounds
displayed good antibacterial performance against S. aureus bacteria.
When we observed the competition between the active compounds
regarding the toxicity, ortho methyl substitution on benzene ring
moiety gave the highest killing nature towards all bacteria. Its anti-
bacterial results were closed to reference drug Ciprofloxacin. Further-
more, 6d, 6 l, and 6n screened compounds showed prominent anti-
bacterial activity, while the other moieties displayed moderate to least
activities.
5

G.M. Reddy, et al.
BioorganicChemistry100(2020)103908
S
O
NC
N
N
N
H2N
O
H
Cl
6a
Docking score -10.0 kcal/mol
6a
S
O
NC
N
N
N
H2N
O
H
Cl
6d
Docking score -9.8 kcal/mol
6d
O
S
O
NC
N
N
N
H2N
O
H
Cl
6n
Docking score -9.8 kcal/mol
6n
Fig. 3. Binding affinity and their energies of compounds 6a, 6d and 6n.
These compounds were also evaluated against two fungi such as A.
nigar and A. flavus and the results are represented in Table 2. The
deadliness examination was only authenticated up to two meditations
of 25 and 50 µg per well and suits our purpose to detect prominent
antifungal active compounds. Molecules tangled within this
investigation (6(a–n)) were determined to be high to minimal toxic
contrary to the two fungal strains. Moreover, total compounds were
displayed high toxic nature towards A. niger than other fungi. Besides,
molecule 6a holds most deadliness than other molecules, while the
composites 6d, 6f, 6k, 6l, and 6n displayed good to moderate
6

G.M. Reddy, et al.
BioorganicChemistry100(2020)103908
Fig. 4. Graphical representation of in vitro toxicity nature of all compounds against bacteria.
Fig. 5. Graphical representation of in vitro toxicity nature of all compounds against fungi.
antifungal properties. On the other hand, remaining molecules con-
tained low to least poisonousness towards fungi
fungicidal volume of more than 2X lowest inhibitory concentration
(i.e.MIC).
3.3. Minimum inhibitory concentration (MIC) and minimum bactericidal/
fungicidal concentration (MBC/MFC)
3.4. Molecular docking studies and SARs analysis
The binding affinity of eight molecules are shown in table 4, to-
wards the DNA-gyrase cleavage complex of S. aureus DNA binding site
surrounded by the protein residues, was analyzed using molecular
docking studies. The binding affinity of the molecule showing strong
interaction energies with the DNA active site and the results were
shown in table 4.
To estimate the MIC and MBC/MFC, most effective compounds 6a,
6d and6n were preferred because of their strong antibacterial effect on
all bacterial and fungal pathogens which were mentioned above. From
the tabulated grades (Table 3), 6a exhibited lowest bacterial MBC result
equal to 2X lowest inhibitory volume (i.e.MIC) towards both Gram-
positive germs, whereas, 6d and 6n have 2X lowest inhibitory con-
centration (i.e.MIC), which was equal to lowest bacterial volume
(i.e.MBC) value against S. aureus pathogen only. It was detected that
three composites delivered the smallest volumes to kill the Gram-po-
sitive pathogens only. Also, the smallest fungicidal volume (i.e. MFC)
which was equal to double to lowest inhibitory concentration (i.e. 2X
MIC) happened. This result belongs to 6d and 6n against A. flavus pa-
thogen. Meanwhile, the other target 6a displayed the lowest bacterial/
The crystal structure of the DNA-gyrase cleavage complex of S.
aureus (PDB_ID:5CDQ) has been considering for the docking of the eight
molecules in Table 4. The crystal structure contains an inhibitor Mox-
ifloxacin intercalation in the E and F chains of DNA. The DNA binding
protein with A and C chains are stabilizing the binding of the DNA with
the bound Moxifloxacin. Compounds were docked into the intercalation
site of DNA of the DNA-gyrase complex. The binding to the DNA was
stabilized by the hydrogen bonds, hydrophobic interactions, and Pi-Pi
7

G.M. Reddy, et al.
BioorganicChemistry100(2020)103908
interaction. In all the eight molecules, the side chain 5-chlor-
obenazoxazole ring stabilized by the thymine ring (DT-8) and Guanine
(DG2009) of the F chain with pi-pi interactions. The 1,4 dihydropyr-
anopyrazole ring NH is forming a stable hydrogen bond with side-chain
carboxylic oxygen of ASP-437 of B chain, it was observed in active
molecules 6a, 6d, 6l, and 6n (Fig. 1&2). The phenyl ring of molecule
6d, 2-methyl phenyl of 6a and 4-hydroxy phenyl of 6n substituted on
dihydropyranopyrazole ring stabilized by the B chain amino acid re-
sidues ASN-476, ARG-458, GLU-477. The molecule 6n cyanide nitrogen
forming hydrogen bond with the oxygen atom of E chain cytosine sugar
ring. The electron-withdrawing groups –NO2, -OMe, -F on the phenyl
ring is stabilized by only ASN476. The loss of hydrogen bond with ASP-
437 observed in molecules 6g, 6h, 6k, and 6f may cause a decrease in
the activity of these molecules. The interaction of DNA bases and the
amino acid side-chains proteins with active 6a and 6d molecules and
less active molecules 6g and 6h were shown in Fig. 2
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103908.
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