Design and biological evaluation of a novel type of potential multi-targeting antimicrobial sulfanilamide hybrids in combination of pyrimidine and azoles

Article abstract of DOI:10.1016/j.bmcl.2020.126982

This work explored a novel type of potential multi-targeting antimicrobial three-component sulfanilamide hybrids in combination of pyrimidine and azoles. The hybridized target molecules were characterized by ¹H NMR, ¹³C NMR and HRMS spectra. Some of the developed target compounds exerted promising antimicrobial activity in comparison with the reference drugs norfloxacin and fluconazole. Noticeably, sulfanilamide hybrid 5c with pyrimidine and indole could effectively inhibit the growth of E. faecalis with MIC value of 1 μg/mL. The active molecule 5c showed low cell toxicity and did not obviously trigger the development of resistance towards the tested bacteria strains. Mechanism exploration indicated that compound 5c could not only exert efficient membrane permeability, but also intercalate into DNA of resistant E. faecalis to form 5c-DNA supramolecular complex, which might be responsible for its antimicrobial action. The further investigation showed that this molecule could be effectively transported by human serum albumins through hydrogen bonds and van der Waals force.

Full text of DOI:10.1016/j.bmcl.2020.126982

Journal Pre-proofs
Design and biological evaluation of a novel type of potential multi-targeting
antimicrobial sulfanilamide hybrids in combination of pyrimidine and azoles
Yan-Fei Sui, Di Li, Juan Wang, Rammohan R. Yadav Bheemanaboina,
Mohammad Fawad Ansari, Lin-Ling Gan, Cheng-He Zhou
PII:
S0960-894X(20)30042-1
DOI:
https://doi.org/10.1016/j.bmcl.2020.126982
BMCL 126982
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
30 November 2019
3 January 2020
17 January 2020
Please cite this article as: Sui, Y-F., Li, D., Wang, J., Yadav Bheemanaboina, R.R., Fawad Ansari, M., Gan, L-L.,
Zhou, C-H., Design and biological evaluation of a novel type of potential multi-targeting antimicrobial sulfanilamide
hybrids in combination of pyrimidine and azoles, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://
doi.org/10.1016/j.bmcl.2020.126982
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will
undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing
this version to give early visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Ltd.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design and biological evaluation of a novel type of potential multi-targeting
antimicrobial sulfanilamide hybrids in combination of pyrimidine and azoles
a,§
a,§
a
a,†
a,‡
Yan-Fei Sui, Di Li, Juan Wang, Rammohan R. Yadav Bheemanaboina, Mohammad Fawad Ansari,
b,
a,
Lin-Ling Gan and Cheng-He Zhou
a
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing, 400715, China
Chongqing Engineering Research Center of Pharmaceutical Sciences, School of Pharmacy, Chongqing Medical University, Chongqing 401331, China
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
This work explored a novel type of potential multi-targeting antimicrobial three-component
sulfanilamide hybrids in combination of pyrimidine and azoles. The hybridized target molecules
1
13
were characterized by H NMR, C NMR and HRMS spectra. Some of the developed target
compounds exerted promising antimicrobial activity in comparison with the reference drugs
norfloxacin and fluconazole. Noticeably, sulfanilamide hybrid 5c with pyrimidine and indole
could effectively inhibit the growth of E. faecalis with MIC value of 1 μg/mL. The active
molecule 5c showed low cell toxicity and did not obviously trigger the development of
resistance towards the tested bacteria strains. Mechanism exploration indicated that compound
Keywords:
Sulfanilamide
Pyrimidine
Azole
Antimicrobial
DNA
5
c could not only exert efficient membrane permeability, but also intercalate into DNA of
resistant E. faecalis to form 5c-DNA supramolecular complex, which might be responsible for
its antimicrobial action. The further investigation showed that this molecule could be effectively
transported by human serum albumins through hydrogen bonds and van der Waals force.
Mechanism
Sulfonamides are the first class of artificial synthetic
antibacterial agents, whose core skeleton is para-aminobenzene
sulfonamide that can compete with dihydrofolate synthetase,
hinder the synthesis of dihydrofolate, effectively prevent the
synthesis of nucleic acids and proteins, and then inhibit the
growth of various microorganisms. The hybridization of core
antibacterial skeleton sulfanilamide with various types of
aromatic heterocycles like six-membered pyrimidine and diazine
as well as five-membered thiazole and oxazole produced a
variety of sulfanilamide types of antibacterial drugs, such as
lower toxicity, less bacterial resistance, higher safety and better
activity.
R1
R
N
R2
N
N
O
S
O
S
N
2
H N
NH
H2N
NH
O
O
Ia: R1 = H, R2 = H
Sulfadiazine
Sulfamerazine
1
2
IIa: R = OCH3 Sulfamethoxypyridazine
Ib: R = H, R = CH3
Ic: R = CH3, R = CH3 Sulfamethazine
1
2
IIb: R = Cl
Sulfachloropyridazine
sulfadiazine,
sulfamethoxypyridazine,
sulfamoxole
and
H3C
sulfathiazole. Especially, the hybrids of sulfanilamide and
pyrimidine with simple substituents have achieved great success,
and a large number of pyrimidine derivatives as clinical
antibacterial drugs are being frequently employed in clinic for the
CH3
N
N
O
S
S
O
S
O
H2N
NH
IV: Sulfathiazole
2
H N
NH
III: Sulfamoxole
1
O
treatment of bacterial infections (Figure 1). However, this kind
O
of antibacterial drugs were found to cause some allergic reactions
and show high toxicity to liver, kidney and central nervous
system of human body. Moreover, their prevalent clinical use or
Figure 1. Sulfanilamide-aromatic heterocycle hybrids as clinical
2
antimicrobial drugs.
abuse developed the resistance with a significant public threat to
health. This highlighted our keen interest to develop new hybrids
of sulfanilamide and pyrimidine as antimicrobial agents with
Pyrimidine with a unique six-membered aromatic framework
containing two nitrogen atoms is an important part in lots of
biomolecules such as deoxyribonucleic acid (DNA) and
3
ribonucleic acid (RNA), and as a crucial block is prevalently
employed in the design of new drug molecules. It has been
extensively investigated towards pyrimidine in combination with
a variety of functional groups and a lot of pyrimidine derived



Corresponding author.
Corresponding author.
4
E-mail addresses: ganlinling2012@163.com (L.-L. Gan),
molecules such as antibacterial trimethoprim and brodimoprim
zhouch@swu.edu.cn (C.-H. Zhou).
†
Postdoctoral researcher from CSIR-Indian Institute of Integrative
Medicine (IIIM), Jammu, India.
‡
Postdoctoral researcher from Jamia Millia Islamia, New Delhi, India.
These authors contributed equally to this work.
§
2
019 Elsevier Ltd. All rights reserved.

as well as antifungal voriconazole5 and pyrimethanil6 were
marketed in currently treating microbial infections. More
importantly, numerous pyrimidine derivatives were reported to
moiety plays a vital role in exerting antimicrobial efficacy
because this kind of heterocyclic fragment is able to make use of
weak noncovalent interactions such as coordination bonds,
hydrogen bonds, ion-dipole, cation-π, π-π stacking and
hydrophobic effect as well as van der Waals force to bind with
the enzymes and receptors in organisms by multiple sites and
thus produce good biological potency. However, so far novel
three-component hybrids of sulfanilamide with pyrimidine and
azole ring or its analogs have been rarely reported.
7
be potential as promising antimicrobial agents. Especially the
combination of pyrimidine and heterocyclic azoles was revealed
to result in strong bioactivity and high safety profile.8
1
0
Nitrogen-containing azole heterocycles and their analogs as
9
antimicrobial agents like metronidazole, ornidazole and
fluconazole have been widely used in clinic to benefit human
health. It has been well validated that nitrogen heterocyclic
An essential moiety with rich electron, which is
able to interact with biomacromolecules like
DNA, RNA, enzymes, receptors
A
core fragment in
Nitrogen-containing
extensively
antimicrobial
clinical
drugs,
heterocycles
are
widely present in
clinical antimicrobial
drugs, in which they
can bind with enzymes
and recptors via weak
noncovalent
which is able to hinder
the synthesis of
dihydrofolate, prevent
the synthesis of nucleic
acids and proteins, and
then inhibit the growth
R
Y
Z
N
N
X
O
S
N
of
various
H2N
NH
interactions
organisms.
in
microorganisms
X = N, C
Y, Z = N, CH
O
The combination of sulfanilamide , pyrimidine and azoles produced novel sulfonamide hybrids,
which possess the potential to target mutiple targets, thus inhibiting the growth of bacteria and fungi
with low probability to induce drug resistance and low toxicity to human normal cells.
Figure 2. Design of novel three-component sulfanilamide hybrids with pyrimidine and azoles as potential multi-targeting antimicrobial agents.
In view of such stimulating overwhelming properties and as
an extension of our studies towards novel antimicrobial agents,
herein a new effort was explored towards a novel type of
azoles like benzimidazoles13 and their analog indoles14 (Figure 2).
These new structural compounds might exert multi-site bindings
with biomolecules, regulate the interaction and affinity between
small molecules and biomolecules, therefore the target molecules
might be conducive to overcome increasingly severe drug
resistance and broaden antimicrobial spectrum.
potential
multi-targeting
antimicrobial
three-component
sulfanilamide hybrids in combination of pyrimidine and azole
ring or its analogs, the target azole derivatives and their analogs
1
1
12
included imidazoles and triazoles as well as benzene-fused
Cl
O
H
1
2
2
, 3: a, Y = CH, R = H, R = H
N
N
N
1
2
S
b, Y = CH, R = H, R = CH
3
R1
Y
N
N
1
2
iii
c, Y = CH, R = H, R = CH CH
2 3
O
N
Y
1
2
d, Y = CH, R = H, R = (CH2)2CH3
N
H2N
1
2
e, Y = CH, R = H, R = CH(CH )
3 2
i
R1
R2
Cl
1
2
N
2a-g
3a-g
f, Y = CH, R = NO , R = CH
2 3
R2
1
2
N
N
g, Y = N, R = H, R = H
N
N
O
Cl
H N
2
S
NH
N
ii
Cl
3
4
1
R3
4, 5: a, Z = N, R = H, R = H
O
3
4
b, Z = N, R = CH , R = CH
3
3
N
N
iii
3
4
R4
R3
c, Z = CH, R = H, R = H
3
4
d, Z = CH, R = H, R = CH
3
N
N
3
4
e, Z = CH, R = H,
R = Br
5
a-f
Z
4a-f
R4
3
4
f, Z = CH, R = H,
R = NO
2
Z
o
Scheme 1. Synthetic route of sulfanilamide derivatives 3 and 5. Reagents and conditions: (i) imidazoles and triazole, potassium carbonate, acetonitrile, 80 C; (ii)
o
o
benzimidazoles and indoles, potassium carbonate, acetonitrile, 80 C; (iii) para-aminobenzene sulfonamide, cesium carbonate, acetonitrile, 80 C.
The desired target sulfanilamide hybrids were constructed via
easy synthetic procedures from commercial para-aminobenzene
sulfonamide, azoles or indoles and 2,4-dichloropyrimidine
according to the preparative route in Scheme 1. The reaction of
with cesium carbonate as base afforded the target three-
component sulfanilamide hybrids 3a-g and 5a-f in moderate to
good yields ranging from 43.8% to 75.1%.
The spectral analysis from H NMR, 13C NMR and HRMS
data supported the structures of synthesized target compounds.
All the protons of NH groups gave downfield singlets at
1
2
,4-dichloropyrimidine with azoles or indoles in the presence of
potassium carbonate efficiently produced five-membered azoles
a-g, fused five-membered azoles and their analog indoles 4a-f
2
1
1.61−11.91 ppm. The chemical shifts of 3-H and 5-H in
1
5
with good yields, and then the coupling of intermediates 2a-g
benzene ring appeared at 6.56–6.62 ppm, while the 2-H and 6-H
protons in benzene ring showed downfield signals at 7.59–7.69
o
and 4a-f respectively with sulfanilamide in acetonitrile at 80 C

ppm due to the existence of electron-withdrawing sulfonyl group.
The electronegativity of nitrogen atom at 1-position in
pyrimidine resulted in larger chemical values at 8.47–8.68 ppm
for the 6-position proton in comparison with the 5-position
proton (6.76–7.38 ppm) in pyrimidine. In 13C NMR spectra, the
chemical shifts of carbons in benzene ring gave expected signals
of 130.3–129.6 ppm and 113.2–112.8 ppm, which should be
assigned to the 2,6-C and 3,5-C, respectively. The 4-C in the
pyrimidine ring gave expected signals at 159.7–159.0 ppm. The
other carbons ideally gave the signals at the expected regions.
drug-resistant strains isolated from infected patients are five
Gram-positive bacteria, six Gram-negative bacteria and five
fungi.
As depicted in Table 1, sulfanilamide derivatives 3a-g and 5a-
f showed inhibitory efficiency against some tested bacterial
strains to some extent, while indolyl sulfanilamide 5c displayed
the most prominent efficiency. Imidazolyl compounds 3a-f could
not effectively inhibit the growth of all the tested bacteria, except
for 2-propyl-imidazolyl derivative 3d, which gave good activities
with MIC value of 8 μg/mL against S. aureus and S. aureus
ATCC 29213, and this might be beneficial from propyl group on
the imidazole ring. The replacement of imidazolyl group in
compound 3a by triazolyl moiety yielded compound 3g, resulting
in decreased efficiency against most of the tested strains but
increased activities towards P. aeruginosa (MIC = 4 μg/mL) and
P. aeruginosa ATCC 27853 (MIC = 32 μg/mL), which might be
attributed to the influence of nitrogen atom in triazolyl ring.
All the synthesized hybrids 3a-g and 5a-f in combination of
pyrimidine and azoles were evaluated for their antimicrobial
activities in vitro by two folds serial dilution technique according
to Clinical and Laboratory Standards Institute (CLSI) with the
positive control of clinical drugs. Minimum inhibitory
concentration (MIC, μg/mL) is defined as the lowest
concentration of new compounds that completely inhibit the
growth of bacteria and fungi. The tested strains including clinical
Table 1. The obtained antibacterial data as MIC (μg/mL) in vitro for sulfanilamide derivatives 3-5 a,b,c
Gram-positive bacteria
Gram-negative bacteria
Compds
S. A.
S. A.
29213
E. C.
P. A.
P. A
27853
MRSA
S. A.
E. F.
K. P.
E.C.
A. B.
2
5923
25922
3
a
32
128
256
256
256
128
256
128
128
256
512
256
128
256
256
128
128
256
256
64
3
b
128
256
3
c
128
16
32
64
128
8
128
8
128
64
256
128
256
512
256
4
64
8
64
64
128
128
256
256
256
1
64
64
256
64
32
256
256
32
8
128
16
64
128
128
128
256
32
8
512
128
128
256
256
256
512
32
128
128
128
256
4
128
128
32
128
32
256
128
1
64
32
3
d
3
e
256
128
512
256
256
32
32
128
64
256
128
8
64
3
f
64
3
5
g
a
128
512
128
16
512
256
8
5
b
64
2
5
c
5
d
4
16
4
16
16
64
2
8
32
4
4
64
5
e
4
64
2
4
32
32
4
4
2
4
4
128
128
8
5
f
16
8
32
4
8
8
128
8
16
2
8
A
0.5
1
4
16
0.5
a
Minimal inhibitory concentrations (MIC, μg/mL) were determined by micro broth dilution method for microdilution plates.
b
MRSA, methicillin-resistant Staphylococcus aureus; S. A., Staphylococcus aureus; S. A. 25923, Staphylococcus aureus ATCC 25923; S. A. 29213,
Staphylococcus aureus ATCC 29213; E. F., Enterococcus faecalis; K. P., Klebsiella pneumonia; E. C., Escherichia coli; E. C. 25922, Escherichia coli ATCC
2
c
5922; P. A., Pseudomonas aeruginosa; P. A. 27853, Pseudomonas aeruginosa ATCC 27853; A. B., Acinetobacter baumanii.
A = Norfloxacin.
It was shown that the benzimidazolyl sulfanilamide hybrids
gave more efficient potency against all the tested bacteria strains,
5
a-b gave relatively lower inhibitory potencies with MIC values
manifesting that the indole ring played a crucial role in
improving antibacterial activity.
ranging from 64 to 512 μg/mL, except for towards MRSA strains.
Benzimidazolyl sulfanilamide hybrid 5a exhibited obvious
suppression to the growth of MRSA with MIC value of 8 μg/mL,
being more active than 5,6-dimethyl-benzimidazolyl derivative
Moreover, MRSA was more sensitive to compounds 5c-e than
norfloxacin (MIC = 8 μg/mL) with MIC values ranging from 2 to
4
μg/mL. Methylindolyl derivative 5d gave good activities
5
b, which indicated that the methyl substitution might be
against MRSA, S. aureus ATCC 25923, P. aeruginosa and P.
aeruginosa ATCC 27853 with MIC value of 4 μg/mL.
Bromoindolyl derivative 5e displayed relatively good inhibitory
potency (MIC = 2 μg/mL) against S. aureus ATCC 25923 and E.
coli ATCC 25922. The displacement of bromoindolyl moiety by
nitroindolyl group would afford compound 5f with good
inhibitory efficiency (MIC = 4 μg/mL) against S. aureus ATCC
25923. Noticeably, Gram-negative A. baumanii was insensitive
to sulfanilamide derivatives 3a-g and 5a-f with MIC values of
32–512 μg/mL, which showed that these substituent groups in
unfavorable for the antibacterial activity.
In comparison with benzimidazolyl sulfanilamide derivatives
5
a-b, most of benzimidazolyl analogous indolyl derivatives 5c-f
showed relatively better activities in inhibiting the growth of the
test bacterial strains and gave broader antibacterial spectrum. It
was noticed that indolyl derivative 5c showed apparent inhibition
with the same MIC value of 1 μg/mL towards E. faecalis and P.
aeruginosa ATCC 27853, which was superior or comparable to
standard drug norfloxacin. In comparison to imidazolyl and
benzimidazolyl sulfanilamides 3a and 5a, indolyl derivative 5c

pyrimidine ring were disadvantageous to improve anti-A.
baumanii potentiality.
much more active than the reference drug fluconazole. Therefore,
they might be potential to become new promising antifungal
agents through deep investigation.
Antifungal assay revealed that the sulfanilamide derivatives
3
a-g and 5a-f showed weak to good antifungal activities in vitro
In order to identify the safety profile, the highly active indolyl
pyrimidine hybridized sulfanilamide derivative 5c was further
evaluated for toxicities against lung cancer cell (A549) and
human normal lung epithelium cells (BEAS-2B) with
doxorubicin (0.5 µM) as a positive standard control using the
colorimetric cell proliferation MTT assay. The results of these
assays are depicted in Figure 3. The cell viability of hybrid 5c
towards these two cell lines was at least 70% after being
incubated at the concentration of 64 μg/mL for 24 h, which
manifested that compound 5c displayed relatively low toxicity to
A549 and BEAS-2B cell lines compared to standard drug
doxorubicin.
against the tested fungi (Table S1). It was noticed that the
antifungal abilities of these compounds displayed similar potency
to their antibacterial efficiencies. Towards C. albicans ATCC
9
0023, 2-propylimidazolyl derivative 3d (MIC = 4 μg/mL) was
found to be more efficient than other imidazolyl compounds
(
MICs = 16–128 μg/mL). In addition, the triazolyl derivative 3g
exerted good antifungal efficacy with MIC value of 8 μg/mL
against C. albicans. It was found that benzimidazolyl derivatives
5
a-b exhibited weak inhibitory activities against the tested fungi.
Among the indolyl derivatives 5c-f, the low MIC values of 2–8
μg/mL were observed towards C. albicans, C. tropical and A.
fumigatus, which suggested that these compounds should be
A549
BEAS-2B
1
1
25
00
1
1
25
00
7
5
5
0
7
5
2
5
0
5
0
25
0
4
2
6
8
4
2
0.5
4
2
6
1
8
4
2
0.5
6
3
1
6
3
Concentration (g/mL)
Concentration (g/mL)
Figure 3. Cell viability of lung cancer cells (A549) and human normal lung epithelium cells (BEAS-2B) incubated with the active compound 5c at different
concentrations.
It is important for potentially antimicrobial agents that the
development of resistance to their action is not readily attained.
results clearly revealed that the active hybrid 5c was capable of
killing Gram-positive bacteria E. faecalis rapidly.
1
6
To evaluate the probability of these molecules to elicit resistance
in bacteria, the drug resistance study of indolyl pyrimidine
hybridized sulfanilamide derivative 5c against E. faecalis was
carried out. As shown in Figure 4, the susceptibility of E. faecalis
bacteria to indolyl derivative 5c remained nearly unchanged even
after 16 passages. By contrast, the drug resistance developing
ability of E. faecalis against norfloxacin was obvious, indicating
that sulfanilamide hybrid 5c with pyrimidine and indole did not
significantly trigger the development of drug resistance as easily
as the reference drug norfloxacin.
DNA-gyrase (PDB code: 2XCS) as an attractive target in
antibacterial drug exploration was applied to be a flexible ligand-
receptor docking investigation to further rationalize the observed
antibacterial activity of indolyl derivative 5c. The molecular
simulation results (Figure 5) showed that nitrogen atom at 3-
position in the pyrimidine ring and the hydrogen atom of the NH
2
group at the 4-position on benzene formed two hydrogen bonds
with DC11 and Asp1083B residues, respectively. The value of
binding energy is 6.9 kcal/mol. These non-covalent binding
might be conducive to stabilize the 5c-enzyme complex, which
might contribute to the superior inhibitory efficacy.
140
120
100
Compound 5c
Norfloxacin
80
60
40
20
0
0
2
4
6
8
10
12
14
16
Passages
Figure 5. Supramolecular binding of compound 5c docked in the bacterial
DNA-gyrase (PDB code: 2XCS).
Figure 4. Development of E. faecalis resistance to compound 5c and
norfloxacin.
Bacteria with biofilms are inherently insensitive to
antimicrobial agents and they are much more resistant to
In order to study the bactericidal action of indolyl pyrimidine
hybridized sulfanilamide derivative 5c, a time-kill kinetics assay
was performed against E. faecalis at different concentrations
MIC and 6 × MIC). As shown in Figure S1, there was more than
log (CFU/mL) reduction in the number of viable bacteria at
concentrations of MIC and 6 × MIC against E. faecalis. These
1
7
conventional antibiotic treatment. Permeabilization of the
bacterial membrane of bacteria E. faecalis was carried out with
propidium iodide (PI) as fluorescent indicator which can only
pass through the membrane of compromised cells and then
fluoresce red after binding with double-stranded DNA.
(
3

Fluorescence intensity enhancement was observed after the
treatment of cells with active molecule 5c. The results of
bacterial membrane permeabilization are shown in Figure 6.
Obvious fluorescence enhancements at 617 nm of the mixtures of
indolyl pyrimidine hybridized sulfanilamide derivative 5c with
PI-treated E. faecalis appeared and became steady after 60 min,
which demonstrated that sulfanilamide hybrid 5c with pyrimidine
and indole could permeate the membranes of E. faecalis strains
efficiently.
intercalate into the double helix of DNA by substituting for NR
in the DNA-NR complex.
Many promising new drugs have been rendered ineffective
because of their unusually high affinity to the protein human
serum albumins (HSA), which is the principal extracellular
protein of the circulatory system. As a result, the investigations
of interactions between drugs or bioactive small molecules and
HSA are not only beneficial to provide a proper understanding of
the absorption, transportation, distribution, metabolism and
excretion properties of drugs, but also significant to design,
1700
1600
1500
1400
1300
1
9
Compound 5c
Control
modify and screen drug molecules.
The binding mode between HSA and the highly active
molecule 5c was investigated with UV-vis spectroscopy on the
molecular level. In the binding experiment (Figure S6), the
aromatic rings in Tryptophan (Trp-214), Tyrosine (Tyr-411) and
Phenylalanine (Phe) residues in HSA gave absorption peak at
2
78 nm and the peak intensity increased with the addition of
hybrid 5c, which indicated the supramolecular interaction of
indolyl pyrimidine hybridized sulfanilamide derivative 5c with
HSA and the extension of HSA peptide strands (Figure S6).
0
20
40
60
80
100
120
Time (min)
The unchanged maximum absorption wavelength implied that
there was a non-covalent interaction between indolyl derivative
Figure 6. Bacterial membrane permeabilization of compound 5c (12 × MIC)
against E. faecalis.
5
c and HSA. The fluorescence intensity of Trp-214 may change
The detection of hyperchromism and hypochromism by
absorption spectroscopy, which are critical spectral features to
distinguish the change of DNA double helical structure, is one of
the most important and useful method in DNA binding studies.
The observed strong hyperchromism might be attributed to the
denaturation of DNA caused by the strong interaction between
the electronic states of intercalating chromophore and that of the
DNA base. As shown in Figure S2, the UV-vis spectra revealed
that the maximum absorption peak of DNA at 260 nm showed
proportional increase and slight blue shift with the increasing
concentration of hybrid 5c. Remarkably, the absorption values of
the 5c-DNA complex were lower than that of sum of free DNA
and free molecule 5c. This hyperchromism effect might be
attributed to the formed binary complexes between 5c and DNA
bases. In general, the observed spectral properties could be
explained by the potent interaction of indolyl derivative 5c with
E. faecalis DNA (Figure S3).
when HSA interacts with other small molecules, which could be
reflected in the fluorescence spectra of HSA in the UV region.
The different mechanisms of quenching are usually classified as
either dynamic quenching or static quenching, which can be
distinguished by their different dependence on temperature
(Figure S7, Table S2).
The Modified Stern-Volmer plots were showed in Figure S8
and the calculated results were depicted in Table S3. As shown in
Figure S9 and Table S3, with the increase of temperature, the
diffusion coefficient increased while the stability of the 5c-HSA
complex reduced. The results suggested that there was a single
class of binding sites on HSA for sulfanilamide hybrid 5c. In
addition, the thermodynamic parameters were obtained in Table
S4. Both hydrophobic interactions and hydrogen bonds played
vital roles in the binding of molecule 5c to HSA and seemed to
2
0
contribute to the stability of the 5c-HSA complex.
In conclusion, this work employed a facile synthetic procedure
to develop a novel type of three-component sulfanilamide
hybrids with pyrimidine and azoles. Some prepared new
sulfanilamide derivatives could significantly inhibit the growth of
the tested strains with equipotent or superior activities to the
current clinical drugs norfloxacin and fluconazole. Notably, the
indolyl pyrimidine hybridized sulfanilamide derivative 5c not
only showed superior inhibitory behaviors against E. faecalis
To further elucidate the supramolecular interaction between
hybrid 5c and E. faecalis DNA, neutral red (NR, a planar
phenazine dye with a confirmed intercalative binding mode with
DNA) was employed as a spectral probe due to its lower toxicity
and higher stability. Therefore, NR was chosen to investigate the
binding mode of compound 5c with DNA in present work, and
the absorption spectra of the NR dye after the addition of DNA
1
8
were shown in Figure S4. The absorption peak of NR at around
60 nm gradually decreased with the increasing concentration of
(MIC = 1 μg/mL) to norfloxacin, but also exhibited low toxicity
4
towards A549 and BEAS-2B cell lines and no obvious propensity
to trigger development of resistance towards E. faecalis.
Moreover, the active sulfanilamide hybrid 5c was also membrane
active and could kill bacteria in short time. The specific
interaction of indoyl derivative 5c with E. faecalis DNA
displayed that it could intercalate into DNA to form 5c-DNA
complex which might further block DNA replication to exert
their powerful antimicrobial activities. Binding investigations
revealed that compound 5c was able to be carried by HSA, which
could generate fluorescent quenching by the active hybrid 5c as a
result of the formation of ground-state, and the calculated
parameters indicated that the binding process was spontaneous.
These results revealed that indolyl pyrimidine hybridized
sulfanilamide derivative 5c could be regarded as a promising
lead compound for the further development as potential
DNA, and a new band at around 530 nm appeared, which could
be attributed to the formation of the new DNA-NR complex. The
isosbestic point at 500 nm provided the evidence for the
formation of DNA-NR complex.
The absorption spectra of a competitive binding between NR
and 5c with DNA in Figure S5 showed that with the increasing
concentration of hybrid 5c, the maximum absorption around 530
nm of the DNA-NR complex decreased, but a slight intensity
increase was observed in the developing band around 460 nm. In
comparison with the absorption band of the free NR in the
presence of the increasing concentrations of DNA (Figure S4),
the spectra in the inset of Figure S5 exhibited the reverse process.
The apparent changes of spectra suggested that indolyl
pyrimidine hybridized sulfanilamide derivative 5c could

antimicrobial agent.
10. (a) Jeyakkumar P, Zhang L, Avula SR, Zhou CH. Design,
synthesis and biological evaluation of berberine-benzimidazole
hybrids as new type of potentially DNA-targeting antimicrobial
agents. Eur J Med Chem. 2016; 122: 205–215. (b) Chen YY,
Gopala L, Bheemanaboina RRY, Liu HB, Cheng Y, Geng RX,
Zhou CH. Novel naphthalimide aminothiazoles as potential
multitargeting antimicrobial agents. ACS Med Chem Lett. 2017; 8:
Acknowledgments
This work was partially supported by National Natural
Science Foundation of China (Nos. 21672173, 21971212), China
1
331–1335; (c) Wang LL, Battini N, Bheemanaboina RRY, Ansari
Postdoctoral
Science
Foundation
(2019M653821XB,
MF, Chen JP, Xie YP, Cai GX, Zhang SL, Zhou CH. A new
exploration towards aminothiazolquinolone oximes as potentially
multi-targeting antibacterial agents: Design, synthesis and
evaluation acting on microbes, DNA, HSA and topoisomerase IV.
Eur J Med Chem. 2019; 179: 166–181. (d) Gong HH, Addla D, Lv
JS, Zhou CH. Heterocyclic naphthalimides as new skeleton
structure of compounds with increasingly expanding relational
medicinal applications. Curr Top Med Chem. 2016; 16: 3303–
2
018M633313), Chongqing Special Foundation for Postdoctoral
Research Proposal (Xm2016039, XmT2018082), Natural Science
Foundation Project of the Chongqing Science, Chongqing
Natural Science Foundation Postdoctoral Science Foundation
Project (cstc2019jcyj-bshX0124) and Technology Committee
(cstc2019jcyj-msxmX0236) and Program of Chongqing
Medicinal and Pharmaceutical College (YGZ 2019102).
3
364.
1
1. (a) Walton T, Zhang W, Li L, Tam CP, Szostak, JW. The
mechanism of nonenzymatic template copying with imidazole-
activated nucleotides. Angew Chem Int Ed. 2019; 58(32): 10812–
References
1
0819 (b) Li ZZ, Maddili SK, Tangadanchu VKR, Bheemanaboina
1
. (a) Brown ED, Wright GD. Antibacterial drug discovery in the
resistance era. Nature. 2016; 529: 336–343. (b) He SC, Ponmani J,
Avula SR, Wang XL, Zhang HZ, Zhou CH. Recent advance in
sulfomide-based medicinal chemistry. Sci Sin Chem. 2016; 46(9):
RRY, Lin JM, Yang RG, Cai gx, Zhou CH. Progress in medicinal
chemistry of nitroimidazole heterocycle. Sci China Chem. 2019;
4
9(2): 230–255. (c) Li ZZ, Tangadanchu VKR, Battini N,
Bheemanaboina RRY, Zang ZL, Zhang SL, Zhou CH. Indole-
nitroimidazole conjugates as efficient manipulators to decrease the
genes expression of methicillin-resistant Staphylococcus aureus.
Eur J Med Chem. 2019; 179: 723–735. (d) Li ZZ, Gopala L,
Tangadanchu VKR, Gao WW, Zhou CH. Discovery of novel
nitroimidazole enols as Pseudomonas aeruginosa DNA cleavage
agents. Bioorg Med Chem. 2017; 25: 6511–6522.
2. (a) Zhang Y, Tangadanchu VKR, Bheemanaboina RRY, Cheng Y,
Zhou CH. Novel carbazole-triazole conjugates as DNA-targeting
membrane active potentiators against clinical isolated fungi. Eur J
Med Chem. 2018; 155: 579–589. (b) Zhang Y, Damu GLV, Cui
SF, Mi JL, Tangadanchu VKR, Zhou CH. Discovery of potential
antifungal triazoles: design, synthesis, biological evaluation, and
preliminary antifungal mechanism exploration. Med Chem
Commun. 2017; 8: 1631–1639. (c) Duan JR, Liu HB, Jeyakkumar
P, Gopala L, Li S, Geng RX, Zhou CH. Design, synthesis and
biological evaluation of novel Schiff base-bridged tetrahydroproto
berberine triazoles as a new type of potential antimicrobial agents.
Med Chem Commun. 2017; 8: 907–916. (d) Cheng Y, Avula SR,
Gao WW, Addla D, Tangadanchu VKR, Zhang L, Lin JM, Zhou
CH. Multi-targeting exploration of new 2-aminothiazolyl
quinolones: Synthesis, antimicrobial evaluation, interaction with
DNA, combination with topoisomerase IV and penetrability into
cells. Eur J Med Chem. 2016; 124: 935–945.
8
23–847. (c) Hu YY, Bheemanaboina RRY, Battini N, Zhou CH.
Sulfonamide-derived four-component molecular hybrids as novel
DNA-targeting membrane active potentiators against clinical
Escherichia coli. Mol Pharm. 2019; 16: 1036–1052.
. Li H, Song HL, Xu H, Lu Y, Zhang S, Yang YL, Yang XL, Lu YX.
Effect of the coexposure of sulfadiazine, ciprofloxacin and zinc on
the fate of antibiotic resistance genes, bacterial communities and
functions in three-dimensional biofilm-electrode reactors.
Bioresour Technol. 2019; 296: 122290.
2
3
1
. (a) Liu HB, Gao WW, Tangadanchu VKR, Zhou CH, Geng RX.
Novel aminopyrimidinyl
benzimidazoles as potentially
antimicrobial agents: Design, synthesis and biological evaluation.
Bioorg Med Chem. 2018; 26: 3707–3715. (b) Fang XF, Li D,
Tangadanchu VKR, Gopala L, Gao WW, Zhou CH. Novel
potentially antifungal hybrids of 5-flucytosine and fluconazole:
Design, synthesis and bioactive evaluation. Bioorg Med Chem Lett.
2
017; 27: 4964–4969.
4
. Jain KS, Arya N, Inamdar NN, Auti PB, Unawane SA, Puranik HH,
Sanap MS, Inamke AD, Mahale VJ, Prajapati CS, Shishoo CJ. The
chemistry and bio-medicinal significance of pyrimidines
condensed pyrimidines. Curr Top Med Chem. 2016; 16: 3133–
174.
&
3
5
6
. Mourad A, Stiber JA, Perfect JR, Johnson MD. Real-world
implications of QT prolongation in patients receiving voriconazole
and amiodarone. J Antimicrob Chemother. 2019; 74(1): 228–233.
. Tang R, Tang T, Tang G, Liang Y, W WC, Yang JL, Niu JF; Tang
JY, Zhou ZY, Cao YS. Pyrimethanil ionic liquids paired with
various natural organic acid anions for reducing its adverse
impacts on the environment. J Agr Food Chem. 2019; 67(40):
1
3. (a) Li ZY, Dong JN, Wang JW, Yang DY, Weng ZQ. Elemental
sulfur-promoted
trifluoroethyl)benzoxazoles and their derivatives. Chem Commun.
019; 55(87): 13132–13135. (b) Zhang L, Addla D, Ponmani J,
one-pot
synthesis
of
2-(2,2,2-
2
Wang A, Xie D, Wang YN, Zhang SL, Geng RX, Cai GX, Li S,
Zhou CH. Discovery of membrane active benzimidazole
quinolones-based topoisomerase inhibitors as potential DNA-
binding antimicrobial agents. Eur J Med Chem. 2016; 111: 160–
1
1018–11024.
7
8
. Hagras M, Abutaleb NS, Ali AO, Abdel-Aleem JA, Elsebaei MM,
Seleem MN, Mayhoub AS. Naphthylthiazoles: targeting
multidrug-resistant and intracellular Staphylococcus aureus with
biofilm disruption activity. ACS Infect Dis. 2018; 4: 1679–1691.
. (a) Liu HB, Gao WW, Tangadanchu VKR, Zhou CH, Geng RX.
1
82. (c) Wang YN, Bheemanaboina RRY, Cai GX, Zhou CH.
Novel purine benzimidazoles as antimicrobial agents by regulating
ROS generation and targeting clinically resistant Staphylococcus
aureus DNA groove. Bioorg Med Chem Lett. 2018; 28: 1621–
Novel aminopyrimidinyl
benzimidazoles as potentially
1
628. (d) Jeyakkumar P, Liu HB, Gopala L, Cheng Y, Peng XM,
antimicrobial agents: Design, synthesis and biological evaluation.
Eur J Med Chem. 2018; 143: 66−84. (b) Li D, Bheemanaboina
RRY, Battini N, Tangadanchu VKR, Fang XF, Zhou CH. Novel
organophosphorus aminopyrimidines as unique structural DNA-
targeting membrane active inhibitors towards drug-resistant
methicillinresistant Staphylococcus aureus. Med Chem Commun.
Geng RX, Zhou CH. Novel benzimidazolyl
tetrahydroprotoberberines: Design, synthesis, antimicrobial
evaluation and multi-targeting exploration. Bioorg Med Chem Lett.
2
4. (a) Veeraveedu P Thanikachalam, Maurya RK, Garg V, Monga V.
An insight into the medicinal perspective of synthetic analogs of
indole: A review. Eur J Med Chem. 2019; 180: 562–612. (b)
Aggarwal T, Sushmita, Verma A K. Recent advances in the
synthesis of carbazoles from indoles. Org Biomol Chem. 2019;
017; 27: 1737–1743.
1
2
018; 9: 1529–1537.
9
. (a) Zhang L, Peng XM, Damu GLV, Geng RX, Zhou CH.
Comprehensive review in current developments of imidazole-
based medicinal chemistry. Med Res Rev. 2014; 34(2): 340−437.
1
7(36): 8330–8342.
(b) Peng XM, Cai GX, Zhou CH. Recent developments in azole
1
1
5. Xiang JB, Leung C, Zhang ZQ, Hu C, Geng D, Liu LL, Yi L, Li
ZW, Berenson J, Bai X. Synthesis and evaluation of 2-alkylthio-4-
compounds as antibacterial and antifungal agents. Curr Top Med
Chem. 2013; 13(16): 1963−2010. (c) Zhang HZ, Damu GLV, Cai
GX, Zhou CH. Current developments in the syntheses of 1,2,4-
triazole compounds. Curr Org Chem. 2014; 18(3): 359−406. (d)
Zhang HZ, Gan LL, Wang H, Zhou CH. New progress in azole
compounds as antimicrobial agents. Mini-Rev Med Chem. 2017;
(N-substituted sulfonamide)pyrimidine hydroxamic acids as anti-
myeloma agents. Chem Biol Drug Des. 2016; 87: 472–477.
6. (a) Cui SF, Addla D, Zhou CH. Novel 3-aminothiazolquinolones:
design, synthesis, bioactive evaluation, SARs, and preliminary
antibacterial mechanism. J Med Chem. 2016; 59: 4488–4510. (b)
Gao WW, Gopala L, Bheemanaboina RRY, Zhang GB, Li S, Zhou
1
7(2): 122–166.

CH. Discovery of 2-aminothiazolyl berberine derivatives as
effectively antibacterial agents toward clinically drug-resistant
Gram-negative Acinetobacter baumanii. Eur J Med Chem. 2018;
higher potency against E. faecalis than
norfloxacin.
1
46: 15–37. (c) Wang LL, Battini N, Bheemanaboina RRY, Zhang
SL, Zhou CH. Design and synthesis of aminothiazolyl norfloxacin
analogues as potential antimicrobial agents and their biological
evaluation. Eur J Med Chem. 2019; 167: 105–123. (d) Maddili SK,
Li ZZ, Kannekanti VK, Bheemanaboina RRY, Tuniki B,
Tangadanchu VKR, Zhou CH. Azoalkyl ether imidazo[2,1-
b]benzothiazoles as potentially antimicrobial agents with novel
structural skeleton. Bioorg Med Chem Lett. 2018; 28: 2426–2431.
7. (a) Benoit DSW, Sims KRJ, Fraser D. Nanoparticles for oral
biofilm treatments. ACS Nano. 2019; 13(5): 4869–4875. (b) Sun H,
Ansari MF, Battini N, Bheemanaboina RRY, Zhou CH. Novel
potential artificial mrsa DNA intercalators: Synthesis and
biological evaluation of berberine-derived thiazolidinediones. Org
Chem Front. 2019; 6: 319–334. (c) Wang YN, Bheemanaboina
RRY, Gao WW, Kang J, Cai GX, Zhou CH. Discovery of
benzimidazole–quinolone hybrids as new cleaving agents toward
drug-resistant Pseudomonas aeruginosa DNA. ChemMedChem.
 Hybrid 5c exhibited low toxicity and no
obvious propensity to trigger development of
resistance.
1

The active molecule 5c could disturb the cell
membrane, intercalate into E. faecalis DNA.
Compound 5c could be effectively transported
by human serum albumins.

2
018; 13: 1004–1017. (d) Gao WW, Zhou CH. Antimicrobial 2-
aminothiazolyl quinolones: what is their potential in the clinic?
Future Med Chem. 2017; 9(13): 1461–1464.
1
8. (a) Zhang GB, Maddili SK, Tangadanchu VKR, Gopala L, Gao
WW, Cai GX, Zhou CH. Discovery of natural berberine-derived
nitroimidazoles as potentially multi-targeting agents against drug-
resistant Escherichia coli. Sci China Chem. 2018; 61(5): 557–568.
(b) Kang J, Gopala L, Tangadanchu VKR, Gao WW, Zhou CH.
Novel naphthalimide nitroimidazoles as multitargeting
antibacterial agents against resistant Acinetobacter baumannii.
Future Med Chem. 2018; 10(7): 711–724. (c) Zhang Y,
Tangadanchu VKR, Cheng Y, Yang RG, Lin JM, Zhou CH.
Potential antimicrobial isopropanol-conjugated carbazole azoles as
dual targeting inhibitors of Enterococcus faecalis. ACS Med Chem
Lett. 2018; 9: 244–249. (d) Cao QC, Wang H, Tangadanchu VKR,
Gopala L, Cai GX, Zhou CH. Design, synthesis, antimicrobial
activity and targeted DNA study of quinazolone imidazoles. Sci
China Chem. 2017; 47(7): 844–858. (e) Liu HB, Gopala L, Avula
SR, Jeyakkumar P, Peng XM, Zhou CH, Geng RX. Chalcone-
benzotriazole conjugates as new potential antimicrobial agents:
design, synthesis, biological evaluation and synergism with clinical
drugs. Chin J Chem. 2017; 35: 483–496.
1
9. (a) Yin BT, Yan CY, Peng XM, Zhang SL, Rasheed S, Geng RX,
Zhou CH. Synthesis and biological evaluation of α-triazolyl
chalcones as a new type of potential antimicrobial agents and their
interaction with calf thymus DNA and human serum albumin. Eur
J Med Chem. 2014; 71: 148–159. (b) Gao WW, Rasheed S,
Tangadanchu VKR, Sun Y, Peng XM, Cheng Y, Zhang FX, Lin
JM, Zhou CH. Design, synthesis and biological evaluation of
amino organophosphorus imidazoles as a new type of potential
antimicrobial agents. Sci China Chem. 2017; 6: 769–785. (c) Kang
J, Tangadanchu VKR, Gopala L, Gao WW, Cheng Y, Liu HB,
Geng RX, Li S, Zhou CH. Novel potentially antibacterial
naphthalimide-derived
metronidazoles:
Design,
synthesis,
biological evaluation and supramolecular interactions with DNA,
human serum albumin and topoisomerase II. Chin Chem Lett.
2
017; 28: 1369–1374.
2
0. (a) Zhou CH, Sui YF. Supermolecules as medicinal drugs. In: Liu
Y, Chen Y, Zhang HY, eds. Handbook of macrocyclic
supramolecular
assembly.
Springer.
Singapore;
2019.
https://doi.org/10.1007/978-981-13-1744-6_66-1. (b) Zhang HZ,
He SC, Peng YJ, Zhang HJ, Gopala L, Tangadanchu VKR, Gan
LL, Zhou CH. Design, synthesis and antimicrobial evaluation of
novel benzimidazole-incorporated sulfonamide analogues. Eur J
Med Chem. 2017; 136: 165–183. (c) Zhang HZ, Jeyakkumar P,
Kumar KV, Zhou CH. Synthesis of novel sulfonamide azoles via
C–N cleavage of sulfonamides by azole ring and relational
antimicrobial study. New J Chem. 2015; 39: 5776–5796. (d) Cheng
Y, Wang H, Dinesh A, Zhou CH. Current researches and
applications of azole-based supermolecules as medicinal agents.
Chin J Org Chem. 2016; 36, 1–42.


Multi-targeting antimicrobial sulfanilamide
hybrids in combination of pyrimidine and
azoles were developed.
Indolyl sulfanilamide derivative 5c displayed

Declaration of interests
The authors declare that they have no known
☒
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☐
The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
Graphical Abstract
Design and biological evaluation of a
novel type of potential multi-targeting
antimicrobial sulfanilamide hybrids in
combination of pyrimidine and azoles
Yan-Fei Sui, Di Li, Juan Wang, Rammohan R. Yadav
Bheemanaboina, Mohammad Fawad Ansari, Lin-Ling



Gan and Cheng-He Zhou
—
——



Corresponding author.
Corresponding author.
E-mail addresses: ganlinling2012@163.com (L.-L. Gan),
zhouch@swu.edu.cn
(
C.-H. Zhou).
†
Postdoctoral researcher from Jamia Millia Islamia, India.
These authors contributed equally to this work.
§
2
019 Elsevier Ltd. All rights reserved.