Aryl fluorosulfate analogues as potent antimicrobial agents: SAR, cytotoxicity and docking studies

Article abstract of DOI:10.1016/j.bioorg.2018.08.001

A series of aryl fluorosulfate analogues (1–37) were synthesized and tested for in vitro antibacterial and antifungal studies, and validated by docking studies. The compounds 9, 12, 14, 19, 25, 26, 35, 36 and 37 exhibited superior antibacterial potency ag

Full text of DOI:10.1016/j.bioorg.2018.08.001

Accepted Manuscript
Aryl fluorosulfate analogues as potent antimicrobial agents: SAR, cytotoxicity
and docking studies
Lekkala Ravindar, S.N.A. Bukhari, K.P. Rakesh, H.M. Manukumar, H.K.
Vivek, N. Mallesha, Zhi-Zhong Xie, Hua-Li Qin
PII:
S0045-2068(18)30595-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.08.001
YBIOO 2458
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
17 June 2018
1 August 2018
1 August 2018
Please cite this article as: L. Ravindar, S.N.A. Bukhari, K.P. Rakesh, H.M. Manukumar, H.K. Vivek, N. Mallesha,
Z-Z. Xie, H-L. Qin, Aryl fluorosulfate analogues as potent antimicrobial agents: SAR, cytotoxicity and docking
studies, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.08.001
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Aryl fluorosulfate analogues as potent antimicrobial agents: SAR,
cytotoxicity and docking studies
1
1
1
2
Lekkala Ravindar , S. N. A. Bukhari , K. P. Rakesh *, H. M. Manukumar , H. K.
3
4
1
1
Vivek , N. Mallesha ，Zhi-Zhong Xie * and Hua-Li Qin *
School of Chemistry, Chemical Engineering and Life Science, Wuhan University of
Technology, Wuhan, 430070, P R China
1
2
Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysuru-
70006, Karnataka, India
5
3
Analytical Research and Development, Syngene International Ltd, Biocon Park,
Bommasandra Industrial Estate, Bangaluru-560099, Karnataka, India
4
SRI RAM CHEM, R & D Centre, Plot No. 31, JCK Industrial Park, Belagola Industrial
Area, Mysore 570016, Karnataka, India
*
Corresponding authors
Email: rakeshasg@gmail.com
Tel: +91 7619540544
Email: qinhuali@whut.edu.cn
Fax: +86 27 87749300.
Email: zhizhong_xie@163.com
Tel: +86 13667111218
Abstract
A series of aryl fluorosulfate analogues (1-37) were synthesized and tested for in vitro
antibacterial and antifungal studies, and validated by docking studies. The compounds 9, 12,
1
4, 19, 25, 26, 35, 36 and 37 exhibited superior antibacterial potency against tested bacterial
strains, while compounds 2, 4, 5, 15, 35, 36 and 37 were found to have better antifungal
activity against tested fungal strains, compared to standard antibiotic gentamicin and
ketoconazole respectively. Among all the synthesized 37 analogs, compounds 25, 26, 35, 36
and 37 displayed excellent anti-biofilm property against Staphylococcus aureus. The
structure-activity relationship (SAR) revealed that the antimicrobial activity depends upon the
presence of –OSO
2
F group and slender effect of different substituent’s on the phenyl rings.
1

The electron donating (OCH
3
) groups in analogs increase the antibacterial activity, and
, F and Br) groups increase the antifungal
interestingly the electron withdrawing (Cl, NO
2
activity (except compound 35, 36 and 37). The mechanism of potent compounds showed
membrane damage on bacteria confirmed by SEM. Compounds 35, 36 and 37 exhibited
highest glide g-scores in molecular docking studies and validated the biocidal property.
Key words: Aryl-fluorosulfates; antimicrobial; docking studies; cytotoxicity.
1
. Introduction:
Aryl fluorosulfates were described more than four decades ago, but the chemistry of
these compounds is quite unexplored [1]. Recently, new interest in the synthesis of aryl
fluorosulfates from the reaction of a phenol with sulfuryl fluoride in the presence of a base
was described by Sharpless and co-workers through a SuFEx click chemistry process [2,3].
Sulfuryl fluoride (SO
2
2
F , bp = −55.4 °C) is produced by Dow Agro Sciences and is most
commonly employed as an insecticide for the control of dry wood termites by whole-
structure fumigation. The sulfuryl fluoride has low toxicity and is relatively inexpensive [4].
Recently, the fluorosulfates based probes have become vital tools in chemical biology and
molecular pharmacology [5,6]. Aryl fluorosulfates probes have also been found to possess
substantial efficacy in chemical and medicinal chemistry [2]. Nowadays bacterial infections,
especially today with the emergence of multidrug-resistant bacteria caused by the misuse of
antibiotics, are becoming a serious problem. Unfortunately, the traditional drugs used in
clinics are exhibiting less effectiveness in the treatment of infections [7]. Therefore, it is of
great significance to develop new types of antimicrobial agents, especially those with new
drug targets or with the ability to overcome drug resistance [8]. However, the chemical
biology toolkit still needs urgent attention to significantly improve and expand the palette of
useful synthetic transformations that can be harnessed to understand biology and drug
discovery. We inspired by the special features of aryl fluorosulfates analogues and our
2

ongoing research program [9-11]. Herein, we report the synthesis of a series of aryl
fluorosulfates (1-37) and their utilization as antimicrobial agents in the field of drug
discovery through exploiting the relative chemical stability of fluorosulfates and carrying out
a number of unique fluorosulfates-sparing functional group modifications for their
antibacterial and antifungal activities studies. In addition, in this work, we also carried out
molecular docking studies of those synthetic compounds in order to correlate their structural
motif with their antibacterial and antifungal activities. The experimental results and docking
studies suggest these compounds will create powerful and valuable chemical tools for
chemical biology research and drug discovery.
2
. Results and Discussion
2
.1. Chemistry
Aryl fluorosulfates were synthesized (Fig. 1) directly from the phenols and SO
F
2 2
following the procedure described by Sharpless and co-workers [2]. All the derivatives were
obtained in good to excellent yield. The formation of the –OSO
2
F was confirmed by the
1
9
1
presence of fluorine (F) peak in F NMR and the absence of -OH peak in H NMR spectra.
1
13
19
All the chemical structures were confirmed by H, C, F NMR and mass spectral analysis
(
Supporting information).
2
2
.2. Biology
.2.1. Antibacterial activity
Aryl fluorosulfates (1-37) were synthesized and screened for their in vitro
antibacterial activity against two Gram-positive strains Staphylococcus aureus and Bacillus
subtilis as well as two Gram-negative strains Escherichia coli and Klebsiella pneumonia by
using the agar well diffusion method [12], as well as micro dilution method [13]. The results
of antibacterial screening were summarized in Table 1 and Table 2. All assays were
performed in triplicate and the results were expressed as the mean of the diameter of
3

inhibition zone in millimeter (mm). Gentamicin was used as the standard drug for the
antibacterial screening. Recently, K. B. Sharpless and his research group was reported
arylfluorosulfates containing analogs showed potential anticancer agents [14]. We inspired
the biological importance of arylfluorosulfates groups, we first time reported to
arylfluorosulfates is good antimicrobial agents. In the present investigations S. aureus used as
a model organism and anti-biofilm activity study was carried out for all 37 compounds.
Among 37 analogs the best was 25 (0.84±0.18), 26 (0.82±0.20), 35 (0.76±0.10), 36
(
0.58±0.31), 37 (0.51±0.23) and they exhibited as classic compounds for the control of
diseases. Among these classic compounds analog 36 and 37 are highly potent in nature (Fig.
). To support the antimicrobial activity, the MIC of compound 37 treated with S. aureus
2
over night and cell morphology was observed in SEM showing cell rupture, variation in the
structure can be visualized (Fig. 3). This indicates that, all potent nature of the compounds
are acting on cell membrane and involved in the destabilization of cell and leads to death of
the bacteria was hypothesized in the present investigation.
The antimicrobial activities those synthetic aryl fluorosulfates were evaluated
accordingly. The results revealed that most of the compounds have shown moderate to
excellent activity against the four tested bacterial strains. Compounds 9, 12, 14, 19, 25, 29,
3
5, 36 and 37 showed superior antibacterial activities compared to the reference drug
gentamicin. These activities may be associated with the presence of both electrons donating
group (OCH ) and sulfonylfluoride (-OSO F) groups in the analogs. Compounds 36 and 37
exhibited excellent antibacterial activities against all of the tested bacterial strains. It may be
explained by the presence of two methoxy groups and two –OSO F groups in the molecules.
3
2
2
Compound 36 was more active than compound 37, in which the presence of double bond may
slightly reduce the antibacterial activity. Interestingly, compound 35 with no electron
donating groups but more -OSO
2
F groups presented in the molecule, displayed good
4

antibacterial activity against all the tested bacterial pathogens, this analog may be
preliminarily prove that the antibacterial activity depends on -OSO F group. Compound 29
2
showed good antibacterial activities against the Gram-negative bacterial strains Escherichia
coli and Klebsiella pneumonia, which may be caused by the antibacterial activity of indole
moiety. Compounds 24 and 25 exhibited excellent antibacterial activity against all the tested
bacterial strains. It may be assumed that antibacterial activity increases along with the
increasing of -OSO
strongly confirmed that the bacterial activities have a significant relationship with the
presence of the –OSO F groups. Compounds 9, 12 and 14 showed good antibacterial activity.
2
F group number on the benzene ring. Both the compounds 24 and 25
2
It may be explained by the presence of methoxy group on the benzene ring. Compound 14
showed good antibacterial activities compared to compounds 9 and 12. The substituent of
ortho, meta and para positions of methoxy groups on the phenyl rings also led to the slight
difference in the antibacterial activity. Compound 20 showed good antibacterial activities
against the Gram positive bacteria Staphylococcus aureus and Bacillus subtilis due to the
presence of another phenyl ring. Compound 24 showed superior antibacterial activity against
the Gram-negative bacteria Escherichia coli and Klebsiella pneumonia and moderate activity
against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. The rest of
the compounds 2-6, 8, 10, 13 and 15-18 showed least antibacterial activity, which may be
explained that electron withdrawing groups (Cl, F, Br and NO
2
) reduced the antibacterial
activity. Compounds 32-34 showed nil antibacterial activity against all the tested bacterial
strains, in which the presence of iodine (I) and CF
reduce the antibacterial activity.
3
groups on the five membered rings may
2
.2.2. Antifungal activity
All the synthesized aryl fluorosulfates (1-37) were screened for their in vitro
antifungal activity against four fungal strains, namely, Aspergillus niger, Fusarium
5

moniliforme, Fusarium oxysporum and Candida albicans by using agar well diffusion
method [15] as well as micro dilution method [16]. The results of antifungal screening were
summarized in Table 2 and Table 3. All assays were performed in triplicate and the results
were presented as the mean of the diameter of inhibition zone in millimeter (mm).
Ketoconazole was used as the standard drug for the antifungal activity screening.
The results revealed that compounds 2, 4, 5, 15, 35, 36 and 37 showed good to
excellent antifungal activities against all the tested fungal strains. Compounds 36 and 37 were
shown excellent antifungal activities, which may be explained in the presence of the two
methoxy and two –OSO
antifungal activities due to the presence of two –OSO
Compounds 2, 4, 5 and 15 exhibited good antifungal activities against all the tested fungal
pathogens. It may be related to the presence of electron withdrawing (Cl, NO and Br) groups
2
F groups on the phenyl ring. Compound 35 displayed good
2
F groups on the two phenyl rings.
2
on the phenyl ring. Compound 3 showed good antifungal activities against the Fusarium
moniliforme and Fusarium oxysporum fungal strains. Compound 17 showed good antifungal
activity against the Fusarium moniliforme and Fusarium oxysporum fungal strains due to the
presence of –CF
compounds (9, 12 and 14) displayed less antifungal activities against the tested fungal
pathogens. It is speculated that the presence of electron donating (OCH ) groups on the
3
groups on the phenyl ring which may increase the antifungal activity. The
3
phenyl ring may reduce the antifungal activity. The remaining compounds showed very less
or poor activity against the tested all pathogens which suggest that other functional groups
(
CN, CH
3
, NH
2
and I) don’t influence the antifungal activity.
Based on their promising antimicrobial activity, these synthetic compounds were
further tested for their minimum inhibitory concentration (MIC). The results showed that,
compounds 35, 36 and 37 exhibited excellent MBC and MFC activity against all the bacterial
and fungal strains (MIC values were below the standards). Compounds 9 exhibited good
6

MBC activity against the bacterial pathogens of Staphylococcus aureus and Bacillus subtilis.
Compounds 14 and 25 displayed good MBC activity against all the tested bacterial
pathogens. Compound 29 showed good MBC activity against the Gram-positive strains
Staphylococcus aureus and Bacillus subtilis as well as Gram-negative strains Escherichia coli
and Klebsiella pneumonia respectively. Compounds 2, 4, 5 and 15 showed good MFC
activity against all the fungal pathogens (MIC values were below the standards). On the basis
of these promising results, we are convinced that aryl fluorosulfates will be widely used in
the near future for the development of antimicrobial drugs.
2
.2.3. Docking studies
The MurB enzyme catalysis repeating disaccharide and pentapeptide units of the
bacterial peptidoglycan layer are connected by a lactyl ether bridge biosynthesized from
phosphoenolpyruvate. It is well known that structure and mechanism of the MurB enzyme
will permit unusual enol ether reduction reaction. Hence disruption at the active side of
MurB, causes inhibition of peptidoglycan. The bacterial Hfq is a protein, which plays an
important role in the regulation of genes in cooperation with sRNAs. Hfq has two or more
sites to bind RNA(s) including U-rich and/or the poly (A) tail of mRNA. Whereas, the AmpC
β-lactamase provide a multi-resistance to the β-lactam antibiotics. These are keys check point
where inhibitors designed to enzymes could work as potent antibacterial compound and may
facilitate as candidate of next-generation antimicrobial agents.
The mevalonate 5-diphosphate decarboxylase produces an isopentenyl diphosphate a
building block for polyisoprenoid synthesis from Mevalonate pathway. Isopentenyl
diphosphate is a critical pathway for growth of the human bacterial pathogen Enterococcus
faecalis. The mevalonate 5-diphosphate decarboxylase suggested as a therapeutic target for
the treatment of drug-resistant bacterial infections. Whereas α-1,2-Mannosyltransferase is an
member of glycosyltransferase family, which actively take part in biosynthesis of cell wall
7

glycoproteins of Saccharomyces cerevisiae. The α-1,2-Mannosyltransferase is known to
implicated in virulence of Candida albicans. The Sec3p-N interacts with Rho1p and
membrane containing PIP in the process of exocytosis. Hence, attempt was focused for
2
potent antimicrobial compound which act on key point of virulent pathogenic bacteria and
fungi which hampers its growth.
Molecular docking depicts the structural geometry, drawn against the interaction of the
protein with ligand binding mode by evaluating the energy scores of different bound poses of
the ligand within functional site of the protein template with a scoring function. The best
scoring reflects the promising targets for drug candidates, whose actions depend upon the
inhibition or regulation of the target protein functions [17]. Among the 37 ligands (Scheme
Fig. 1), M-36 was found to be more potent than the standards used in the in vitro
methodologies. The docking studies results were represented against potent compound M-36
(
Fig. 4 and 5). Antibacterial potency of M-36 was also found potent against MurB. The
structure of ligand M-36 is depicted as structure analog of curcumin [18] which is known to
possess antimicrobial as well as antioxidant activities, not surprisingly, M-36 in our docking
result also showed tremendous bioactivity. It is worthy to note, a M-36 formed hydrogen
bond with the hydroxyl group of hydrophilic amino acids in MurB (1MBT) and AmpC β-
lactamase (Supporting information Table S1). Hence, it can be reasonably speculated that
compound M-36 inhibited the bacterial peptidoglycan bio-synthesis by restricting the vital
MurB enzyme from carrying out its function [19]. MurB, a critical enzyme involved in
bacterial cell wall synthesis, we have established one plausible target for the synthesized
molecules M-36. Bacterial Hfq is a protein that plays an important role in the regulation of
genes in cooperation with sRNAs. An RNA chaperone Hfq acts as a central player in post-
transcriptional gene regulation in several Gram-negative bacteria [20]. Ligand M-37 was
8

found to be slightly more potent than M-36 because M-37 obstruct the RNA binds in the
post-transcriptional process of protein synthesis (Supporting information Table S2).
2
.2.4. Molecular Dynamics
The molecular dynamic simulations of MurB complex with compound 36 (Fig 6)
simulated their interaction under explicit solvent conditions. The OPLS 2005 potential energy
analysis of the docked complex showed a gradual decrease and appeared to remain stable
after 5 ns to MD simulations. In MD simulation, there was change in the positioning of
compound 36 in relation to the protein indicating conformational change of the ligand. This
clearly deduces the compound 36 closely interact with MurB active and causes
conformational changes as the compound binds to the protein. The VMD package revealed
that, MurB structure formed number of salt bridges involving residues. These salt bridges
were formed intra as well as inter bond with MurB polypeptide. Thus, from the present study
it is concluded that MurB from Escherichia coli docked with Compound 36 is a stable
complex when subjected to MD simulation in presence of explicit solvent.
The antifungal property of M-36 showed good antifungal activity against those chosen
protein templates (Table 5). 1FI4 is responsible for sterol/isoprenoid biosynthesis, 3A58
where Rho- and phosphoinositide-dependent localization is present and 1S4N is involved in
the biosynthesis of yeast cell walls glycoproteins [21-24], Docking studies showed that the
amino acid residues of enzyme 1FI4 played an active role in the interaction with ligand M-36
(
Table 5). Ligand M-36 was found to be more potent with the glide XP score of -5.74
kcal/mol (Supporting information Table S1 and S2) showing strong interaction with Lys123
via the hydrogen bond and salt-bridge, whereas the hydrogen bond between M-36 and Val40
was formed via a catalytical water molecule. Thereby we can assume that M-36 might have
interference Rho- and phosphoinositide-dependent localization. On the other hand, in case the
of 1FI4, M-37 was better than M-36 with the glide XP score of -6.84 kal/mol and forming a
9

hydrogen bond with Thr75. Ligand, M-36 had glide XP score of -6.21kcal/mol in case of
S4N, forming a hydrogen bond with Val282. Our antifungal docking studies demonstrated
1
that M-36 is connected more effectively with above mentioned targets than the known
antifungal compounds.
QikProp, the prediction program was used to calculate pharmacokinetic ADME
(
absorption, distribution, metabolism and excretion) properties consisting of principal
descriptors and physiochemical properties. Qikprop modules predict the range of molecular
properties for the newly synthesized compounds to compare them with 95% of those known
drugs. All the ligands obey the Lipinski’s rules: QPpolrz, QPlogPC16, QPlogPoct, QPlogPw,
QPlogPo/w, QPlogS, CIQPlogS, QPlogHERG, QPPCaco, QPlogBB, QPPMDCK, QPlogKp,
QPlogKhsa, Human Oral Absorption, Percent Human Oral Absorption, SAfluorine, SA
amide O, number of Nitrogen and Oxygen and Lipinski Rule of Five. Based on the above
results, it may be concluded that M-36 possesses the potentiality to be utilized as a better
antibacterial and antifungal drug candidate (Supporting information Table S3).
2
.2.5. Cytotoxicity assay
The cytotoxicity evaluation of the synthesized compounds were carried out using
MDA-MB-231 is a triple negative breast cancer cell line. In order to ascertain the likely
safety of compounds for their potential use [25,26] a standard cell-based toxicity analysis was
performed and showed different performance of synthesized compounds response to the
MDA-MB-231 cells in the present investigations. The compounds 9, 12, 36 and 37 are less
cytotoxic and compounds 4, 5, 13, 23, 24, 29 and 35 are moderate and compounds 6, 16, 22,
3
2 and 33 showed high toxicity to the cells. But rest of the compounds is negligible in the
effect against MDA-MB-231 cells. The greater effect of compounds 9 (36±0.13), 12
48±0.12), 36 (38±0.36) and 37 (40±0.28) µg/mL has IC50 value compared to the standard
doxorubicin having IC50 value of 21 µg/mL. This significant effect represents the
(
10

permeability of the synthesized compounds to the cell. It also confirms the effect of
compound can pass through the membrane to cause the death of the cell and consistence with
the reported literatures [26].
3
. Conclusion
In the present investigation, a series of simple aryl fluorosulfates were synthesized in
good yields and tested for their preliminary in vitro antibacterial and antifungal activities. It
was that found compounds (9, 12, 14, 19, 25, 26, 35, 36 and 37) displayed good antibacterial
activity while compounds (5, 9, 10, 11, 12, 28 and 30) demonstrated good antifungal
activities compared to that of the standard antibiotics gentamicin and ketaconazole. Among
3
7 analogs, the compounds 36 and 37 are highly potent in nature. Further, SAR study showed
that the presence of -OSO F groups, electrons donating (OCH ) and withdrawing (Cl, NO , F
2
3
2
and Br) groups on the phenyl ring played an important role in the antimicrobial activities.
Molecular docking studies were performed for all the synthesized compounds, among them
compounds 35, 36 and 37 showed the highest glide g-scores. On the basis of these promising
results, we convinced that aryl fluorosulfates will be widely used in the near future for
antimicrobial drugs.
4
. General Experimental details
All reactions were carried out under an air atmosphere. Unless otherwise specified,
1
NMR spectra were recorded in CDCl
3
on a 500 or 400 MHz (for H), 471 or 376 MHz (for
1
9
13
F), or 126 MHz (for C) spectrometer. All chemical shifts are reported in ppm relative to
1
TMS ( H NMR, δ ppm) as an internal standard. All chemicals and reagents were purchased
from Energy Chemicals, China and used without further purification. The coupling constants
are reported in Hertz (Hz). The following abbreviations are used to explain the multiplicities:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. High resolution
mass spectroscopic analysis was performed on a Bruker MicroTOF QII mass spectrometer in
11

positive mode. Progress of the reaction was monitored by TLC using silica gel coated on
glass plates the compounds on the TLC plates were detected under UV light.
4
.1. Procedure for the synthesis of aryl fluorosulfates (1-37)
In a 50 mL three-necked flask equipped with a stirring bar, phenol (2 mmol, 1.0
equiv) and Et
3
N (1.2 equiv) were dissolved in 10 mL CH
2
Cl
2
. SO
2
2
F was introduced by
bubbling through the solution. The reaction was monitored by TLC. After 4-12 h at room
temperature before concentrating under vacuum. The crude product was purified by silica gel
chromatography by gradient elution with 5−20% EtOAc / Petroleum ether to give pure
product (1-37).
1
19
13
Note: All the aryl fluorosulfates are identical to those reported regarding the H, F and C
NMR to those reported in the previous papers [26-29]. Syntheses and characterization of the
unprecedented new compounds are reported as below.
Full characterization of the new compounds
4
.2. 3-(Trifluoromethyl)phenyl sulfofluoridate (13)
Petroleum ether / ethyl acetate = 20 : 1 (v / v) as eluent for column chromatography.
1
white solid, 84% Yield. H NMR (500 MHz, CDCl
3
) δ 7.72 (d, J = 7.8 Hz, 1H), 7.66 (t, J =
1
3
8
1
.1 Hz, 1H), 7.63 (s, 1H), 7.57 (d, J = 8.2 Hz, 1H); C NMR (126 MHz, CDCl
3
) δ 149.8,
33.2 (q, J = 34.6 Hz), 131.3, 125.7 (q, J = 3.6 Hz), 124.5, 122.8 (q, J = 273.4 Hz), 118.5 (q,
1
9
J = 4.6 Hz); F NMR (471 MHz, CDCl3) δ: 38.4 (1F), -62.9 (3F); HRMS m/z [M+]: Calcd
for C S: 243.9817; Found: 243.9816
.3. 4-(Tert-butyl)phenyl sulfofluoridate (18)
7
H
4
F
4
O
3
4
Petroleum ether / ethyl acetate = 20 : 1 (v / v) as eluent for column chromatography.
1
Colorless oil, 89% Yield. H NMR (500 MHz, CDCl
3
) δ 7.49 (d, J = 8.8 Hz, 2H), 7.27 (d, J =
) δ 152.0, 147.9, 127.3, 120.2, 34.7,
1
3
8
.6 Hz, 2H), 1.35 (s, 9H); C NMR (126 MHz, CDCl
3
12

1
9
3
2
4
1.2; F NMR (471 MHz, CDCl
32.0569; Found: 232.0562
.4. 3,4-Dimethylphenyl sulfofluoridate (19)
3
) δ: 37.1; HRMS m/z [M+]: Calcd for C
8
H
13FO S:
3
Petroleum ether / ethyl acetate = 20 : 1 (v / v) as eluent for column chromatography.
1
Colorless oil, 81% Yield. H NMR (500 MHz, CDCl
3
) δ 7.22 (d, J = 8.4 Hz, 1H), 7.13 (s,
1
3
1
1
H), 7.08 (d, J = 7.7 Hz, 1H), 2.32 (s, 3H), 2.30 (s, 3H); C NMR (126 MHz, CDCl
3
) δ:
1
9
48.2, 139.3, 137.5, 131.1, 121.5, 117.8, 19.9, 19.3; F NMR (471 MHz, CDCl
FO S: 204.0256; Found: 204.0261
.5. 4-(Benzyloxy)phenyl sulfofluoridate (21)
3
) δ: 37.1;
HRMS m/z [M+]: Calcd for C
8
H
9
3
4
Petroleum ether / ethyl acetate = 20 : 1 (v / v) as eluent for column chromatography.
1
White solid, 85% Yield. H NMR (500 MHz, CDCl
3
) δ 7.41-7.38 (m, 4H), 7.35 (t, J = 6.6
1
3
Hz, 1H), 7.25 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 9.1 Hz, 2H); 5.07 (s, 2H); C NMR (126
1
9
MHz, CDCl
MHz, CDCl3) δ: 36.5; HRMS m/z [M+]: Calcd for C13
.6. 4’-Cyano-[1,1’-biphenyl]-4-yl sulfofluoridate (22)
3
) δ 158.5, 143.8, 136.2, 128.8, 128.3, 127.5, 122.0, 116.1, 70.6; F NMR (471
H11FO
4
S: 282.0362; Found: 282.0370
4
Petroleum ether / ethyl acetate = 10 : 1 (v / v) as eluent for column chromatography.
1
White solid, 85% Yield. H NMR (500 MHz, CDCl
3
) δ 7.77 (d, J = 8.4 Hz, 2H), 7.78 (t, J =
19
13
8
.5 Hz, 4H), 7.47 (d, J = 8.4 Hz, 2H); F NMR (471 MHz, CDCl
3
) δ 38.1; C NMR (126
MHz, CDCl
3
) δ 150.2, 143.6, 140.0, 132.8, 129.3, 127.9, 121.7, 118.5, 112.0; HRMS m/z
M+]: Calcd for C13 FNO S: 277.0209; Found: 277.0215
.7. Benzene-1,3,5-triyl trisulfofluoridate (25)
Petroleum ether / ethyl acetate = 10 : 1 (v / v) as eluent for column chromatography.
[
H
8
3
4
1
13
White solid, 86% Yield. H NMR (500 MHz, CDCl
3
) δH: 7.53 (s, 3H); C NMR (126 MHz,
) δ: 150.2, 115.6; F NMR (471 MHz, CDCl ) δ: 40.1; HRMS m/z [M+]: Calcd for
: 371.8891; Found: 371.8899
1
9
CDCl
3
3
C
6
H
3
F
3
O S
9 3
13

4
.8. 2-Iodocyclopenta-1,4-dien-1-yl sulfofluoridate (32)
Petroleum ether / ethyl acetate = 20 : 1 (v / v) as eluent for column chromatography.
1
Colorless oil, 84% Yield. H NMR (500 MHz, CDCl
3
) δ 7.93 (d, J = 8.0 Hz, 1H), 7.46 (t, J =
1
9
7
.5 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H); F NMR (471 MHz, CDCl )
3
1
3
δ: 42.2; C NMR (126 MHz, CDCl
M+]: Calcd for C FIO S: 301.8910; Found: 301.8915
.9. (Z)-4-(7-(4-((fluorosulfonyl)oxy)-3-methoxyphenyl)-3-hydroxy-5-oxohept-3-en-1-yl)-2-
3
) δ 150.4, 140.8, 130.2, 130.0, 121.7, 88.4; HRMS m/z
[
5
H
4
3
4
methoxyphenyl sulfurofluoridate (36)
Petroleum ether / ethyl acetate = 5 : 1 (v / v) as eluent for column chromatography.
1
Off white solid, 85% yield. H NMR (500 MHz, CDCl
3
) δ 15.3 (s, 0.7H), 7.19 (d, J = 8.1 Hz,
2
0
H), 6.86 (d, J = 1.5 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 5.39 (s, 0.7H), 3.87 (s, 6H), 3.55 (s,
1
3
.3H), 2.92 (t, J = 7.6 Hz, 3.3H), 2.88-2.81 (m, 1.6H), 2.58 (J = 7.5 Hz, 3.3H); C NMR
(
126 MHz, CDCl
3
) δ 202.5, 192.4, 151.0, 142.8, 137.4, 122.2, 120.5, 113.7, 113.6, 99.9, 56.1,
1
9
4
4.8, 39.6, 31.2, 29.1; F NMR (471 MHz, CDCl
: 536.0622; Found: 536.0630
.10. 4-((1E,3Z,6E)-7-(4-((fluorosulfonyl)oxy)-3-methoxyphenyl)-3-hydroxy-5-oxohepta-
3
) δ: 39.5; HRMS m/z [M+]: Calcd for
C
21
H
22
F
2
O S
10 2
4
1
,3,6-trien-1-yl)-2-methoxyphenyl sulfurofluoridate (37)
Petroleum ether / ethyl acetate = 5 : 1 (v / v) as eluent for column chromatography.
1
Off white solid, 83% yield. H NMR (500 MHz, CDCl
3
) δ 15.7 (s, 1H), 7.62 (d, J = 15.9 Hz,
2
H), 7.35 (d, J = 8.2 Hz, 2H), 7.21-7.19 (m, 4H), 6.61 (d, J = 15.9 Hz, 2H), 5.89 (s, 1H), 3.98
1
3
(
s, 6H); C NMR (126 MHz, CDCl
3
) δ 182.8, 151.5, 139.7, 139.0, 136.6, 125.9, 122.9,
20.6, 112.5, 56.3; F NMR (471 MHz, CDCl ) δ: 40.3; HRMS m/z [M+]: Calcd for
: 532.0309; Found: 532.0315
1
9
1
3
C
21
H
18
F
2
O
10 2
S
5
. Biology
.1. Antibacterial activity
5
14

In vitro antibacterial activity was evaluated against human pathogens of both gram
positive organisms namely S. aureus and B. substilis as well as gram negative organisms
namely E. coli and K. pneumoniae by agar well diffusion method and micro dilution method
with slight modifications [12].
5
.1.1. Agar Well Diffusion Method [3]
The microorganisms were inoculated into the sterilized nutrient broth and maintained
at 37 °C for 24 hours. On the day of testing, bacteria were subcultured separately in 100 mL
of sterilized nutrient broth. Inoculated subcultured broths were kept at room temperature for
the growth of inoculums. Using the sterile cork borer, wells (6 mm) were made into each
petriplate. The compounds were dissolved in DMSO of 5 mg/mL and from this 5, 10, 15 and
2
0 µL (25, 50, 75, 100 µg/well) were added into the wells by sterile pipettes. The antibiotic
standard gentamicin for antibacterial activity (as positive control) was tested against the
pathogens. The samples were dissolved in DMSO and showed no inhibition act as negative
control. The plates were incubated at 37 °C for 24 h for bacteria growth. After appropriate
incubation the inhibition zone diameter of each well was measured. Duplicates were
maintained and the average values were calculated for eventual assessing of antimicrobial
activity.
5
.1.2. Microdilution Method [30]
All the microorganisms were grown in Muller-Hinton broth. After cultivation for 16–
8 h at 37 °C, bacteria were harvested and their density was determined by measuring OD at
1
A600. MIC of the compounds was determined by agar dilution method. Suspension of each
4
4
microorganism was prepared to contain approximately (1 x 10 - 2 x 10 CFU/mL) and
applied to the plates with serially diluted compounds to be tested (dissolved in DMSO) and
the reference drug gentamicin incubated at 37 °C overnight. Minimum inhibitory
concentration was considered to be the lowest concentration that completely inhibited the
15

growth of microorganisms on the plates. Diameter of Zone of inhibition (mm) was measured
after 24 h and MIC values were determined.
5
.2. Antifungal activity
In vitro antifungal activity was evaluated against human pathogens of A. niger, F.
moniliforme and F. oxysporum by agar well diffusion method well as microdilution method
with slight modifications [15].
5
.2.1. Agar well diffusion method [31]
The microorganisms were inoculated into the sterilized nutrient broth and maintained
at 37 °C for 24 hours. On the day of testing, bacteria were subcultured separately into 100 mL
of sterilized nutrient broth. Inoculated subcultured broths were kept at room temperature for
the growth of inoculums. Using the sterile cork borer, wells (6 mm) were made into each
petriplate. The compounds were dissolved in DMSO of 5 mg/mL and from this 5, 10, 15 and
2
0 µL (25, 50, 75, 100 µg/well) were added into the wells by using sterile pipettes.
Simultaneously the antifungal standards ketoconazole for antifungal activity (as positive
control) were tested against the pathogens. The samples were dissolved in DMSO and
showed no inhibition act as negative control. The plates were incubated at 28 °C for 48 h for
fungi growth. After appropriate incubation the diameter of zone of inhibition zone diameter
of each well was measured. Duplicates were maintained and the average values were
calculated for eventual antimicrobial activity.
5
.2.2. Microdilution Method [15]
Sabouraud agar was used for the preparation of plates. Suspension of each
5
microorganism was prepared to contain 10 CFU/mL. The agar plates were inoculated with
fungal strains and serially diluted test compounds and reference drug dissolved in DMSO.
The plates were incubated at 25 °C for 48–72 h. Minimum inhibitory concentration was
considered to be the lowest concentration that completely inhibited the growth of
16

microorganisms on the plates. Diameter of inhibition zone (mm) was measured after 48 h and
MIC values were determined.
Action of compounds on Shigella flexneri biofilm growth
Inoculum preparation
The S. flexneri was grown in nutrient agar at 35 °C for 18-20 h and cells were
harvested by centrifuging at 5000 rpm for 8 min at 4 °C. Wash the cells thrice in sterile saline
solution and re-suspend pelleted cells in saline solution. Cell density adjusted to an optical
density at 600 nm (OD600) of 0.1 using UV-visible spectrometer and viable counts of
approximately 6 log cfu/mL.
5
.3. Anti-biofilm activity of compounds
The qualitative and quantitative test was performed for determination of biofilm
production using microtiter plate method (MtP) [32]. The experiment was set according to
reported method (dos Santos Rodrigues et al., 2017) with slight modifications, 20 µl aliquots
of cell suspension was inoculated into each one of six well polystyrene microtiter
plate containing 180 µl of nutrient agar supplemented with glucose (10 g/100 mL). MtP was
covered and incubated at static condition of 37 °C for 18 h to favors greater adherence S.
flexneri. After, each well was washed thrice with saline solution; cells were fixed with 150 µl
of methanol for 20 min and dry the MtP at room temperature. The cells were stained with
crystal violet (0.5%) for 15 min then discard the contents and wash thrice with 200 µl of
saline solution. Dry the MtP, using 150 µl of 95% ethanol then dye bound to the cells was
eluted for 30 min and absorbance at 490 nm was determined using microplate
spectrophotometer.
5
.4. Docking Studies
The co-ordinates of 1F1H (Mevalonate 5-diphosphate decarboxylase), 3A58 (Sec3p -
Rho1p complex), 1S4N (α1,2-Mannosyltransferase), 3HSB (YmaH (Hfq), 1MBT (MurB),
17

1
KE4 (AmpC β-lactamase) from Bacillus substilis were obtained from the Brookhaven
Protein Data Bank9 Ligand were drawn using Maestro 2D sketcher and energy minimize was
computed by OPLS 2005. Proteins were prepared by retrieving into Maestro platform
(
Schrödinger, Inc.). Protein structure was corrected, by using Prime software module of
Schrödinger to correct the missing loops and in the protein. Water molecules from YmaH
were removed beyond 5 Å from the hetero atom respectively. Water molecules which are
thought to be important in aiding the interaction between the receptor were optimized during
protein pepwizard. Automated, necessary bonds, bond orders, hybridization, explicit
hydrogens and charges were assigned. OPLS 2005 force field was applied to the protein to
restrained minimization and RMSD of 0.30 Å was set to converge heavy atoms during the
pre-processing of protein before starting docking. Using Extra-precision (XP) docking and
scoring each compound were docking into the receptor grid of radii 20Å × 20Å × 20Å and
the docking calculation were judge based on the Docking score and Glide score. Molecular
visualization was done under Maestro workspace [33].
5
.5. Molecular Dynamics
Molecular dynamics (MD) simulations were performed to validate the stability of
ligand at the active site using Desmond program inbuilt with OPLS 2005 module. The MurB-
Compound 36 complex was positioned in an orthorhombic cell soaked with a pre-equilibrated
box of explicit solvent including 0.15M NaCl salt with single-point charge (SPC) water
model in a cubic box, with geometrical dimension of 10 Å×10 Å× 10 Å. All overlapping
solvent molecules were removed and an appropriate number of counter ions were added to
maintain charge neutrality. The energy minimization for both SPC water models and the
protein before subjecting the system to MD simulation performed using default relaxation
protocol with an interface for 10 ns. The average structure of the simulated complex that was
18

generated was used for computing the distances of interacting residues as well as salt bridge
formation.
5
.6. Cytotoxicity assay
Cell culture
The Human triple negative breast cancer cells (MDA-MB- 231) was purchased from
the National Center for Cell Sciences (NCCS), Pune, India. They were maintained in DMEM
under standard cell culture conditions at 37 °C and 5% CO in a humidified environment.
.7. MTT assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was
2
5
used to determine the cytotoxicity of the compounds as previously described [34]. The optical
density was measured at 620 nm in an ELISA multiwell plate reader (Thermo Multiskan EX,
USA). The OD value was used to calculate the percentage of viability using the following
formula.
%
of Viability = OD value of experimental sample x 100
OD value of experimental control
Statistical analysis
All studies were implemented in triplicate. All data are expressed as the mean ± standard
deviation (SD) and P values of less than 5% were considered statistically significant.
Acknowledgements
We are grateful to the National Natural Science Foundation of China (Grant No.
2
1772150) and Wuhan University of Technology for financial support.
Competing financial interests: The authors declare no competing financial interests.
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22

Figure 1: Scheme representation of synthesis of compounds (1-37).
23

Figure 2: The antibiofilm activity of aryl fluorosulfate analogs. The all 37 synthetic
analogs were test for anti-biofilm activity agasint S. aureus in 96 well palte using crustal
violet method. The obtained results showed tat, the compounds 25, 26, 35, 36, and 37 are
highly potent in nature and compounds 9, 12, 14, and 19 are moderate in nature compared to
the others had negligeble activity agaisnt S. aureus biofilm growth was depicted.
Figure 3: The SEM image of S. aureus. The S. aureus was treated with MIC concentration
of compound 37 overnight and processed sample was observed in SEM. The results showed
that, the highly potent compound 37 act on cell membrane of S. aureus leading to structural
variations and death of the organism was indicated in arrow in the depicted figure.
24

Figure 4: (A) Molecular interaction of MurB (PDB ID: 1MBT), (B) YmaH (Hfq) (PDB ID:
HSB) and (C) AmpC β-lactamase (PDB ID: 1KE4) with compound 36, showing the
3
hydrogen bond with the backbone amino groups on left side and secondary structure
respective protein depicting the best docked pose for compound 36. RNA is represented in
CPK colored in red.
25

Figure 5: (A) Molecular interaction of Mevalonate 5-diphosphate decarboxylase (PDB ID:
FI4), (B) α1,2-Mannosyltransferase (PDB ID: 1S4N) and (C) Sec3p - Rho1p complex (PDB
1
ID: 3A58) with compound 36, showing the hydrogen bond with the backbone amino groups
on left side and secondary structure respective protein depicting the best docked pose for
compound 36 at the catalytical site.
26

Figure 6: Simulative interactive diagram showing MurB docked with compound 36
simulated in water environment using Desmond application of Schrondinger software. The
complex was subjected for 10ns MD simulations. Green color represent backbone donor
whereas blue color represent side-chain acceptor.
27

Table 1: Antibacterial activity of synthesized compounds (1-37)
a
Zone of Inhibition (mm)
Bacillus substilis
Entry
Gram-positive bacteria
Gram-negative bacteria
Escherichia coli Klebsiella pneumonia
Staphylococcus aureus
2
5µg/mL
07±1
04±1
07±1
-
50µg/mL
11±0
09±1
10±1
-
75µg/mL
15±2
14±1
13±1
-
100µg/mL
18±0
16±2
16±0
-
25µg/Ml
06±1
05±1
06±1
-
50µg/mL
11±2
09±1
09±2
-
75µg/mL
16±1
12±1
12±1
-
100µg/mL
19±1
16±0
16±1
-
25µg/mL 50µg/mL 75µg/mL
100µg/mL
20±1
-
25µg/mL 50µg/mL
75µg/mL 100µg/mL
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
10±1
-
05±1
-
13±1
-
17±2
-
07±0
-
11±0
-
16±0
-
19±1
-
-
08±1
-
11±1
-
13±0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
07±1
04±1
04±1
-
13±1
-
11±1
06±1
06±1
-
19±2
-
15±2
10±4
10±2
19±1
15±1
14±0
-
31±1
-
09±1
07±1
-
13±1
10±2
-
15±1
14±2
-
17±1
17±1
-
05±1
-
09±2
-
12±1
-
16±0
-
06±1
-
10±1
-
12±1
-
14±2
-
-
-
-
-
-
15±1
05±1
07±1
17±1
-
18±1
-
10±1
07±1
-
15±1
14±1
09±1
12±1
10±2
12±1
20±1
09±1
-
08±1
15±1
10±1
07±2
-
22±1
09±1
10±1
24±1
-
24±2
-
14±2
10±2
-
20±
19±1
14±2
15±2
16±1
15±2
27±2
12±1
-
12±1
22±1
15±1
11±2
-
27±2
11±2
13±1
28±2
-
30±1
-
17±0
14±1
-
24±1
22±1
17±1
20±2
21±1
19±1
33±1
19±1
-
15±2
27±2
21±1
15±1
-
31±0
15±2
16±0
30±1
-
34±1
-
21±1
16±1
-
30±1
26±1
20±1
26±1
23±1
27±1
38±1
26±1
-
18±1
30±2
24±0
18±2
-
15±1
07±2
06±0
16±1
-
17±2
-
09±1
-
23±2
10±1
10±1
21±2
-
25±1
-
12±1
-
28±2
15±1
13±1
27±1
-
31±2
-
17±1
-
31±1
19±1
17±0
31±0
-
34±1
-
21±0
-
26±2
-
-
27±2
-
30±1
11±0
14±1
-
15±1
04±1
-
17±1
-
19±1
06±1
10±1
06±1
-
08±1
07±1
-
10±1
09±1
11±0
19±1
08±2
08±1
-
16±2
06±1
-
21±2
10±1
-
22±1
-
24±2
10±2
12±0
09±1
-
11±2
11±2
-
14±2
14±2
16±1
26±2
10±2
12±1
07±0
19±1
09±1
-
27±3
14±1
-
28±2
-
30±1
14±1
17±0
13±1
-
14±0
15±0
10±1
19±1
18±1
20±1
31±1
14±1
14±2
10±1
25±1
14±1
-
31±1
17±1
-
32±1
-
34±1
17±0
21±1
16±1
-
16±1
17±1
13±1
23±1
21±1
28±1
38±1
19±2
17±2
15±1
30±1
18±1
-
-
-
-
15±1
-
16±1
05±1
06±1
-
23±1
-
22±2
09±1
10±1
-
31±2
-
33±2
14±1
18±0
-
-
-
-
-
-
-
-
-
20±1
13±1
18±1
13±1
11±1
09±1
19±1
06±1
07±1
07±1
14±1
08±1
06±2
-
24±1
17±1
11±2
19±2
16±2
13±1
26±1
14±1
11±2
12±2
17±1
14±2
12±1
-
30±1
23±2
15±2
22±4
21±1
17±1
32±1
21±2
15±1
16±1
21±2
20±1
16±2
-
34±2
27±1
19±1
27±1
24±1
26±0
36±2
24±2
17±2
18±1
25±1
23±1
19±1
-
07±1
13±1
-
10±2
10±1
08±1
19±1
07±1
08±1
-
10±1
17±2
-
19±2
15±1
13±2
25±1
10±0
12±1
-
12±1
21±1
-
24±1
19±2
18±1
29±1
12±1
15±1
-
14±0
26±1
-
29±1
23±1
25±1
33±2
16±1
19±1
-
16±2
07±1
07±1
-
24±2
14±1
10±2
-
30±1
19±1
11±1
-
35±2
21±1
15±0
-
-
-
-
-
28

3
3
3
3
3
3
4
5
6
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
05±1
-
09±2
-
11±2
-
14±0
-
07±1
-
10±2
-
12±1
-
14±2
-
20±1
22±1
18±1
19±1
-
27±1
31±1
26±1
25±2
-
35±1
37±1
34±1
31±1
-
41±2
42±2
40±2
34±2
-
21±2
20±1
18±2
18±1
-
28±2
29±2
27±2
24±2
-
37±1
39±2
36±2
30±1
-
43±2
44±1
41±1
33±1
-
19±1
19±1
17±1
21±1
-
28±3
27±2
25±2
27±2
-
36±1
36±1
34±1
31±1
-
41±2
41±1
39±2
34±2
-
18±2
20±1
19±1
19±1
-
27±1
30±1
28±1
25±
-
37±2
39±2
37±1
31±1
-
44±3
44±2
43±1
34±2
-
Std
Control
DMSO
a
Values are mean of three determinations, the ranges of which are <5% of the mean in all cases.
Std: Gentamicin, NA: No activity, (±) Standard deviation
29