Benzodioxane-Benzamides as Antibacterial Agents: Computational and SAR Studies to Evaluate the Influence of the 7-Substitution in FtsZ Interaction

Article abstract of DOI:10.1002/cmdc.201900537

FtsZ is a crucial prokaryotic protein involved in bacterial cell replication. It recently arose as a promising target in the search for antimicrobial agents able to fight antimicrobial resistance. In this work, going on with our structure-activity relationship (SAR) study, we developed variously 7-substituted 1,4-benzodioxane compounds, linked to the 2,6-difluorobenzamide by a methylenoxy bridge. Compounds exhibit promising antibacterial activities not only against multidrug-resistant Staphylococcus aureus, but also on mutated Escherichia coli strains, thus enlarging their spectrum of action toward Gram-negative bacteria as well. Computational studies elucidated, through a validated FtsZ binding protocol, the structural features of new promising derivatives as FtsZ inhibitors.

Full text of DOI:10.1002/cmdc.201900537

Accepted Article
Title: Benzodioxane-benzamides as antibacterial agents:
Computational and SAR studies to evaluate the influence of the
7-substitution in FtsZ interaction
Authors: Valentina Straniero, Victor Sebastián Pérez, Martina Hrast,
Carlo Zanotto, Andrea Casiraghi, Lorenzo Suigo, Irena Zdovc,
Antonia Radaelli, Carlo De Giuli Morghen, and Ermanno
Valoti
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900537
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900537
A Journal of
www.chemmedchem.org

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
Benzodioxane-benzamides as antibacterial agents: Computational
and SAR studies to evaluate the influence of the 7-substitution in
FtsZ interaction
[
a]
[b]
[c]
[d]
[a]
Valentina Straniero ,*, Victor Sebastián Pérez , Martina Hrast , Carlo Zanotto , Andrea Casiraghi ,
[
a]
[e]
[d]
[f]
[a]
Lorenzo Suigo , Irena Zdovc , Antonia Radaelli , Carlo De Giuli Morghen and Ermanno Valoti
[
a]
Dr. V. Straniero, orcid.org/0000-0002-5089-0879; Mr. A. Casiraghi, orcid.org/0000-0002-6256-5694, Mr. L. Suigo, orcid.org/0000-0002-8958-1547, Prof. E.
Valoti, orcid.org/0000-0002-5608-3875
Department of Pharmaceutical Sciences
Università degli Studi di Milano
Via Luigi Mangiagalli 25, 20133 Milano, Italy
valentina.straniero@unimi.it
[
[
b]
c]
Dr. V. Sebastián Pérez, orcid.org/0000-0002-8248-4496
Centro de Investigaciones Biológicas (CSIC)
Ramiro de Maeztu 9, 28040 Madrid, Spain
Dr. M. Hrast, orcid.org/0000-0003-0488-2445
Faculty of Pharmacy
University of Ljubljana
Aškerčeva cesta 7, SI-1000 Ljubljana, Slovenia
Dr. C. Zanotto, orcid.org/0000-0003-0222-6102, Prof. A. Radaelli, orcid.org/0000-0002-5683-6216;
Department of Medical Biotechnologies and Translational Medicine
Università degli Studi di Milano;
via Vanvitelli 32, 20129 Milano, Italy;
Prof. I. Zdovc, orcid.org/0000-0002-5419-2779;
Faculty of Veterinary Medicine
University of Ljubljana
Gerbičeva 60, SI-1000 Ljubljana, Slovenia
Prof. C. De Giuli Morghen, orcid.org/0000-0002-4127-3596;
Catholic University “Our Lady of Good Counsel”,
Rr. Dritan Hoxha, Tirana, Albania.
[d]
[e]
[f]
Supporting information for this article is given via a link at the end of the document.
Abstract: FtsZ is a crucial prokaryotic protein involved in bacterial cell
replication. It recently arose as a promising target in the search for
antimicrobial agents able to fight antimicrobial resistance. In this work,
going on with our SAR study, we develop differently 7-substituted 1,4-
benzodioxane compounds, linked to the 2,6-difluoro-benzamide by a
methylenoxy bridge. Compounds exhibit promising antibacterial
activities not only vs multi drug resistant Staphylococcus aureus, but
also on mutated Escherichia coli strains, thus enlarging their spectrum
of action also towards Gram-negative bacteria. Computational studies
elucidate, through a validated FtsZ binding protocol, the structural
features of new promising derivatives as FtsZ inhibitors.
Glycopeptides as vancomycin, [4] tetracyclines, aminoglycosides
as gentamicin, lincosamides as clindamycin, macrolides as
erythromycin, fluoroquinolones as ciprofloxacin and several other
drugs showed their inefficacy towards Sa. [5]
Moreover,
Enterococcus faecalis
a
large number of both Gram-positive, as
[6]
[7]
and Streptococcus pneumonia , and
Gram-negative strains, as Escherichia coli (E. coli), [ became
resistant to the originally potent antimicrobials. This general
inefficacy stressed the urgent need for the development of new
antibacterial agents with an innovative and effective mechanism
of action.
8]
Among the possible targets, one critical process is the bacterial
cell division and its crucial proteins, considering the essentiality of
the division process in the growth and in the replication of
bacterial cells. Filamentous temperature-sensitive protein Z
Introduction
(
FtsZ) is one of the most promising, well-defined and druggable
Antimicrobial resistance is in continuous growing, reaching
extreme levels. The World Health Organization (WHO) estimates
by 2050 10 million deaths each year caused by this problem. This
number definitely surpasses people died by cancer. [1]
This alarming progression renders commonly used antibiotics
less or none effective. Novel molecules for the treatment of many
bacterial infections are thus required.
[
9]
targets. It has a mandatory role in cell division,
conserved in bacteria
homology with human β-tubulin, even if they are functionally
similar. Any perturbation on the protein proved deleterious on
cytokinetic assembling and bacterial cell survival.
is highly
[
10]
and has less than 20% structural
Starting from these considerations, in the last decade, a wide
number of researchers focused their worked in the development
of FtsZ inhibitors. The firstly discovered FtsZ compounds derived
from natural molecules and from their structural modifications.
Then, synthetic molecules gave better results; the most
interesting antimicrobial classes were 3-alkoxylbenzamides
Among the most common multi-drug resistant bacteria emerged
Staphylococcus aureus (Sa). Indeed, Sa isolated from clinics
revealed now not only to be resistant to methicillin, ampicillin,
oxacillin and other β-lactam antibiotics, [2, 3] but also to a wider
number of antibiotics, independently of their targets.
1
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
(
PC190723, its derivative TXY707 and the derived prodrugs
TXY541, TXY436 and TXY709 in Figure 1), benzopyridines,
pyrido- and pyrimido- pyrazines, benzimidazoles, naphtalenes
[
11-18]
and sulfonamides.
All these compounds proved their
inhibition towards the prokaryotic protein, resulting in
a
bactericidal effect on a wide number of bacterial strains,
especially Gram-positive ones.
Figure 2 – Reference in-house FtsZ inhibitors
We profusely demonstrated the importance of the substituent in
- position of the 1,4-benzodioxane ring, and we evaluated how
7
the simplification of the estereal function (eluding one or the other
Oxygen atoms as for IV and V) results in the maintenance of the
antibacterial activity.
In this work, as a continuation of our SAR study and starting from
reference compounds III, IV and V, we developed novel
derivatives, modifying the substituent in position 7 of the 1,4-
benzodioxane moiety and achieving compounds 1-11.
Specifically, the carboxymethyl group was hydrolyzed obtaining
the corresponding carboxylic acid (1). 1 was a versatile tool, used
for its conversion into the morpholinamide (2) and for the
bioisosteric replacement. Both the carboxymethyl and the
carboxylic acid were substituted by introducing a tetrazole (3-5)
or variously 5-substituted 1,2,4-oxadiazoles (6-11).
Figure 1 – Literature FtsZ inhibitors
Moreover, FtsZ crystal structures of different strains was
intensively studied, alone or in association with known inhibitors,
In addition to the synthesis and SAR study, we report a broader
antibacterial activity. In detail, we tested the compounds against
several Gram-positive Sa strains: methicillin sensitive, methicillin
resistant and multi-drug resistant. Furthermore, we evaluate their
activity not only vs Extended-spectrum beta-lactamase-positive
E. coli, but also on mutated E. coli strains, as done for PC190723
and its prodrug TXY436. [ The best derivatives were also proved
for their cytotoxicity towards human MRC-5 cells.
[
19-22]
and they were used as important starting points for designing
novel inhibitors. In the latest years, our research group profusely
investigate the SARs of different molecules, acting on peculiar
biological targets. [23-26] In this context, we recently identified a
copious class of FtsZ inhibitors. They potently inhibited both
methicillin-sensitive and methicillin-resistant S. aureus and M.
tuberculosis. [27-29]
15]
Moreover, we performed in silico studies, aimed at defining a
docking protocol for this protein; we validated the model using the
literature crystal structures of PC190723 and TXY707 and we
then applied the protocol on our compounds. Hotspots analysis
were accomplished to identify the main binding pockets in the
protein and the main key interactions for each binding cavity.
Docking studies allow us to decipher the binding mode of our
family of derivatives and to define the chemical and structural
properties of the binding cavity.
Among them, the most interesting derivatives, here called I, II and
[27,28]
III,
showed MIC vs MRSA between 0.5 and 0.6 µg/mL (see
Figure 2). Morphometric analysis performed on Sa cells incubated
with our derivatives showed the typical alterations of cell division
inhibition.
Besides, we confirmed FtsZ as the target of this class of
derivatives [29] using a GTPase activity assay and a polymerization
activity assay. The two in vitro assays are the ones widely used
to investigate changes in FtsZ enzymatic function, as suggested
by Kusuma and Tripathy in recent reviews on this topic. [30, 31] FtsZ
inhibition of PC190723 and of other FtsZ inhibitors was confirmed
in this way.
Besides, calculations of a huge number of physicochemical
properties revealed the general good profile in terms of drug-like
properties for the vast majority of the compounds.
Specifically, using the GTPase assay, we determined the effect of
our derivatives on Sa FtsZ GTPase activity by incubating each Chemistry
compound with Sa FtsZ and by kinetically measuring inorganic
phosphate release, as already seen for PC190723.
Compounds 1-11 (Figure 3) were designed starting from the
Conversely, we evaluated by SDS-page the increase in
polymerization after the progressive addition of our derivatives (5,
chemical structures of III, IV and V.
The general synthetic pathway for their synthesis is shown in the
Scheme 1 and it started from the commercially available 3,4-
dihydrobenzoic acid for all the derivatives. The synthesis for each
compound follows a particular branch that is better explained and
reported in Scheme 2, Scheme 3 and Scheme 4.
The first common step for whole series of compounds (see
Scheme 2) is the esterification of the starting carboxylic acid (12)
and the consecutive reaction with epibromohydrin, yielding the 7-
substituted intermediate (13). The 6- substituted isomer was
10, and 20 µg/mL) and we quantified the percentage FtsZ in the
pellets by densitometric analysis.
We assessed our class of compounds is able to modify the
GTPase activity of Sa FtsZ and to stabilize FtsZ polymers,
suppressing polymer dynamics and thus blocking bacterial cell
division.
2
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
obtained only in traces, which were successfully removed via
purification by flash chromatography. In order to achieve 1,
compound 13 was firstly mesylated (14) and sequentially reacted
with 2,6-difluoro-3-hydroxybenzamide, accomplishing the
intermediate 15, which was then hydrolyzed under basic and
warm conditions, achieving 1 as a white solid.
two intermediates were isolated by flash chromatography and
characterized by NMR, their 1H-NMR were significantly diverse
and such a difference let to define the right methylation position
on the tetrazole ring. Specifically, the chemical shift of the methyl
group was of 4.15 ppm for derivative 23 and of 4.37 ppm for 24.
Literature data [32,33] helped us in the identification of the two
compounds. In particular, the ratio between the 1- and the 2-
methyl tetrazole derivatives resulted to be of 25/75, respectively.
23 and 24 followed the same synthetic pathway, consisting in the
debenzylation of the ether (25 and 26) and the consequent
mesylation of the hydroxyl function, affording 27 and 28, and the
final reaction with 2,6-difluoro-3-hydroxybenzamide, obtaining 4
and 5.
The simple condensation of 1 with morpholine, in the presence of
HOBt and EDC hydrochloride as coupling agents, produce the
compound
2 in quantitative yields. The synthesis of the
bioisosters 3-11 differ from one product to the other, but required
the accomplishment of a common intermediate, 18 (see Scheme
3). For its obtainment, the 7-substituted intermediate 13
underwent the simultaneous protection of the hydroxyl function
with benzyl bromide and the hydrolysis of the ester, yielding 16.
Its carboxylic function was converted into the primary amide 17,
via formation of the acyl chloride. The sequential treatment with
TFAA allow the dehydration and the consecutive formation of the
nitrile group, obtaining 18.
Scheme 1 – General synthetic pathway for the synthesis of 1-11
Scheme 2 – Synthesis of 1 and 2
Figure 3 – Compounds object of the present work
The three tetrazoles 3-5 were prepared from 18, following two
different pathways, as reported in Scheme 1. In case of 3 (better
explained in Scheme 4), the intermediate 18 was firstly
debenzylated (19) and mesylated (20), then reacted with 2,6-
difluoro-3-hydroxybenzamide (21) and finally treated with sodium
azide. This last step let the conversion of the nitrile function into
the tetrazole ring and thus the accomplishing of 3.
Reactives and solvents: (a) SOCl
epibromohydrin, TEA, DCM, RT; (c) Mesyl chloride, TEA, DCM, RT (d) 2,6-
Difluoro-3-hydroxybenzamide, K CO , DMF, 80°C; (e) NaOH, MeOH, 50°C; (f)
morpholine, HOBt hydrate, EDC*HCl, DIPEA, DCM, RT.
2
, trimethyl ortoformate, MeOH, reflux; (b)
On the other hand (see Scheme 4), in case of 4 and 5, the
tetrazole 22 was formed from the nitrile function, treating 18 with
sodium azide as previously done. This was further methylated,
obtaining both the 1- and 2- substituted tetrazoles 23 and 24. The
2
3
3
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
Scheme 3 – Synthesis of 18
The synthesis of 6-11 required an additional common
intermediate, called 29 (see Scheme 5), which was easily
obtained from 21, through treatment with hydroxylamine
hydrochloride in basic conditions. Starting from 29, 6 and 8 were
quickly obtained through treatment with CDI and TCDI,
respectively, in the presence of DBU as a base. The methylated
derivatives 7 and 9 were obtained from treatment of 6 and 8 with
methyl iodide.
Even 10 and 11 were easily accomplished from 29, treating it
firstly with acetic or propionic anhydride, in a suitable solvent, and
then with NaOH, in order to let the cyclization into the required
ring.
Reactives and solvents: (a) I. Benzyl bromide, NaH, THF, reflux; II. NaOH,
MeOH, 50°C; (b) I. SOCl
pyridine, RT.
2 3
, 50°C; II. NH 30%, DCM, RT; (c) TFAA, dioxane,
Scheme 4 – Synthesis of 4 and 5
Reactives and solvents: (a) BBr
3
, DCM, 0°C; (b) Mesyl chloride, TEA, DCM, RT; (c) 2,6-Difluoro-3-hydroxybenzamide,
K
2
CO , DMF, 80°C; (d) NaN , ZnCl
3
3
2
, H O/DMF, reflux; (e) MeI, K CO , DMF, 50°C.
2
2
3
Scheme 5 – Synthesis of 6-11
Reactives and solvents: (a) NH
5°C; (d); TCDI, DBU, ACN, reflux; (e) I. acetic anhydride, DCM, RT; II. NaOH, DMF, RT; (f) I. propionic anhydride, ethyl
acetate, RT; II. NaOH, DMF, RT.
2 2 3 2 2 3
OH*HCl, K CO , EtOH/H O, reflux; (b) CDI, DBU, ACN, reflux; (c) MeI, K CO , ACN/DMF,
4
cytotoxicity on human MRC-5 cells. We determined TD90 as the
concentration (µg/mL) able to reduce by 90% the viability of these
cells; furthermore, we calculated the therapeutic index (TI),
expressed as the ratio between TD90 and MBC values.
Results and Discussion
We initially tested compounds 1-11 as done for the reference
compounds III, IV and V, as a continuation of our previous studies.
Specifically, we chose a methicillin-sensitive S. aureus (MSSA,
ATCC 29213) and a methicillin-resistant S. aureus (MRSA, ATCC
When tested on ESBL E. coli, none of the compounds resulted in
inhibiting cell growth at 100 µg/mL, which was the highest dose
proven. These data were in line with previously obtained results
for the reference compounds III-V, suggesting a complete
inactivity on this strain, even modifying the substituent in 7-
position on the 1,4-benzodioxane ring (Table 1).
4
3300) as Gram-positive bacteria and an extended-spectrum of
beta-lactamase-positive (ESBL) E. coli as Gram-negative one
Table 1). Their inhibitory ability was evaluated determining the
MIC, i.e. the lowest compound dose (µg/mL) at which growth is
(
Differently, the MSSA and MRSA inhibition values were promising
inhibited, as well as the Minimal Bactericidal Concentration (MBC), and interesting, considering the distinctive behavior of structurally
i.e. the minimal dose (µg/mL) at which cell growth is irreversibly
blocked after compound removal. The compounds showing the
most promising activities vs MRSA were also assessed for their
similar compounds.
Derivative 9 resulted to have the most promising antibacterial
activities (see Table 1). We conducted morphometric analyses
4
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
(
Figure 4) to assess its effect on bacterial cell division process,
as reference FtsZ inhibitor (positive control). No morphological
changes were noticed in untreated S. aureus.
showing significant alterations in cellular dimension. Bacterial
swelling and cell desegregation commonly resulted in cytocidal
activity.
Specifically, by light microscopy (upper images in Figure 4), we
observed an increase in cell volume of S. aureus, when cultured
in the presence of 9 and difluorononyloxy benzamide (DFNB) [34]
The same morphometric changes were confirmed by
transmission electron microscopy (lower images in Figure 4),
which showed also alterations in the septum formation and
bacterial desegregation, with consequent loss of the
cytoplasmatic content.
Figure 4 – Phase-contrast light microscopy (Top) and ultrastructural analysis by transmission electron microscopic
Bottom) of S. aureus after overnight incubation: Cells with no compound (NT as negative control); cells in the
(
presence of compound DFNB (0.25 µg/mL) as positive control and of compound 9 (0.6 µg/mL). Scale bar: 1 micron.
Desegregations and loss of cytoplasmatic content: black solid arrows; bacterial swelling: white arrows; alterations
in septum formation: black dashed arrows.
MSSA and MRSA inhibition values of all the derivatives, reported
in Table 1, give interesting insights in their SAR.
methyl group resulted in two opposite effects on antimicrobial
activity. Specifically, compound 4, having the methyl in position 2-,
revealed to be a potent inhibitor, while 5, with the methyl in
position 1-, is even less active than the tetrazole 3.
It is suggested that the difference in antimicrobial activity could be
a consequence of the substitution position and thus of the steric
hindrance of the methyl group, which affects FtsZ interaction.
In case of 3, the maintenance of the weak inhibitory activity could
be justified by the charge delocalization of the tetrazole ring that
influences the tautomeric balance, thus affecting the whole
lipophilicity of the molecule. The formation of both the two
regioisomers (4 and 5), during alkylation reaction, corroborated
our hypothesis.
Indeed, the simple transformation of the methyl ester of III into the
carboxylic acid 1 or into the morpholinamide 2 resulted in the
complete loss of activity.
The detrimental result of the free carboxylic acid 1 underlined how
the inhibitory potency of III was strictly related to the maintenance
of the ester moiety, and not to the supposed metabolic hydrolytic
product.
Furthermore, compound 2 was suitably designed from III as
morpholine amide in order to include in its structure peculiar
requirements. Firstly, the replacement of the Oxygen atom with
the Nitrogen one as isoster. Secondly, the insertion of a
disubstituted amide in order to avoid any competition with the
active site of the 2,6-difluorobenzamide and, at the same time, to
keep to ability of acting as HBA, as the reference ester III.
Unfortunately, the loss of antibacterial activity of 2 confirmed the
importance of hydrophobic substituents in 7- position of the
benzodioxane moiety, similarly with what previously obtained for
A similar behavior was seen also for the bioisosteric 5-substituted
oxadiazoles 6-11. Particularly, non-methylated rings 6 and 8
showed
a loss of the antimicrobial activity. Conversely,
methylated oxadiazoles gave also in this case two different
results: 7 was completely inactive, while 9-11 brought promising
results.
The just mentioned differences in FtsZ interaction of the
methylated oxadiazoles, and consequently the discrepancy in the
inhibitory activities, confirmed the importance of the substitution
position. Specifically, when the methyl is close to the 1,4-
benzodioxane, as for 7, the derivative is inactive. Otherwise, the
presence of 2- methylated rings, as for 9, 10 and 11, results in
potent antimicrobial agents.
7
- butyl aminocarbonyl derivative. [24] Moreover, in order to better
elucidate the importance of the methyl ester of III, we introduced
different 5-membered rings, in their free or methylated species
(
see compound 3-11, Figure 3), which are commonly used as
bioisosters of the carboxylic and carboxymethyl function,
respectively.
Considering tetrazoles 3 (bioisoster of the carboxylic acid 1) and
4, 5 (both bioisosters of the methyl ester III), the presence of the
5
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
Table 1. Inhibitory activity of compounds III-V and 1-11 against MSSA, MSSA and MRC-5
MSSA ATCC 29213
MRSA ATCC 43300
MRC-5
ESBL E. coli
Cpd
MIC
µg/mL)
MBC
(µg/mL)
MIC
(µg/mL)
MBC
(µg/mL)
TI
TI
TD90 (µg/mL)
MIC (µg/mL)
(
III
IV
V
1
0.6
0.6
5
> 1280
N.D
2.5
N.D.
5
/
> 800
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
2.5
> 160
> 160
> 800
5
10
40
5
10
40
400
> 100
> 100
80
> 100
> 100
100
/
> 100
> 100
80
> 100
> 100
100
/
/
2
/
/
/
3
/
/
/
4
2.5
2.5
36
2.5
2.5
36
90 ± 0.7
5
> 100
> 100
> 100
> 100
0.6
> 100
> 100
> 100
> 100
0.6
/
> 100
> 100
> 100
> 100
0.6
> 100
> 100
> 100
> 100
0.6
/
/
6
/
/
/
/
/
7
/
/
8
/
/
9
1184
> 320
> 320
1184
> 320
> 320
740 ± 0.17
> 800
> 800
1
0
1
2.5
2.5
2.5
2.5
1
2.5
2.5
2.5
2.5
In order to confirm the importance of the structural features in the
interaction with FtsZ and consequently in the antibacterial
potency, we followed a diverse approach.
moiety bind. The hotspots results will allow us to assess the
binding interactions of the docking studies. Considering the
hotspots results, it will be possible to further identify and design
novel derivatives that meet the interactions criteria.
Using computational studies, we aimed at deciphering the
potential binding mode of this family of derivatives and at gaining
more insights into the binding cavities of Sa FtsZ protein. At the
same time, we performed an analysis considering the main
pharmaceutical relevant properties of these derivatives, in order
to study their drug-likeness. With the objective of obtaining more
information related to the main catalytic pocket in the protein as
well as identifying potential additional binding cavities, hotspot
analysis was carried out. According to the results obtained using
Fragment Hotspots maps application, [35] two druggable binding
pockets were identified; these pockets are the GTP binding site
and the ligand binding site. Both pockets showed a hydrophobic
profile depicted with the yellow hotspot and several additional
polar hotspots represented in blue and red (Figure 5).
Before carrying out the docking studies, our first aim was the
definition and the consequent validation of the docking protocol
for FtsZ, in order to decipher the binding mode of the class of
compounds described in this work. For this purpose, two potent
inhibitors, PC190723 and TXA707 that had been previously
crystallized with the FtsZ protein of S. aureus (PDB code: 4DXD
[
14]
and 5XDT [22]) were considered as reference compounds.
Moreover, the two compounds crystallized (PC190723 and
TXA707 respectively) contained a 2,6-difluorobenzamide moiety,
also found in the chemical family described in this work. For this
reason, both compounds were redocked into the binding site of
the FtsZ crystal structure. As a result, the poses obtained from the
docking studies were almost identical to the ones found in the
crystal structures (Figure 6). Moreover, it is important to remark
that the main interactions, three key hydrogens bonds with Val207,
Leu209 and Gln263, are maintained in the docking results
compared with the crystal structure. Additionally, the hydrophobic
interaction profile is also very similar. These results validate the
docking protocol, which will be further applied in the docking
studies.
Once the protocol was validated, the binding mode of the
compounds synthesized in this work was assessed. In order to
optimize the active site of the protein considering this new
chemical family of benzodioxane derivatives, an induced fit
docking study was performed with the most potent compound of
this series (compound 9). This docking method allows
conformational changes in the lateral chains of the residues that
are part of the binding site induced by the bound ligand. According
to the results, it is possible to confirm the three key hydrogen
bonds between the 2, 6-difluorobenzamide moiety and the FtsZ
protein. On the other hand, modeling data highlighted the specific
FtsZ region involved in binding the benzodioxane scaffold. This
Figure 5 – Hotspots analysis of the FtsZ protein. 2 pockets were identified, the
ligand and the GTP binding site (left and right side of the protein, respectively)
For this study, we are going to focus mainly on the ligand binding
site since previous studies have identified this pocket as the cavity
in which most of the ligands containing 2, 6-difluorobenzamide
6
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
scaffold extends its conformation towards the inner part of the
pocket and the benzodioxane substituent, the 5-membered ring
moiety, is inserted into the deepest part of the cavity characterized
accommodated in the inner hydrophobic subpocket surrounded
by hydrophobic residues such as Met98, Phe100, Val129, Ile162,
Gly193, Ile197, Val214, Met218, Met226, Leu261 and Ile311. The
high hydrophobicity of this subpocket suggests that polar
substituents in the benzodioxane moiety are not well tolerated.
This finding might explain the absence of activity of molecules
containing a hydrophilic substituent such as the ones that are
present in compounds 1, 2, 3 and 6-8.
[
22]
by its narrowness and hydrophobicity.
These chemical
features that characterized the binding mode of these derivatives
can be observed in Figure 7. The docking results for the rest of
the active derivatives such as compound 4, 10 or 11 revealed, as
expected, very similar binding poses compared to reference
compound 9. In this sense, the 5-membered ring substituent is
Figure 6 - TXA707 docking results in the crystal structure of FtsZ 5XDT. A superposition of the ligand in the crystal structure (purple) and the results of the
docking studies (yellow) are depicted
Figure 7 - Binding mode of the most active compound, 9, of this family of derivatives
Additionally, we calculated in silico the most significant physical-
Also, the narrowness of the pocket and its steric restrictions might
chemical and drug-like properties of this class of compounds, in
order to achieve further information and to understand any
influence on cellular activity and FtsZ inhibition. Table 2 reports
the calculations of the following predicted parameters: dipole
moment of the molecule (Dipole), octanol/water partition
coefficient (QPlogPo/w), apparent Caco-2 cell permeability in
nm/sec (QPPCaco) and apparent MDCK cell permeability in
nm/sec (QPPMDCK).
explain that substitutions in position 1 of the 5-membered ring are
not preferred such as in compound 5 compared to 2 substituents
(
compound 4). The former is indeed more linear and less steric
hindered than the latter, which therefore seems to be not tolerated
by the task. The general steric requirements of the cavity can also
validate the promising activities of compounds 9-11, differently
from the null inhibition of 7.
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
Table 3. Inhibitory activity of compounds III-V and 1,3-5, 9-11 against MRSA
Table 2. Key drug-like and permeability properties calculated for this family of
12.1 and MRSA 11.7
compounds.
Cpd
III
IV
V
1
Dipole
9.399
10.188
5.871
9.115
10.805
13.998
6.783
13.311
15.685
11.064
8.206
8.596
6.78
QPlogPo/w
2.663
2.809
3.073
2.223
2.490
1.543
2.279
2.23
QPPCaco
295.021
384.081
1.036.487
26.502
QPPMDCK
321.39
Cpd
MRSA 12.1
MRSA 11.7
MIC (µg/mL)
MIC (µg/mL)
427.37
III
IV
V
≤ 1
4
≤ 1
8
1249.93
30.202
4
4
2
460.630
61.419
520.197
58.907
1
> 128
> 128
4
> 128
> 128
4
3
3
4
201.657
118.901
38.547
213.008
120.357
35.623
4
5
5
> 128
≤ 1
2
> 128
≤ 1
4
6
1.250
1.831
2.701
3.173
2.55
9
7
75.493
73.685
10
11
8
233.639
305.372
249.607
290.952
745.038
628.525
268.238
316.567
2
4
9
Moreover, the good permeability profiles of most of our novel
derivatives encourage us to explore more in depth also Gram-
negative strains.
1
0
1
1
6.657
2.96
Indeed, the promising permeability values (in particular the ones
of V, 2, 4, 8-11) seem to be in conflict with their intrinsically
inactivity on ESBL E. coli, prompting us in evaluating suitably
mutated strains. Indeed the two strains, E. coli D22 and E. coli
N43, are characterized by differences in permeability and in
bacterial cell drug-retention, respectively.
Considering these results, the general in silico permeability
profiles confirmed that the higher the permeability values are, the
more potent are the derivatives. Regarding dipole values, it is
important to remark the relationship between high dipole values
and low antibacterial activity, such as in the case of compounds
3
, 5, 6 and 7. According to the recommended values by the
[36]
Qikprop software,
the higher the computed dipole moment is
Specifically the former bears a mutation in the LpxC gene that
increases membrane permeability, while the latter is knockout of
AcrAB cell membrane pump.
(
mainly above 12), the lower the ability to show properties in the
range of drug-like compounds. Moreover, the high dipole value of
, confirms the initial suggestion about its inactivity, which was
5
Since the poor activity on E. coli ESBL may reflect an inability of
our compounds to target E. coli FtsZ effectively, we aimed in
understanding if the reason could be due to an impossibility to
penetrate Gram-negative membrane or, as for PC190723 and its
prodrug TXY436 [ , to being a substrate of the resistance-
nodulation-cell division (RND)-type efflux pump AcrAB.
previously explained in terms of steric hindrance in the target-
approach. Despite the high similarity between 4 and 5, there is a
great difference in terms of dipole values, suggesting that 4
displays better drug-like properties with a better permeability
15]
profile. Similarly, the inactivity of derivative
6
and
7
is
a
demonstrated not only by the inadequate presence of
hydrophilic 5-membered ring substitution for the interaction profile
in the enzyme, but also by the low permeability profile combined
with the high dipole values.
Table 4. Inhibitory activity of compounds III-V and 1,3-5, 9-11 against mutated
E. coli
E. coli D22
MIC (µg/mL)
> 128
E. coli N43
Cpd
The promising drug-like properties of this class of derivatives,
together with the interesting data on MRSA and MSSA, prompted
us in enlarging the Gram-positive strains to be tested. We firstly
evaluated the derivatives on two clinical isolates of S. aureus,
called MRSA 12.1 and MRSA 11.7, which show resistance
towards antibiotics belonging to different classes and thus
directed to disparate targets. MRSA 12.1 is resistant to
MIC (µg/mL)
III
IV
V
1
16
32
> 128
> 128
64
> 128
> 128
> 128
32
3
> 128
gentamicin,
kanamycin,
rifampicin,
streptomycin,
sulfamethoxazole and tetracycline, while MRSA 11.7 proved its
resistance vs ciprofloxacin, clindamycin, erythromycin,
quinupristin and dalfopristin in association, tetracycline, tiamulin
and trimethoprim.
4
> 128
5
> 128
> 128
16
9
> 128
1
0
1
> 128
16
Table 3 reports the obtained antimicrobial activities, expressed as
MICs. Data show how 4, 9, 10 and 11 confirmed their promising
activity, and thus their potentiality towards these multi-drug
resistant strains.
1
> 128
32
The resulting MICs, summarized in Table 4, revealed how only 1,
and 5 confirm their complete inactivity towards these mutated
3
8
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
Gram-negative bacteria, as previously seen for Gram-positive Sa
ABBREVIATIONS
strains, due to their structural features.
CDI= 1,1’-Carbonyldidimidazole, DBU= 1,8-Diazabicyclo[5.4.0]undec-7-
ene, DCM= Dichloromethane, DIPEA= N,N-Diisopropylethylamine, DMF=
N,N-Dimethylformamide, DMSO= Dimethyl sulphoxide, EDC*HCl= N-(3-
Dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride, HOBt= 1-
Hydroxybenzotriazole hydrate, TCDI= 1,1′-Thiocarbonyldiimidazole, TEA=
Triethylamine, TFAA= Trifluoroacetic anhydride, THF= Tetrahydrofuran.
On the contrary, the other derivatives, together with the reference
compounds III, IV and V, resulted active, with antimicrobial
potency almost comparable to the one shown by TXY436. It
suggests that III, IV, V, 4, 9, 10 and 11, as well as TXY436, are
substrates of the AcrAB efflux pump and that they are thus able
to target FtsZ function and disrupt cell division also in this
bacterium.
General Procedure for Mesylation (Method A). Mesyl chloride (1.30
equiv) was added dropwise to a solution of the alcohol (1.00 equiv) and
TEA (1.30 equiv) in DCM (15 mL) at 0 °C. The mixture was stirred at that
temperature for 1 to 3 hours, diluted with DCM (20 mL), washed firstly with
1
0% aqueous NaHCO
3
(10 mL), secondly with 10% aqueous HCl (10 mL)
SO , filtered
Conclusions
and finally with 10% aqueous NaCl (10 mL), dried over Na
2
4
and concentrated under vacuum to give a residue, which was used as
crude or purified by flash chromatography on silica gel.
The aims of the present work could be summarized as follows: 1)
deepening the SAR analysis of this chemical class, starting from
reference compound III; 2) enlarging the variety of bacterial
strains; 3) performing a computation study in order to better
elucidate the FtsZ binding site features; 4) understanding the
reasons of inactivity towards Gram-negative E. coli.
General Procedure for the Final Condensation (Method B). Potassium
carbonate (1.10 equiv) was added to
a solution of 2,6-difluoro-3-
hydroxybenzamide (1.05 equiv) in dry DMF (5 mL). After stirring at room
temperature for 30 min, a solution of the mesyl derivative (1.00 equiv) in
DMF (5 mL) was added. The reaction mixture was stirred at 80 °C for 16h,
concentrated under vacuum, diluted with Ethyl Acetate (15 mL), washed
with brine (3x10 mL), dried over Na SO , filtered and concentrated, to give
2 4
a residue, which was purified by flash chromatography on silica gel and/or
by crystallization.
Among all the synthetized derivatives, we obtained a series of
potent bioisosters of III. The correlation of the chemical structures
with the antimicrobial activities, the predicted physical chemical
properties and docking analysis let us observing that both the
molecular dipole value and the molecular shape are directly
related to the bactericidal properties.
General Procedure for the Debenzylation (Method C). BBr
DCM (1.00 equiv) was added dropwise to a solution of the benzyl ether
1.00 equiv) in DCM (5/10 mL) at -5 °C. The mixture was stirred at -5 °C
for 2 hours, then it was diluted with DCM (10 mL) and quenched with 10%
aqueous NaHCO (10 mL), stirring at 0 °C for 20 minutes. The organic
layer was then dried over Na SO , filtered and concentrated under vacuum.
3
1.0 M in
(
Indeed, also the molecular geometry, and specifically the
presence of regioisomers, revealed to be determinant in FtsZ
interaction. The strong differences in antimicrobial activity
between 4 and 5, 7 and 9-11 highlighted both the permeability
issue and that some substituents are not well tolerated by the FtsZ
binding cavity. This consideration will be useful for further
structural modifications, because it help us in defining the
chemical requirements of a potent FtsZ inhibitor with antimicrobial
cellular activity.
3
2
4
General Procedure for the Methylation (Method D). Potassium
carbonate (1.20 equiv) was added to a solution of the tetrazole in DMF or
ACN (10 mL). After stirring for 30 minutes at room temperature, methyl
iodide (1.20 equiv) was added dropwise. The reaction mixture was stirred
at 50 °C for 3 to 18 hours, concentrated under vacuum, diluted with Ethyl
Acetate (10 mL), washed firstly with 10% aqueous NaHCO
3
(1x10 mL),
SO , filtered
a residue, which was purified by flash
secondly with 10% aqueous NaCl (1x10mL), dried over Na
2
4
and concentrated to give
chromatography on silica gel.
Finally, the whole results coming from the Gram-negative strains
highlights the capability of these compounds to penetrate E. coli
membrane and to have an antibacterial activity on this bacteria.
The genetic inactivation of RND-type efflux pump AcrAB renders
these derivatives as potent as PC190723 on this pathogen,
confirming that they are substrates of the efflux pump.
Methyl 3,4-dihydroxybenzoate (12): Trimethyl orthoformate (3.9 mL,
35.68 mmol) and thionyl chloride (4.73 mL, 64.88 mmol) was added to a
solution of 3,4-dihydroxybenzoic acid (5 g, 32.44 mmol) in methanol (50
mL) at 0 °C. The reaction mixture was heated at reflux and stirred for 16
hours, concentrated under vacuum, diluted with Ethyl Acetate (50 mL),
washed firstly with 10% aqueous NaHCO
3
(2x15 mL), secondly with 10%
aqueous NaCl (2x15 mL), dried over Na SO
give 4.36 g of 12 (80%) as a white solid. mp: 130 °C. H NMR (300 MHz,
2
4
, filtered and concentrated, to
1
Experimental Section
CDCl
3
): δ 7.64 (d, J = 2.0 Hz, 1H), 7.57 (dd, J = 8.3, 2.0 Hz, 1H), 6.91 (d,
Chemical synthesis
J = 8.3 Hz, 1H), 3.89 (s, 3H) ppm.
Methyl
3-(hydroxymethyl)-1,4-benzodioxan-6-carboxylate
(13):
All starting materials were obtained from commercial suppliers and used
without further purification. H and 13C NMR spectra were taken on Varian
Potassium Carbonate (4.30 g, 31.11 mmol) was added to a solution of 12
4.36 g, 25.93 mmol) in DMF. After stirring at room temperature for 1 hour,
1
(
3
7
00 Mercury NMR spectrometer operating at 300 MHz for 1H NMR, and
epibromohydrin (2.66 mL, 31.11 mmol) was added. The reaction mixture
was stirred at 40 °C for 18 hours, concentrated under vacuum, diluted with
Ethyl Acetate (50 mL), washed firstly with 10% aqueous NaCl (1x30 mL),
secondly with 10% aqueous NaOH (1x30 mL) and finally with 10%
aqueous NaCl (1x30 mL), dried over Na
give 4.6 g of 13 (79%) as a yellow oil. H NMR (300 MHz, CDCl
5 MHz for 13C NMR. Chemical shifts (δ) are reported in ppm relative to
residual solvent (CDCl
3 6
or DMSO-d ) as internal standard. Signal
multiplicity is used according to the following abbreviations: s= singlet, d=
doublet, dd= doublet of doublets, t= triplet, td= triplet of doublets, q=
quadruplet, m= multiplet, sept= septuplet and bs= broad singlet.. Silica gel
F254 was used in analytical thin-layer chromatography (TLC), and silica
gel (particle size 40-63 µm, Merck) was used in flash chromatography;
visualizations were accomplished with UV light (λ 254 nm). Melting point
were determined by DSC (Q20 TA INSTRUMENTS) or Büchi Melting Point
2
SO
4
, filtered and concentrated, to
1
3
): δ 7.58
(
(
3
m, 2H), 6.91 (dd, J = 8.7, 4.2 Hz, 1H), 4.36 (dd, J = 11.1, 2.1 Hz, 1H), 4.26
m, 1H), 4.16 (dd, J = 11.1, 7.8 Hz, 1H), 3.93 (dd, J = 12.1, 4.1 Hz, 1H),
.87 (s, 3H), 3.85 (dd, J = 12.1, 1.5 Hz, 1H) ppm.
(
B-540). Elemental analyses of the new substances are within 0.40% of
theoretical values, therefore the purity of all the final products proved to be
95%.
Methyl 3-(methylsulfonyloxymethyl)-1,4-benzodioxan-6-carboxylate
(14): Method A from 13 (4.6 g, 20.52 mmol), purifying the crude by flash
chromatography on silica gel and eluting with 7/3 Cyclohexane/Ethyl
>
9
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
Acetate, giving 5.65 g (91%) of 14 as a yellow oil. 1H NMR (300 MHz,
over Na
SO
, filtered and concentrated to give 11.06 g (87%) of 17 as a
2
4
CDCl
3
): δ 7.59 (m, 2H), 6.93 (dd, J = 8.9, 2.0 Hz, 1H), 4.52 (m, 3H), 4.36
yellow oil. 1H NMR (300 MHz, CDCl
3
): δ 7.42–7.28 (m, 7H), 6.90 (d, J =
8.4 Hz, 1H), 4.60 (s, 2H), 4.35 (m, 2H), 4.12 (m, 1H), 3.75 (dd, J = 10.7,
.9 Hz, 1H), 3.67 (dd, J = 10.7, 6.3 Hz, 1H) ppm.
(m, 1H), 4.15 (m, 1H), 3.88 (s, 3H), 3.10 (s, 3H) ppm.
4
2,6-Difluoro-3-(7-methoxycarbonyl-1,4-benzodioxan-2-
yl)methoxybenzamide (15): Method B from 14 (1.25 g, 4.15 mmol),
3-(benzyloxymethyl)-1,4-benzodioxan-6-carbonitrile
(18):
purifying the crude by crystallization from methanol and yielding 0.45 g
Trifluoroacetic anhydride (22.62 mL, 162.74 mmol) was added to a solution
of 17 (11.06g, 36.99 mmol) in dioxane/pyridine (4/1, 100 mL tot.) at 0 °C.
The reaction mixture was stirred at room temperature for 16 hours, diluted
with Ethyl Acetate (50 mL), washed firstly with 10% aqueous HCl (2x15
1
(29%) of 15 as a white solid. mp= 154.7 °C. H NMR (300 MHz, CDCl
3
): δ
7
4
3
.59 (m, 2H), 7.08 (td, J = 9.1, 5.2 Hz, 1H), 6.90 (m, 2H), 5.99 (bs, 2H),
.58 (m, 1H), 4.46 (dd, J = 11.5, 2.4 Hz, 1H), 4.36–4.18 (m, 3H), 3.88 (s,
H) ppm.
3
mL), secondly with 10% aqueous NaHCO (15 mL), finally with 10% NaCl,
dried over Na
2
SO
4
, filtered and concentrated under vacuum to give 8.32 g
2
,6-Difluoro-3-(7-carboxy-1,4-benzodioxan-2-yl)methoxybenzamide
1
(80%) of 18 as a dark brown oil. H NMR (300 MHz, CDCl
3
): δ 7.42–7.28
(
1): 10% aqueous Sodium hydroxide (0.8 mL, 2 mmol) was added to a
(m, 5H), 7.17 (m, 2H), 6.92 (d, J = 8.3 Hz, 1H), 4.60 (s, 2H), 4.37 (m, 2H),
solution of 15 (0.3 g, 0.79 mmol) in methanol (8 mL). The reaction mixture
was stirred at 50 °C for 4 hours, concentrated under vacuum and diluted
with DCM and H O. The further treatment of the aqueous phase with
2
4
1
.12 (m, 1H), 3.75 (dd, J = 10.4, 4.7 Hz, 1H), 3.67 (dd, J = 10.4, 5.6 Hz,
H) ppm.
concentrated HCl let the precipitation of 0.27 g of 1 (92%) as a white solid.
3-(hydroxymethyl)-1,4-benzodioxan-6-carbonitrile (19): Method
from 18 (3.72 g, 13.23 mmol), giving 2.47 g (quantitative) of 19 as a brown
oil. 1H NMR (300 MHz, CDCl
): δ 7.17 (m, 2H), 6.94 (d, J = 8.3 Hz, 1H),
4.39 (dd, J = 11.2, 1.8 Hz, 1H), 4.27 (m, 1H), 4.18 (dd, J = 11.2, 7.8 Hz,
1H), 3.95 (dd, J = 12.1, 4.1 Hz, 1H), 3.87 (dd, J = 12.1, 4.8 Hz, 1H) ppm.
C
1
mp: 219°C with partial decomposition. H NMR (300 MHz, d
6
-DMSO): δ
1
5
4
1
1
2.74 (bs, 1H), 8.13 (bs, 1H), 7.86 (bs, 1H), 7.43 (m, 2H), 7.30 (td, J = 9.4,
.3 Hz, 1H), 7.06 (m, 2H), 4.63 (m, 1H), 4.51 (dd, J = 11.6, 2.2 Hz, 1H),
.34 (m, 2H), 4.19 ppm (m, 1H). 13C -NMR (75 MHz, d
-DMSO): δ 167.1,
61.7, 152.6 (dd, J = 240.2, 6.7 Hz), 148.4 (dd, J = 247.2, 8.3 Hz), 147.3,
43.1 (dd, J = 10.8, 2.9 Hz), 142.6, 124.6, 123.6, 118.7, 117.5, 117.1 (dd,
3
6
(
7-cyano-1,4-benzodioxan-2-yl)methylmethansulfonate (20): Method
A from 19 (2.47 g, 12.92 mmol), giving 3.30 g (95%) of 20 as a brown oil.
1H NMR (300 MHz, CDCl
): δ 7.20 (m, 2H), 6.97 (d, J = 8.8 Hz, 1H), 4.49
m, 3H), 4.37 (dd, J = 10.6, 2.4 Hz, 1H), 4.18 (dd, J = 11.7, 6.7 Hz, 1H),
J = 24.9, 20.2 Hz), 116.3 (d, J = 9.1 Hz), 111.5 (dd, J = 22.7, 3.6 Hz), 71.7,
3
6
8.6, 65.2 ppm. Elemental analysis calculated (%) for C17
13 2 6
H F NO : C
(
48.41; H 6.38; N 4.03. Found: C 48.35; H 6.50; N 3.97.
3.10 (s, 3H) ppm.
2
,6-Difluoro-3-(7-(N-morpholylcarbonyl)-1,4-benzodioxan-2-
3
-(7-cyano-1,4-benzodioxan-2-yl)methoxy-2,6-difluorobenzamide
yl)methoxybenzamide (2): DIPEA (0.15 mL, 0.86 mmol) was added
dropwise to a solution of 1 (0.15 g, 0.41 mmol), HOBt Hydrate (0.061 g,
(
21): Method B from 20 (0.89 g, 3.30 mmol), purifying the crude by flash
chromatography on silica gel, eluting with 1/1 Cyclohexane/Ethyl Acetate
and yielding 0.61 g (53%) of 21 as a white solid. mp: 158.2°C. 1H NMR
0.45 mmol) and EDC hydrochloride (0.086 g, 0.45 mmol) in DCM (5 mL).
After stirring at room temperature for 30 min, morpholine (0.04 mL, 0.45
mmol) was added. The reaction mixture was stirred at that temperature for
(
1
300 MHz, d
H), 7.32 (dd, J = 8.4, 2.1 Hz, 1H), 7.26 (dt, J = 9.3, 5.1 Hz, 1H,), 7.07 (dt,
J = 9.3, 2.0 Hz, 1H), 7.1 (d, J = 8.4 Hz, 1H,), 4.67 (m, 1H), 4.52 (dd, J =
6
-DMSO): 8.10 (br s, 1H), 7.82 (br s, 1H), 7.40 (d, J = 1.9 Hz,
16 hours , washed firstly with 10% aqueous HCl, secondly with 10%
aqueous NaHCO , and finally with 10% aqueous NaCl, dried over Na SO ,
3
2
4
11.6, 2.5 Hz, 1H), 4.27 (m, 3H) ppm.
filtered and concentrated, to give a residue which was purified by a flash
chromatography on silica gel. Elution with 3/7 Cyclohexane/Ethyl Acetate
gave 0.073 g (41%) of 2 as a white solid. mp: 268°C with partial
3-(7-(1H-tetrazol-5-yl)-1,4-benzodioxan-2-yl)methoxy-2,6-
difluorobenzamide (3): Sodium azide (0.19 g, 1.81 mmol) and ZnCl2
0.13 g, 0.99 mmol) were added to a solution of 21 (0.2 g, 0.58 mmol) in
/1 water/DMF (5 mL). The reaction mixture was stirred at 100 °C for 24
hours, concentrated under vacuum, diluted with Ethyl Acetate and treated
with 10% NaHCO (15 mL). The pH was brought to 1 treating with
concentrated HCl and the aqueous phase was extracted with Ethyl Acetate
3x15 mL). The organic phase was dried over Na SO , filtered and
decomposition. 1H NMR (300 MHz, d
-DMSO): δ 8.13 (bs, 1H), 7.86 (s,
H), 7.29 (td, J = 9.4, 5.3 Hz, 1H), 7.08 (td, J = 9.0, 1.8 Hz, 1H), 6.92 (m,
H), 4.64 (m, 1H), 4.47 (dd, J = 11.5, 2.3 Hz, 1H), 4.32 (m, 1H), 4.18 (dd,
6
(
1
1
3
J = 11.5, 7.1 Hz, 1H), 4.02 (q, J = 7.2 Hz, 1H), 3.52 (d, J = 32.1 Hz, 10H)
ppm. 13C NMR (75 MHz, d
-DMSO): δ 168.9, 161.8, 152.5 (dd, J = 240.3,
.6 Hz), 148.3 (dd, J = 247.2, 8.5 Hz), 144.4, 143.1 (dd, J = 10.8, 3.1 Hz),
42.6, 129.2, 121.1, 117.4, 116.7, 117.0 (dd, J = 25.0, 20.2 Hz), 116.3 (d,
3
6
6
1
(
2
4
concentrated under vacuum to give 0.1 g (44%) of 3 as a yellowish solid.
mp = 198 °C. H NMR (300 MHz, d
J = 9.1 Hz), 111.5 (dd, J = 22.9, 3.9 Hz), 71.9, 68.6, 66.5, 65.0 ppm.
Elemental analysis calculated (%) for (C21 ): C 58.06; H 4.64; N
1
6
-DMSO): δ 8.12 (bs, 1H), 7.85 (bs, 1H),
20 2 2 6
H F N O
7
(
.56 (m, 2H), 7.31 (dt, J = 9.0, 5.2 Hz, 1H), 7.14 (d, J = 9.0 Hz, 1H), 7.09
dt, J = 9.0, 3.0 Hz, 1H), 4.76-4.62 (m, 1H), 4.53 (dd, J = 11.7, 2.5 Hz, 1H),
6.45. Found: C 57.90; H 4.70; N 6.40.
4
=
1
1
2
.40 (dd, J = 11.0, 4.0 Hz, 1H), 4.34 (dd, J = 11.0, 5.8 Hz, 1H), 4.24 (dd, J
3
-(benzyloxymethyl)-1,4-benzodioxan-6-carboxylic acid (16):
A
11.5, 7.1 Hz, 1H) ppm. 13C-NMR (75 MHz, d
6
-DMSO): δ 161.7, 155,
solution of 13 (10.31 g, 46.43 mmol) in dry THF (50) mL) was added to a
suspension of NaH (2.78 g, 69.64 mmol) in THF (15 mL) at 0 °C. After
stirring at room temperature for 30 minutes, benzyl bromide (8.28 mL,
52.6 (dd, J = 240.3, 6.7 Hz), 148.4 (dd, J = 247.4, 8.6 Hz), 145.8, 143.5,
43.1 (dd, J = 10.9, 3.2 Hz), 116.4 (d, J = 9.1 Hz), 116.2, 111.5 (dd, J =
2.8, 3.9 Hz), 71.9, 68.6, 65.1 ppm. Elemental analysis calculated (%) for
69.64 mmol) was added. The reaction mixture was heated at reflux and
(
17 13 2 5 4
C H F N O ): C, 52.45; H, 3.37; N, 17.99. Found: C, 52.39; H, 3.51; N,
stirred for 16 hours, concentrated under vacuum, diluted with MeOH (50
mL) and treated with 10% aqueous NaOH (36 mL, 90 mmol). The solution
was heated at 50 °C, stirred for 2 hours, concentrated under vacuum,
17.68.
5-((3-benzyloxymethyl)-1,4-benzodioxan-6-yl)-1H-tetrazole (22).
A
washed with Et
extracted with Ethyl Acetate (3x25mL). The organic phase was dried over
Na SO , filtered and concentrated under vacuum to give 12.76 g (91%) of
6 as a yellow oil. 1H NMR (300 MHz, CDCl
): δ 7.64 (m, 2H), 7.35 (m,
H), 6.93 (dd, J = 8.7, 6.6 Hz, 1H), 4.61 (s, 2H), 4.38 (m, 2H), 4.16 (m, 1H),
.77 (dd, J = 10.4, 4.8 Hz, 1H), 3.69 (dd, J = 10.4, 5.7 Hz, 1H) ppm.
2
O (30 mL), treated with concentrated HCl and finally
solution of 18 (0.60 g, 2.11 mmol) in 1/1 water /DMF (10 mL) was added
with sodium azide (0.43 g, 6.59 mmol) and ZnCl2 (0.49 g, 3.58 mmol), at
room temperature. The reaction was then heated to 100 °C and left stirring
for 16 hours. At completion, the mixture was then concentrated under
reduced pressure, resumed with Ethyl Acetate (20 mL) and washed with
aqueous 10% NaCl (3 x 15 mL); the organic layer was then dried over
2
4
1
5
3
3
Na
2
SO
4
, filtered and concentrated under reduced pressure, accomplishing
3
-(benzyloxymethyl)-1,4-benzodioxan-6-carboxamide (17): A solution
1
650 mg (95%) of 22 as a brownish oil. H NMR (300 MHz, CDCl
3
): δ= 7.51
of 16 (12.76 g, 42.49 mmol) in SOCl
at 50 °C for 3 hours, concentrated under vacuum, diluted with DCM (100
mL), added with 30% aqueous NH (25 mL) and stirred at room
temperature for 24 hours. The reaction mixture was then concentrated,
diluted with Ethyl Acetate, washed with 10% NaHCO (2x15 mL), dried
2
(15.5 mL, 212.45 mmol) was stirred
(m, 2H), 7.37-7.31 (m, 5H), 6.97 (d, J = 8.63 Hz, 1H), 4.62 (s, 2H), 4.42-
4
2
.29 (m, 2H), 4.15 (dd, J = 10.6, 7.1 Hz, 1H), 3.75 (dd, J = 10.6, 5.3 Hz,
H) ppm.
3
3
1
0
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
5
-((3-benzyloxymethyl)-1,4-benzodioxan-6-yl)-1-methyl-tetrazole (23)
and 5-((3-benzyloxymethyl)-1,4-benzodioxan-6-yl)-2-methyl-tetrazole
24). Method D from 22 (0.65 g, 2.00 mmol) in DMF and stirring for 3 hours.
2,6-difluoro-3-(7-(N'-hydroxycarbamimidoyl)-1,4-benzodioxan-2-
yl)methoxybenzamide (29): A solution of potassium Carbonate (2.55 g,
18.48 mmol) in H O (5 mL) was added to a solution of 21 (1.28 g, 3.70
2
(
Purification by flash chromatography on silica gel, eluting with 7/3
Cyclohexane/Ethyl Acetate, yielded 120 mg of 23 as white wax and 380
mg of 24 as light yellow oil. Cumulative yield: 77.3%. 5-((3-
mmol) and Hydroxylamine hydrochloride (1.28 g, 18.48 mmol) in ethanol
(45 mL). The reaction mixture was stirred at reflux for 18 hours,
concentrated under vacuum, diluted with Et
10% aqueous HCl (2x10mL). The aqueous phase was neutralized with
10% aqueous NaHCO and extracted with Ethyl Acetate (3x10 mL). The
organic phase was dried over Na SO , filtered and concentrated under
vacuum to give 1.20 g (85%) of 29 as a brown oil. 1H NMR (300 MHz,
DMSO-d ) δ= 9.49 (s, 1H), 8.12 (bs, 1H), 7.85 (bs, 1H), 7.30 (m, 1H), 7.18
2
O (10 mL) and extracted with
1
benzyloxymethyl)-1,4-benzodioxan-6-yl)-1-methyl-tetrazole (23): H NMR
(
300 MHz, CDCl
3
): δ= 7.34 (m, 5H), 7.28 (m, 2H), 7.04 (d, J = 8.3 Hz, 1H),
.62 (s, 2H), 4.36 (m, 2H), 4.18 (dd, J = 10.4, 7.6 Hz, 1H), 4.15 (s, 3H),
.78 (dd, J = 10.4, 4.8 Hz, 1H), 3.70 ppm (dd, J = 10.4, 5.5 Hz, 1H). 5-((3-
3
4
3
2
4
benzyloxymethyl)-1,4-benzodioxan-6-yl)-2-methyl-tetrazole (24): 1H NMR
6
(
1
300 MHz, CDCl
3
): δ= 7.64 (m, 2H), 7.35-7.29 (m, 5H), 6.98 (d, J = 8.2 Hz,
(m, 2H), 7.07 (dt, J = 9.1, 1.9 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 4.58 (m,
1H), 4.43 (dd, J = 11.3, 2.4 Hz, 1H), 4.32 (m, 2H), 4.13 (dd, J = 11.3, 7.1
Hz, 1H) ppm.
H), 4.62 (s, 2H), 4.45-4.33 (m, 2H), 4.37 (s, 3H), 4.16 (dd, J = 10.3, 7.1
Hz, 1H), 3.78 (dd, J = 10.4, 4.3 Hz, 1H), 3.70 (dd, J = 10.4, 5.8 Hz, 1H)
ppm.
2,6-difluoro-7-((1,2,4-oxadiazol-5-one-3-yl)-1,4-benzodioxan-2-
5
-((3-hydroxymethyl)-1,4-benzodioxan-6-yl)-1-methyl-tetrazole (25):
yl)methoxybenzamide (6): DBU (0.13 mL, 0.86 mmol) and CDI (0.14 g,
0.86 mmol) were added to a solution of 29 (0.25 g, 0.66 mmol) in dioxane
(10 mL). The reaction mixture was stirred at reflux for 18 hours, diluted
with DCM (15 mL), washed with 10% aqueous HCl (2x5 mL), dried over
Method C from 23 (0.38 g, 0.98 mmol), giving 190 mg (99.2%) of 25 as a
yellowish oil. H NMR (300 MHz, CDCl
1
3
): δ= 7.35 (m, 2H), 7.06 (d, J = 8.4
Hz, 1H), 4.36 (m, 2H), 4.17 (dd, J = 12.7, 7.5 Hz, 1H), 4.17 (s, 3H), 3.97
dd, J = 12.1, 4.2 Hz, 1H), 3.89 ppm (dd, J = 12.1, 4.9 Hz, 1H).
(
2 4
Na SO , filtered and concentrated under vacuum. The residue was purified
by crystallization from DCM/Ethyl Acetate 1/3 to give 21o mg (79%) of 6
5
-((3-hydroxymethyl)-1,4-benzodioxan-6-yl)-2-methyl-tetrazole (26):
1
6
as a white solid. mp: 250 °C H NMR (300 MHz, DMSO-d ): δ= 12.79 (s,
Method C from 24 (0.12 g, 0.31 mmol), giving 50 mg (99%) of 26 as a
yellowish oil. H NMR (300 MHz, CDCl
1
H), 8.12 (bs, 1H), 7.85 (bs, 1H), 7.30 (m, 3H), 7.05 (m, 1H), 7.08 (d, J =
Hz, 1H), 4.67 (m, 1H), 4.51 (dd, J = 11.7, 2.4 Hz, 1H), 4.46 (m, 2H), 4.21
): δ= 161.7,
1
3
): δ= 7.69 (d, J = 2.0 Hz, 1H), 7.63
9
(dd, J = 8.4, 2.0 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 4.32 (m, 2H), 4.37 (s,
dd, J = 11.6, 7.2 Hz, 1H) ppm. 13C NMR (75 MHz, DMSO-d
(
6
3H), 4.18 (dd, J = 10.9, 7.3 Hz, 1H), 3.95 (dd, J = 12.0, 4.2 Hz, 1H), 3.87
160.4, 157.3, 152.6 (dd, J = 240.2, 6.8 Hz), 148.3 (dd, J = 247.3, 8.6 Hz),
146.5, 143.3, 143.1 (dd, J = 11.0, 3.3 Hz), 120.0, 118.5, 117.0 (dd, J =
25.0, 20.4 Hz), 116.9, 116.3 (dd, J = 9.1, 1.5 Hz), 115.5, 111.5 (dd, J =
22.8, 4.0 Hz), 71.9, 68.5, 65.1 ppm. Elemental analysis calculated (%) for
(dd, J = 12.0, 5.0 Hz, 1H) ppm.
5-((3-mesyloxymethyl)-1,4-benzodioxan-6-yl)-1-methyl-tetrazole (27):
Method A from 25 (0.19 g, 0.97 mmol), giving 200 mg (83.3%) of 27 as a
brown oil. H NMR (300 MHz, CDCl
1
13 2 3 6
(C18H F N O ): C, 53.34; H, 3.23; N, 10.37. Found: C, 53.31; H, 3.28; N,
3
): δ= 7.31 (m, 2H), 7.08 (d, J = 8.4 Hz,
10.31.
1H), 4.55 (m, 1H), 4.47 (m, 2H), 4.41 (m, 1H), 4.22 (dd, J = 11.7, 7.5 Hz,
1H), 4.17 (s, 3H), 3.11 (s, 3H) ppm.
2,6-difluoro-7-((N’-methyl-1,2,4-oxadiazol-5-one-3-yl)-1,4-
benzodioxan-2-yl)methoxybenzamide (7): Method D from 6 (0.15 g,
.38 mmol) in ACN and stirring for 18 hours. Purification by flash
chromatography on silica gel, eluting with 7/3 Cyclohexane/Ethyl Acetate,
5
-((3-mesyloxymethyl)-1,4-benzodioxan-6-yl)-2-methyl-tetrazole (28):
0
Method A from 26 (0.05 g, 0.28 mmol), giving 60 mg (83%) of 28 as a
yellow oil. H NMR (300 MHz, CDCl
1
3
): δ= 7.67 (m, 1H), 7.36 (m, 1H), 6.98
yielded 70 mg of 7 as a white solid. mp: 230 °C with partial decomposition.
(
1
d, J= 8.4 Hz, 1H), 4.85 (m, 2H), 4.36 (m, 2H), 4.37 (s, 3H), 4.18 (dd, J =
1.4, 6.4 Hz, 1H), 3.11 (s, 3H) ppm.
1H NMR (300 MHz, DMSO-d
): δ= 8.11 (bs, 1H), 7.84 (bs, 1H), 7.28 (m,
H), 7.12 (m, 2H), 4.69 (m, 1H), 4.54 (dd, J = 11.6, 2.3 Hz, 1H), 4.37 (m,
H), 4.24 (dd, J = 11.6, 7.1 Hz, 1H), 3.18 (s, 3H) ppm. 13C NMR (75 MHz,
): δ= 161.9, 159.8, 159.0, 152.6 (dd, J = 240.0, 6.8 Hz), 148.4
dd, J = 247.1, 8.6 Hz), 146.3, 143.3, 143.1 (dd, J = 10.5, 3.0 Hz), 122.4,
6
3
2
3
-(7-(1-methyl-tetrazol-5-yl)-1,4-benzodioxan-2-yl)methoxy-2,6-
difluorobenzamide (4): Method B from 27 (0.20 g, 0.19 mmol), purifying
DMSO-d
(
6
the crude by crystallization from chloroform and yielding 170 mg (62.5%)
of 4 as a white solid. mp: 190-192 °C. 1H NMR (300 MHz, DMSO-d
) δ=
.09 (bs, 1H), 7.81 (bs, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.32 (dt, J = 8.4, 2.1
Hz, 1H), 7.26 (d, J = 9.0 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.05 (dt, J = 9.0,
6
118.4, 117.7, 116.9 (dd, J = 24.8, 19.5 Hz), 116.6, 116.5 (dd, J = 9.8, 1.5
8
Hz), 111.5 (dd, J = 22.9, 4.1 Hz), 72.0, 68.6, 65.1, 29.9 ppm. Elemental
analysis calculated (%) for (C19 ): C, 54.42; H, 3.61; N, 10.02.
Found: C, 54.22; H, 3.84; N, 9.92.
15 2 3 6
H F N O
2
.0 Hz, 1H), 4.67 (m, 1H), 4.52 (dd, J = 11.6, 2.4 Hz, 1H), 4.34 (dd, J =
8.5, 4.8 Hz, 1H), 4.34 (dd, J = 11.1, 4.8 Hz, 1H), 4.22 (dd, J = 11.6, 7.2
2
Hz, 1H), 4.10 (s, 3H) ppm. 13C NMR (75 MHz, DMSO-d
2,6-difluoro-7-((5-mercapto-1,2,4-oxadiazol-3-yl)-1,4-benzodioxan-2-
yl)methoxybenzamide (8): DBU (0.20 mL, 1.37 mmol) and TCDI (0.24 g,
6
): δ= 161.6, 153.9,
52.6 (dd, J = 240.2, 6.8 Hz), 148.4 (dd, J = 247.3, 8.5 Hz), 145.6, 143.3,
43.1 (dd, J = 10.8, 3.1 Hz), 122.7 (d, J = 9.1 Hz), 118.3, 117.3, 117.1 (dd,
1
1
1.37 mmol) were added to a solution of 29 (0.4 g, 1.05 mmol) in ACN (10
mL). The reaction mixture was stirred at reflux for 18 hours, diluted with
DCM (15 mL), washed with 10% aqueous HCl (2x5 mL), dried over
Na SO , filtered and concentrated under vacuum. The residue was purified
2 4
J = 24.8, 20.6 Hz), 116.3 (dd, J = 9.2, 2 Hz), 111.5 (dd, J = 22.8, 3.9 Hz),
2, 68.6, 65.1, 35.4 ppm. Elemental analysis calculated (%) for
): C, 53.60; H, 3.75; N, 17.36. Found: C, 53.41; H, 3.91; N,
7
(
18 15 2 5 4
C H F N O
by crystallization from Ethyl Acetate to give 0.1 g (23%) of 8 as a yellow
17.20.
1
solid. mp= 173 °C with decomposition. H NMR (300 MHz, DMSO-d
6
): δ=
3
-(7-(2-methyl-tetrazol-5-yl)-1,4-benzodioxan-2-yl)methoxy-2,6-
8
7
1
.14 (bs, 1H), 7.87 (bs, 1H), 7.43 (m, 2H), 7.31 (td, J = 9.3, 5.2 Hz, 1H),
difluorobenzamide (5): Method B from 28 (0.06 g, 0.23 mmol), purifying
the crude by flash chromatography on silica gel, eluting with
Cyclohexane/Ethyl Acetate 7/3 and affording 33 mg (37.4%) of 5 as a white
.09 (m, 2H), 4.69 (m, 1H), 4.54 (dd, J = 11.6, 2.3 Hz, 1H), 4.39 (dd, J =
1.1, 3.8 Hz, 1H), 4.33 (dd, J = 11.1, 5.7 Hz, 1H), 4.24 (dd, J = 11.6, 7.1
Hz, 1H) ppm. 13C NMR (75 MHz, DMSO-d
6
): δ= 188.2, 161.9, 159.6, 152.6
solid. mp: 187 °C. 1H NMR (300 MHz, DMSO-d
) δ= 8.11 (bs, 1H), 7.83
bs, 1H), 7.53 (td, J = 8.4, 2.1 Hz, 2H), 7.32 (dt, J = 9.3, 5.2 Hz, 1H), 7.08
td, J = 9.1, 1.9 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 4.67 (m, 1H), 4.51 (dd, J
6
(
(
(
dd, J = 240.8, 6.8 Hz), 148.4 (dd, J = 247.5, 8.2 Hz), 146.7, 143.4, 143.1
dd, J = 10.9, 3.4 Hz), 120.9, 118.6, 116.9 (dd, J = 24.7, 20.2 Hz), 116.5
dd, J = 9.0, 2.3 Hz), 116.2, 115.8, 111.5 (dd, J = 23.3, 3.7 Hz), 71.9, 68.6,
(
(
=
1
2
1
11.6, 2.4 Hz, 1H), 4.35 (m, 2H), 4.39 (s, 3H), 4.23 (dd, J = 11.6, 7.1 Hz,
65.1 ppm. Elemental analysis calculated (%) for (C18
13 2 3 5
H F N O S): C,
H) ppm. 13C NMR (75 MHz, DMSO-d
40.3, 6.7 Hz), 148.4 (dd, J = 247.3, 8.4 Hz), 145.2, 143.4, 143.1 (dd, J =
0.8, 3.3 Hz), 121, 120.2, 117.1 (dd, J = 24.8, 20.3 Hz), 116.4 (d, J = 7.2
6
): δ= 164.1, 161.7, 152.6 (dd, J =
51.31; H, 3.11; N, 9.97. Found: C, 51.01; H, 3.28; N, 9.87.
2,6-difluoro-7-((5-methylthio-1,2,4-oxadiazol-3-yl)-1,4-benzodioxan-
2
-yl)methoxybenzamide (9): Method D from 8 (0.14 g, 0.33 mmol) in
Hz), 115.3 (dd, J = 22.9, 3.9 Hz), 71.9, 68.6, 65.1, 40.0 ppm. Elemental
analysis calculated (%) for (C18 ): C, 53.60; H, 3.75; N, 17.36.
Found: C, 53.50; H, 3.87; N, 17.12;
ACN and stirring for 18 hours. Purification by flash chromatography on
silica gel, eluting with 7/3 Cyclohexane/Ethyl Acetate, yielded 70 mg of 9
as a white solid. mp: 165 °C. H NMR (300 MHz, DMSO-d
15 2 5 4
H F N O
1
6
): δ= 8.12 (bs,
1
1
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
1
=
4
1
H), 7.84 (bs, 1H), 7.49 (dd, J = 8.4, 2.0 Hz, 1H), 7.45 (m, 1H), 7.31 (dt, J
9.3, 5.3 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.08 (dt, J = 8.9, 1.8 Hz, 1H),
.68 (m, 1H), 4.52 (dd, J = 11.6, 2.3 Hz, 1H), 4.38 (dd, J = 11.2, 4.2 Hz,
H), 4.33 (dd, J = 11.2, 5.8 Hz, 1H), 4.24 (dd, J = 11.6, 7.2 Hz, 1H), 2.80
Cells were grown over night in the presence of increasing concentrations
of the compounds showing an evident inhibitory activity at concentrations
lower than 1 µg/ml, starting from the previously determined MIC. Data are
reported for S. aureus cultured in the presence of 9 (0.6 µg/ml) and DFNB
(0.25 µg/ml) as a positive control. Untreated S. aureus cultures were used
as negative control. Samples were analysed by phase contrast under a
Zeiss Axioskop microscope.
(
(
(
s, 3H) ppm. 13C NMR (75 MHz, DMSO-d
6
): δ= 179.0, 167.8, 161.7, 152.6
dd, J = 240.8, 6.8 Hz), 148.4 (dd, J = 247.1, 8.6 Hz), 146.2, 143.4, 143.1
dd, J = 11.2, 3.0 Hz), 121.1, 119.6, 118.4, 117.1 (dd, J = 24.7, 20.2 Hz),
1
6
9
16.4 (dd, J = 9.4, 1.9 Hz), 116.2, 111.5 (dd, J = 22.5, 3.7 Hz), 71.9, 68.6,
5.2, 15.1 ppm. Anal. Calcd for (C19 S): C, 52.41; H, 3.47; N,
.65. Found: C, 52.39; H, 3.48; N, 9.64.
Transmission electron microscopy
15 2 3 5
H F N O
S. aureus (109 cells/ml) were cultured in the presence of 9 and DFNB
(
positive control), at the same concentrations used for optical microscopy
and, after 16 hours incubation at 37 °C, the cells were harvested and
processed for transmission electron microscopy, as already described [37]
Untreated S. aureus was used as negative control. After centrifugation at
2
,6-difluoro-7-((5-methyl-1,2,4-oxadiazol-3-yl)-1,4-benzodioxan-2-
yl)methoxybenzamide (10): Acetic anhydride (0.1 mL, 1.05 mmol) was
added to a solution of 29 (0.4 g, 1.05 mmol) in DCM/DMF (10 mL + 2 mL).
The reaction mixture was stirred at room temperature for 2 hours, diluted
with DCM, washed with 10% aqueous NaHCO , dried over Na SO ,
3 2 4
filtered and concentrated under vacuum. The residue was diluted with
DMF (5 mL), added with NaOH (0.2 g, 5 mmol) and stirred for 18 hours.
.
3
2
100 x g for 5 min at room temperature, pelleted bacteria were fixed in
.5% glutaraldehyde (Polysciences, Warrington, PA) in 0.1 Na
M
cacodylate buffer, pH 7.4, for 1 h at 4 °C, rinsed twice, and post-fixed in
Na cacodylate-buffered 1% OsO , for 1 h at 4 °C. The samples were
4
2
The reaction mixture was then diluted with H O and filtered to give a solid.
dehydrated through a series of graded ethanol solutions and propylene
oxide and embedded in Poly/Bed 812 resin mixture. Ultrathin sections
were obtained using a Reichert-Jung ultramicrotome equipped with a
diamond knife. Samples were then stained with water-saturated uranyl
acetate and 0.4% lead citrate in 0.1 M NaOH. The specimens were viewed
under a Philips CM10 electron microscope.
The residue was purified crystallizing from ethanol, obtaining 40 mg (9%)
1
of 10 as a white solid. mp: 155 °C. H NMR (300 MHz, DMSO-d
6
): δ= 8.14
(bs, 1H), 7.86 (bs, 1H), 7.50 (dd, J = 8.4, 2.0 Hz, 1H), 7.45 (dd, J = 4.8, 2.0
Hz, 1H), 7.31 (dt, J = 9.5, 3.9 Hz, 1H), 7.09 (m, 1H), 7.07 (dd, J = 8.4, 4.8
Hz, 1H), 4.68 (m, 1H), 4.51 (ddd, J = 11.6, 4.5, 2.5 Hz, 1H), 4.39 (dd, J =
1
1.5, 4.5 Hz, 1H), 4.33 (dd, J = 11.5, 5.9 Hz, 1H), 4.23 (dd, J = 11.6, 7.0
Hz, 1H), 2.63 (s, 3H) ppm. 13C NMR (75 MHz, DMSO-d6): δ= 177.7, 167.5,
Antibacterial activity
1
1
61.7, 152.6 (dd, J = 240.0, 6.8 Hz), 148.4 (dd, J = 247.5, 8.3 Hz), 143.3,
43.1 (dd, J = 10.9, 3.4 Hz), 121.0, 120.2, 118.4, 117.1 (dd, J = 24.8, 20.3
The antibacterial activity of compounds 1-11 and III-V was tested by using
both a methicillin-sensitive and a methicillin-resistant S. aureus strain, and
an ESBL E. coli clinical isolate. All of the compounds were dissolved at the
final concentration of 20 mg/mL in dimethyl sulphoxide (DMSO) and
serially diluted in LB. After incubation at 37 °C for 16 hours in aerobic
Hz), 116.4 (dd, J = 9.4, 1.9 Hz), 116.0, 111.5 (dd, J = 22.9, 4.1 Hz), 71.9,
8.6, 65.1, 12.5 ppm. Elemental analysis calculated (%) for
): C, 56.58; H, 3.75; N, 10.42; Found: C, 56.49; H, 3.83; N,
6
(
19 15 2 3 5
C H F N O
10.34.
culture tubes, the concentration of prokaryotic cells was determined by
optical density measurement, at 600 nm (OD600) in a SmartSpecTM 3000
2
,6-difluoro-7-((5-ethyl-1,2,4-oxadiazol-3-yl)-1,4-benzodioxan-2-
yl)methoxybenzamide (11): Propionic anhydride (0.12 mL, 0.9 mmol)
was added to a solution of 29 (0.34 g, 0.9 mmol) in DCM/DMF (10 mL + 2
mL). The reaction mixture was stirred at room temperature for 2 hours,
spectrophotometer (Bio-Rad, Oceanside, CA, USA). Fresh cell cultures
5
were used at 10 cells/mL in a final volume of 2 mL. Each bacterial sample
was grown with different compound concentrations that ranged from 100
to 0.1 µg/mL for the first screening of all the compounds.
3 2 4
diluted with DCM, washed with 10% aqueous NaHCO , dried over Na SO ,
filtered and concentrated under vacuum. The residue was diluted with
DMF (5 mL), added NaOH (0.2 g, 5 mmol), stirred for 18 hours,
concentrated under vacuum, diluted with Ethyl Acetate (10 mL), washed
with 10% aqueous NaCl (3x5 mL), dried over Na SO , filtered and
2 4
concentrated to give a residue which was purified by flash chromatography
For compounds 4, 9, 10 and 11, that were active up to 10 µg/mL but not at
0.1 µg/mL further analyses with intermediate concentrations were
performed. After incubation of each sample overnight at 37 °C, an aliquot
was collected under sterile conditions and the OD600 was measured to
determine the MIC. To determine the MBC, the bacteria were then washed
three times with LB, centrifuged at 900× g for 10 min at 4 °C, and the pellet
resuspended in fresh LB. After overnight incubation at 37 °C, the absence
of growth was confirmed by OD measurement. All tests were performed in
triplicate and for each series of experiments, both positive (no compounds)
and negative (no bacteria) controls were included.
on silica gel. Elution with 3/7 Cyclohexane/Ethyl Acetate gave 0.14 g (37%)
1
of 10 as a white solid. mp: 164 °C H NMR (300 MHz, DMSO-d
6
): δ= 8.14
(
(
bs, 1H), 7.87 (bs, 1H), 7.51 (dd, J = 8.4, 1.9 Hz, 1H), 7.46 (m, 1H), 7.32
dt, J = 9.4, 5.3 Hz, 1H), 7.10 (m, 1H), 7.08 (dd, J = 8.4, 1H), 4.68 (m, 1H),
4.52 (dd, J = 11.6, 2.3 Hz, 1H), 4.39 (dd, J = 11.2, 4.5 Hz, 1H), 4.33 (dd, J
= 11.2, 5.8 Hz, 1H), 4.24 (dd, J = 11.6, 7.1 Hz, 1H) ,2.98 (q, J = 7.6 Hz,
2
H), 1.32 (t, J = 7.6 Hz, 3H) ppm. 13C NMR (75 MHz, DMSO-d6): = 181.4,
167.4, 161.7, 152.6 (dd, J = 240.0, 6.8 Hz), 148.4 (dd, J = 247.1, 8.6 Hz),
146.0, 143.3, 143.1 (dd, J = 10.5, 3.0 Hz), 121.0, 120.2, 118.3, 117.1 (dd,
Antimicrobial activities of compounds against MRSA 11.7, MRSA 12.1 and
mutated E. coli were determined by the broth microdilution method in 96-
well plate format following the Clinical and Laboratory Standards Institute
J = 25.1, 19.9 Hz), 116.3, 116.0 (d, J = 14.6 Hz), 111.5 (dd, J = 23.6, 2.6
Hz), 71.9, 68.6, 65.1, 20.0, 10.9 ppm. Elemental analysis calculated (%)
for (C20H F N O ): C, 57.56; H, 4.11; N, 10.07. Found: C, 57.55; H, 4.11;
17 2 3 5
(CLSI) guidelines and European Committee on Antimicrobial Susceptibility
Testing (EUCAST) recommendations. Bacterial suspension of specific
bacterial strain equivalent to 0.5 McFarland turbidity standard was diluted
with cation-adjusted Mueller Hinton broth with TES (Thermo Fisher
N, 10.04.
5
Scientific), to obtain a final inoculum of 10 CFU/ml. Compounds dissolved
Cells
in DMSO and inoculum were mixed together and incubated for 20 h at
Normal human lung fibroblasts (MRC-5) were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% heat-
inactivated calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin
37 °C. After incubation the minimal inhibitory concentration (MIC) values
were determined by visual inspection as the lowest dilution of compounds
showing no turbidity. Tetracycline was used as a positive control on every
assay plate.
2
in an incubator at 5% CO atmosphere and 37 °C. The Gram-positive
Staphylococcus aureus (methicillin-sensitive MSSA ATCC 29213, and
methicillin-resistant MRSA ATCC 43300) and the Gram-negative
Escherichia coli (ESBL, extended-spectrum beta-lactamase-positive E.
coli) bacterial cells were grown in Luria-Bertani Broth (LB) medium at 37 °C
under constant shaking at 300 rpm.
Thiazolyl blue tetrazolium bromide cytotoxicity assay
Compounds 4, 9, 10 and 11, showing an antibacterial activity at a
concentration lower than 10 µg/ml, were serially diluted in DMEM and
tested on MRC-5 cells by the thiazolyl blue tetrazolium bromide (MTT)
cytotoxicity assay (Sigma, St Louis, MO, USA). Cells (104 cell/well) were
tested in a 96-well plate using serially two-fold-diluted concentrations of
Optical microscopy
1
2
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
the compound in 100 µl DMEM medium. After a 24-h incubation, the
compound was removed and the cells were overlaid with 1 mg/ml MTT in
docking score and an energy window of 2.5 kcal/mol was used for ring
sampling. In the energy minimization step, distance dependent dielectric
constant was 4.0 with a maximum number of minimization steps of
100,000. In the clustering, poses were considered as duplicates and
discarded if both: RMS deviation is less than 0.5 Å and maximum atomic
displacement is less than 1.3 Å.
100 µl serum-free DMEM for 3 h at 37 °C. The MTT solution was then
replaced with DMSO for 10 min, and the absorbance was measured at 570
nm. The percentage of cytotoxicity was calculated by the formula 100 –
(sample OD/untreated cells OD) x 100. The compound concentration
reducing cell viability by 50 or 90% was defined as the TD50 or TD90 toxic
dose. The therapeutic index (TI) was also determined and defined as the
ratio between TD90 and the minimal bactericidal concentration (MBC)
values.
Hotspots maps. The Fragment Hotspot maps software [36] identifies the
location and environment of binding sites on the protein by first calculating
atomic hotspots and then producing Fragment Hotspot maps using simple
molecular probes. These maps specifically highlight fragment-binding sites
and their corresponding pharmacophores. H-Bond acceptor (red), donor
Computational studies
(
blue), and apolar/aromatic interactions (yellow), identified by this tool, can
Ligand preparation. The preparation of the compounds synthesized in this
work was performed before carrying out further computational studies. The
preparation together with the 2D-to-3D conversion was performed using
LigPrep tool, [38] that is included in the Schrödinger software package. This
application allows the preparation of molecules following different steps
required for ligand-protein analysis such as the addition of hydrogen atoms,
the calculation of the ionization state of the molecules at a specific pH or
the generation of potential tautomers. Also, low-energy ring conformations
are generated for every molecule, followed by a final energy minimization
using the OPLS-2005 force field. [39,40] In order to perform the studies,
physiological pH conditions were used to prepare the molecules, all of
them were desalted and in the last step the compounds were minimized
as default.
assist medicinal chemists in the search of interesting interactions. In this
sense, it allows predicting the bind or improve the binding affinities for
different ligands, and suggest modifications to the molecules. In this work,
the structure of FtsZ from S. aureus was analyzed according the hotspots
maps with the objective of obtaining more insights of the binding pocket in
the protein. Also, the full structure of the protein was inspected in order to
identify potential alternative or allosteric pockets useful in future drug
discovery programs.
Induced Fit Docking. This study is carried out by fitting the ligand to the
protein and permitting changes in the active site geometry. [47,48] This was
done by docking the compound 9, the reference compound in the study,
to the protein in the Glide program [49,50] and generating several poses in
the active site. Then, Prime [51] predicts the protein structure using the pose
of the corresponding ligand and rearranging nearby side chains of the
active site and minimizing the overall energy of the protein. [49, 52] Finally,
each ligand is re-docked into its corresponding low energy protein
structures and the resulting complexes are ranked according to docking
score. No constraints were set, XP (extra precision) mode was used in a
standard protocol, the induced fit was optimized to 5.0 Å of the ligand
poses and the rest of the parameters were set to default.
Protein preparation. In order to obtain the crystal structure of the protein to
[
41]
carry out the computational studies, a search on the PDB
was
performed. According to the crystal structures available in the PDB of the
FtsZ protein from S. aureus, two entries were retrieved: 4DXD [14] and
5
XDT [22]. The selection was based on the experimental quality of the
structure and the presence of a ligand in the active site. In this case, both
crystals are holo structures with a potent inhibitor bound to the protein.
These inhibitors displayed a 2,6-difluorobenzamide scaffold identical to
one of the moieties that is present in the family of inhibitors described here.
The structures of the proteins were preprocessed adding the
corresponding hydrogens, deleting water molecules and assigning bond
orders. Also the proteins were refined carrying out H-bond assignment and
calculation of the protonation state of the residues at physiological pH with
Keywords:
FtsZ
inhibition;
1,4-benzodioxane-2,6-
difluorobenzamide; Mutated E. coli; Molecular Modelling; Cavity
Detection
a final restraint minimization, using the Protein Preparation Wizard tool [42]
implemented on Maestro software. [43]
References:
Ligand Characterization. The compounds previously prepared were
analyzed using the Qikprop [35] module of the small molecule drug
discovery suite in the Schrödinger software package. Among other
parameters, ADME Tox properties were predicted using the Qikprop
program. Qikprop allowed us to calculate and predict a total of 44
pharmaceutical relevant properties. These properties include both simple
molecular descriptors and relevant computational predictions for drug
discovery. Among other parameters it is possible to calculate the following
descriptors: molecular weight (MW), molecular volume, number of
hydrogen bond acceptor and donor (HBA, and HBAs respectively), PSA,
QPlogPo/w (predicted octanol/water partition coefficient), dipole values
and violations related to Lipinski’s rule of five [44] and Jorgensen’s rule of
three. [45]
[
[
1]
2]
González-Bello, C. Recently developed synthetic compounds with anti-
infective activity. Current Opinion in Pharmacology 2019, 48, 17-23.
Solomon, S. L.; Oliver, K. B. Antibiotic Resistance Threats in the United
States: Stepping Back from the Brink. American Family Physician 2014
89, 938-941.
[
[
[
3]
4]
5]
Li, Y.; Yang, L.; Fu, J.; Yan, M.; Chen, D.; Zhang, L. Microbial
pathogenicity and virulence mediated by integrons on Gram-positive
microorganisms. Microbial Pathogenesis 2017, 111, 481-486.
McGuinness, W. A.; Malachowa, N.; DeLeo, F. R. Vancomycin
Resistance in Staphylococcus aureus. Yale journal of Biology and
Medicine, 2017, 269-281.
Foster, T. J. Antibiotic resistance in Staphylococcus aureus. Current
status and future prospects. FEMS Microbiology Reviews 2017, 41, 430-
449.
Docking studies. Docking studies were performed employing Glide module
[
46]
[6]
Rengaraj, R.; Mariappan, S.; Sekar, U.; Kamalanadhan, A. n. Detection
of Vancomycin Resistance among Enterococcus faecalis and
Staphylococcus aureus. Journal of Clinical and Diagnostic Research
included in the Schrödinger software package. As a first approach,
docking studies with the previously crystallized ligands were performed to
validate the protocol. Once, the protocol was validated. The selected 3D
target structures of FtsZ from S. aureus and the compounds synthesized
in this work were used as a starting point of the analysis. Docking studies
were carried out applying a similar protocol in terms of conformational
search and evaluation parameters for all the compounds. In all the cases,
the center of the previously crystallized ligand in the catalytic pocket was
selected as the centroid of the grid. In the grid generation, a scaling factor
of 1.0 in van der Waals radius scaling and a partial charge cutoff of 0.25
were used. The xtra precision (XP) mode was used in the docking studies
for all the molecules and no constraints were applied during the process.
The ligand sampling was flexible, epik state penalties were added to the
2016, 10, DC04-DC06.
[
[
7]
8]
Brauers, J.; Ewig, S.; Kresken, M. β-Lactam-Antibiotika in der Therapie
von ambulant erworbenen Atemwegsinfektionen mit Penicillin-
resistenten Pneumokokken. Pneumologie 2002, 56, 605-609.
Saenz, Y.; Brinas, L.; Domínguez, E.; Ruiz, J.; Zarazaga, M.; Vila, J.;
Torres, C. Mechanisms of Resistance in Multiple-Antibiotic-Resistant
Escherichia coli Strains of Human, Animal, and Food Origins.
Antimicrobial Agents and Chemotherapy 2004, 48, 3996-4001.
[
9]
Panda, D.; Bhattacharya, D.; Gao, Q. H.; Oza, P. M.; Lin, H.-Y. J.;
Hawkins, B.; Hibbs, D. E.; Groundwater, P. W. Identification of agents
1
3
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
targeting FtsZ assembly. Future Medicinal Chemistry 2016, 8, 1111-
132.
diastereoisomeric histidine-containing dipeptides by property space
analysis. Chirality 2012, 24, 566-576.
1
[
[
10] Vaughan, S.; Wickstead, B.; Gull, K.; Addinall, S. G. Molecular Evolution
of FtsZ Protein Sequences Encoded Within the Genomes of Archaea,
Bacteria, and Eukaryota. Journal of Molecular Evolution 2004, 58, 19-29.
11] Haydon, D. J.; Stokes, N. R.; Ure, R.; Galbraith, G.; Bennett, J. M.; Brown,
D. R.; Patrick J. Baker; Barynin, V. V.; Rice, D. W.; Sedelnikova, S. E.;
Heal, J. R.; Sheridan, J. M.; Aiwale, S. T.; Chauhan, P. K.; Srivastava,
A.; Taneja, A.; Collins, I.; Errington, J.; Czaplewski, L. G. An Inhibitor of
FtsZ with Potent and Selective Anti-Staphylococcal Activity. Science
[26] Fumagalli, L.; Pallavicini, M.; Budriesi, R.; Bolchi, C.; Canovi, M.; Chiarini,
A.; Chiodini, G.; Gobbi, M.; Laurino, P.; Micucci, M.; Straniero, V.; Valoti,
E. 6-Methoxy-7-benzofuranoxy and 6-Methoxy-7-indolyloxy Analogues
of
2-[2-(2,6-Dimethoxyphenoxy)ethyl]aminomethyl-1,4-benzodioxane
(WB4101):1 Discovery of a Potent and Selective α1D-Adrenoceptor
Antagonist. Journal of Medicinal Chemistry 2013, 56, 6402-6412.
[27] Chiodini, G.; Pallavicini, M.; Zanotto, C.; Bissa, M.; Radaelli, A.; Straniero,
V.; Bolchi, C.; Fumagalli, L.; Ruggeri, P.; Morghen, C. D. G.
Benzodioxane–benzamides as new bacterial cell division inhibitors.
European journal of medicinal chemistry 2015, 89, 252-265.
2008, 321, 1673-1675.
[
[
12] Kaul, M.; Mark, L.; Zhang, Y.; Parhi, A. K.; LaVoie, E. J.; Pilch, D. S.
Pharmacokinetics and in vivo antistaphylococcal efficacy of TXY541, a
[28] Straniero, V.; Pallavicini, M.; Chiodini, G.; Zanotto, C.; Volontè, L.;
Radaelli, A.; Bolchi, C.; Fumagalli, L.; Sanguinetti, M.; Menchinelli, G. 3-
1-methylpiperidine-4-carboxamide prodrug of PC190723. Biochemical
Pharmacology 2013, 86, 1699-1707.
(Benzodioxan-2-ylmethoxy)-2,
6-difluorobenzamides
bearing
13] Kaul, M.; Mark, L.; Zhang, Y.; Parhi, A. K.; Lyu, Y. L.; Pawlak, J.;
Saravolatz, S.; Saravolatz, L. D.; Weinstein, M. P.; LaVoie, E. J.; Pilch,
D. S. TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved
Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-
Resistant Staphylococcus aureus. Antimicrobial Agents and
Chemotherapy 2015, 59, 4845-4855.
hydrophobic substituents at the 7-position of the benzodioxane nucleus
potently inhibit methicillin-resistant Sa and Mtb cell division. European
journal of medicinal chemistry 2016, 120, 227-243.
[29] Straniero, V.; Zanotto, C.; Straniero, L.; Casiraghi, A.; Duga, S.; Radaelli,
A.; De Giuli Morghen, C.; Valoti, E. 2, 6‐Difluorobenzamide inhibitors of
the bacterial cell division protein FtsZ: design, synthesis and Structure
Activity Relationship study. ChemMedChem 2017, 12, 1303-1318.
[30] Kusuma, K. D.; Payne, M.; Ung, A. T.; Bottomley, A. L.; Harry, E. J. FtsZ
as an Antibacterial Target: Status and Guidelines for Progressing This
Avenue. ACS Infectious Diseases 2019, 5, 1279-1294.
[
14] Tan, C. M.; Therien, A. G.; Lu, J.; Lee, S. H.; Caron, A.; Gill, C. J.;
Lebeau-Jacob, C.; Benton-Perdomo, L.; Monteiro, J. M.; Pereira, P. M.;
Elsen, N. L.; Wu, J.; Deschamps, K.; Petcu, M.; Wong, S.; Daigneault,
E.; Kramer, S.; Liang, L.; Maxwell, E.; Claveau, D.; Vaillancourt, J.;
Skorey, K.; Tam, J.; Wang, H.; Meredith, T. C.; Sillaots, S.; Wang-
Jarantow, L.; Ramtohul, Y.; Langlois, E.; Landry, F.; Reid, J. C.;
Parthasarathy, G.; Sharma, S.; Baryshnikova, A.; Lumb, K. J.; Pinho, M.
G.; Soisson, S. M.; Roeme, T. Restoring methicillin-resistant
Staphylococcus aureus susceptibility to β-lactam antibiotics. Science
Translational Medicine 2012, 4, 1-11.
[31] Tripathy, S.; Sahu, S. K. FtsZ inhibitors as a new genera of antibacterial
agents. Bioorganic Chemistry, 2019, 91, 103169.
[32] Xie, A.; Zhang, Q.; Liu, Y.; Feng, L.; Hu, X.; Dong, W. An Environmentally
Friendly Method for N-Methylation of 5-Substituted 1H-Tetrazoles with a
Green Methylating Reagent: Dimethyl Carbonate. Journal of
Heterocyclic Chemistry 2015, 52, 1483-1487.
[
[
15] Kaul, M.; Zhang, Y.; Parhi, A. K.; Voie, E. J. L.; Pilch, D. S. Inhibition of
RND-type efflux pumps confers the FtsZ-directed prodrug TXY436 with
activity against Gram-negative bacteria. Biochemical Pharmacology
[33] Begtrup, M. Introduction of substituents into 5‐membered aza‐
heteroaromatics. Bulletin des Societes Chimiques Belges 1988, 97, 573-
598.
2014, 89, 321-328.
[34] Czaplewski, L. G.; Collins, I.; Boyd, E. A.; Brown, D.; East, S. P.; Gardiner,
M.; Fletcher, R.; Haydon, D. J.; Henstock, V.; Ingram, P.; Jones, C.;
Noula, C.; Kennison, L.; Rockley, C.; Rose, V.; Thomaides-Brears, H. B.;
Ure, R.; Whittaker, M.; Stokes, N. R. Antibacterial alkoxybenzamide
inhibitors of the essential bacterial cell division protein FtsZ, Bioorganic
& Medicinal Chemistry Letters, 2009, 19, 524-527.
16] Awasthi, D.; Kumar, K.; Knudson, S. E.; Slayden, R. A.; Ojima, I. SAR
Studies on Trisubstituted Benzimidazoles as Inhibitors of Mtb FtsZ for
the Development of Novel Antitubercular Agents. Journal of Medicinal
Chemistry 2013, 56, 9756-9770.
[
[
[
17] Xin, L.; Shutao, M. Advances in the discovery of novel antimicrobials
targeting the assembly of bacterial cell division protein FtsZ. European
journal of medicinal chemistry 2015, 95, 1-15.
̈
[35] QikProp, v. 4.1; Schrodinger, LLC: New York, 2014.
[36] Radoux, C. J.; Olsson, T. S.; Pitt, W. R.; Groom, C. R.; Blundell, T. L.
Identifying interactions that determine fragment binding at protein
hotspots. J Med Chem 2016, 59, 4314-4325.
18] SunMing-Hui, J.; LiXin-Yi; WangYang; ZhangRong-Ju; YuanHan-Yu;
Zhu, L.-L. Vanillin derivatives as the selective small molecule inhibitors
of FtsZ. 2014, 23, 2985-2994.
[37] Bissa, M.; Zanotto, C.; Pacchioni, S.; Volonté, L.; Venuti, A.; Lembo, D.;
De Giuli Morghen, C.; Radaelli, A. The L1 protein of human papilloma
virus 16 expressed by a fowlpox virus recombinant can assemble into
virus-like particles in mammalian cell lines but elicits a non-neutralising
humoral response, Antivir. Res. 2015, 116, 67-75.
19] Leung, A. K. W.; White, E. L.; Ross, L. J.; Borhani, D. W. Crystallization
of the Mycobacterium tuberculosis cell-division protein FtsZ. Acta
Crystallographica Section D 2000, D56, 1634-1637.
[
[
20] Lowe, J.; Amos, L. A. Crystal structure of the bacterial cell-division
protein FtsZ. Nature 1998, 391, 203-206.
[38] Schrödinger Release 2015-4: LigPrep, Schrödinger, LLC, New York, NY,
2015.
21] Matsui, T.; Yamane, J.; Mogi, N.; Yamaguchi, H.; Takemoto, H.; Yao, M.;
Tanaka, I. Structural reorganization of the bacterial cell-division protein
FtsZ from Staphylococcus aureus. Acta Crystallographica Section D
[39] Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development and
testing of the OPLS all-atom force field on conformational energetics and
properties of organic liquids. J Am Chem Soc 1996, 118, 11225-11236.
[40] Banks, J. L.; Beard, H. S.; Cao, Y.; Cho, A. E.; Damm, W.; Farid, R.;
Felts, A. K.; Halgren, T. A.; Mainz, D. T.; Maple, J. R.; Murphy, R.; Philipp,
D. M.; Repasky, M. P.; Zhang, L. Y.; Berne, B. J.; Friesner, R. A.;
Gallicchio, E.; Levy, R. M. Integrated modeling program, applied
chemical theory (IMPACT). J Comput Chem 2005, 26, 1752-1780.
[41] Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig,
H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank. Nucleic acids
research 2000, 28, 235-242.
2012, D68, 1175-1188.
[
[
[
[
22] Fujita, J.; Maeda, Y.; Mizohata, E.; Inoue, T.; Kaul, M.; Parhi, A. K.;
LaVoie, E. J.; Pilch, D. S.; Matsumura, H. Structural Flexibility of an
Inhibitor Overcomes Drug Resistance Mutations in Staphylococcus
aureus FtsZ. ACS Chemical Biology 2017, 12, 1947-1955.
23] Straniero, V.; Pallavicini, M.; Chiodini, G.; Ruggeri, P.; Fumagalli, L.;
Bolchi, C.; Corsini, A.; Ferri, N.; Ricci, C.; Valoti, E. Farnesyltransferase
inhibitors: CAAX mimetics based on different biaryl scaffolds. Bioorganic
&
Medicinal Chemistry Letters 2014, 24, 2924-2927.
24] Bolchi, C.; Pallavicini, M.; Bernini, S. K.; Chiodini, G.; Corsini, A.; Ferri,
N.; Fumagalli, L.; Straniero, V.; Valoti, E. Thiazole- and imidazole-
containing peptidomimetic inhibitors of protein farnesyltransferase.
Bioorganic & medicinal chemistry letters 2011, 21, 5408-5412.
[42] Sastry, G. M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W.
Protein and ligand preparation: parameters, protocols, and influence on
virtual screening enrichments. Journal of computer-aided molecular
design 2013, 27, 221-234.
25] Vistoli, G.; Straniero, V.; Pedretti, A.; Fumagalli, L.; Bolchi, C.; Pallavicini,
M.; Valoti, E.; Testa, B. Predicting the physicochemical profile of
[43] Schrödinger Release 2015-4: Maestro, Schrödinger, LLC, New York, NY,
2015.
1
4
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
[
44] Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental
and computational approaches to estimate solubility and permeability in
drug discovery and development settings. Advanced drug delivery
reviews 2001, 46, 3-26.
[
[
[
45] Jorgensen, W. L. Efficient drug lead discovery and optimization.
Accounts of chemical research 2009, 42, 724-733.
46] Schrödinger Release 2015-4: Glide, Schrödinger, LLC, New York, NY,
2015.
47] Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R., Novel
procedure for modeling ligand/receptor induced fit effects. J Med Chem
2006, 49 (2), 534-53.
[
48] Farid, R.; Day, T.; Friesner, R. A.; Pearlstein, R. A., New insights about
HERG blockade obtained from protein modeling, potential energy
mapping, and docking studies. Bioorg Med Chem 2006, 14 (9), 3160-
3173.
[49] Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood,
J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T., Extra precision
glide: docking and scoring incorporating
enclosure for protein-ligand complexes. J Med Chem 2006, 49 (21),
177-6196.
a model of hydrophobic
6
[
50] Glide, S., Schrödinger Release 2015-4; LLC, New York, NY, 2015
51] Jacobson, M. P.; Friesner, R. A.; Xiang, Z.; Honig, B., On the role of the
crystal environment in determining protein side-chain conformations. J
Mol Biol 2002, 320 (3), 597-608.
[
[
52] Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.;
Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw,
D. E.; Francis, P.; Shenkin, P. S., Glide: a new approach for rapid,
accurate docking and scoring. 1. Method and assessment of docking
accuracy. J Med Chem 2004, 47 (7), 1739-1749.
1
5
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900537
FULL PAPER
Entry for the Table of Contents
In this work, we summarize our recent findings in the SAR of a series of benzamide inhibitors of the prokaryotic cell division protein
FtsZ. The proposed compounds exhibit promising activity against Gram-positive and Gram-negative strains, including multi drug
resistant bacteria. We also validate a FtsZ binding protocol and highlight the key structural features for the optimal interaction with the
target protein.
1
6
This article is protected by copyright. All rights reserved.