Design, Synthesis, Antimicrobial Evaluation and in Silico Studies of Eugenol-Sulfonamide Hybrids

Article abstract of DOI:10.1002/cbdv.202100066

Using molecular hybridization, specific sulfonamide derivatives of eugenol were synthesized with subtle modifications in the allylic chain of the eugenol subunit (and also in the nature of the substituent group in the sulfonamide aromatic ring) which allo

Full text of DOI:10.1002/cbdv.202100066

DOI: 10.1002/cbdv.202100066
FULL PAPER
Design, Synthesis, Antimicrobial Evaluation and in Silico Studies of
Eugenol-Sulfonamide Hybrids
Helloana Azevedo-Barbosa,a Bianca Pereira do Vale,a Grazielle Guidolin Rossi,b
Fallon dos Santos Siqueira,b Kevim Bordignon Guterres,b Marli Matiko Anraku de Campos,b
Thiago dos Santos,c Jamie Anthony Hawkes,a Danielle Ferreira Dias,d Stefânia Neiva Lavorato,e
Thiago Belarmino de Souza,f and Diogo Teixeira Carvalho*a
a
Faculdade de Ciências Farmacêuticas, Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva 700,
Alfenas 37130-001, MG, Brazil, e-mail: diogo.carvalho@unifal-mg.edu.br
Departamento de Análises Clínicas e Toxicológicas, Universidade Federal de Santa Maria, Av. Roraima No. 1000,
b
Cidade Universitária, Camobi, Santa Maria 97105-900, RS, Brazil
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, Ribeirão
c
Preto 14040-903, SP, Brazil
d
Instituto de Química, Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700, Alfenas 37130-001,
MG, Brazil
e
Centro das Ciências Biológicas e da Saúde, Universidade Federal do Oeste da Bahia, Rua Professor José Seabra
de Lemos, 316, Recanto dos Pássaros, Barreiras 47808-021, BA, Brazil
Escola de Farmácia, Universidade Federal de Ouro Preto, Morro do cruzeiro, Bauxita, Ouro Preto 35400-000
f
MG, Brazil
Using molecular hybridization, specific sulfonamide derivatives of eugenol were synthesized with subtle
modifications in the allylic chain of the eugenol subunit (and also in the nature of the substituent group in the
sulfonamide aromatic ring) which allowed us to study the influence of structural changes on the antimicrobial
potential of the hybrids. Antimicrobial test results showed that most of the synthesized hybrid compounds
showed good activity with better results than the parent compounds. Molecular docking studies of the hybrids
with the essential bacterial enzyme DHPS showed complexes with low binding energies, suggesting that DHPS
could be a possible target for the antibacterial sulfonamide-eugenol hybrids. Furthermore, most of the final
compounds presented similar docking poses to that of the crystallographic ligand sulfamethoxazole. The results
obtained allow us to conclude that these are promising compounds for use as new leads in the search for new
antibacterial sulfonamides.
Keywords: sulfonamides, eugenol, antimicrobial, molecular hybridization, dihydropteroate synthase, molecular
docking.
surpassed the discovery of new active substances for
drugs, whilst at the same time the rise in the misuse of
Introduction
Research aimed at discovering new antimicrobial antibacterial compounds has led to a worrying
agents, especially new antibacterial agents, represents increase in microbial resistance.[1–3] Several strategies
one of the most urgent aspects in the field of new are employed in the discovery of new antibacterial
drug research. The evolution of microorganisms has drug candidates; i) the systematic screening of
synthetic compound libraries; ii) the investigation of
natural products guided by ethnopharmacology; iii)
the structural modification of known drugs; and iv) the
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.202100066
in silico design of new molecules.[4–6]
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Chem. Biodiversity 2021, 18, e2100066
In this context, the process known as molecular and sulfonamide-based drugs separately) herein we
hybridization has great appeal and importance, en- present for the first time the antibacterial activity of
abling the discovery of excellent candidates for anti- eugenol and dihydroeugenol-based hybrids bearing
infectious agents. This approach is characterized by the sulfonamide functionality (Scheme 1).
the connection (or fusion) of distinct pharmacophoric
In order to better understand the influence of
subunits, derived from synthetic or natural bioactive structural changes on the antibacterial potential of the
substances, in order to create new chemical entities hybrid molecules, certain derivatives were also synthe-
which are referred to as ‘hybrids’, These hybrids can sized with subtle modifications in the allylic chain of
present improved properties compared to the pre- the eugenol subunit, and in the nature of the
existing pharmacophores individually.[7–9]
substituent group in the aromatic ring of sulfonamide.
The introduction of sulfonamide-based antibacteri- Furthermore, we present molecular docking results
al compounds into the therapeutic arsenal repre- that evaluate the ability of these compounds to
sented one of the most important milestones in interact with dihydropteroate synthase (an enzyme
advancing the treatment of infectious diseases, and essential for bacterial multiplication), which gives a
contributed to the reduction in mortality rates.[10] direct indication of the antibacterial properties of the
Numerous sulfonamide-based compounds have been tested compound.
used to develop potent lead substances with better
efficacy and less toxicity.[11] Indeed, bioactive mole-
cules possessing the sulfonamide group have been Results and Discussion
reported to demonstrate potent properties such as
Chemistry
antibacterial,[12]
antifungal,[13]
antiviral,[14]
antiparasitic,[15] and anti-inflammatory properties[16] The substances evaluated in this study were synthe-
amongst others. Pharmacological properties of euge- sized according to the synthetic route shown in
nol have been extensively highlighted, especially Scheme 2, and this route has previously been reported
involving
its
antibacterial
and
antifungal by Azevedo-Barbosa et al.[30] In summary, the starting
properties.[17–22] Our research group has previously molecules 1 and 2 were nitrated to produce inter-
demonstrated that synthetic compounds obtained mediate compounds 3a–3d that were further reduced
from natural phenylpropanoids, such as eugenol or its to aryl amines 4a–4d with tin chloride. These amines,
analogs (dihydroeugenol and isoeugenol), have impor- which are key intermediates for obtaining the desired
tant antimicrobial activity and that hybrids obtained products, were reacted with the respective sulfonyl
from phenylpropanoids and sulfonamide moieties chlorides to form the known compounds 5a–5d and
have important antitumor activity.[23–30] Taking ad- 6a–6d, as well as the newly presented compounds 6e
vantage of the molecular hybridization strategy (and and 6f.
the well-established antibacterial potential of eugenol
Scheme 1. Rationale for the design of new sulfonamide-based molecular hybrids as antibacterial compounds.
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Chem. Biodiversity 2021, 18, e2100066
°
Scheme 2. Synthetic route to the phenylpropanoid-based sulfonamide derivatives: i) Bi(NO3)3 ·5H2O, SiO2, CHCl3, 70 C; ii) NaNO3,
°
°
°
KHSO4, SiO2/H2O (1:1), CH2Cl2, 25 C; iii) SnCl2 ·2H2O, EtOH, 80 C; iv) 4-acetamidobenzenesulfonyl chloride, pyridine, 100 C; v)
°
°
°
SOCl2, MeOH, 25 C; vi) benzenesulfonyl chloride, triethylamine, 25 C, vii) 4-nitrobenzenesulfonyl chloride, triethylamine, 25 C.
The characterization data for the previously known tween the sulfonamide and the hydroxy group. Finally,
compounds are in agreement with the literature. The the HR-MS data is in strict agreement with the
structural characterization of the newly reported calculated molecular masses.
compounds 6e and 6f was confirmed by Fourier
Therefore, from the analytical data collected (a
transform infrared spectroscopy (FT-IR), 1H and 13C supplementary file is available containing all of the
nuclear magnetic resonance spectroscopy (NMR) and relevant spectra) it can be concluded that the
high-resolution mass spectrometry (HR-MS) as de- structures of the synthesized compounds are those
scribed in the experimental section. In summary, the shown in Scheme 2. These compounds were then
successful formation of sulfonamide linkages was evaluated in antibacterial studies as well as molecular
confirmed by its characteristic bands in FT-IR spectra docking studies as shown in the following sections.
(3441 or 3485 cmÀ 1 for NH and 1168 or 1225 cmÀ 1 for
SO2). The presence of the propyl side chain in these
compounds was confirmed (using NMR) by the
Antibacterial In Vitro Studies
presence of the group of signals between 2.46– The susceptibility profiles against Gram-negative (Kleb-
0.87 ppm for 6e and 2.49–0.90 for 6f. The existence of siella pneumoniae, Pseudomonas aeruginosa), Gram-
seven aromatic hydrogens in each compound may be positive (Staphylococcus aureus, Staphylococcus sapro-
observed as signals between 7.80–6.42 for 6e and phyticus) and non-tuberculosis Mycobacterium micro-
8.23–6.48 for 6f. Small values of coupling constants organisms (Mycobacterium abscessus, Mycobacterium
(<2) were found for the doublets related to the massiliense) were determined for sulfamethoxazole,
aromatic hydrogens in the dihydroeugenol subunit. eugenol and the synthesized hybrid compounds
This confirms that the ortho relationship exists be- following known protocols.[31,32] The minimum inhib-
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Chem. Biodiversity 2021, 18, e2100066
itory concentration (MIC) values are shown in Table 1 any of the tested microorganisms (both resulting in
with the best results shown in the shaded boxes.
As a typical antimicrobial agent of the sulfonamide
high MIC values).
In contrast, most of the tested hybrid compounds
class, sulfamethoxazole was one of the first systemic showed low MIC values–indicating good antimicrobial
bacteriostatic drug used in the treatment of different activity. It is possible to see that compounds 5a, 5c
human infections.[33] Sulfamethoxazole is one of the and 6a–6e had low MIC values against at least one
broadest spectrum sulfonamide drugs, and has been studied pathogen. As the best results, we cite
widely used to control infections caused by fast compounds 5c, 6b and 6d against S. aureus, which
growing non-tuberculosis mycobacteria, mainly due to show MIC values lower than 40 μgmLÀ 1. Therefore, we
its easy oral administration.[34] It is worth noting that conclude that the inhibition of the microorganisms in
on first appearance, the MIC figures for sulfamethox- their planktonic form occurred due to its new
azole above are relatively high against K. pneumoniae, structural pattern, since the MIC values were much
P. aeruginosa, S. aureus and S. saprophyticus which lower than the MIC values of sulfamethoxazole and
may be mistaken for potentially resistant species. eugenol alone (3125 and 2500 μgmLÀ 1, respectively).
However, sulfamethoxazole (whilst being a broad-
The same was detected for compounds 6a, 6b, 6c
spectrum antibacterial agent) is not used to treat these and 6e with MIC values below 80 μgmLÀ 1 against S.
species due to its low level of activity. In the case of M. saprophyticus, whilst eugenol and sulfamethoxazole
abscessus and M. massiliense, resistance mechanisms had values higher than 2500 μgmLÀ 1. For the Gram-
are known to have been developed by microorgan- negative microorganisms, it is important to note that
isms of the genus Mycobacterium against sulfamethox- none of the hybrids showed good (i.e., low) MIC
azole. This is confirmed by our results, which show a values, although all of them inhibited P. aeruginosa
resistance profile with M. massiliense and a sensitivity with lower MIC values than the parent compounds.
profile with M. abscessus.
For microorganisms of the genus Mycobacterium, we
Overall, the results obtained in our study demon- observed a satisfactory inhibitory effect of the new
strate high MIC values for sulfamethoxazole against compounds against M. massiliense, highlighting the
Gram-negative and Gram-positive microorganisms values obtained for 6a, 6c and 6e, all of them lower
(3125 μgmLÀ 1). Moreover, considering the dosage than those obtained for sulfamethoxazole and eugenol
breakpoints listed in the standard protocols,[31] we alone.
observed a resistance profile to sulfamethoxazole
The general aspects that could be observed
presented by M. massiliense, as already reported in the regarding the influence of the structure on antimicro-
studies by Siqueira et al.[35] and Flores et al.[36] These bial activity were that the substitution of aromatic
values corroborate the decrease in the clinical use of amino or acetamide groups by the electron-with-
sulfamethoxazole, alone or in combination with drawing nitro group was detrimental, as can be seen
trimethoprim, due to the development of resistance to between derivatives 6d and 6f, which showed the
this agent.[37] As seen with the sulfamethoxazole, overall highest MIC results. On the other hand, the
eugenol also did not show noteworthy activity against removal of both the amino and acetamide groups still
allowed for good activity, as seen by the antibacterial
Table 1. Minimum inhibitory concentration (MIC) values for the synthesized compounds and parent compounds.
Compounds
Minimum Inhibitory Concentration (μgmLÀ 1
)
Gram-negative
K. pneumoniae
Gram-positive
S. aureus
Mycobacteria
M. abscessus
P. aeruginosa
S. saprophyticus
M. massiliense
Eugenol
Sulfamethoxazole
625
2500
2500
1250
1250
2500
1250
625
2500
2500
1250
625
625
1250
625
312
625
625
2500
3125
78
39
156
19
312
39
78
2500
3125
312
312
78
78
19
625
78
156
156
8
2500
625
156
78
156
39
78
156
128
1250
312
78
312
78
312
78
312
5a
5c
6a
6b
6c
6d
6e
6f
625
1250
156
312
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Chem. Biodiversity 2021, 18, e2100066
action of 6e against S. aureus, S. saprophyticus, M. experimental antibacterial activity and the in silico
abscessus and M. massiliense. The interchange from an enzyme inhibition potential. It is important to reaffirm
allyl to a propyl side chain in 6a to 6b and 6c to 6d, that whilst ‘low’ MIC values indicate a ‘high’ inhibitory
positively affected the antimicrobial action against S. activity, ‘low’ binding energy values (i.e., more
aureus and M. abscessus. Considering the relative negative) refer to a ‘high’ affinity between the enzyme
position of the sulfonamide group, its change from and ligand.
meta to ortho position in the phenolic ring increased
Compound 5a was one of the least active com-
antibacterial potency, as seen for 6c against S. pounds against the evaluated bacterial strains (other
saprophyticus and for 6d against K. pneumoniae, P. than against K. pneumoniae where it showed some
aeruginosa and M. abscessus.
activity), which correlates with the docking studies. In
general, the in vitro studies indicate that the com-
pounds with free amines tend to have greater
antibacterial activity than the corresponding amides,
Molecular Docking and Prediction Studies
Since classic sulfonamides are known as inhibitors of which was also detected via the in silico studies and
the essential bacterial enzyme dihydropteroate syn- corroborates what is known from structure–activity
thase (DHPS), it was hypothesized that the antibacte- relationships in sulfa drugs.[42] Although compounds
rial activity exhibited by the sulfonamide-eugenol 6e and 6d present low in vitro MIC values and low in
hybrids described in this work may be correlated to silico binding energies complexing with DHPS, com-
their ability to inhibit DHPS. Thus, the interaction of pounds 5a and 6a do not follow this correlation.
the designed compounds with this enzyme was
Considering the conformations assumed by the
predicted by docking studies and the main results are ligands into the enzyme active site, we found three
shown in Table 2. The crystal structure coordinates of different interaction profiles (Figure 1A–C). Com-
Y. pestis dihydropteroate synthase complexed with pounds 6a–6e present similar docking poses (Fig-
sulfamethoxazole and dihydropteroate diphosphate ure 1A). In addition, their poses are similar to the pose
(DHP) were obtained from the Protein Data Bank (PDB) of crystallographic ligand sulfamethoxazole (Fig-
online database using the identification number ure 1D). Since complexes with the lowest binding
‘3TZF’,[38]
energies were found for DHPS and these ligands, and
It is worth noting that although we are comparing considering their pose similarity with sulfamethoxazole
data from cell-based experimental biological activities (a well-known DHPS inhibitor), suggesting that DHPS
with theoretical enzyme-based studies (which do not could be a possible target of the sulfonamide-eugenol
take into account physico-chemical properties, mem- hybrids.
brane transposition capacity or the differences be-
For compounds 5a, 5c and 6f, another two
tween Gram-positive and Gram-negative bacteria), interaction profiles were found (shown in Figure 1B
previously published studies show a good correlation and 1 C), resulting in the highest binding energy
between MIC-based experimental trends and theoret- complexes among the evaluated ones. The energy
ical ones.[39–41] From the results shown in Table 2, the differences could be explained based on the extra
comparison between the binding energies of the interaction between the sulfonamide group and the
enzyme-ligand complexes and the MIC data in the enzyme or the π-π stacking interaction with PHE190
in vitro assays shows a partial correlation between the
Figure 1. Docking results for the most stable complexes formed from sulfonamides and the dihydropteroate synthase (DHPS) active
site (PDB ID: 3TZF),[38] where (A) are sulfonamides 6a–6e, (B) is sulfonamide 5c, (C) is sulfonamide 5a and (D) is sulfonamide 6c and
crystallographic sulfamethoxazole (green carbons). The DHPS active site is represented by green ribbons, and atoms are white=
carbon, yellow=sulfur, blue=nitrogen, and red=oxygen.
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Table 2. Docking results in Y. pestis DHPS active site
Compound Binding Energy (kcal/mol) Ligand-Enzyme Interaction
Hydrogen bond
π-π T-shaped
π-π Stacking
Cation-π
5a
À 6.9
H3CO:GLN226
SO:ARG235
SO:ARG235
NH2:THR62
SO2:SER222
NH2:THR62
SO2:SER222
HO:ARG235
SO2:SER222
SO2:SER222
–
CONHPh:PHE28
–
5c
6a
À 6.9
À 7.7
HOPh:PHE28
NH2Ph:PHE28
NH2Ph:PHE190
NH2Ph:PHE28
NH2Ph:PHE190
NH2Ph:PHE28
NH2Ph:PHE190
NH2Ph:PHE28
NH2Ph:PHE190
Ph:PHE28
–
–
–
–
6b
6c
6d
6e
À 7.6
À 7.7
À 7.6
À 7.8
–
–
–
–
–
HOPh:PHE28
HOPh:PHE28
HOPh:PHE28
HO:ARG235
SO2:SER222
Ph:PHE190
6f
À 7.0
SO:ARG235
Heteroaromatic NH2:ASP185,
NH:ASP185,
HOPh:PHE28
–
–
–
–
6a-DHP
conjugate
À 10.3
Ar:ARG255
NH2:ASN115,
NH:ASP96,
C=N:LYS221,
C=O:LYS221
HO:ARG235
SO:SER222
Sulfonamide
moiety
NHPh:PHE28,
NHPh:PHE190
–
–
NHPh:LYS221
–
6b-DHP
conjugate
À 10.2
À 10.1
À 10.0
Heteroaromatic NH:ASP185
NH:ASP96
–
Ar:ARG255
C=N:LYS221
C=O:LYS221
Sulfonamide
moiety
SO:SER222
–
NHPh:PHE28,
NHPh:PHE190
–
–
NHPh:LYS221
–
6c-DHP
conjugate
Heteroaromatic NH2:SER27
NH2:GLU60
–
Ar:ARG255
NH: SER27
NH:ASP96
Sulfonamide
moiety
HO:LYS192,
HO:GLY189
SO:SER222
NHPh:PHE28
NHPh:PHE190
–
–
–
6d-DHP
conjugate
Heteroaromatic NH2:SER27
–
Ar:ARG255
NH2:GLU60
NH: SER27
NH:ASP96
Sulfonamide
moiety
HN:GLY189
HO:GLY189
SO:SER222
NHPh:PHE28
NHPh:PHE190
–
–
–
predicted to the five other compounds (Figure 2 and
Table 2).
Roland et al.[42] demonstrated that sulfonamides
could act as substrates for DHPS, being recognized by
For compounds 6a–6d we also proposed the the p-aminobenzoic acid (PABA) binding site and
interaction between DHPS and the reaction products conjugated to dihydropteroate diphosphate. Recently,
between dihydropteroate diphosphate, one of its Zhao et al.[43] demonstrated the potential of structure-
substrates, and the 4-aminobenzenesulfonamides, related pterin-sulfa conjugates to inhibit such enzyme,
which we refer to as dihydropterin-sulfonamide con- which inspired us to evaluate the activity of these
jugates (Figure 3).
conjugates possibly generated in situ.
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Figure 2. Interaction of compounds 6a, 5a and 6f (A–C, respectively) with the dihydropteroate synthase (DHPS) active site. The
carbon chain of the DHPS active site is represented as green ribbons, and the atoms are white=carbon, yellow=sulfur, blue=
nitrogen, and red=oxygen.
Figure 3. Structures of dihydropteroate diphosphate (DHP)-aniline adducts.
For the proposed conjugates, two interaction described by Zhao et al.[43] (Figure 6D). The DHP unit,
profiles were found: one for compounds 6a and 6b as well as the central aromatic ring and the sulfona-
(Figure 4A) and another for compounds 6c and 6d mide group, assume similar positions differing slightly
(Figure 4B). This can be expected due to the structural only in the position of the N-phenyl group of the
similarity between these pairs. The complexes formed sulfonamides.
by compound 6a-DHP or 6b-DHP with DHPS were
Subsequently, the physicochemical properties and
more energetically stable than with 6c-DHP or 6d- toxicity risks of the tested compounds were predicted
DHP. The heteroaromatic systems of compounds 6a- (Table 3) using the OSIRIS Property Explorer.[44] The
DHP and 6b-DHP were docked in a similar way to the toxicity risks predicted for eugenol seem to be sup-
same group in crystallographic DHP, as well as its pressed in the hybrids, since none of eugenol-
aromatic rings found in the same position of crystallo- sulfonamide hybrids presents mutagenic, tumorigenic,
graphic sulfamethoxazoles aromatic system (Figure 4C). or irritant potential like eugenol. In comparison to
This pose is also similar to the one found for pterin- sulfamethoxazole, all of the synthesized sulfonamides
sulfa conjugate compound 16 in DHPS active site as present higher lipophilicity (ClogP) than this drug. Clog
P
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Figure 4. Interaction of DHP-sulfonamide conjugates (white=carbon, yellow=sulfur, blue=nitrogen, red=oxygen) with Y. pestis
DHPS active site (green ribbon, PDB ID: 3TZF)[38] where (A) is the conformation of 6a-DHP and 6c-DHP found in their most stable
complex with DHPS active site. (B) is the conformation of 6c-DHP and 6d-DHP found in their most stable complex with DHPS
active site. (C) is the conformation comparison among ligands 6a-DHP and 6b-DHP (white=carbon) and crystallographic
sulfamethoxazole (green=carbon) and DHP (pink=carbon) interacting with DHPS active site. (D) is the pose of pterin-sulfa
conjugate compound 16 in Y. pestis DHPS active site (green ribbon, PDB ID: 5JQ9).[40]
Table 3. Predicted physicochemical properties, toxicity risks and drug score of eugenol-sulfonamide hybrids, eugenol, and
reference drug sulfamethoxazole [*values closer to 1 indicate the greater the potential of a compound to become a drug].
Compound
Toxicity Risks
Physicochemical
properties
Drug Likeness Drug Score*
Mutagenic Tumorigenic Irritant Reproductive effect ClogP
Solubility MW
Eugenol
Sulfamethoxazole –
+
+
–
–
–
–
–
–
–
–
–
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2.27
0.44
2.28
2.28
1.89
2.08
1.89
2.08
2.76
1.84
À 2.05
À 3.02
À 3.72
À 3.72
À 3.46
À 3.52
À 3.46
À 3.52
À 3.44
À 3.90
164
253
376
376
334
336
334
336
321
366
À 2.78
2.77
1.30
0.11
0.88
0.70
0.77
0.61
0.66
0.77
0.79
0.41
0.39
5a
5c
6a
6b
6c
6d
6e
6f
–
–
–
–
–
–
–
–
3.09
À 0.17
0.38
1.67
2.22
À 7.29
À 8.55
values close to (or above) 2 indicate that the designed none of these groups. In general, the best Drug Score
compounds may have better gastrointestinal absorp- values are associated to the sulfonamides that also
tion profiles, as well as having greater potential to demonstrated the best (i.e., lowest) MIC values: 5c, 6b,
access the central nervous system, which are impor- 6c and 6d.
tant features for the treatment of systemic and central
bacterial infections, respectively. The combination of
values obtained from drug likeness, ClogP, solubility,
molecular weight (MW) and the toxicity risks calcu-
Conclusions
lation, results in the Drug Score parameter, which is A set of sulfonamides, designed as hybrid compounds
important to assess the potential of a new substance from either eugenol or dihydroeugenol were screened
to become a drug. It is important to remember that for the first time as antibacterial agents. Most of the
the closer the value is to 1, the greater the potential of tested hybrid compounds showed good antimicrobial
a compound has to become a drug. The eugenol- activity, with the best results being demonstrated by
sulfonamide hybrids present Drug Score values which the compounds with a free amino group-as is usual
are higher than eugenol itself, indicating an improve- with the classic antibacterial sulfonamide drugs. How-
ment in their general properties compared to the ever, this seems not to be a requirement since the
parent compound. It is possible to observe that the unsubstituted compound 6e had good (i.e., low) MIC
aromatic amino and amide groups contribute pos- values against S. aureus, S. saprophyticus, M. abscessus
itively to this parameter, since lower values were and M. massiliense.
predicted for compounds 6e and 6f, which present
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In general, the most active compounds showed Waters Xevo G2-S QTOF and Bruker Daltonics micro
excellent (i.e., low) MIC values when compared to TOF QII/ESI-TOF, both equipped with ESI positive and
eugenol alone, which had an MIC greater than negative ion sources.
2000 μgmLÀ 1 against S. aureus and S. saprophyticus.
For the Gram-negative microorganisms, it is important
to note that none of the hybrids showed good (i.e.,
Synthesis and Characterization
low) MIC values, although all of them inhibited P. The synthesis and characterization of the amino key
aeruginosa with lower MIC values than the parent intermediates 4a–4d and the sulfonamide com-
compounds.
pounds 5a–5d and 6a–6d has previously been
Docking studies of these sulfonamides with the published by this research group.[30] The synthesis of
essential bacterial enzyme DHPS showed complexes the new compounds 6e and 6f were done as follows:
*
with favorable binding energies and these results
suggest that DHPS could be a possible target of the
antibacterial sulfonamide-eugenol hybrids. Further-
more, compounds 6a–6e presented similar docking
poses which are also very close to that with the
crystallographic ligand sulfamethoxazole. Docking
studies also indicate that dihydropterin-sulfonamide
conjugates interact with DHPS active site in a similar
way to pterin-sulfa conjugates known to act as
competitive DHPS inhibitors.[40] These results indicate
promise, which leads us to conclude that compounds
6a–6e are the most promising compounds for use as
new leads in the search for new antibacterial sulfona-
mides.
The amino derivative 4b (1 equiv.) and the respec-
tive benzenesulfonyl chloride (3 equiv.) were solubi-
lized in triethylamine (5 ml) and vigorously stirred
overnight at room temperature.
The progress of the reaction was monitored by TLC
using a hexane/ethyl acetate solvent mixture (8:2
ratio v/v).
The reaction mixture was poured into ice/water and
the pH was adjusted to pH 1–2 using hydrochloric
acid before being extracted with ethyl acetate (3×
50 ml aliquots).
The organic phase was dried over anhydrous
sodium sulfate and concentrated under reduced
pressure to produce the crude product, which in
turn was purified by column chromatography (silica
gel, hexane/ethyl acetate (6:4 v/v) solvent mixture).
*
*
*
Experimental Section
The analytical results generated for the newly
presented synthesized hybrid compounds 6e and 6f
General
Eugenol, dihydroeugenol and other reagents were are as follows:
purchased from Sigma (São Paulo, Brazil) and were
used as supplied. Thin-layer chromatography (TLC) on
N-(2-Hydroxy-3-methoxy-5-propylphenyl)
silica gel plates (Macherey–Nagel, DC-Fertigfolien benzenesulfonamide (6e). Beige soÀli1d. Yield: 30%.
°
ALUGRAM® Xtra Sil G/UV254) was used to monitor the Melting Point: 120–125 C. FT-IR (cm ): 3441 (NÀ H),
progress of reactions and to prove purity. Column 3275 (OÀ H), 2953–2847 (CÀ H sp3), 1616, 1512 (C=C),
chromatography was performed using column grade 1330, 1168 (S=O), 1311 (CÀ OÀ C). 1H-NMR (δ ppm;
silica gel (Sorbiline; 0.040–0.063 mm mesh size). CDCl3, 300 MHz): 7.80–7.76 (m; 2H; H2’), 7.51–7.46 (m;
Melting points of the compounds were obtained on a 1H; H4’), 7.41–7.35 (m; 2H; H3’), 6.95 (d; 1H; J=1.8;
Büchi 535 melting-point apparatus and are uncor- H4), 6.94 (s; 1H; OH), 6.42 (d; 1H; J=1.8; H6), 5.49 (s;
rected. Fourier transform infrared spectroscopy (FT-IR) 1H; NHSO2), 3.78 (s; 3H; OCH3), 2.46 (t; 2H; J=7.6;
spectra were recorded on a Shimadzu FT-IR Affinity-1 CH3À CH2À CH2), 1.62–1.49 (sex; 2H; CH3À CH2À CH2),
spectrophotometer. Nuclear magnetic resonance 0.87 (t; 3H; J=7.3; CH3À CH2À CH2). 13C-NMR (δ ppm;
(NMR) spectra were recorded on a Bruker AC-300 CDCl3, 75 MHz): 146.1 (C3), 139.0 (C1’), 134.5 (C1),
1
spectrometer (300 MHz for H-NMR and 75 MHz for 134.1 (C5), 132.8 (C4’), 128.7 (C2’), 127.2 (C3’), 123.4
13C-NMR experiments) using either deuterated chloro- (C2), 113.8 (C4), 107.8 (C6), 56.0 (OCH3), 37.8
form or deuterated dimethyl sulfoxide. Chemical shifts (CH3À CH2À CH2),
24.6
(CH3À CH2À CH2),
13.5
(δ) were reported in parts per million (ppm) with (CH3À CH2À CH2). HR-MS ESI/Q-TOF (m/z): [M+Na]+
reference to tetramethylsilane (TMS) as the internal calculated for C16H19NNaO4S=344.0927; Found
standard and coupling constants (J) were reported in 344.0925.
Hertz (Hz). High resolution mass (HR-MS) spectra were
obtained on a quadrupole time-of-flight instruments
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Chem. Biodiversity 2021, 18, e2100066
N-(2-Hydroxy-3-methoxy-5-propylphenyl)-4-ni-
trobenzenesulfonamide (6f). Yellow solid. Yield: 33%.
Determination of the Minimum Inhibitory Concentration
(MIC)
À 1
°
Melting Point: 176–180 C. FT-IR (cm ): 3485 (NÀ H),
3240 (OÀ H), 2958–2859 (CÀ H sp3), 1613, 1528 (C=C), Susceptibility tests were performed using the broth
1
1514, 1462 (NO2), 1340, 1225 (S=O), 1314 (CÀ OÀ C). H- microdilution method according to the Clinical and
NMR (δ ppm; CDCl3, 300 MHz): 8.23–8.19 (m; 2H; H3’), Laboratory Standards Institute (CLSI) M24-A2
7.95–7.90 (m; 2H; H2’), 6.98 (d; 1H; J=1.7; H4), 6.95 (s; standard[31] protocol for the mycobacteria group and
1H; OH), 6.48 (d; 1H; J=1.7; H6), 5.40 (s; 1H; NHSO2), according to CLSI M100-S23 standard[32] for the other
3.79 (s; 3H; OCH3), 2.49 (t; 2H; J=7.6; CH3À CH2À CH2), bacteria. The compounds were used at different
1.64–1.52 (sex.; 2H; CH3À CH2À CH2), 0.90 (t; 3H; J=7.3; concentrations, according to the mass obtained in the
CH3À CH2À CH2). 13C-NMR (δ ppm; CDCl3, 75 MHz): 150.1 synthesis stage and the quantity available for the test,
(C4’), 146.2 (C3), 144.8 (C1’), 134.9 (C1), 134.7 (C5), from serial dilutions in Mueller–Hinton Broth (Hi
128.5 (C3’), 123.9 (C2’), 122.1 (C2), 114.7 (C4), 107.8 Media Laboratories Pvt. Ltd., India). The density of the
(C6), 56.0 (OCH3), 37.7 (CH3À CH2À CH2), 24.6 inoculum was standardized according to the 0.5
(CH3À CH2À CH2), 13.6 (CH3À CH2À CH2). HR-MS ESI/Q- MacFarland scale and transferred to the microplates.
TOF (m/z): [M+K]+ calculated for C16H18KN2O6S= The plates were incubated at 30 C for 72 h for the
°
°
405.0517; Found 405.0518.
mycobacteria group and at 35 C during 24 h for the
Gram-positive and Gram-negative bacteria. MIC was
From the analytical data generated (a supplemen- considered as the lowest concentration, given as
tary file is available containing all of the relevant μgmLÀ 1 values, of the compound that inhibited visible
spectra), it can be concluded that the structures of the bacterial growth. The compound 2,3,5-triphenyltetra-
synthesized compounds are those shown in Figure 2. zolium chloride (TTC) (Vetec®) was used as an indicator
These compounds were then evaluated in antibacterial of microbial growth.
studies as well as molecular docking studies as shown
in the following sections.
Molecular Docking and Prediction Studies
The structures of the sulfonamide derivatives and the
Antimicrobial Evaluation
Microorganisms
dihydropteroate diphosphate (DHP)-sulfonamide con-
jugates were drawn and optimized with a Dreiding-
like force field in the Biovia Discovery Studio software
For this study three groups of standard strains were program.[45] The crystal structure coordinates of Y.
used from the Bacteria Bank of the Mycobacteriology pestis dihydropteroate synthase complexed with sulfa-
Laboratory (LABMYCO), Department of Clinical and methoxazole and dihydropteroate diphosphate (DHP)
Toxicological Analysis, Federal University of Santa were obtained from the Protein Data Bank (PDB)
Maria. The first group comprised of the standard online database using the identification number
strains of rapidly growing mycobacteria (M. fortuitum ‘3TZF’,[38]
ATCC 6841 and M. abscessus ATCC 19977). The second
group included Gram-positive bacteria (Staphylococcus
AutoDock PDBQT files of macromolecules and
aureus ATCC 29213 and Staphylococcus saprophyticus ligands were prepared using AutoDock Tools Software
ATCC 15305). The third group comprised the Gram- v1.5.2[46] using the method described by Morris
negative bacteria, Pseudomonas aeruginosa (PAO1) et al.[47] For the docking studies with the sulfonamide
and Klebsiella pneumonia (KPC-type carbapenemase). derivatives, the macromolecule structure was prepared
The mycobacteria strains were kept in a Löwenstein- by keeping only chain B and their crystalized DHP and
Jensen agar (Hi Media Laboratories Pvt. Ltd., India) and Mg2+ ligands. The dimensions of the docking grid
the Gram-positive and Gram-negative strains remained were 1.6 nm×1.6 nm×1.6 nm (16 Å×16 Å×16 Å) and
stored in BHI broth (brain heart infusion broth) the coordinates of the center point were (29.951)×
°
supplemented with glycerol at À 20 C until use.
(À 1.452)×(8.959), covering the area occupied by the
crystallographic ligand sulfamethoxazole. For the
docking studies with the DHP-sulfonamide adducts,
crystal DHPS was prepared by keeping only the chain
B and removing all ligands, water molecules and chain
A. The dimensions of the docking grid were 2.4 nm×2
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Chem. Biodiversity 2021, 18, e2100066
References
nm×20 nm (24 Å×20 Å×20 Å) and the coordinates of
the center point were (24.878)×(À 1.871)×(8.959),
covering the area occupied by crystallographic ligands
sulfamethoxazole and DHP.
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The conception and design of the work was carried
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Received January 25, 2021
Accepted March 4, 2021
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