Searching for Novel Inhibitors of the S. aureus NorA Efflux Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-benzothiazine Analogues

Article abstract of DOI:10.1002/cmdc.201700286

Bacterial resistance to antimicrobial agents has become an increasingly serious health problem in recent years. Among the strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sub-lethal concentration of the antibacterial agent at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei. None of the new synthesized compounds showed any appreciable intrinsic antibacterial activity, and, in particular, 2-(3,4-dimethoxyphenyl)quinoline (6 c) was able to decrease, in a concentration-dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S. aureus SA-K2378 (norA++) and SA-1199B (norA+/A116E GrlA).

Full text of DOI:10.1002/cmdc.201700286

Accepted Article
Title: Searching for Novel Inhibitors of the S. aureus NorA Efflux
Pump: Synthesis and Biological Evaluation of the 3-Phenyl-1,4-
benzothiazine Analogues
Authors: Tommaso Felicetti, Rolando Cannalire, Maria Sole Burali,
Serena Massari, Giuseppe Manfroni, Maria Letizia Barreca,
Oriana Tabarrini, Bryan D. Schindler, Stefano Sabatini, Glenn
W Kaatz, and Violetta Cecchetti
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To be cited as: ChemMedChem 10.1002/cmdc.201700286
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10.1002/cmdc.201700286
ChemMedChem
Searching for Novel Inhibitors of the S. aureus
NorA Efflux Pump: Synthesis and Biological
Evaluation of the 3-Phenyl-1,4-benzothiazine
Analogues
Tommaso Felicetti,[a] Rolando Cannalire,[a] Maria Sole Burali,[a] Serena Massari,[a]
Giuseppe Manfroni,[a] Maria Letizia Barreca,[a] Oriana Tabarrini,[a] Bryan D. Schindler,[b]
Stefano Sabatini,[a]* Glenn W. Kaatz,[b] and Violetta Cecchetti[a]
[a] Tommaso Felicetti, Dr. Rolando Cannalire, Maria Sole Burali, Dr. Serena Massari, Dr.
Giuseppe Manfroni, Prof. Maria Letizia Barreca, Prof. Oriana Tabarrini, Prof. Stefano
Sabatini, Prof. Violetta Cecchetti
Department of Pharmaceutical Sciences, University of Perugia, I-06123 Perugia, Italy
E-mail: stefano.sabatini@unipg.it
[b] Dr. Bryan D. Schindler, Prof. Glenn W. Kaatz
Department of Internal Medicine, Division of Infectious Diseases, School of Medicine,
Wayne State University and the John D. Dingell Department of Veteran Affairs Medical
Center, Detroit, Michigan 48201 USA
E-mail: gkaatz@juno.com
Keywords: antimicrobial resistance (AMR), inhibitors, medicinal chemistry, NorA efflux
pump, Staphylococcus aureus.
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Abstract
Bacterial resistance to antimicrobial agents has become an increasingly serious health
problem in recent years. Among the strategies by which resistance can be achieved,
overexpression of efflux pumps such as NorA of S. aureus leads to a sub-lethal
concentration of the antibacterial at the active site that in turn may predispose the
organism to the development of high-level target-based resistance. With the aim to
improve both the chemical stability and the potency of our previously reported 3-phenyl-
1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei.
None of the new synthesized compounds showed any appreciable intrinsic antibacterial
activity and, in particular, the 2-phenylquinoline 6c was able to reduce, in a concentration-
dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S.
aureus SA-K2378 (norA++) and SA-1199B (norA+/ A116E GrlA).
Introduction
Antimicrobial resistance (AMR) is not a recent phenomenon, but it is a critical health
issue today. Bacteria causing common infections have developed resistance to each class
of antibacterials[1] and AMR has evolved to become a worldwide health threat.[2] With the
dearth of new antibiotics coming to the market, the need for strategies to avert a
developing global crisis in health care is increasingly urgent.[2a, 3]
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common
antimicrobial-resistant pathogens causing invasive infections; indeed, about 80,000 cases
with 11,000 deaths were observed in 2013 in the US, even more than HIV/AIDS (7,638
deaths).[4] In addition, MRSA strains are constantly accumulating further antibiotic
resistance genes, thereby creating highly virulent and difficult to treat ‘superbugs’. Thus,
resistance to the entire class of β-lactam antibiotics and to additional drugs such as
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erythromycin, clindamycin, ciprofloxacin (CPX), tetracycline, etc.[5] makes MRSA infections
difficult to treat. In addition, vancomycin, which represents the standard of care for
complicated MRSA infections, is losing its efficiency owing to increasingly emergence of
vancomycin-resistant S. aureus (VRSA) strains.[6]
AMR is achieved by four main mechanisms: enzymatic inactivation of the drug,[7]
modification of the drug target(s),[8] reduction of intracellular drug concentration by either
changes in membrane permeability[9] or overexpression of efflux pumps,[10] and biofilm
formation.[11]
With regard to efflux pumps, they are membrane-based proteins providing a self-
defense mechanism by which substances, natural or synthetic, that could potentially harm
the microorganism are actively removed from the cell. When expressed at baseline, these
transporters confer moderate levels of resistance. However, their overexpression can
widely affect susceptibility to chemotherapeutic agents by reducing the antibacterial
concentration inside bacterial cells.[12] Interestingly, efflux pumps seem also to be involved
in biofilm formation, further contributing to increase the resistance levels.[13]
Instead of, or in addition to, the search for new antibacterials, a fruitful strategy may be
to find a molecule able to circumvent resistance mechanisms. Efflux pump inhibitors (EPIs)
could be able to restore the susceptibility of the resistant strains to those antibacterial
agents that are efflux pumps substrates.[14]
One of the most studied efflux pumps in S. aureus is NorA, a protein belonging to the
Major Facilitator Superfamily.[15] Using energy supplied by the proton motive force, this
transporter is capable of extruding multiple structurally dissimilar substrates such as
hydrophilic fluoroquinolones, tetracyclines, monovalent cation and dyes (such as ethidium
bromide – EtBr).[16]
To date, some non-antibiotic drugs such as reserpine[17] and verapamil[18] have been
identified as NorA EPIs but concentrations required for pump inhibition activity are too high
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for them to be clinically relevant. Thus, great efforts have been made to identify potent
NorA EPIs. This work has included studies by phytochemists, who have reported
numerous natural EPI compounds including flavones, isoflavones, porphyrin
phaeophorbide A, and acylated glycosides.[19] Additionally, academic groups have also
contributed and have identified new classes of NorA inhibitors. To date, there have been a
few examples of rational drug design and structure−activity relationships (SAR) studies
with NorA EPIs.[20]
Although the therapeutic utility of EPIs has yet to be validated in the clinical setting, this
approach holds promise for improving the efficacy and/or extending the clinical utility of
existing antibacterials, giving new life to old drugs with secure social and economic
benefit.[21]
By a structure minimizing of the phenothiazine moiety of known active EPIs, we
previously reported a series of substituted 3-phenyl-2H-benzo[1,4]thiazine and 3-phenyl-
3,4-dihydro-2H-benzo[1,4]thiazine as NorA EPIs.[22] These two classes demonstrated the
capability of completely restoring the antibacterial activity of ciprofloxacin against a NorA
overexpressing strain (SA-K2378 norA++)[23] and to reduce the EtBr efflux of SA-1199B
(norA+ and A116E GrlA).[24] Despite these promising results, their further development as
EPIs was precluded because the 3-phenyl-2H-benzo[1,4]thiazines suffer from chemical
instability in polar solvents due to a spontaneous pheomelanin-like oxidation with
dimerization at the C-2 position.[25]
In this work, starting from the promising results in terms of EPI activity of the
benzo[1,4]thiazine series, we report the design, synthesis, and biological evaluation of six
different sets of benzothiazine derivatives having an alternative scaffold in order to
improve chemical stability and EPI activity.
Results & Discussions
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Rational design
The lack of the crystal structure for NorA and the structural heterogeneity of known
26]
NorA EPIs[20c-e,
prompted us to pursue a classical medicinal chemistry approach
entailing modifications of the benzothiazine nucleus aimed at the substitution of the
sulphur atom with the goals being to: i) resolve the chemical stability issues, ii) retain the
EPI activity, and iii) contribute to better delineate the SAR of this class of NorA EPIs. Thus,
the sulphur atom in position 1 of the 2H-benzo[1,4]thiazine scaffold was substituted by an
oxygen (-O-), a nitrogen (-NH-), or a methylene (-CH2-) to obtain the corresponding 3-
phenyl-2H-benzo[1,4]oxazine (1), 2-phenyl-1,2,3,4-tetrahydroquinoxaline (2), and 2-
phenyl-1,2,3,4-tetrahydroquinoline (3) nuclei, respectively. Then, with the aim to expand
the series, 3-phenyl-3,4-dihydro-2H-benzo[1,4]oxazine (4), in which the double bond in the
ring B of the 3-phenyl-2H-benzo[1,4]oxazine (1) scaffold has been reduced, was
synthesized. Finally, by aromatization of the
B
ring of 2-phenyl-1,2,3,4-
tetrahydroquinoxaline (2) and 2-phenyl-1,2,3,4-tetrahydroquinoline (3) nuclei, 2-
phenylquinoxaline (5) and 2-phenylquinoline (6) derivatives, respectively, were also
obtained as potential NorA EPIs (Figure 1).
Figure 1. From the 3-phenyl-2H-benzo[1,4]thiazines to 3-phenyl-2H-benzo[1,4]oxazine (1), 2-phenyl-1,2,3,4-
tetrahydroquinoxaline
(2),
2-phenyl-1,2,3,4-tetrahydroquinoline
(3),
3-phenyl-3,4-dihydro-2H-
benzo[1,4]oxazine (4), 2-phenylquinoxaline (5), and 2-phenylquinoline (6) EPIs analogues.
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All the obtained nuclei were further decorated at the meta or para positions in the
exocyclic phenyl (ring C) with those substituents (methoxy or propoxy groups) according
with the previously delineated SAR of the 3-phenyl-2H-benzo[1,4]thiazine NorA EPIs
(Table 1).[22]
Before proceeding with the synthesis of the new planned compounds, an accurate
search for possible dimers of the new nuclei has been performed. Although dimers were
found only for the 3-phenyl-2H-benzo[1,4]oxazine[27] and the 2-phenylquinoxaline[28] nuclei,
none of these was the result of a spontaneous dimerization as previously seen for the 3-
phenyl-2H-benzo[1,4]thiazines.
Chemistry
Three principal synthetic strategies were carried out for the construction of the 2H-
benzo[1,4]oxazine, quinoxaline, and quinoline nuclei.
Synthesis of 3-phenyl-2H-benzo[1,4]oxazines and 3-phenyl-3,4-dihydro-2H-
benzo[1,4]oxazines.
Compounds 1a,[29] 1b,[30] 1c,[31] 1d, 1e, and 4a,[29] 4b,[31] 4c,[31] 4d, 4e were synthesized
following the procedure depicted in Scheme 1. The synthesis of the key intermediate
10a[32] was carried out by following the procedure reported by Baraldi et al.[32] The same
procedure was then used to synthesize also the intermediates 10b,[33] 10c and 10e by
reacting 2-nitrophenol 7 with properly substituted 2-bromo-1-phenylethanones 9a, 9b,
9c,[34] and 9e[22] in presence of K2CO3 in dry acetone. Starting from compounds 10a[32] and
10b,[33] by using Raney-Ni/H2 at 0°C to reduce the nitro group, compounds 1a[29] and 1b[30]
were obtained while compounds 1c-e could not be isolated by this procedure. On the other
hand, compounds 1c,[31] 1d, and 1e were directly obtained by reacting 2-aminophenol 8
with properly substituted 2-bromo-1-phenylethanones 9c,[34] 9d,[35] and 9e[22] in dry DMF
and using NaH. Using Raney-Ni/H2 at 70°C both the nitro group of compounds 10a,[32]
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10b,[33] 10c, 10e and the endocyclic C=N double bond were reduced to obtain compounds
4a,[29] 4b,[31] 4c[31] and 4e. Finally, the subsequent reduction of compound 1d with
BH3∙THF in dry THF provided derivative 4d (Scheme 1 and Table 1).
Scheme 1. Reagents and conditions: a) K2CO3, dry acetone, r.t., 2 h, 42-95%; b) NaH, dry DMF, r.t., 1 h, 12-
48%; c) H2, Raney-Ni, dioxane, 0°C, 5 h, 57-80%; d) H2, Raney-Ni, dioxane, 70°C, 5 h, 42-95%; e) 1M
BH3∙THF, dry THF, r.t., 3 h, 47%.
Synthesis of 2-phenyl-1,2,3,4-tetrahydroquinoxalines and 2-phenylquinoxalines.
Compounds 2a,[36] 2b,[37] 2c-e and 5a,[38] 5b,[39] 5c,[40] 5d,[41] 5e were obtained following
the procedure depicted in Scheme 2, by the reaction of benzene-1,2-diamine 11 with
properly substituted 2-bromo-1-phenylethanones 9a, 9b, 9c,[34] 9d[35] and 9e[22] using
anhydrous sodium acetate and dry MeOH. The spontaneous oxidation of the 2-phenyl-3,4-
dihydroquinoxaline intermediates 12a-e in the corresponding 2-phenylquinoxaline
derivatives gave compounds 5a,[38] 5b,[39] 5c,[40] 5d,[41] and 5e. Compounds 2a,[36] 2b,[37]
and 2c-e were finally obtained, after reduction in anhydrous condition of 5a,[38] 5b,[39] 5c,[40]
5d[41] and 5e using BH3·THF in dry THF (Scheme 2 and Table 1).
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Scheme 2. Reagents and conditions: a) CH3COONa, dry MeOH, r.t., 6 h, 20-69%; b) 1M BH3·THF, dry THF,
r.t., 4 h, 14-70%.
Synthesis
of
2-phenyl-1,2,3,4-tetrahydroquinolines
and
2-phenyl-3,4-
dihydroquinolines.
Compounds 3a,[42] 3b,[43] 3c,[44] 3d,[45] 3e, and 6a,[46] 6b,[46] 6c,[47] 6d,[48] 6e were
obtained according to the procedure depicted in Scheme 3. Following the procedure
described from Mierde et al[46] for the synthesis of compounds 6a[46] and 6b,[46] the
derivatives 6c,[47] 6d,[48] and 6e were obtained in a single step by the reaction of 2-
(aminophenyl)methanol 13[49] with properly substituted 1-phenylethanones 14c, 14d, and
14e[22] and t-BuOK in dioxane.
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Scheme 3. Reagents and conditions: a) t-BuOK, dioxane, 80°C, 4 h, 7-62%; b) Na0, n-PrOH, reflux, 3 h, 8-
70%; c) Raney-Ni/H2, EtOAc, r.t., 1 h, 47%.
The successive reduction of the double bonds was carried out with Na0 using n-PrOH at
reflux to give the 2-phenyl-1,2,3,4-tetrahydroquinolines 3a,[42] 3b,[43] 3c,[44] and 3e. Due to
the very low yield observed for compound 3d,[45] a different reduction method was carried
out using H2 and using Raney-Ni as catalyst (Scheme 3 and Table 1).
Biological data and discussion
EtBr efflux inhibition assays. All the synthesized EPI analogues were initially
evaluated for their ability to inhibit the NorA efflux pump and then for their capability to act
synergistically with the antibacterial CPX. The ability of putative EPIs to interfere with EtBr
efflux was assessed using a screening concentration of 50 µM. SA-1199B was used as
the test strain in these studies and a previously-described fluorometric approach was
used.[24] This strain overexpresses NorA by way of a promoter mutation and also
possesses a mutation in topoisomerase IV-A116E GrlA that reduces its affinity to CPX
(Table 1). The 3-(4-methoxyphenyl)-2H-benzo[1,4]thiazine hit (PT3[22]) and the alkaloid
reserpine,[17] reported in literature as a potent NorA inhibitor, were included in the test as
reference compounds.
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Table 1. EtBr efflux inhibition (%) of 3-phenyl-2H-1,4-benzothiazine analogues, PT3, and
reserpine at 50 µM concentration against SA-1199B (norA+/A116E GrlA) strain.
EtBr efflux
inhib. (%)
Compd.
R3
R4
R5
1a[29]
1b[30]
1c[31]
1d
H
H
H
OMe
H
H
H
0
0
OMe OMe
OMe OMe
H
2.4±1.2
31.6±2.4
22.1±1.8
OMe
H
1e
On-Pr
2H-benzo [1,4]oxazines
2a[36]
2b[37]
2c
H
H
H
H
0
OMe
H
0
OMe OMe
OMe OMe
H
OMe
H
0
8.1±0.5
0
2d
2e
H
On-Pr
1,2,3,4-tetrahydroquinoxalines
1,2,3,4-tetrahydroquinolines
3a[42]
3b[43]
3c[44]
3d[45]
3e
4a[29]
4b[31]
4c[31]
4d
H
H
H
OMe
H
H
15.2±1.1
5.8±1.0
OMe OMe
OMe OMe
H
OMe
H
8.6±1.7
16.2±2.1
32.6±2.1
H
On-Pr
H
H
H
H
0
OMe
H
H
0
0
0
OMe OMe
OMe OMe
H
OMe
H
4e
On-Pr
51.0±1.4
3,4-dihydro-2H-benzo[1,4]oxazines
5a[38]
5b[39]
5c[40]
5d[41]
5e
H
H
H
OMe
H
H
0
41.1±3.1
0
8.1±1.9
12.5±2.5
OMe OMe
OMe OMe
H
H
OMe
H
On-Pr
quinoxalines
6a[46]
6b[46]
6c[47]
6d[48]
6e
H
H
H
OMe
H
H
41.1±0.9
16.3±1.4
49.3±2.3
32.1±2.1
15.5±1.8
OMe OMe
OMe OMe
H
OMe
H
H
On-Pr
quinolines
Reserpine
PT3[22]
H
OMe
H
19.5±0.4
82±2.1
Experiments were performed in triplicate, and the results were expressed as mean total
efflux over a 5 min time course.
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Despite the fact that none of the tested compounds displayed an EtBr efflux inhibition
activity against SA-1199B comparable to that of reserpine, eight of thirty compounds (1d,
1e, 3e, 4e, 5b,[39] 6a,[46] 6c[47] and 6d[48] highlighted in Table 1) showed an activity better
than that of the hit progenitor PT3;[22] moreover, four of them (4e, 5b,[39] 6a,[46] and 6c[47])
were more than the twice as active as PT3[22] activity (Table 1).
MIC evaluation, synergistic activity, and cytotoxicity assessment. To avoid a
deceptive interpretation in assigning NorA inhibitory activity and before assaying their
synergistic activity with CPX, the most promising compounds of each sub-series (1d, 2d,
3e, 4e, 5b,[39] and 6c[47] – Table 1) were tested for potential intrinsic antibacterial activity
against different S. aureus strains. The strains included S. aureus ATCC 25923 norA wild-
type (wt), SA-K1902 in which norA gene was deleted (norA-),[23] SA-K2378 overexpressing
norA (norA++),[23] and SA-1199B (norA+/A116E GrlA).[24] The selected compounds
showed no intrinsic antibacterial activity up to the highest concentration tested (100 µg/mL)
(data not shown).
Compounds 1d, 2d, 3e, 4e, 5b,[39] and 6c[47] were then investigated at a concentration
of 25 µg/mL (1/4 of the highest concentration used in the intrinsic antibacterial activity
assays) for their synergistic activity with the antibacterial fluoroquinolone CPX against S.
aureus ATCC 25923 (wt), SA-K1902 (norA-),[23] and SA-K2378 (norA++).[23] The 50 %
cytotoxic concentration (CC50) against human epithelial HeLa (ATCC CCL2) cells for the
above cited compounds, was also evaluated (Table 2).
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Table 2. Synergistic activity with CPX of compounds 1d, 2d, 3e, 4e, 5b,[39] 6c,[47] and reserpine at 25
µg/mL concentration against selected S. aureus strains and CC50 against HeLa cells.
CPX MICs (µg/mL)
HeLa
Cytotoxicity
CC50 (µg/mL)
Modified S. aureus
strains
S. aureus
ATCC 25923
Compd.
1d
Structure
K1902
K2378
0.31
0.31
0.16
0.16
0.16 (8)
36.6±1.4
2d
0.63 (2)
140.2±2.1
3e
4e
0.31
0.31
0.16
0.16
0.31 (4)
0.31 (4)
135.7±1.7
57.3±0.9
5b[39]
0.31
0.16
0.16
0.16
0.31 (4)
0.16 (8)
0.16 (8)
28.1±2.9
158.0±2.1
-
6c[47]
0.16 (2)
0.08 (4)
PT3[22]
Reserpine
CPX
0.16 (2)
0.31
0.08 (2)
0.16
0.31 (4)
-
1.25
86.0±1.6
Values in brackets show the fold reduction of CPX MIC. Results are expressed as 50% cytotoxic
concentration (CC50). The CC50 was defined as the concentration required to reduce the cell number by
50% compared to that for the untreated controls. Each concentration was tested in triplicate.
As expected, negligible or no synergistic activity between any of the tested compounds
and CPX has been observed against the S. aureus strains ATCC 25923 (wt) and SA-
K1902 (norA-)[23] (Table 2). These data are in agreement with a low level of expression of
the NorA efflux pump in these strains. On the contrary, all but one compound showed a
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good or moderate synergistic activity with CPX (4- to 8-fold CPX MIC reduction) against
the modified S. aureus strain (SA-K2378) in which the norA gene was overexpressed.[23]
The activities of compounds 1d and 6c[47] were comparable to that of reference compound
PT3[22] and definitely better than reserpine. At the same time, compounds 3e, 4e, and 5b,
although less active than PT3,[22] retained an activity comparable to reserpine.
Furthermore, compounds 2d, 3e, and 6c[47] resulted the less cytotoxic compounds against
HeLa cells (CC50>100 µg/mL). In particular, compound 6c,[47] showed a CC50 value about
the twice that of the CPX.
To better delineate the synergistic activity with CPX for the more interesting
compounds, the 2H-benzo[1,4]oxazine 1d and the quinoline 6c,[47] checkerboard assays in
combination with CPX have been performed against S. aureus ATCC 25923 (wt) and SA-
K2378 (norA++)[23] (Figure 2). The 2H-benzo[1,4]thiazine progenitor PT3[22] and reserpine
were included for comparative purposes.
Figure 2. Effect of compounds 1d, 6c[47], PT3[22] and reserpine on the CPX MICs against S. aureus ATCC
25923 (wt) and SA-K2378 (norA++) strains.
As expected, minimal synergistic activity with CPX against S. aureus ATCC 25923 (wt)
has been observed for compounds 6c[47] and 1d. However, against the SA-K2378
(norA++)[23] there was a gradual reduction of CPX MIC across all concentrations of tested
compounds.
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In particular, both 6c[47] and 1d displayed a good synergistic activity with CPX; in fact
they were able to reduce the CPX MIC against SA-K2378 (norA++)[23] by 8-fold at
concentrations as low as 12.5 and 25 μg/mL, respectively. In particular, compound 6c[47]
showed the best activity with a CPX MIC reduction of 4-fold at 3.13 μg/mL, one dilution
below that necessary for the reference compounds PT3[22] and reserpine. Therefore,
isobolograms highlight that the tested compounds showed the same (compound 1d) or
a slightly better (compound 6c[47]) synergistic activity with CPX with respect to those of
PT3[22] and reserpine (Figure 2).
PAINS and aggregators filters. The most active compounds (1d, 2d, 3e, 4e, 5b,[39]
and 6c[47]) by the EtBr assays were examined for known classes of pan-assay
interference compounds (PAINS)[50] and aggregators by using the ZINC15[51] remover
filter at http://zinc15.docking.org. None of the compounds was found as potential PAINS
or aggregator, with the exception of 2d resulting as potential PAINS. Indeed, compound
2d, which was capable of reducing EtBr efflux albeit by a small amount (8.1%), did not
have any synergistic effect with CPX in checkerboard assays. Moreover, since
molecules possessing a LogP value higher than 5 are more likely to produce
aggregates, for the six compounds LogP values have been predicted by ACD/LogP DB
and all of them had a LogP value lower than 5.
Conclusions
In this work we have synthesized six new series of derivatives obtained by the classical
replacement of the 2H-benzo[1,4]thiazine sulphur atom, to be tested for their S. aureus
NorA EPI activity and showing an improved chemical stability compared to their
benzothiazine progenitors. In all but one series were identified compounds able to reduce
EtBr efflux on the SA-1199B S. aureus strain (Table 1). Compounds 1d, 1e, 3e, 4e, 5b,[39]
6a,[46] 6c,[47] and 6d[48] showed an improved EtBr efflux inhibition activity with respect to
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that of PT3[22] even if it is still lower than that of reserpine. Compounds 1d, 3e, 4e, 5b,[39]
and 6c[47] also displayed good synergistic activity with CPX similar to the mentioned
reference compounds when tested against SA-K2378 (norA++).[23] Among them, 6c[47]
showed the best EPI activity in synergism with CPX and also a low toxicity on HeLa cells
(CC50 = 158 µg/mL). Therefore, since 6c[47] shows an EPI activity at a concentration 50-
fold lower than its CC50, it can be considered as new hit for future optimizations. Definitely,
the quinoline nucleus emerged to be the best scaffold to design and synthesize a new
class of promising EPIs. Noteworthy, these results are in accordance with the parallel
findings in our previous study[26g] where the quinoline core emerges as an excellent
scaffold from an optimization of the flavone nucleus. Thus, further studies could be carried
out around this nucleus in an attempt to improve EPI activity and to explore SAR.
Experimental Section
Bacterial Strains. The strains of S. aureus employed were ATCC 25923 (wild-type),
SA-K1902 (norA-deleted),[23] SA-1199B (overexpressing norA and also possessessing
an A116E GrlA substitution).[24] In addition, SA-K2378, which overexpresses norA from
a multicopy plasmid, was also used.[23] This strain was produced by cloning norA and its
promoter into plasmid pCU1 and then introducing the construct into SA-K1902.[23]
EtBr Efflux. The loss of EtBr from S. aureus SA-1199B was determined
fluorometrically as previously described.[52] Experiments were performed in triplicate,
and the results were expressed as mean total efflux over a 5 min time course.
Microbiologic Procedures. MICs were determined by microdilution techniques
according to CLSI guidelines.[53] The effect of combining reserpine, PT3,[22] or scalar
dilutions of freshly prepared solutions of each selected compound on the MICs of CPX
was also determined. Checkerboard combination studies using CPX and the tested
compounds were performed as described previously.[54]
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Cell viability assay. All compounds were tested on human epithelial HeLa cell line
(ATCC CCL-2). HeLa cells were grown in RPMI 1640 supplemented with 10% heat-
inactivated fetal calf serum, 10,000 units penicillin and 10 µg streptomycin/mL overnight
to confluence. Monolayers were treated for 24 h at 37°C with scalar concentration of
tested compounds (0-250 µg/mL). Cell viability was then evacuate by the use of an ATP
bioluminescence kit (Via Light kit; Cambrex). Results are expressed as 50% cytotoxic
concentration (CC50). The CC50 was defined as the concentration required to reduce the
cell number by 50% compared to that for the untreated controls. Each concentration
was tested in triplicate.
Synthesis. All reactions were routinely checked by thin-layer chromatography (TLC)
on silica gel 60F254 (Merck) and visualized using UV illumination. Flash column
chromatography was performed on Merck Silica Gel 60 (mesh 230-400) using the
indicated solvents. Yields were of purified product and were not optimized. Melting
points were determined in capillary tubes (Mettler PF62 apparatus) and are
1
uncorrected. H NMR spectra were recorded at 200 or 400 MHz, with a Bruker
Advance-DRX-200 or 400 instrument and with Me4Si as the internal standard. 13C NMR
spectra were recorded at 101 MHz with a Bruker Advance-DRX 400 instrument. The
chemical shift (δ) values are reported in ppm, and the coupling constants (J) are given
in Hz. The abbreviations used are as follows: s, singlet; bs, broad singlet; d, doublet; dd,
double doublet; t, triplet; m, multiplet. The spectral data are consistent with the assigned
structures. Reagents and solvents were purchased from common commercial suppliers
and were used as received. For routine aqueous workup, the reaction mixture was
extracted with CH2Cl2 or EtOAc. The organic layer was washed with brine, dried over
anhydrous Na2SO4, and concentrated with a Büchi rotary evaporator at low pressure. All
starting materials were commercially available unless otherwise indicated. The purity of
the tested compounds was evaluated by combustion analysis using a Fisons elemental
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analyzer, model EA1108CHN, and data for C, H, and N are within 0.4% of the
theoretical values (≥95% sample purity).
13
All melting points, spectral data (1H NMR and C NMR), and elemental analysis are
shown in the Supporting Information.
Synthesis of the 3-phenyl-2H-benzo[1,4]oxazines (1a,[29] 1b,[30] 1c,[31] 1d, 1e) and the
3-phenyl-3,4-dihydro-2H-benzo[1,4]oxazines (4a,[29] 4b,[31] 4c,[31] 4d, 4e).
General procedure for the synthesis of compounds 10b,[33] 10c, and 10e.
Under N2 atmosphere, to a solution of 2-nitrophenol 7 (1.1 eq) in dry acetone (1.80 mL per
mmol), K2CO3 (1.8 eq) was added. After 20 min the appropriate 2-bromo-ethanone 9b, 9c,
or 9e (1.0 eq) was added portionwise and the mixture was stirred at room temperature for
2h. The reaction was then poured in H2O and the solid obtained was solubilised in EtOAc,
dried over Na2SO4 and the organic layer evaporated under reduced pressure to afford
derivatives 10b,[33] 10c, and 10e.
1-(4-methoxyphenyl)-2-(2-nitrophenoxy)ethanone (10b).[33] By using 2-bromo-1-(4-
methoxyphenyl)ethanone 9b (6.55 mmol, 1.50 g), compound 10b[33] was obtained as a
white solid (0.79 g, 42% yield).
1-(3,4-dimethoxyphenyl)-2-(2-nitrophenoxy)ethanone (10c). By using 2-bromo-1-(3,4-
dimethoxyphenyl)ethanone 9c[34] (3.86 mmol, 1.00 g), compound 10c was obtained as a
yellow solid (1.16 g, 95% yield).
2-(2-nitrophenoxy)-1-(4-propoxyphenyl)ethanone (10e). By using 2-bromo-1-(4-
propoxyphenyl)ethanone 9e[22] (3.89 mmol, 1.00 g), compound 10e was obtained as a
yellowish oil (1.07 g, 87% yield).
General procedure for the synthesis of compounds 1a[29] and 1b[30].
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To a solution of compound 10a[32] or 10b[30] in 1,4-dioxane (5.00 mL per mmol), Raney-Ni
(2.00 g per mmol) was added at 0 °C. Then H2 was bubbled into the mixture and stirred for
5h. The mixture was filtered over Celite® and the organic phase evaporated under
reduced pressure. The crude mixture was then purified to obtain compound 1a[29] and
1b.[30]
3-phenyl-2H-1,4-benzoxazine (1a).[29] Starting from 10a[32] (1.00 g, 3.89 mmol) compound
1a[29] was obtained as a white solid after purification by flash column chromatography
(100% benzene) (0.30 g, 57% yield).
3-(4-methoxyphenyl)-2H-1,4-benzoxazine (1b).[30] Starting from derivative 10b[33] (2.09
mmol, 0.60 g), compound 1b[30] was obtained after crystallization from Et2O as a white
solid (0.40 g, 80% yield).
General procedure for the synthesis of compounds 4a,[29] 4b,[31] 4c,[31] and 4e.
To a solution of compound 10a,[32] 10b,[33] 10c, or 10e in dioxane (8.00 mL per mmol),
Raney-Ni (2.00 g per mmol) was added at and then H2 was bubbled into the mixture and
stirred for 5h at 70° C. The mixture was filtered over Celite® and the organic phase
evaporated under reduced pressure. The crude mixture was then purified to obtain
compound 4a,[29] 4b,[31] 4c,[31] and 4e.
3-phenyl-3,4-dihydro-2H-1,4-benzoxazine (4a).[29] Starting from derivative 10a[32] (1.94
mmol, 0.50 g), compound 4a[29] was obtained after purification by flash column
chromatography (EP/EtOAc 99/1) as a colourless oil (0.39 g, 95% yield).
3-(4-methoxyphenyl)-3,4-dihydro-2H-1,4-benzoxazine (4b).[31] Starting from derivative
10b[33] (0.69 mmol, 0.20 g), compound 4b[31] was obtained after crystallization by Et2O as
a white solid (0.16 g, 94% yield).
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3-(3,4-dimethoxyphenyl)-3,4-dihydro-2H-1,4-benzoxazine
(4c).[31]
Starting
from
derivative 10c (1.58 mmol, 0.50 g), compound 4c[31] was obtained after purification by
column chromatography (cyclohexane/EtOAc 95/5→90/10) as a yellowish solid (0.18 g,
42% yield).
3-(4-propoxyphenyl)-3,4-dihydro-2H-1,4-benzoxazine (4e). Starting from derivative 10e
(2.22 mmol, 0.70 g), compound 4e was obtained after crystallization from Et2O as a
yellowish solid (0.47 g, 78% yield).
General procedure for the synthesis of compounds 1c,[31] 1d, and 1e.
In anhydrous condition, to a solution of 2-aminophenol 8 (1.0 eq) in dry DMF (2.00 mL per
mmol), NaH 60% (1.2 eq) was added. After stirring for 10 min the properly substituted 2-
bromo-ethanone (9c,[34] 9d,[35] or 9e[31]) dissolved in dry DMF (1.00 mL per mmol) was
added dropwise and the resulting solution was stirred at room temperature for 1h. The
reaction mixture was poured in cold water and the solid was filtrated to afford after
purification compounds 1c,[31] 1d, and 1e.
3-(3,4-dimethoxyphenyl)-2H-1,4-benzoxazine
(1c).[31]
By
using
2-bromo-1-
phenylethanone 9c[34] (1.36 g, 5.25 mmol), after purification by flash column
chromatography (benzene/EtOAc 95/5) compound 1c[31] was obtained as a yellow solid
(0.27 g, 19% yield).
3-(3,4,5-trimethoxyphenyl)-2H-1,4-benzoxazine
(1d).
By
using
2-bromo-1-
phenylethanone 9d[35] (6.92 mmol, 2.00 g), after purification by flash column
chromatography (benzene/EtOAc 95/5) compound 1d was obtained as a yellow solid (0.25
g, 12% yield).
3-(4-propoxyphenyl)-2H-1,4-benzoxazine (1e). By using 2-bromo-1-phenylethanone
9e[22] (4.55 mmol, 1.17 g), after purification by flash column chromatography
(benzene/EtOAc 95/5) compound 1e was obtained as a yellow solid (0.51 g, 48% yield).
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3-(3,4,5-trimethoxyphenyl)-3,4-dihydro-2H-1,4-benzoxazine
(4d).
In
anhydrous
conditions 1M BH3 ·THF (1.25 mmol, 1.25 mL) was maintained at 0 °C and a solution of
compound 1d (0.15 g, 0.50 mmol) in 20 mL of dry THF, was added dropwise. The reaction
mixture was stirred for 3 h at room temperature. The mixture was then added of about
10.00 mL of MeOH, poured in H2O, extracted with EtOAc and dried over Na2SO4. From
the evaporation of the organic phases and purification of the crude solid by flash column
chromatography (CH2Cl2/MeOH 99.5/0.5) compound 4d was obtained as yellow solid
(0.70 g, 47% yield).
Synthesis of the 2-phenyl-1,2,3,4-tetrahydroquinoxalines (2a,[36] 2b,[37] 2c-e) and the
2-phenylquinoxalines (5a,[38] 5b,[39] 5c,[40] 5d,[41] 5e).
General procedure for the synthesis of compounds 5a,[38] 5b,[39] 5c,[40] 5d,[41] and 5e.
To a stirring solution of benzene-1,2-diamine 7 (1.2 eq) in dry MeOH (5.00 mL per mmol,
anhydrous sodium acetate (1.2 eq) was added. Properly substituted 2-bromo-ethanone
(9a, 9b, 9c,[34] 9d[35] or 9e[22]) (1.0 eq) was then added dropwise and the mixture was
hence stirred at room temperature for 6 h. The solvent was removed under reduced
pressure and the reaction mixture poured in water, neutralised and extracted with CH2Cl2.
The organic layer was dried over Na2SO4 and evaporated under reduced pressure to
obtain, after purification, compounds 5a,[38] 5b,[39] 5c,[40] 5d[41] and 5e.
2-phenylquinoxaline (5a).[38] By using 2-bromo-1-phenylethanone 9a (10.05 mmol, 2.00
g), after purification by column chromatography (EP/EtOAc 95/5), compound 5a[38] was
obtained as a white solid (1.43 g, 69% yield).
2-(4-methoxyphenyl)quinoxaline (5b).[39] By using 2-bromo-1-phenylethanone 9b (9.26
mmol, 2.12 g), after purification by flash column chromatography (cyclohexane/EtOAc
90/10) compound 5b[39] was obtained as a yellow solid (1.03 g, 47% yield).
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2-(3,4-dimethoxyphenyl)quinoxaline (5c).[40] By using 2-bromo-1-phenylethanone 9c[34]
(1.16 mmol, 0.30 g), after purification by flash column chromatography
(cyclohexane/EtOAc 90/10) compound 5c[40] was obtained as a yellow solid (0.12 g, 39%
yield).
2-(3,4,5-trimethoxyphenyl)quinoxaline (5d).[41] By using 2-bromo-1-phenylethanone
9d[35] (3.45 mmol, 1.00), after purification by column chromatography (EP/EtOAc
95/5→90/10) compound 5d[41] was obtained as a yellowish solid (0.21 g, 20% yield).
2-(4-propoxyphenyl)quinoxaline (5e). By using 2-bromo-1-phenylethanone 9e[22] (3.89
mmol, 1.00 g), after purification by flash column chromatography (EP/EtOAc 60/40)
compound 5e was obtained as a yellowish solid (0.29 g, 28% yield).
General procedure for the synthesis of compounds 2a,[36] 2b,[37] 2c-e.
In anhydrous conditions, 1M BH3 ·THF (3.0 eq) was maintained at 0 °C and the properly
substituted 2-phenylquinoxaline 5a,[38] 5b,[39] 5c,[40] 5d[41] or 5e (1.0 eq), dissolved in dry
THF (10.00 mL per mmol), was added by dropping. The reaction mixture was then stirred
for 4 h at room temperature. Then, the mixture was quenched with MeOH and the crude
was poured in H2O, extracted with EtOAc and dried over Na2SO4. After evaporation of the
organic phases and purification compounds 2a,[36] 2b,[37] 2c-e were obtained.
2-phenyl-1,2,3,4-tetrahydroquinoxaline (2a).[36] Starting from derivative 5a[38] (1.50
mmol, 0.31 g), after purification by column chromatography (EP/EtOAc 95/5) compound
2a[36] was obtained as a pink solid (0.15 g, 46% yield).
2-(4-methoxyphenyl)-1,2,3,4-tetrahydroquinoxaline (2b).[37] Starting from derivative
5b[39] (0.42 mmol, 0.1 g), after purification by column chromatography (EP/EtOAc 90/10)
compound 2b[37] was obtained as a yellowish solid (0.71 g, 70% yield).
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2-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydroquinoxaline (2c). Starting from derivative
5c[40] (1.13 mmol, 0.30 g), after purification by column chromatography (EP/EtOAc
90/10→80/20) compound 2c was obtained as a yellow solid (0.11 g, 36% yield).
2-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydroquinoxaline (2d). Starting from derivative
5d[41] (0.67 mmol, 0.20 g), after purification by column chromatography (EP/EtOAc
90/10→80/20) compound 2d was obtained as a white solid (0.07 g, 34% yield).
2-(4-propoxyphenyl)-1,2,3,4-tetrahydroquinoxaline (2e). Starting from derivative 5e
(0.68 mmol, 0.18 g), after purification by flash column chromatography (EP/Et2O 95/5)
compound 2e was obtained as a white solid (0.03 g, 14% yield).
Synthesis of the 2-phenyl-1,2,3,4-tetrahydroquinolines (3a,[42] 3b,[43] 3c,[44] 3d,[48] 3e)
and the 2-phenylquinolines (6c,[47] 6d,[48] 6e).
General procedure for the synthesis of compounds 6c,[47] 6d[48] and 6e.
To a solution of properly substituted 1-phenylethanones 14c-d and 14e[22] (1.5 eq) in 1,4-
dioxane (2.50 mL per mmol), t-BuOK (2.5 eq) and 2-(aminophenyl)methanol 13[49] (1.0 eq)
were added and stirred at 80 °C for 4h. The solvent was removed and the reaction mixture
poured in water and extracted with EtOAc. The organic layer was dried over Na2SO4 and
evaporated under reduced pressure to obtain after purification compounds 6c,[47] 6d[48] and
6e.
2-(3,4-dimethoxyphenyl)quinoline (6c).[47] By using 1-(3,4-dimethoxyphenyl)ethanone
14c (6.09 mmol, 1.10 g), after purification by flash column chromatography
(cyclohexane/EtOAc 90/10) compound 6c[47] was obtained as a white solid (0.07 g, 7%
yield).
2-(3,4,5-trimethoxyphenyl)quinoline
(6d).[48]
By
using
1-(3,4,5-
trimethoxyphenyl)ethanone 14d (4.04 mmol, 0.85 g), after purification by flash column
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ChemMedChem
chromatography (cyclohexane/EtOAc 95/5) compound 6d[48] was obtained as a white solid
(0.42 g, 35% yield).
2-(4-propoxyphenyl)quinoline (6e). By using 1-(4propoxyphenyl)ethanone 14e[22] (3.65
mmol, 0.65 g), after purification by column chromatography (EP/Et2O 93/7) compound 6e
was obtained as a white solid (0.40 g, 62% yield).
General procedure for the synthesis of compounds 3a,[42]3b,[43] 3c[44] and 3e.
A stirring solution of properly substituted 2-phenyl-quinoline 6a,[46] 6b,[46] 6c[47] or 6e (1.0
eq) in propan-1-ol (8.20 mL per mmol) was heated at reflux. After 15 min, Na0 (20.0 eq)
was added and stirred for 3h. The solvent was removed and the reaction mixture poured in
water and extracted with EtOAc. The organic layer was dried over Na2SO4 and evaporated
under reduced pressure to obtain after purification compounds 3a,[42] 3b,[43] 3c[44] and 3e.
2-phenyl-1,2,3,4-tetrahydroquinoline (3a).[42] Starting from the compound 6a[46] (2.44
mmol, 0.50 g), after purification by column chromatography compound 3a[42] was obtained
as a colourless oil (0.35 g, 70% yield).
2-(4-methoxyphenyl)-1,2,3,4-tetrahydroquinoline (3b).[43] Starting from the compound
6b[46] (0.89 mmol, 0.21 g), after purification by column chromatography (EP/Et2O 95/5)
compound 3b[43] was obtained as a yellow solid (0.07 g, 33% yield).
2-(3,4-dimethoxyphenyl)-1,2,3,4-tetrahydroquinoline (3c).[44] Starting from the
compound 6c[47] (1.43 mmol, 0.38 g), after purification by column chromatography
(EP/EtOAc 95/5) compound 3c[44] was obtained as a yellow solid (0.03 g, 8% yield).
2-(4-propoxyphenyl)-1,2,3,4-tetrahydroquinoline (3e). Starting from the compound 6e
(0.38 mmol, 0.10 g), after purification by flash column chromatography (EP/Et2O 98/2)
compound 3e was obtained as a white solid (0.04 g, 40% yield).
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ChemMedChem
2-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydroquinoline (3d).[45] To a solution of
compound 6d[48] (0.14 g, 0.47 mmol) solubilised in 50.00 mL of EtOAc, Raney-Ni was
added at room temperature, and H2 was bubbled into the mixture and stirred for 1h. The
mixture was filtered over Celite® and the organic phase evaporated under reduced
pressure to obtain compound 3d[45] as a yellowish oil after purification by flash column
chromatography (EP/EtOAc 95/5) (0.15 g, 47% yield).
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Table of contents text
Contrasting bacterial resistance to antimicrobial agents by efflux pumps inhibition is a
promising strategy aimed to restore antibacterial MIC in resistant strains. In an attempt of
improving the benzothiazine class of Staphylococcus aureus NorA inhibitors, in terms of
efflux inhibition activity and chemical stability, the synthesis of six different sets of
derivatives was performed. The 2-phenylquinoline series provided the best derivatives
thereby suggesting this scaffold as the best one in order to further optimizations.
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