Regulation of the Escherichia coli AtoSC two component system by synthetic biologically active 5;7;8-trimethyl-1;4-benzoxazine analogues

Article abstract of DOI:10.1016/j.bmc.2011.06.029

The Escherichia coli AtoSC two component system;upon acetoacetate induction;regulates the expression of the atoDAEB operon;through His → Asp phopshotransfer;thus modulating important cellular processes. In this report the effect of seven 5,7,8-trimethyl-1

Full text of DOI:10.1016/j.bmc.2011.06.029

Bioorganic & Medicinal Chemistry 19 (2011) 5061–5070
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Regulation of the Escherichia coli AtoSC two component system
by synthetic biologically active 5;7;8-trimethyl-1;4-benzoxazine analogues
Panagiota S. Filippou a, , Eftychia N. Koini b, , Theodora Calogeropoulou b, Panagiota Kalliakmani a,
Christos A. Panagiotidis c, Dimitrios A. Kyriakidis a,
⇑
a Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
b National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, 48 Vas. Constantinou Ave., 11635 Athens, Greece
c Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 December 2010
Revised 1 June 2011
Accepted 8 June 2011
Available online 21 June 2011
The Escherichia coli AtoSC two component system;upon acetoacetate induction;regulates the expression
of the atoDAEB operon;through His ? Asp phopshotransfer;thus modulating important cellular pro-
cesses. In this report the effect of seven 5,7,8-trimethyl-1,4-benzoxazine derivatives on the regulation
of the E. coli AtoSC system was studied. The new compounds were tested for their effectiveness on the
expression of the atoC and the regulated atoDAEB operon. The non-substituted 5,7,8-trimethyl-1,4-ben-
zoxazine (4a), was the most potent inducer on atoC transcription;resulting in accumulation of AtoC pro-
tein. The induction of atoC by 4a was specific;since no effect was observed on the other genes of the
system (atoS and atoDAEB). Moreover;compound 4a was shown to significantly up-regulate the
in vitro kinase activity of the histidine kinase AtoS without altering the protein levels in the cell. Inter-
estingly;this compound appeared to modulate the acetoacetate-mediated induction of the atoDAEB pro-
moter by the AtoSC system. These data provide the first evidence for a differential modulator role of 5,7,8-
trimethyl-1,4-benzoxazine;on the AtoSC two component system mediated signaling.
Keywords:
AtoSC two component system
atoDAEB operon
Kinase activity
5,7,8-Trimethyl-1,4-benzoxazine
derivatives
Escherichia coli
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
mechanisms.10–12 Putrescine, the non-natural diamine 1,3-diami-
nopropane and their synthetic analogues elicited a pronounced atoC
AtoSC is a well characterized two component system (TCS) in
Escherichia coli, which relies on transmembrane sensor AtoS histi-
dine kinase (HK) autophosphorylation and subsequent AtoC re-
sponse regulator (RR) phosphorylation. AtoS was reported to be
in trans-phosphorylated between the monomers of a homodimer
in a conserved H-box containing the phosphorylation site His-
398 residue.1 His-398 is lying in proximity to the AtoC Asp-55 res-
idue upon AtoSC interaction for the subsequent phosphotransfer
and AtoC phosphorylation necessary for AtoSC regulatory actions,2
the transcriptional regulation of the atoDAEB operon that encode
for proteins involved in short-chain fatty acid metabolism.2–5 By
governing the atoDAEB operon;this system regulates;amongst oth-
ers;the biosynthesis and the intracellular distribution of cPHB,6,7
the flagella synthesis;8 the chemotactic behaviour and the involve-
ment of extracellular Ca2+ on cPHB synthesis regulated by the
AtoSC system.9
transcriptional activation, without activating neither the atoDAEB
operon promoter nor atoS.11 Histamine, a biogenic amine, elicited
a polyamine-mediated inductive phenotype and induced atoC
expression, without activating the atoDAEB operon promoter.12 Fur-
thermore, the involvement of TCSs including AtoSC, in specialized
functions including the bacterial pathogenicity, virulence and
host-microbe interactions3,4 warrant the investigation of a number
of synthetic amine-containing compounds, including 1,4-benzoxa-
zines, on their possible involvement on the signaling cascade.
The 2H-1,4-benzoxazine-3-(4H)-one and 3,4-dihydro-2H-1,4-
benzoxazine systems have been studied extensively for building
natural and designed biologically active compounds, which span
from herbicides, fungicides, cardiovascular agents, KATP channel
openers, compounds against diabetes, neuroprotectants and agents
against anxiety and depression.13–17 Thus, the above mentioned
heterocycles can be considered as privileged scaffolds for the
development of potential new drugs. We recently reported, the
synthesis of 5,7,8-trimethyl-1,4-benzoxazine derivatives encom-
passing the pharmacophore aminoamide functionality of lidocaine
and their biological evaluation as novel antiarrhythmics against
ischemia-reperfusion injury.18 Furthermore, the 5,7,8-trimethyl-
1,4-benzoxazine moiety can be considered as a bioisostere of the
5,7,8-trimethyl-1,4-benzopyran nucleus of the well known antiox-
idant vitamin E.
We have recently reported the regulation of the AtoSC system by
polyamines and histamine, as cellular components that enable path-
ogenic bacteria to survive and overcome host defence
⇑
Corresponding author. Fax: +30 2310997689.
E-mail address: kyr@chem.auth.gr (D.A. Kyriakidis).
PSF and ENK contributed equaly in this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.029

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P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
As a continuation of our studies on the effect of amines on the
Bromination of 2,3,5-trimethyl-6-nitrophenol with a mixture of
acetic acid/hydrogen peroxide/hydrogen bromide in petroleum
ether afforded 9, which upon cross-coupling reaction with 4-
methoxyphenyl boronic acid in the presence of Pd(PPh3)4 in DMF
at 100 °C gave 6-(4-methoxyphenyl)-2,3,5-trimethylnitrophenol
(10), which was alkylated with ethyl-2-bromoacetate in the pre-
sense of Cs2CO3 and a catalytic amount of TBAI to give ether 11,
which was hydrogenated to the corresponding aniline, which
spontaneously cyclized to benzoxazinone 12, which upon subse-
quent reduction with BH3.SMe2 in THF afforded the desired 6-
aryl-benzoxazine 13 (Scheme 4). Compound 4c was synthesized
as previously reported.18
AtoSC system and its implication in important biological path-
ways;we investigated the effect of a series of 5,7,8-trimethyl-1,4-
benzoxazine derivatives (Fig. 1) on the regulation of the E. coli
AtoSC system. We investigated their effect on: (i) the transcription
of atoSC system genes, (ii) the modulation of AtoS and AtoC endog-
enous protein levels, (iii) the alteration of activity of AtoS HK, (iv)
the dimerization/oligomerization ability of its cytoplasmic AtoS
form and (v) the effect on the acetoacetate-mediated atoDAEB
operon induction.
In order to obtain initial information on structure–activity rela-
tionships we chose to include in our study the unsubstituted 5,7,8-
trimethyl-1,4-benzoxazine (4a) as well as 2-, 3-, and 6-substituted
derivatives. More specifically, we introduced a 4-methoxyphenyl
substituent at positions C2, C3 and C6, compounds 4b, 8, and 13,
respectively. In addition, we incorporated a halogen substituent
at C6, compounds 6a, b as well as a phenyl group at C2 and a
chloro-substituent at C6, compound 4c. Compounds 4b, c, and 8
were obtained as racemic mixtures and were studied as such.
2.2. Effect of compounds 4a–c, 6a, b, 8, and 13 on the
transcription of the atoC and atoDAEB operon genes
Compounds 4a–c, 6a, b, 8, and 13 were initially tested for their
effect on the growth of E. coli strain BW25113 (atoSC+). Following a
longer lag period in the presence of the compounds, the bacteria
displayed similar doubling times during the logarithmic phase irre-
spective of the compound used (data not shown).
2. Results and discussion
2.1. Chemistry
Subsequently compounds 4a–c, 6a, b, 8, and 13 were tested for
their effect on atoC gene and the atoDAEB operon transcription. The
ability of the previously synthesized constructs11,21 carrying lacZ
fused to either of the promoters of the atoSC system (i.e., the atoC
and the atoDAEB promoter) to respond to the compounds of the
present study was evaluated in E. coli strain BW25113 (Fig. 2).
The compounds were added in the growth medium at a final con-
centration of 1 mM. To facilitate comparisons between different
sets of data, the b-galactosidase activity measured in the wild-type
strain BW25113 growing in the absence of the compounds, was
arbitrarily defined as 100%. The new compounds, demonstrated a
differential effect on gene expression and more specifically, com-
pounds 4a–c and 13 activated the transcription of the atoC, while
analogues 6a and 8 reduced it. Compounds 6b and 13 did not ex-
hibit any significant effect on atoC or atoDAEB, respectively
(Fig. 3a and b). Compounds 4a, 6a, and 6b induced a repression
of atoDAEB, while compounds 4b, 4c, and 8 stimulated this
promoter.
These data may provide an indication on the effect of the substi-
tution pattern on the 5,7,8-trimethyl-1,4-benzoxazine scaffold on
activity on both promoters. More specifically, the presence of a
4-methoxyphenyl substituent at position C2 or C6 (compounds
4b and 13, respectively) results in atoC activation which is also
the case for the C2 and C6 disubstituted derivative 4c. The presence
of a halogen group at C6 exerts opposite effects with the chloro
derivative 6a to repress atoC while, the bromo analogue 6b to in-
duce slight activation. Finally, the presence of a 4-methoxyphenyl
substituent at C3 (compound 8) induces a repression of atoC simi-
lar to compound 6a. Interestingly, the non-substituted derivative
4a results in pronounced stimulation of atoC.
The synthesis of the compounds of the present study 4a, b, 6a,
b, 8, and 13 is depicted in Schemes 1–4. Compounds 4a, 4c, and 6a
were prepared as previously reported.18
More specifically, 2,3,5-trimethyl-6-nitro-phenol (1)18 was
alkylated with the appropriate
a-bromoester in the presence of
Cs2CO3 and a catalytic amount of TBAI to give ethers 2a, b. Hydro-
genation of 2a gave the corresponding aniline which spontane-
ously cyclized to benzoxazinone 3a. Reduction of the nitro group
in compound 2b was effected using CuCl/NaBH4 in ethanol19 to
give benzoxazinone 3b after spontaneous cyclization. Subsequent
reduction of benzoxazinones 3a, b with BH3.SMe2 in THF afforded
the desired compounds 4a, b (Scheme 1). Treatment of benzoxaz-
inone 3a with a mixture of acetic acid/hydrogen peroxide/hydro-
gen chloride in petroleum ether afforded the 6-chloro-derivative
5a, while bromination with a mixture of acetic acid/hydrogen per-
oxide/hydrogen bromide in petroleum ether afforded the 6-bromo-
benzoxazinone 5b.20 Reduction of compound 5a with BF3.Et2O and
NaBH4 in THF, at 0–5 °C afforded the desired 6-chloro-benzoxazine
6a, while reduction with BH3.SMe2 in THF at rt afforded 6-bromo
benzoxazine 6b (Scheme 2). The synthesis of the 3-aryl-substituted
benzoxazine derivative 8 was effected through alkylation of
2,3,5-trimethyl-6-nitrophenol (1) with 2-bromo-40-methoxyaceto-
phenone in the presence of Cs2CO3 and TBAI in DMF to afford
compound 7, followed by reduction of the nitro group with CuCl,
NaBH4 and cyclization to the corresponding imine which was re-
duced further in situ to afford compound 8 (Scheme 3).
Concerning the atoDAEB promoter the effects were not as dras-
tic. The presence of a halogen substituent at C6 (compounds 6a and
b) induced repression of the atoDAEB, while the presence of a 4-
methoxyphenyl group at C2 or C3 or C6 (compounds 4b, 8, and
13, respectively) resulted in stimulation. Interestingly, compounds
4b, 4c, 6a, and 13 activate the transcription of both promoters,
while, compounds 4a, 6b, and 8 activate atoC and repress atoDAEB.
Overall, the most potent transcriptional activator of the atoC
gene was the non-substituted derivative 4a (Fig. 3a). The specific-
ity of this effect was demonstrated through the lack of activation
(with
a slight repression) of the atoDAEB operon promoter
(Fig. 3b). In the cases where both atoC and atoDAEB were activated
by the compounds (i.e., 4b and c), the activation of atoDAEB could
not be attributed to the enhanced intracellular accumulation of
AtoC, the product of atoC, but rather to their direct effects on
Figure 1. Structures of 5,7,8-trimethyl-1,4-benzoxazine derivatives.

P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
5063
H
N
H
N
O
d
O
O
b
3a
4a
NO2
COOR
NO2
OH
a
H
N
H
N
O
c
R1
R1=H, R=C2H5
O
d
2a
2b R1=4-CH3O-C6H4, R=CH3
1
O
O
OCH3
3b
OCH3
4b
Scheme 1. Reagents and conditions: (a) R1CH(Br)COOR (R = Me or Et), Cs2CO3, TBAI, DMF, rt; (b) H2, Pd/C, EtOH, 70 °C; (c) CuCl, NaBH4, EtOH, 85 °C; (d) BH3ꢀSMe2, THF, rt.
H
N
H
N
H
N
activation of transcription of the atoC gene in a concentration-
dependent manner (Fig. 4).
O
X
O
X
a
b
2.3. Effect of compound 4a on atoSC two component system
genes transcription
O
O
O
3a
5a X=Cl
5b X=Br
6a X=Cl
6b X=Br
The next issue we addressed was whether compound 4a affects
the transcription of other genes in a similar manner. Thus, we
investigated genes that share a topological and/or functional rele-
vance with atoC, that is, the neighboring atoS gene, encoding the
AtoS kinase and the regulated atoDAEB operon.
Scheme 2. Reagents and conditions: (a) CH3COOH-H2O2-HCl or HBr, petroleum
ether, 60 °C; (b) BF3ꢀEt2O, NaBH4, THF, 0–5 °C or BH3.SMe2, THF, rt.
Therefore, the same experiment was performed using E. coli
BW25113/pCPG6 (atoC-lacZ), BW25113/pCPG4 (atoS-lacZ) and
BW25113/pCPG5 (atoA-lacZ) cells grown in the presence of com-
pound 4a at 1 and 2.5 mM concentration, respectively. Taking into
consideration the ability of the construct pCPG6 (atoC-lacZ) to re-
spond to the natural precursor, putrescine, for atoC activation in
E. coli strain BW25113,11 putrescine was used as a positive control
of induction in this experiment (data not shown).
atoDAEB transcription. Accordingly, this is a possible explanation of
the fact that the potent activation of the atoC by 4a does not en-
hance but rather slightly repress the atoDAEB operon expression
(Fig. 3).
In an effort to investigate the concentration-depended activa-
tion of the atoC by 4a, we repeated the same experiment in cells
of E. coli BW25113 carrying plasmid pCPG6 (atoC-lacZ) in the pres-
ence of increasing concentrations of compound 4a (Fig. 4). The re-
sults of the b-galactosidase assay confirmed that 4a caused
As shown in Figure 5 compound 4a activated in a concentra-
tion-dependent manner the atoC gene, while neither of the other
OCH3
H
N
NO2
O
NO2
OH
OCH3
a
b
O
OCH3
O
1
Br
8
7
O
Scheme 3. Reagents and conditions: (a) Cs2CO3, TBAI, DMF, rt; (b) CuCl, NaBH4, EtOH, 85 °C.
H3CO
Br
NO2
OH
NO2
OH
NO2
OH
a
c
b
10
1
9
H3CO
H
H3CO
H
H3CO
N
N
O
NO2
d
e
O
O
OC2H5
O
O
12
13
11
Scheme 4. Reagents and conditions: (a) CH3COOH-H2O2-HBr;petroleum ether, rt; (b) 4-OCH3C6H5B(OH)2, Cs2CO3, Pd(PPh3)4, DMF, 100 °C; (c) BrCH2COOC2H5, Cs2CO3, TBAI,
DMF, rt; (d) H2, Pd/C, EtOH, 70 °C; (e) BH3ꢀSMe2, THF.

5064
P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
Figure 2. Structural organization of the atoSC and atoDAEB operon genes and constructs of plasmid pMLB1034 carrying various promoters fused to a promoterless lacZ gene.
pCPG4 (atoS-lacZ) represents the promoter of atoS and a part of the atoS gene, pCPG5 (atoA-lacZ) represents the promoter of the atoDAEB operon extending 460 bp upstream
the translational start of the atoD gene and pCPG6(atoC-lacZ) represents the promoter of atoC and a part of the atoC gene (⁄denotes part of the gene).
Figure 4. Induction of atoC mediated by compound 4a. The transcriptional
activation of the atoC was measured by assaying b-galactosidase expression in
BW25113/ pCPG6 (atoC-lacZ) cells growing in the presence of increasing concen-
trations of 4a (0, 0.25, 0.5, 1.0, 2.5 and 5 mM) (see Section 4). The results are
presented from two independent experiments, while in each experiment two clones
from each transformant were tested.
activation leads to AtoC protein accumulation in the cell, thus
affecting the translational mechanism. E. coli BW25113 cells trans-
formed with the multicopy plasmid pUC-Az, overexpressing the
AtoS and AtoC proteins22 were grown and compound 4a was added
Figure 3. Effect of 5,7,8-trimethyl-1,4-benzoxazine derivatives in the transcription
of atoC gene and the atoDAEB operon in T. coli strain BW25113. The E. coli K12 cells
BW25113 carrying plasmids (a) pCPG6 (atoC-lacZ) and (b) pCPG5 (atoA-lacZ) were
cultured in the presence of 1 mM of the indicated 5,7,8-trimethyl-1,4-benzoxazine
derivatives, respectively. The transcriptional activity of the constructs was mea-
sured by assaying b-galactosidase activity.
half at OD600 0.2 and the other half at OD600 0.4, respectively and
the growth was continued to OD600 0.9.
Surprisingly, under these culture conditions, cells exposed to
higher concentrations of 4a indicated a reduction on AtoC protein
levels (Fig. 6a). Similar observations were obtained for AtoS protein
levels, by immunoblot analysis of the same cell extracts with the
purified polyclonal anti-AtoS (Fig. 6b). The protein levels of AtoS
and AtoC under the specific cell culture conditions is irreversibly
proportional to the concentration of 4a added in the growth cul-
tures (Fig. 6).
two promoters (atoS and atoDAEB) was affected in a similar man-
ner. Specifically, the results of the b-galactosidase assay suggested
that 4a elicited a pronounced effect in the transcriptional activa-
tion of the atoC gene, whereas failed to induce atoS gene and
slightly suppressed the atoDAEB operon.
This result could be attributed to a broad effect of this com-
pound under the specific culture conditions causing stress/ heat
shock response and the subsequent protein degradation23,24 since
a concentration-dependent increase of the heat shock protein
DnaK was observed whereas the protein levels of the heat shock
Hsp60 remained unchanged (Fig. 6c and d). This result is also
2.4. Effect of compound 4a on the expression of AtoSC protein
levels
Since compound 4a activated the transcription of atoC in a con-
centration-dependent manner, it was investigated whether this

P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
5065
Figure 5. Effect of 5,7,8-trimethyl-1,4 benzoxazine (4a) on atoSC and the regulated
atoDAEB operon gene expression. The E. coli K12 strain BW25113 was transformed
with plasmids pCPG4(atoS-lacZ), pCPG6(atoC-lacZ) and pCPG5(atoA-lacZ). Induction
mediated by compound 4a was measured by assaying b-galactosidase expression in
the presence of the indicated concentrations of the compound (1.0 and 2.5 mM,
respectively).
indicative of the fact that upregulation of the Hsp proteins under
stress/heat shock-induced E. coli cells is critical for survival and
Hsp70 (DnaK) acts as
a
negative modulator for Hsp60
expression.25,26
Furthermore, the effect of 4a on the protein levels of the AtoC
was observed in cells growing in different culture conditions.
E. coli BW25113/pUC-Az cells were exposed to final concentra-
tions 1 and 2.5 mM of 4a at the beginning of the growth phase
and grown to an OD600 0.5–0.6. The protein levels of AtoC in the
cell extracts were monitored by immunoblotting using affinity-
purified rabbit polyclonal anti-AtoC antibody.2 As shown in Fig-
ure 7a, atoC transcriptional activation also produced increased
AtoC accumulation. This indicates that compound 4a under these
specific culture conditions does not enhance AtoC degradation.
On the other hand, AtoS protein levels remained constant with
a decrease at the higher concentration of the tested compound
(Fig. 7b).
Figure 6. (a, b) Effect of compound 4a on AtoSC protein levels. E. coli K12 cells
BW25113/pUC-Az were grown in the presence of various concentrations of 4a to a
cell density of 0.9 (see Section 4). The total cell extracts from each culture (15 lL)
were subjected to SDS–PAGE 10% w/v and immunostained with (a) anti-AtoC
(1:500) and (b) anti-AtoS (1:1000). BW28878 cells, not expressing the AtoC and
AtoS proteins, were used as negative control. (c, d) Effect of compound 4a on
endogenous DnaK and Hsp60 expression. E. coli K12 cells BW25113/ pUC-Az were
grown in the presence of the indicated increasing concentrations of compound 4a to
an OD600 0.9. Western blot of total cell extracts (15 lL) was performed to detect the
expression of the heat shock proteins: (c) DnaK (1:2000) and (d) Hsp60 (1:1000).
2.5. Effect of the 5,7,8-trimethyl-1,4-benzoxazine derivatives on
AtoS kinase activity
It is conceivable that the possible effect of the 5,7,8-trimethyl-
1,4-benzoxazine derivatives on the biological output of the AtoS–
AtoC two-component system (i.e., the activation of the atoDAEB op-
eron expression) could be due to their influence on AtoS kinase
activity. To test whether these compounds could indeed influence
the autophosphorylation of AtoS, the in vitro autophosphorylation
assay was carried out with the purified cytosolic protein, cytoAtoS
(see Section 4). Equal concentration (7 lV) of cytoAtoS autophos-
phorylated in the absence or presence of each of the 5,7,8-tri-
methyl-1,4-benzoxazine derivatives of the present study at a
final concentration of 1 mM.
Interestingly, of all the compounds tested, 4a, resulted in an
enhancement of the in vitro autophosphorylation activity of the
truncated cytosolic protein of AtoS (cytoAtoS), whereas the other
5,7,8-trimethyl-1,4-benzoxazine derivatives 4b, c, 6a, b, 8, and
13 had no/ or slight effect on the induction of AtoS autophosphory-
lation (Fig. 8a).
Figure 7. Effect of compound 4a on AtoSC protein expression. The E. coli K12 cells
BW25113/pUC-Az were cultured in the absence or presence of 1.0 and 2.5 mM of
compound 4a. Total cell extracts (15 lL) of each culture grown to an OD600 0.5–0.6
were analyzed. Immunoblot analysis was performed using the purified polyclonal
rabbit antibody of (a) AtoC and (b) AtoS respectively. BW28878 cells, not expressing
the AtoS and AtoC proteins, were used as negative control.

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P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
Figure 8. Effect of the 5,7,8-trimethyl-1,4-benzoxazine derivatives on AtoS autoph-
oshorylation activity. (a) Autophosphorylation activity of AtoS kinase was measured
when purified cytoAtoS (7 lV) was incubated with [c-
32P]-ATP for 30 min at room
temperature, in the absence or presence of the indicated 5,7,8-trimethyl-1,4-
benzoxazine derivative at the final concentration of 1 mM or (b) purified cytoAtoS
(4
absence or presence of 0.1, 0.2, 0.5 and 1.0 mM compound 4a, respectively. Prior
the addition of [
32P]-ATP each protein sample was preincubated for 15 min with
the appropriate compound. The reactions were stopped by the addition of SDS
loading buffer followed by separation on SDS–PAGE 12% w/v and autoradiography.
lV) was incubated with [c-
32P]-ATP for 30 min at room temperature, in the
Figure 9. Chemical cross-linking of purified cytoAtoS in the presence of compound
4a. Purified cytoAtoS (2 g) was cross-linked with glutaraldehyde 0.1% v/v for
30 min at room temperature in the absence or presence of 0.5 and 1 mM 4a,
respectively (see Section 4). Each sample (25 L) was withdrawn at the time
indicated, subjected to SDS–PAGE 8.5% w/v and immunostained with anti-His
(1:500). Lane 1: cytoAtoS with no glutaraldehyde added, 2: cytoAtoS treated with
glutaraldehyde, 3 and 4: cytoAtoS treated with glutaraldehyde in the presence of
0.5 and 1 mM 4a, respectively.
l
c
-
l
2.6. Compound 4a affects AtoS kinase activity
Since it was demonstrated that the AtoS protein levels remained
constant in cells exposed to 4a at the final concentration of 1 mM
(Fig. 7b), we next examined the involvement of this compound on
the activation of AtoS kinase activity.
In order to test whether 4a could induce the autophosphoryla-
tion of AtoS sensor histidine kinase, the in vitro autophosphoryla-
tion assay was carried out with the purified cytosolic protein,
the biological output of the AtoSC system, that is, the acetoacetate
dependent activation of the atoDAEB operon expression. To test
this possibility the effect of acetoacetate on the activity of the ato-
DAEB promoter was measured in the presence of compound 4a.
Thus, E. coli BW25113 cells carrying plasmid pCPG5 (atoA-lacZ)
were grown in the presence of 2.5 or 10 mM acetoacetate and their
effects on acetoacetate-dependent induction of atoDAEB promoter
were compared for various concentrations of compound 4a. Aceto-
acetate (10 mM) strongly induced atoDAEB promoter activity in
cells of atoSC+ genetic background. In contrary, acetoacetate
cytoAtoS (4 lV), in the presence of increasing concentrations of
compound 4a. As shown in Figure 8b, the presence of increasing
concentrations of the compound 4a in the reaction mixture stimu-
lated the enzyme autophosphorylation activity. AtoS kinase activa-
tion by compound 4a occurs at higher concentrations of the tested
compound (Fig. 8b).
2.7. Determination of the effect of compound 4a on AtoS
dimerization/oligomerization
Since the mechanism of AtoS activation suggests that AtoS mol-
ecules function as dimers1 we examined if the kinase dimerization
is affected by compound 4a.
The ability of AtoS to form dimers/oligomers in vitro in the ab-
sence or presence of 4a was determined by glutaraldehyde cross-
linking of its recombinant, purified cytosolic domain (cytoAtoS).
Western blot analysis of the products of such cross-linking exper-
iments indicated that the presence of 4a at the final concentra-
tion of 0.5 mM in the reaction mixture does not enhance
cytoAtoS dimerization. Conversely, the dimeric form of AtoS ap-
peared to slightly decrease when 4a was added at the concentra-
tion of 1 mM, suggesting that the AtoS oligomers (probably
remaining in the stacking gel) may be stimulated by this com-
pound (Fig. 9). The possibility that 4a may influence AtoS ATP
binding ability, thus leading to the kinase activation remains to
be elucidated.
Figure 10. Effect of compound 4a on acetoacetate-activation of the atoDAEB
operon. Effect of the simultaneous presence of acetoacetate and 4a on the activation
of the atoDAEB promoter compared to the acetoacetate effect in BW25113 cells. The
activity of the atoDAEB-lacZ construct (pCPG5) was measured in the presence of
increasing concentrations of compound 4a (0.1, 0.5, 1.0, 2.5 mM) in E. coli BW25113
cells. The cells were grown in a modified M9 mineral medium in the absence or
presence of 2.5 or 10 mM acetoacetate respectively and the b-galactosidase activity
was measured.
2.8. Compound 4a enhances the acetoacetate effect on atoDAEB
induction
The effects of compound 4a on atoC transcription and AtoS
in vitro kinase activity raised the question if they could influence

P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
5067
(2.5 mM) induced to a lesser extent the atoDAEB operon (Fig. 10).
4. Experimental
Compound 4a added at increasing concentrations in the cultures
produced further activation of the atoDAEB expression for both
acetoacetate concentrations (2.5 and 10 mM, respectively).
More specifically, cells growing in the presence of both acetoac-
etate 10 mM and compound 4a demonstrated the most potent ef-
fect on atoDAEB promoter activation (Fig. 10). This result indicates
the cooperative effect of 4a and acetoacetate-mediated TCS induc-
tion, in contrast to the competition between acetoacetate and sper-
midine-mediated TCS induction occurred previously.7
The enhancement of acetoacetate-mediated TCS induction was
not due to alterations on either AtoS (Fig. 7b) or AtoC accumulation
protein levels (Fig. 3b, and 7a), but rather to AtoS kinase activity
enhancement, or even to an increase of the ability of the phosphor-
ylated AtoC to bind to the atoDAEB operon promoter in the pres-
ence of this compound. The compound itself on the other hand
does not promote the activation of atoDAEB transcription, indicat-
ing that neither 4a nor one of its metabolic products in the cell may
influence AtoSC in the absence of the natural inducer, acetoacetate
(Fig. 3b). This effect might be due to the opposite activities of his-
tidine kinases that can phosphorylate or dephosphorylate their re-
sponse regulators.27 Therefore, compound 4a in the absence of the
acetoacetate-binding may influence the phosphatase activity of the
AtoS leading to AtoC dephosphorylation that slightly suppresses
the atoDAEB operon expression (Fig. 3b).
In general, acetoacetate-binding further promotes AtoS auto-
phosphorylation in the cell without affecting the AtoS dimeriza-
tion/oligomerization state.1 As stated, ligand binding alters the
conformation of the histidine kinase thus leading to the ‘on kinase
active state’.28 It is therefore possible the kinase domain of AtoS to
be ‘switched on’ and 4a binding to the kinase domain may induce
conformational changes in order to facilitate ATP binding, thus
activating AtoS, leading to further atoDAEB activation. After all,
4a was indeed shown to be an activator of AtoS enzymatic activity
in vitro with the enhancement of the autophosphorylation activity
of the truncated protein of AtoS (Fig. 8) that is even in the absence
of the natural inducer in the ‘active kinase state’, thus similarly
leading to 4a binding.
Taken together, the above data provide evidence that multiple
regulatory interactions may occur between the recognized signal
by the AtoS histidine kinase and its cognate AtoC response regulator
phosphorylation site and the mechanisms underlying the enhance-
ment of acetoacetate-mediated induction by compound 4a remain
to be elucidated. Overall, the prominent role of the 5,7,8-tri-
methyl-1,4-benzoxazine (4a) in the bacterial microenvironment
may provide the lead for the elucidation of unresolved implications
of AtoSC in pathophysiology and the development of novel thera-
peutic drugs possibly through cross-regulation with other systems.
4.1. Materials and methods
NMR spectra were recorded on a Bruker AC 300 and a Varian
600 MHz spectrometer operating at 300 MHz for 1H and
75.43 MHz for 13C, and at 600 MHz for 1H, respectively. 1H NMR
spectra are reported in units of d relative to the internal standard
of signals of the remaining protons of deuterated chloroform, at
7.26 ppm. 13C NMR shifts are expressed in units of d relative to
CDCl3 at 77.0 ppm. 13C NMR spectra were proton noise decoupled.
All NMR spectra were recorded in CDCl3. Silica gel plates Mache-
rey-Nagel Sil G-25 UV254 were used for thin layer chromatography.
Chromatographic purification was performed with silica gel (200–
400 mesh). Mass spectra were recorded on a Varian Saturn 2000
GC–MS instrument in the EI mode. High resolution mass spectra
were obtained by the Department of Chemistry and Biochemistry,
University of Notre Dame, USA
4.1.1. (4-Methoxy-2,3,5-trimethyl-6-nitro-phenoxy)-phenyl-
acetic acid methyl ester (2b)
To a solution of 2-nitro-3,5,6-trimethylphenol (1) (0.182 g,
1 mmol) in dry DMF (3.33 mL) was added cesium carbonate
(0.358 g, 1.1 mmol), TBAI (catalytic amount) and methyl 2-bro-
mo-2-(4-methoxyphenyl) acetate (0.285 g, 1.1 mmol) under nitro-
gen. The mixture was stirred for 5 min at room temperature and
the mixture was diluted with water and was extracted with ethyl
acetate. The organic phase was washed with brine and was dried
over anhydrous Na2SO4. The solvent was evaporated in vacuo
and the crude product was purified by flash column chromatogra-
phy using (cyclohexane/acetone 9:1) as eluting solvent to afford 4-
methoxy-2,3,5-trimethyl-6-nitro-phenoxy)-phenyl-acetic
acid
methyl ester, as a viscous oil in 95% yield (341 mg). Rf 0.38 (petro-
leum ether 40–60 °C/acetone 4:1); 1H NMR (300 MHz, CDCl3): d
7.34 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.79 (s, 1H), 5.26
(s, 1H), 3.74 (s, 3H, –OCH3), 3.67 (s, 3H, –OCH3), 2.17 and 2.16 (s,
6H, ArCH3), 1.94 (s, 3H, ArCH3). 13C NMR (75.5 MHz, CDCl3): d
170.0, 160.7, 147.6, 144.6, 141.1, 129.7, 129.4, 128.2, 127.9,
127.5, 114.3, 84.4, 55.4, 52.9, 20.4, 17.2, 13.5.
4.1.2. 2-(4-Methoxyphenyl)-5,7,8-trimethyl-2H-1,4-benzoxazin-
3(4H)-one (3b)
To a solution of 4-methoxy-2,3,5-trimethyl-6-nitro-phenoxy)-
phenyl-acetic acid methyl ester (0.359 g, 1 mmol) in absolute eth-
anol (5 mL) at 0 °C was added CuCl (0.495 g, 5 mmol) followed by
NaBH4 (0.378 g, 10 mmol) over 5 min. The resulting mixture was
refluxed for 20 min to 3 h and the reaction was cooled to room
temperature and was diluted with CH2Cl2. The mixture was filtered
through celite and the filtrate was evaporated in vacuo. The crude
product was purified by flash column chromatography (petroleum
ether 40–60 °C/ethyl acetate 9:1). Yield: 252 mg (85%); white so-
lid; mp 176–177 °C; Rf 0.31 (petroleum ether 40–60 °C/acetone
4:1); 1H NMR (300 MHz, CDCl3): d 8.75 (s, 1H, –NHCO–), 7.34 (d,
J = 8.5 Hz, 2H, ArH), 6.85 (d, J = 8.5 Hz, 2H, ArH), 6.60 (s, 1H, ArH),
5.64 (s, 1H, CH), 3.77 (s, 3H, OCH3), 2.19 (s, 3H, ArCH3), 2.18 (s,
3H, ArCH3), 2.15 (s, 3H, ArCH3); 13C NMR (75.5 MHz, CDCl3): d
166.3, 159.8, 140.8, 132.3, 128.1, 127.4, 125.0, 122.7, 122.2,
120.5, 113.9, 77.8, 55.2, 19.4, 16.1, 11.6; MS (EI), m/z 297 (M+,
100); HRMS (ESI+) calcd for C18H20O3N [M+H]+ 298.1438, found
298.1438.
3. Conclusions
A series of seven 5,7,8-trimethyl-1,4-benzoxazine derivatives
have been tested for their effectiveness on the expression of the
atoC, as well as that of the atoDAEB (ato) operon. 5,7,8-Trimethyl-
1,4-benzoxazine (4a), activated atoC transcription in a specific
manner since none of the other tested genes of the system (atoS
and atoDAEB) showed similar effect. In addition, compound 4a re-
sulted in accumulation of the AtoC protein and was also shown to
significantly up-regulate the in vitro kinase activity of the histidine
kinase AtoS without altering the protein levels in the cell. Conse-
quently, compound 4a appeared to modulate the acetoacetate-
mediated atoDAEB operon induction indicating a modulator role
of 5,7,8-trimethyl-1,4-benzoxazine on AtoSC two component sys-
tem-mediated signaling.
4.1.3. 2-(4-Methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-
1,4-benzoxazine (4b)
To a solution of 2-(4-methoxyphenyl)-5,7,8-trimethyl-2H-1,4-
benzoxazin-3(4H)-one (3b) (0.297 g, 1 mmol) in THF (50 mL) was

5068
P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
added dropwise at 0 °C a solution of borane. dimethylsulfide
(24 mmol, 2.28 mL). The reaction mixture was stirred at room tem-
perature until completion. The reaction was stopped by dropwise
addition of water at 0 °C until gas evolution has ceased. The result-
ing mixture was extracted with ethyl acetate. The organic layer
was extracted with brine and was dried over anhydrous Na2SO4.
The solvent was evaporated in vacuo and the residue was purified
by flash column chromatography using (cyclohexane/acetone 9:1)
as elution solvent to afford 2-(4-methoxyphenyl)-5,7,8-trimethyl-
3,4-dihydro-2H-1,4-benzoxazine (4b) in 89% yield (251 mg) as a
white solid. mp 99–100 °C; 1H NMR (300 MHz, CDCl3): d 7.46 (d,
J = 8.5 Hz, 2H, Ar H), 7.04 (d, J = 8.5 Hz, 2H, ArH), 6.66 (s, 1H,
ArH), 5.10 (dd, J = 2.4 Hz, J = 8.9 Hz, 1H, CH), 3.92 (s, 3H, OCH3),
3.61 (dd, J = 2.4 Hz, J = 11.8 Hz, 1H, CH2), 3.38 (dd, J = 8.9 Hz,
J = 11.8 Hz, 1H, CH2), 2.31 (s, 3H, ArCH3), 2.27 (s, 3H, ArCH3), 2.22
(s, 3H, ArCH3); 13C NMR (75.5 MHz, CDCl3): d 159.5, 142.7, 132.0,
128.6, 127.5, 126.7, 123.4, 122.4, 120.4, 114.0, 75.3, 55.4, 48.2,
19.4, 16.7, 11.6; HRMS (FAB+) calcd for C18H21O2N [M]+ 283.1572,
found 283.1551.
mixture was stirred for 5 min at room temperature and the mix-
ture was diluted with water and was extracted with ethyl acetate.
The organic phase was washed with brine and was dried over
anhydrous Na2SO4. The solvent was evaporated in vacuo and the
crude product was purified by flash column chromatography
(petroleum ether 40–60 °C/ethyl acetate 9:1) to afford 1-(4-
methoxyphenyl)-2-(2,3,5-trimethyl-6-nitrophenoxy)ethanone (7)
in 98% yield (322 mg) as a pale yellow sticky solid. Rf 0.44 (petro-
leum ether 40–60 °C/ethyl acetate 4:1); 1H NMR (300 MHz, CDCl3):
d 7.80 (d, J = 8.9 Hz, 2H, ArH), 6.84 (d, J = 8.9 Hz, 2H, ArH), 6.79 (s,
1H, ArH), 5.08 (s, 2H, CH2), 3.76 (s, 3H, OCH3), 2.17 (s, 3H, ArCH3),
2.15 (s, 3H, ArCH3), 2.10 (s, 3H, ArCH3); 13C NMR (75.5 MHz,
CDCl3): d 191.5, 164.2, 148.4, 144.7, 141.4, 130.4, 129.2, 128.2,
127.9, 127.4, 114.1, 76.8, 55.7, 20.3, 17.1, 12.5; MS (EI), m/z: 283
(38, M+-NO2), 135 (100); HRMS (ESI+) calcd for C18H20O5N
[M+H]+ 330.1336, found 330.1337.
4.1.7. 3-(4-Methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-
benz]oxazine (8)
To a solution of 1-(4-methoxyphenyl)-2-(2,3,5-trimethyl-6-
nitrophenoxy) ethanone (7) (0.329 g, 1 mmol) in absolute ethanol
(5 mL) at 0 °C was added CuCl (0.495 g, 5 mmol) followed by
NaBH4 (0.378 g, 10 mmol) over 5 min. The resulting mixture was
refluxed for 20 min and the reaction was cooled to room tempera-
ture and was diluted with CH2Cl2. The mixture was filtered
through celite and the filtrate was evaporated in vacuo. The crude
product was purified by flash column chromatography (petroleum
ether 40–60 °C/ethyl acetate 4:1) to afford compound 8. Yield:
260 mg (92%); pale yellow solid; mp 90 °C; Rf 0.41 (petroleum
ether 40–60 °C/ethyl acetate 3:2); 1H NMR (300 MHz, CDCl3): d
7.33 (d, J = 8.5 Hz, 2H, ArH), 6.88 (d, J = 8.5 Hz, 1H, ArH), 6.67 (s,
1H, ArH), 5.01 (dd, J = 8.7 Hz, J = 2.8 Hz, 1H, CH), 4.03 (dd,
J = 10.4 Hz, J = 2.9 Hz, 1H, CH2), 3.86–3.80 (m, 4H, CH2 and OCH3),
3.63 (br s, 1H. NH), 2.15 (s, 3H, ArCH3), 2.14 (s, 6H, ArCH3); 13C
NMR (75.5 MHz, CDCl3): d 159.5, 145.1, 135.6, 132.7, 127.8,
127.3, 127.2, 126.9, 121.1, 114.1, 78.1, 73.1, 55.5, 19.5, 17.2, 12.8;
HRMS (FAB+) calcd for C18H22O2N [M+H]+ 284.1651, found
284.1627.
4.1.4. 6-Bromo-5,7,8-trimethyl-2H-1,4-benzoxazin-3(4H)-one
(5b)
To a solution of 5,7,8-trimethyl-2H-1,4-benzoxazin-3(4H)-one
(3b) (0.191 g, 1 mmol) in petroleum ether 40–60 °C (10 mL) was
added a mixture of CH3COOH/H2O2 (30%) /HBr (48%) (6.5 mmol:
6.5 mmol:2.5 mmol) and the resulting mixture was stirred at room
temperature for 3 h. The reaction mixture was diluted with ethyl
acetate and the organic layer was washed with brine, was dried
over anhydrous Na2SO4, and the solvent was evaporated in vacuo.
The residue was purified by flash column chromatography (petro-
leum ether 40–60 °C/ethyl acetate 85:15) to afford compound 5b
as a white solid. Yield: 202 mg (75%); mp 234–237 °C; Rf 0.24
(petroleum ether 40–60 °C/ethyl acetate 4:1); 1H NMR (600 MHz,
CDCl3): d 7.54 (br s, 1H, NHCO), 4.56 (s, 2H, CH2), 2.37 (s, 3H,
ArCH3), 2.34 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3); 13C NMR
(75.5 MHz, CDCl3): d 165.9, 141.1, 132.3, 124.0, 123.1, 121.2,
120.9, 67.0, 20.3, 17.2, 12.9; MS(EI), m/z 269 [M+,91], 271 ([M
+2]+,100); HRMS (ESI+) calcd for C11H13O2NBr [M+H]+ 270.0124,
found 270.0121.
4.1.8. 4-Bromo-2,3,5-trimethyl-6-nitrophenol (9)
4.1.5. 6-Bromo-3,4-dihydro-5,7,8-trimethyl-2H-1,4-benzoxaz
ine (6b)
To a solution of 2,3,5-trimethyl-6-nitrophenol (1) (0.181 g,
1 mmol) in petroleum ether 40–60 °C (10 mL) was added a mixture
of CH3COOH/H2O2 (30%) /HBr (48%) (6.5 mmol: 6.5 mmol:2.5 m-
mol) and the resulting mixture was stirred at room temperature
for 3 h. The reaction mixture was diluted with ethyl acetate and
the organic layer was washed with brine, was dried over anhy-
drous Na2SO4, and the solvent was evaporated in vacuo. The resi-
due was purified by flash column chromatography (petroleum
ether 40–60 °C/ethyl acetate 95:5) to afford compound 9 as a yel-
low solid. Yield: 166 mg (64%); mp 156–157 °C; Rf 0.38 (petroleum
ether 40–60 °C/ethyl acetate 95:5); 1H NMR (300 MHz, CDCl3): d
9.64 (s, 1H, OH), 2.64 (s, 3H, ArCH3), 2.50 (s, 3H, ArCH3), 2.32 (s,
3H, ArCH3); 13C NMR (75.5 MHz, CDCl3): d 150.4, 144.7, 135.5,
131.6, 125.4, 120.6, 22.0, 21.9, 13.3; MS(EI), m/z 259 [M+,74], 261
([M +2]+,68); HRMS (ESI-) calcd for C9H9O3Br [M-1]- 257.9771,
found 257.9779.
To a solution of 6-bromo-5,7,8-trimethyl-2H-1,4-benzoxazin-
3(4H)-one (5b) (0.270 g, 1 mmol) in THF (20 mL) at 0 °C was added
dropwise borane dimethylsulfide (24 mmol, 2.28 mL). The reaction
mixture was stirred at room temperature until completion. The
reaction was stopped by dropwise addition of water at 0 °C until
gas evolution has ceased. The organic layer was extracted with sat-
urated aqueous NaHCO3 and brine and was dried over anhydrous
Na2SO4. The solvent was evaporated in vacuo and the residue
was purified by flash column chromatography (hexane/acetone
9:1). Yield: 250 mg (98%); white solid; mp 99–100 °C; Rf 0.24
(petroleum ether 40–60 °C/acetone 4:1); 1H NMR (300 MHz,
CDCl3): d 4.20 (t, J = 4.2 Hz, 2H, CH2), 3.42 (br s, 2H, CH2), 3.30
(br s, 1H, NH), 2.32 (s, 3H, ArCH3), 2.24 (s, 3H, ArCH3), 2.15 (s,
3H, ArCH3); 13C NMR (75.5 MHz, CDCl3): d 141.1, 129.9, 126.2,
123.4, 120.1, 119.5, 64.8, 41.3, 20.0, 17.2, 12.7; MS (EI), m/z 255
[M+, 100], 257 ([M+2]+, 100); HRMS (FAB+) calcd for C11H14ONBr
[M]+ 255.0259, found 255.0255.
4.1.9. 40-Methoxy-2,3,6-trimethyl-5-nitro[1.10-biphenyl]-4-ol
(10)
A mixture of tetrakis(triphenylphosphine)palladium (0.115 g,
0.1 mmol), 4-methoxyphenyl boronic acid (0.167 g, 1.1 mmol), 4-
bromo-2,3,5-trimethyl-6-nitrophenol (9) (0.260 g, 1.0 mmol), and
Cs2CO3 (0.489 g, 1.5 mmol) in THF (5 mL) was degassed and then
was heated under reflux overnight. After cooling to room temper-
ature, the reaction mixture was partitioned between water and
diethyl ether. The organic extracts were washed with brine and
4.1.6. 1-(4-Methoxyphenyl)-2-(2,3,5-trimethyl-6-nitrophenoxy)
ethanone (7)
To a solution of 2-nitro-3,5,6-trimethylphenol (1) (0.182 g,
1 mmol) in dry DMF (3.33 mL) was added cesium carbonate
(0.358 g, 1.1 mmol), TBAI (catalytic amount) and 2-bromo-40-
methoxyacetophenone (0.252 g, 1.1 mmol) under nitrogen. The

P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
5069
dried over anhydrous Na2SO4, the solvent was evaporated and the
residue was purified by flash column chromatography (petroleum
ether 40–60 °C/diethyl ether/dichloromethame 97:2:1) to afford
the desired product 10 in 75% yield (215 mg) as a yellow sticky so-
lid; Rf = 0.30 (petroleum ether 40–60 °C/diethyl ether 95:5); 1H
NMR d 10.16 (s, 1H,JY), 6.97 (s, 4H, ArH), 3.86 (s, 3H, OCH3),
2.26 (s, 3H, ArCH3), 2.17 (s, 3H, ArCH3), 1.97 (s, 3H, ArCH3); 13C
NMR (75.5 MHz, CDCl3): d 158.9, 151.4, 144.7, 135.7, 132.6,
131.1, 130.9, 124.2, 114.4, 55.5, 19.5, 19.3, 12.6; MS (EI), m/z:
methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine
(13) in 84% yield (237 mg) as a white solid. mp 127–129 °C; Rf 0.37
(petroleum ether 40–60 °C/ethyl acetate 4:1); 1H NMR (300 MHz,
CDCl3): d 7.03 (d, J = 8.4 Hz, 2H, ArH), 6.94 (d, J = 8.4 Hz, 2H, ArH),
4.30 (t, J = 4.3 Hz, 2H, CH2), 3.85 (s, 3H, OCH3), 3.49 (br s, 2H,
CH2), 2.16 (s, 3H, ArCH3), 1.88 (s, 3H, ArCH3), 1.79 (s, 3H, ArCH3);
13C NMR (75.5 MHz, CDCl3): d 158.0, 141.1, 134.5, 134.1, 130.9,
128.7, 125.4, 121.8, 119.3, 113.5, 65.0, 55.2, 41.5, 17.1, 14.2, 11.8;
MS (EI), m/z: 283 (100, V+); HRMS (FAB+) calcd for C18H21O2N
[M]+ 283.1572, found 283.1562.
287
[
M+, 100]; HRMS (ESI-) calcd for C16H16O4N [M–1]ꢁ
286.1085, found 286.1085.
4.2. Biological assays
4.1.10. Ethyl 2-[(40-methoxy-2,3,6-trimethyl-5-nitro[1,10-biphe
nyl]-4-yl)oxy]acetate (11)
4.2.1. Bacterial strains and plasmids
To a solution of 40-methoxy-2,3,6-trimethyl-5-nitro[1,10-biphe-
nyl]-4-ol (10) (0.287 g, 1 mmol) in dry DMF (3.33 mL) was added
cesium carbonate (0.358 g, 1.1 mmol), TBAI (catalytic amount)
and ethyl bromoacetate (0.184 g, 1.1 mmol) under nitrogen. The
mixture was stirred for 5 min at room temperature and the mix-
ture was diluted with water and was extracted with ethyl acetate.
The organic phase was washed with brine and was dried over
anhydrous Na2SO4. The solvent was evaporated in vacuo and the
crude product was purified by flash column chromatography
(cyclohexane/diethyl ether 9:1) to afford ethyl 2-[(40-methoxy-
2,3,6-trimethyl-5-nitro[1,10-biphenyl]-4-yl)oxy]acetate (11) in
98% yield (346 mg) as a pale yellow oil; Rf = 0.52 (petroleum ether
40–60 °C/ethyl acetate 4:1); 1H NMR (300 MHz, CDCl3): d 6.98 (br
s, 4H, ArH), 4.54 (s, 2H, CH2), 4.31 (m, 2H, CH2CY3), 3.86 (s, 3H,
OCH3), 2.27 (s, 3H, ArCH3), 1.97 (s, 3H, ArCH3), 1.91 (s, 3H, ArCH3),
1.34)m, 3H, CH2CY3); 13C NMR (75.5 MHz, CDCl3): d 168.1, 158.8,
146.3, 144.8, 139.7, 139.2, 131.7, 130.1, 128.7, 126.5, 114.1, 71.5,
61.4, 55.2, 18.2, 15.7, 14.1, 13.0; MS (EI), m/z: 373 [ M+, 25], 327
([ MꢁNO2]+, 78).
E. coli strain BW25113 [lacIqrrnB3 dlacZ4787 hsdR514 DE(ara-
BAD)567 DE(rhaBAD)568 rph-1] (a gift from Dr. Hirofumi Aiba, Na-
goya University, Japan) were transformed with recombinant
plasmids.
The plasmids were carrying various promoters of genes, fused
to a promoterless lacZ gene on pMLB1034 vector, that is, pCPG4
(atoS-lacZ), pCPG5 (atoA-lacZ) and pCPG6 (atoC-lacZ), carrying the
atoS promoter, the atoDAEB promoter and atoC promoter, respec-
tively (Fig. 2). These constructs, carrying the promoter of the ato-
DAEB operon and the regulatory atoS–atoC two component
system genes, can direct the synthesis of the lacZ gene under the
control of their cognate promoters.11,21 The ability of these con-
structs to respond to various substances induction, driving the
expression of lacZ gene, reflects the transcription of the reporter
genes. Plasmid pMLB1034 was used as negative control.
Plasmid pUC-Az containing the atoS, atoC genes and a part of
the atoDAEB operon (atoD, atoA, and two-thirds of atoE) has been
described previously.22
4.2.2. Culture conditions
4.1.11. 6-(4-Methoxyphenyl)-5,7,8-trimethyl-2H-1,4-benzoxaz
in-3(4H)-one (12)
E. coli cells carrying the appropriate plasmids (Fig. 2) were
grown at 37 °C with vigorous shaking in M9 minimal medium,29
supplemented with 0.1 mM CaCl2, 1 mM MgSO4, 0.5% w/v glucose,
To
a
solution of ethyl 2-[(40-methoxy-2,3,6-trimethyl-5-
nitro[1,10-biphenyl]-4-yl)oxy]acetate (11) (0.373 g, 1 mmol) in
dry ethanol (10 mL) was added 10% palladium on carbon (25% w/
w of starting material) and the mixture was hydrogenated at
70 °C overnight. After filtration and evaporation in vacuo, the crude
product was purified by flash column chromatography (petroleum
ether 40–60 °C/ethyl acetate 85:15) to afford 6-(4-methoxy-
phenyl)-5,7,8-trimethyl-2H-1,4-benzoxazin-3(4H)-one (12). Yield:
267 mg (90%); white crystals; mp 211–213 °C; Rf 0.29 (petroleum
ether 40–60 °C/ethyl acetate 3:1); 1H NMR (600 MHz, CDCl3): d
7.50 (s, 1H, -NHCO-), 6.98 (d, J = 8.8 Hz, 2H, ArH), 6.95 (d,
J = 8.8 Hz, 2H, ArH), 4.61 (s, 2H, CH2), 3.85 (s, 3H, OCH3), 2.19 (s,
3H, ArCH3), 1.90 (s, 3H, ArCH3), 1.87 (s, 3H, ArCH3); 13C NMR
(75.5 MHz, CDCl3): d 165.8, 158.3, 141.0, 136.0, 133.2, 131.3,
130.6, 122.6, 122.1, 119.9, 113.8, 67.1, 55.2, 17.3, 14.0, 12.0; HRMS
(FAB+) calcd for C18H19O3N [M]+ 297.1365, found 297.1352; MS
(EI), m/z: 297 [M+, 100].
1.7 ꢂ 10ꢁ3 mM FeSO4, 1
l
g/mL thiamine and 80
l
g/mL DL-proline.
Ampicillin was used at the final concentration of 100
lg/mL. The
strains to be tested were grown overnight, then diluted 1:15 in
the growth medium containing various substances (1 mM concen-
tration), or various concentrations of 4a or acetoacetate (2.5 or
10 mM) in combination with 4a. Cultures were grown to OD600
0.4–0.7 and the cells were harvested by centrifugation at 8000ꢂg
and washed twice with M9 before the b-galactosidase assay.
In addition, BW25113 cells carrying plasmid pUC-Az were
grown in supplemented M9 mineral medium, in the absence or
presence of various concentrations of compound 4a added at the
beginning of the growth or in portions of ½ of the compound tested
at an OD600 0.2 and 0.4, respectively. The cells were harvested
when cultures reached logarithmic phase (JD600 0.5–0.6) or late
logarithmic phase (JD600 0.9) by centrifugation at 8000ꢂg for
10 min. The cell pellets were washed twice with ice-cold buffer sal-
ine and stored at ꢁ20 °C until use.
4.1.12. 6-(4-Methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-
1,4-benzoxazine (13)
4.2.3. b-Galactosidase assay
To a solution of 6-(4-methoxyphenyl)-5,7,8-trimethyl-2H-1,4-
benzoxazin-3(4H)-one (12) (0.297 g, 1 mmol) in THF (50 mL) was
added dropwise at 0 °C a solution of borane. dimethylsulfide
(24 mmol, 2.28 mL). The reaction mixture was stirred at room tem-
perature until completion. The reaction was stopped by dropwise
addition of water at 0 °C until gas evolution has ceased. The organic
layer was extracted with saturated aqueous NaHCO3 and brine and
was dried over anhydrous Na2SO4. The solvent was evaporated in
vacuo and the residue was purified by flash column chromatogra-
phy using (hexane/acetone 9:1) as elution solvent to afford 6-(4-
b-Galactosidase activity assays were performed using the E. coli
BW25113 (atoSC+) carrying the appropriate plasmids, as de-
scribed.29 In all experiments, BW25113 carrying plasmid
pMLB1034 was used as negative control.
4.2.4. Immunoblot analysis
An aliquot of 1 mL of each culture was centrifuged and resus-
pended in SDS loading buffer of 1.5ꢂ (75 mM Tris–HCl pH 6.8,
3% w/v SDS, 0.15% w/v bromophenol blue, 15% v/v glycerol and

5070
P. S. Filippou et al. / Bioorg. Med. Chem. 19 (2011) 5061–5070
b-mercaptoethanol at a final concentration of 10% v/v), boiled for
5 min and kept at ꢁ20 °C for immunoblot analysis.
immunostained with the rabbit polyclonal antibodies against,
DnaK, Hsp60, AtoC and AtoS, respectively, prepared as
described.2,11,33
4.2.5. Purification of recombinant cytosolic AtoS protein form
The recombinant His6-tagged protein cytoAtoS was purified as
described (5) by immobilized metal affinity chromatography
(IMAC) on Ni2+-NTA agarose (Qiagen) columns and dialyzed
against a storage buffer containing 50 mM Tris–HCl pH 7.6,
0.5 mM DTT and 50% v/v glycerol. Following the determination of
the cytoAtoS-His6 concentration and an analysis of its purity by
SDS–polyacrylamide gel electrophoresis, the protein was stored
in aliquots at ꢁ80 °C.
Supplementary data
Supplementary data (1H and 13C NMR spectra for compounds
2a, b, 3a, b, 4a–c, 5a, b, 6a, b, and 7–13) associated with this article
can be found, in the online version, at doi:10.1016/j.bmc.2011.
06.029.
References and notes
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4.2.6. Protein concentration
Protein concentrations were determined by the Bradford
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30
method,
dard.
using bovine serum albumin as the reference stan
4.2.7. Kinase activity assay
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Purified recombinant histidine kinase cytoAtoS-His6 was incu-
bated at room temperature for approximately 30 min with
1.6
(pH 7.6), 10 mM MgCl2, 100 mM KCl, 1 mM DTT and 10 mM NaF
at a final reaction volume of 45 L. Equal concentrations of cytoA-
lM [c-
32P]-ATP (4 nmoles per reaction) in 50 mM Tris-HCl
l
toS were pre-incubated at room temperature with the phosphory-
lation buffer in the absence or presence of each of the 5,7,8-
trimethyl-1,4-benzoxazine derivatives (at the final concentration
of 1 mM) or compound 4a (in increasing molar concentrations,
0.1, 0.2, 0.5 and 1.0 mM) for 15 min, prior the addition of [c-
32P]-
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4ꢂ SDS sample buffer and analyzed by SDS–PAGE and
autoradiography.
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Purified cytoAtoS-His6 protein (2 lg) was initially incubated
with the cross-linking buffer (20 mM Hepes-KOH, pH 7.9, 0.2 mM
EDTA, 0.1 M NaCl, 20% v/v glycerol and 0.5 mM DTT) and the indi-
cated concentrations of compound 4a (0.5 and 1 mM) for 15 min at
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with 0.1% v/v glutaraldehyde added in the reaction. The reaction
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volume was 25 lL and the cross-linking was terminated by addi-
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SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was
performed using 8.5, 10 and 12% w/v polyacrylamide gels.31
Proteins were transferred to immobilon PVDF membranes32 and