Synthesis and evaluation of halogenated nitrophenoxazinones as nitroreductase substrates for the detection of pathogenic bacteria

Article abstract of DOI:10.1016/j.tet.2013.07.047

The synthesis and microbiological evaluation of 7-, 8- and 9-nitro-1,2,4-trihalogenophenoxazin-3-one substrates with potential in the detection of nitroreductase-expressing pathogenic microorganisms are described. The 7- and 9-nitrotrihalogenophenoxazinon

Full text of DOI:10.1016/j.tet.2013.07.047

Tetrahedron 69 (2013) 8456e8462
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Synthesis and evaluation of halogenated nitrophenoxazinones as
nitroreductase substrates for the detection of pathogenic bacteria
Alexandre F. Bedernjak a, Paul W. Groundwater b, Mark Gray a, Arthur L. James c,
,
Sylvain Orenga d, John D. Perry e, Rosaleen J. Anderson a
*
a Sunderland Pharmacy School, University of Sunderland, Sunderland SR1 3SD, UK
b Faculty of Pharmacy, The University of Sydney, Sydney NSW 2006, Australia
c School of Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK
d
ꢀ
Department of Microbiology, bioMerieux, 38390 La Balme-les-Grottes, France
e Department of Microbiology, Freeman Hospital, Newcastle upon Tyne NE7 7DN, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and microbiological evaluation of 7-, 8- and 9-nitro-1,2,4-trihalogenophenoxazin-3-one
substrates with potential in the detection of nitroreductase-expressing pathogenic microorganisms are
described. The 7- and 9-nitrotrihalogenophenoxazinone substrates were reduced by most Gram-negative
microorganisms and were inhibitory to the growth of certain Gram-positive bacteria; however, the
majority of Gram-positive strains that were not inhibited by these agents, along with the two yeast
strains evaluated, did not reduce the substrates. These observations suggest there are differences in the
active site structures and substrate requirements of the nitroreductase enzymes from different strains;
such differences may be exploited in the future for differentiation between pathogenic microorganisms.
The absence of reduction of the 8-nitrotrihalogenophenoxazinone substrates is rationalized according to
their electronic properties and correlates well with previous findings.
Received 15 February 2013
Received in revised form 5 July 2013
Accepted 15 July 2013
Available online 20 July 2013
Keywords:
Chromogenic substrates
Nitroreductase
Phenoxazinones
Microbial identification
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction
the nitroso intermediate and then follows the two-electron re-
duction process, or is rapidly reoxidised to a nitro group in the
The ability of nitroreductase and other enzymes to reduce an
aromatic nitro group to the corresponding hydroxylamine and/or
amine is of significant interest, with great potential for biomedical,
biocatalyst and bioremediation applications.1e4 For example, the
reduction of a nitroaromatic prodrug by a bacterial nitroreductase
is central to the GDEPT anticancer therapeutic approach,5,6 for
which the mechanism of the Escherichia coli nitroreductase acti-
vation has been elucidated.7
Nitroreductase enzymes have been subdivided into two different
classes,8,9 namely type I and type II nitroreductases, based on their
relative sensitivity to oxygen. The type I nitroreductases are oxygen
insensitive and can reduce nitro compounds to their corresponding
amines via the nitroso and hydroxylamine intermediates, using
a two-electron transfer mechanism.9e11 With the type II nitro-
reductases, which are oxygen sensitive, the reduction of nitro
groups is initiated via a single electron transfer process, forming
a nitro radical anion, which either accepts a second electron to form
presence of oxygen.8,9
The widespread distribution of nitroreductase enzymes in
pathogenic bacteria and yeasts offers the potential to enhance the
detection of such microorganisms in both clinical and food appli-
cations.11,12 The successful detection of pathogenic bacteria as
a result of their nitroreductase action has been demonstrated in
over 30 Gram-negative and Gram-positive microorganisms,11 using
the fluorogenic 7-nitrocoumarin-3-carboxylic acid 1a or 7-nitro-4-
methylcoumarin 1b (Fig. 1). These compounds emit a strong
O2N
O
O
X
N
R
R1
O2N
R2
O2N
1a R1 = COOH, R2 = H
1b R1 = H, R2 = CH3
2a X = O
2b X = S
2c X = N
3a R =
3b R =
3c R =
N
N
S
N
* Corresponding author. Sunderland Pharmacy School, Science Complex, Uni-
versity of Sunderland, Sunderland SR1 3SD, UK. Tel.: þ44 191 515 2591; e-mail
address: roz.anderson@sunderland.ac.uk (R.J. Anderson).
Fig. 1. Examples of established fluorogenic and chromogenic nitroreductase substrates.
0040-4020/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.07.047

A.F. Bedernjak et al. / Tetrahedron 69 (2013) 8456e8462
8457
fluorescent signal upon exposure to UV light (
l
¼365 nm) after in-
X
X
O
O
NH2
X
X
X
X
N
O
X
i)
cubation with the bacteria. Moreover, other fluorogenic com-
pounds, such as the 4-nitrobenzylcarbamate derivatives of 7-
aminocoumarins,13 and derivatives of nitrobenzoxazole 2a, nitro-
benzothiazole 2b and nitrobenzimidazole 2c (Fig. 1), were also
shown to respond to nitroreductase activity, although with greater
selectivity for Gram-negative bacteria.14,15 The extension of this
approach to chromogenic nitroaromatic substrates, including de-
rivatives of 4-(40-nitrostyryl)-pyridine 3a,16 4-(40-nitrostyryl)-
quinoline 3b17 and 2-(40-nitrostyryl)-benzothiazole 3c18 (Fig. 1),
suggests a general application of nitroreductase enzyme substrates
as detection agents, particularly for Gram-negative pathogenic
bacteria. It is interesting to note that more spatially compact
nitroreductase substrates, such as nitrobenzenes and nitro-
coumarins 1a and 1b,11,13 are reduced by a wider range of both
Gram-negative and Gram-positive bacteria, while the more bulky
nitroaromatic substrates, for example, the nitrobenzoxazole 2a,
nitrobenzothiazole 2b and nitrobenzimidazole 2c derivatives, are
less well reduced by the Gram-positive bacteria,14e18 which may
suggest differences in the active sites of Gram-negative nitro-
reductases compared to those of Gram-positive bacteria.
O2N
O2N
OH
O
8
9
5-NO2
4-NO2
10 3-NO2
11a X = F
11b X = Cl
11c X = Br
5a-c 7-NO2
6a-c 8-NO2
7a,b 9-NO2
a X = F; b X = Cl; c X = Br
Scheme 1. Synthesis of nitro-1,2,4-trihalogenophenoxazin-3-ones 5e7. Reagents and
conditions: (i) EtOH, NaOAc, rt.
Table 1
Yields of synthesized nitrohalogenophenoxazin-3-ones substrates 5aec, 6aec and
7a,b
Compound
Nitro group position
X
Yield (%)
5a
5b
5c
6a
6b
6c
7a
7b
7-
7-
7-
8-
8-
8-
9-
9-
F
12
90
95
39
99
72
15
45
Cl
Br
F
Cl
Br
F
Cl
We previously reported the synthesis and the microbiological
evaluation of the chromogenic 7-aminophenoxazinones 4 and their
corresponding aminopeptidase substrates, which indicate the
presence of certain bacteria by the intense colour released upon
bacterial enzymatic action.19 By analogy, the corresponding 7-
nitrophenoxazinone was a desirable target substrate as a potential
marker of nitroreductase activity. Herein we report the preparation
and evaluation of 7-, 8- and 9-nitro-1,2,4-trihalogenophenoxazin-
3-ones 5e7, (Fig. 2), for their ability to detect pathogenic microor-
ganisms through nitroreductase activity.
a highly insoluble solid, suspected to be triphenodioxazines 12aec,
respectively. IR spectroscopy and mass spectrometry evidence for
structure 12a was obtained alongside nitrophenoxazinone 5a. Mital
and Jain have described the formation of triphenodioxazines under
conditions similar to those described here (Scheme 1).21
F
10
N
12
O
11
1
13
10
2NO2
O2N
9
3
N
O
F6
8
4
7
5
R1
X
2
10
N
9
12a 3,10-NO2
12b 2,9-NO2
12c 1,8-NO2
1
N
O
R2
O
X
8
O2N
3
4
X
7
H2N
O
O
6
R3
5
A reaction mechanism for an analogous condensation of various
aminophenols with 2,3-dichloro-1,4-naphthoquinone in an etha-
4a R1 = pentyl, R2 = R3 = H
4b R1 = R2 = R3 = CH3
4c R1 = R2 = CH3, R3 = H
5a-c 7-NO2
6a-c 8-NO2
7a,b 9-NO2
nolic solution containing sodium or potassium acetate has been
22
€
reported by Agarwal and Schafer. Deprotonation of the nitro-
a X = F; b X = Cl; c X = Br
aminophenol 8e10 is followed by a 1,4-Michael-type nucleophilic
attack of the resulting phenoxide on tetrahalogenobenzoquinone
11aec, resulting in the formation of the corresponding 2-phenoxy-
3,5,6-trihalogenoquinones 13aec, 14aec and 15a,b. These in-
termediates then undergo ring closure to form the nitro-1,2,4-
trihalogenophenoxazin-3-ones 5aec, 6aec and 7a,b (Scheme 2).
Fig. 2. 7-Aminophenoxazin-3-one chromogens 4 and nitrotrihalogenophenoxazinones
5e7.
2. Results and discussion
2.1. Synthesis
X
X
O
X
The nitrotrihalogenophenoxazinones were synthesized using the
route described previously for the synthesis of 7-nitrohalogeno
phenoxazinones 5b, 5c and 8-nitro-1,2,4-trichlorophenoxazinone
6b20 without modification. With the availability of several 2-
aminonitrophenol isomers, the opportunity was taken to include
the synthesis of 8-nitro and 9-nitrophenoxazinones in this study.
Following the general procedure, 2-aminonitrophenols 8e10 were
condensed with tetrahalogeno-1,4-benzoquinones 11aec in an
ethanolic solution containing sodium acetate (Scheme 1). 7-Nitro
5aec, 8-nitro 6aec and 9-nitro-1,2,4-trihalogenophenoxazin-3-
ones 7a,b were isolated in low to high yield (Table 1). The forma-
tion of the desired products was confirmed mostly by 13C NMR
spectroscopy, with the characteristic CeF coupling observed in the
13C NMR spectra of fluorinated nitrophenoxazin-3-ones 5a, 6a and
7a assisting the characterization of these novel products.
NH2
O
X
X
O2N
O2N
O
O
O
O
NH
X
2 X
X
Na
8
9
5-NO2
4-NO2
10 3-NO2
11a X = F
11b X = Cl
11c X = Br
X
X
X
O
O
X
N
O
X
-H2O
O2N
O2N
O
O
NH2
X
13a-c 5'-NO2
14a-c 4'-NO2
15a,b 3'-NO2
a
b
c
X = F
X = Cl
X = Br
5a-c 7-NO2
6a-c 8-NO2
7a,b 9-NO2
The synthesis of fluorinated derivatives 5a, 6a and 7a proved to
be problematic and all were isolated in poor yield, along with
Scheme 2. Suggested mechanism for the formation of nitrohalogenophenoxazin-3-
ones19 5aec, 6aec and 7a,b.

8458
A.F. Bedernjak et al. / Tetrahedron 69 (2013) 8456e8462
2.2. Microbiological evaluation
these organisms. The most toxic substrate was 8-nitro-1,2,4-
trifluorophenoxazin-3-one 6a, which was universally toxic to all
Gram-positive bacteria and the yeasts, while 9-nitro-1,2,4-
trifluorophenoxazin-3-one 7a was toxic to most of these strains
and 7-nitro-1,2,4-trifluorophenoxazin-3-one 5a also exhibited
toxicity across many of the Gram-positive bacteria. The growth of
the two Enterococcal strains tested and Listeria monocytogenes (No.
18) was not inhibited by any of the substrates except 6a. The tri-
fluorinated derivatives 5a, 6a and 7a were the most toxic com-
pounds in the series.
The chromogenic substrates 5e7 were evaluated for their suit-
ability as nitroreductase substrates for the detection of microbial
nitroreductase activity. The reduction of the weakly coloured
compounds 5e7 by an unspecified nitroreductase was expected to
produce the corresponding, intensely coloured, aminophenoxazin-
3-ones 16aec, 17aec and 18a,b. This would result in a dramatic
and readily visualised change in colour at the site of reduction
(Scheme 3).
Most Gram-negative bacteria successfully reduced substrates
5aec and substrates 7a,b. The enzymatic activity was witnessed by
the generation of a red to orange colouration in the bacterial col-
onies for substrates 5aec, brown for substrate 7a and lilac for
substrate 7b. The most encouraging results were obtained with 7-
nitro-1,2,4-trifluorophenoxazinone 5a, as this substrate produced
the brightest and most strongly contrasting colour in the bacterial
colonies, with minimal diffusion into the medium (Fig. 3). Absence
(or low diffusion) of the reduced substrate in the medium is es-
sential for distinction and accurate localization of individual bac-
terial colonies expressing nitroreductase enzymes when several
microorganisms are present in the culture medium.
None of these substrates were reduced at all by Burkholderia
cepacia (No. 4), which can also metabolise certain nitroaromatic
compounds, such as 2,4-dinitrotoluene, by an alternative defined
oxidative pathway, leading to oxidative cleavage of the aromatic
system.2 Burkholderia species exhibit high-level intrinsic re-
sistance to many antibacterial agents, even to colistin and genta-
micin, due to their particularly impermeable membrane,23e26 so
these substrates may not even reach the cytoplasm, where nitro-
X
X
10
N
9
1
N
O
X
2
X
8
i)
H2N
O2N
3
4
X
7
O
O
O
6
5
X
5a-c 7-NO2
6a-c 8-NO2
7a,b 9-NO2
a
b
c
X = F
X = Cl
X = Br
16a-c 7-NH2
17a-c 8-NH2
18a,b 9-NH2
Scheme 3. Expected nitroreductase enzyme activity and reduction of substrates 5aec,
6aec and 7a,b. Conditions: (i) bacterial nitroreductase enzymatic activity.
The halogenated nitrophenoxazin-3-ones 5e7 were evaluated
for their suitability as nitroreductase substrates with a range of
pathogenic microorganisms from the National Collection of Type
Cultures (London, UK) and from the Freeman Hospital Microbiology
Department (Newcastle upon Tyne, UK), including 10 Gram-nega-
tive and 8 Gram-positive strains and 2 yeasts (Table 2). All of these
microorganisms had previously been shown to express nitro-
reductases and to have the ability to reduce a nitro-substituted
aromatic fluorogenic compound.11
Table 2
Reduction of substrates 5e7 by selected clinically relevant Gram-positive and Gram-negative bacteria, and yeasts
Bacterial strain
Substratea
Reference
5a
5b
5c
6a
6b
6c
7a
7b
Visualization of colouration after 24 hb
Gram-negative bacteria
1 Escherichia coli
c
NCTC 10418
NCTC 10211
NCTC 10662
LMG 1222
NCTC 11176
NCTC 74
Wild type 46262
Wild type 462403
NCTC 11936
NCTC 7475
þ
þ
þ
ꢀ
þ
þ
þ
þ
þ
þ
þ
þ
þ
ꢀ
ꢀ
þ
þ
þ
þ
þ
þ
þ
ꢀ
ꢀ
ꢀ
þ
ꢀ
þ
þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
þ
þ
þ
ꢀ
ꢀ
þ
þ
þ
þ
þ
þ
þ
ꢀ
ꢀ
þ
þ
þ
þ
þ
þ
2 Serratia marcescens
3 Pseudomonas aeruginosa
4 Burkholderia cepacia
5 Yersinia enterocolitica
6 Salmonella typhimurium
7 Citrobacter freundii
8 Morganella morganii
9 Enterobacter cloacae
10 Providencia rettgeri
Gram-positive bacteria
11 Bacillus subtilis
NCTC 9372
NCTC 775
NGd
ꢀ
ꢀ
ꢀ
NG
NG
NG
NG
NG
NG
NG
NG
ꢀ
ꢀ
NG
ꢀ
ꢀ
12 Enterococcus faecalis
13 Enterococcus faecium
14 Staphylococcus epidermidis
15 Staphylococcus aureus
16 Staphylococcus aureus (MRSA)
17 Streptococcus pyogenes
18 Listeria monocytogenes
ꢀ
ꢀ
ꢀ
ꢀ
þ
NCTC 7171
NCTC 11047
NCTC 6571
NCTC 11939
NCTC 8306
NCTC 11994
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
NG
NG
NG
NG
þ
NG
NG
ꢀ
NG
NG
ꢀ
ꢀ
NG
ꢀ
NG
NG
NG
NG
ꢀ
NG
NG
þ
ꢀ
ꢀ
ꢀ
NG
ꢀ
NG
ꢀ
NG
ꢀ
ꢀ
NG
ꢀ
ꢀ
Yeasts
19 Candida albicans
20 Candida glabrata
ATCC 90028
NCPF 3943
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
NG
NG
ꢀ
ꢀ
ꢀ
ꢀ
NG
ꢀ
ꢀ
ꢀ
a
Each substrate was tested at a concentration of 50 mg/L.
Results after 24 h of incubation at 37 ꢁC.
Positive indicates colour observed; negative indicates no colour observed.
NG, no growth.
b
c
d
None of the substrates were toxic to the Gram-negative bacteria
reductase activity would be expected to occur. B. cepacia (No. 4) was
the only Gram-negative strain that did not reduce 7-nitro-1,2,4-
trifluorophenoxazin-3-one 5a and thus distinguishes this bacte-
rium from the other pathogenic strains tested. This substrate 5a
and growth was observed in the presence of all substrates; how-
ever, toxicity was observed towards some of the Gram-positive
bacteria and yeasts, resulting in the inhibition of the growth of

A.F. Bedernjak et al. / Tetrahedron 69 (2013) 8456e8462
8459
imine system in the para-position. In compounds 7a,b, the 9-
nitro group is conjugated at the ortho-position to the quinone-
imine system and C9 may be considered to be similarly electron
deficient to C7 in 5aec. Conversely, in compounds 6aec, the 8-nitro
group is para to the potentially electron releasing ether oxygen.
Overall then, the electron density at C8 in the case of substrates
6aec can be expected to be considerably higher than C7 and C9 in
the case of substrates 5aec and 7a,b, respectively. The electron
withdrawing effect of the quinone-imine portion of the phenox-
azinone ring affects the 7- and 9-positions primarily and would
thus be expected to facilitate the reduction of substrates 5 and 7
(Scheme 5), the oxygen bridging atom can contribute to some ex-
tent to the higher electron density at the 8-position, thereby re-
ducing the propensity for reduction of substrates 6aec (Scheme 5).
Fig. 3. (a) Inoculation pattern, referenced to the numbers of strains in Table 2; (b)
screening of 7-nitro-1,2,4-trifluorophenoxazinone 5a with 10 Gram-negative and 8
Gram-positive bacterial strains and 2 yeasts.
X
X
X
N
O
X
N
O
X
(a)
O2N
O2N
O
O2N
O2N
O
also differentiates L. monocytogenes (No. 18) from the other Gram-
positive strains, being the only substrate reduced by this bacterium.
The chlorinated and brominated derivatives of 7-nitropheno
xazinone 5b and 5c and the chlorinated derivative of 9-nitro
X
X
X
N
O
X
N
X
(b)
O
phenoxazinone 7b are able to differentiate MRSA (No. 16) from
b
-
O
O
X
X
lactam susceptible Staphylococcus aureus (No. 15) and Staphylo-
coccus epidermidis (No. 14); of these three strains, only MRSA was
not inhibited by substrates 5b, 5c and 7b. Although few of the
Gram-positive bacteria and the yeasts were unable to reduce the
nitro substrates, it was not due to a lack of expression of nitro-
reductase, as it has been previously shown that they reduce other
nitroreductase substrates.11
The three 8-nitrophenoxazinone substrates 6aec were not re-
duced by nitroreductases from Gram-negative bacteria, unlike their
analogues 5aec and 7a,b, despite good bacterial growth (Table 2).
Catalytic reduction of the 8-nitrophenoxazinones 6aec using hy-
drogen and either Pd/C or PtO2, followed by re-oxidation of the
initial aminophenol product (19aec) with manganese (IV) oxide,
produced deeply coloured 8-aminophenoxazinones (17aec), of
which the most soluble example, 1,2,4-trifluoro-8-aminopheno
xazin-3-one 17a, was isolated and characterized (Scheme 4). The
lack of any colour after incubation of the 8-nitrophenoxazinones
6aec with bacteria thus provides evidence that the three com-
pounds were not substrates for nitroreductase activity.
Scheme 5. Key resonance forms that contribute to differentiation of nitroreductase
activity in (a) 7-nitrohalogenophenoxazin-3-ones 5aec and (b) 8-
nitrohalogenophenoxazin-3-ones 6aec.
The 13C NMR data provide support for these electronic differ-
ences (Table 3): in compounds 5aec and 7a,b, the chemical shifts of
C7 and C9, respectively, show greater deshielding than C8 of the
Table 3
13C NMR data for CeNO2 of nitrotrihalogenophenoxazin-3-ones 5aec, 6aec, and
7a,b
Compound Chemical shift of C bearing nitro group (DMSO-d6;
d
/ppm)
CeNO2
aX¼F
bX¼Cl
149.8
145.3
147.8
cX¼Br
149.7
145.3
N.D.b
5
6
7
C7
C8
C9
149.1 (147.9a)
145.2 (143.4a)
147.8 (146.4a)
a
NMR solvent was THF-d8.
N.D.: not done.
b
X
X
X
X
X
H
N
O2N
N
O
X
H2N
X
H2N
N
O
X
i)
ii)
O
O
OH
O
X
6a X = F
6b X = Cl
6c X = Br
19a X = F
19b X = Cl
19c X = Br
17a X = F Blue-violet
17b X = Cl Blue
17c X = Br Blue
Scheme 4. Catalytic reduction of 8-nitrophenoxazinones 6aec and oxidation of the resulting aminophenols 19aec to 8-aminophenoxazinones 17aec. Reagents and conditions: (i)
H2, Pd/C 10% or PtO2 (used for the reduction of 6c), EtOAc/MeOH (1:1); (ii) MnO2, MeOH.
Early work by McCormick27 and later studies by Kitamura,28
testing a wide range of variously substituted nitro compounds for
their suitability as nitroreductase substrates, showed that the po-
sition of the nitro group with respect to other substituents,
alongside their electronic properties, is a determining factor in the
bacterial reduction of nitroaromatic compounds. For example,
nitroreductase activity was generally higher for nitrobenzenes
bearing a para-electron withdrawing group. Consideration of the
substituents at the para-position to the nitro group in each of the
present substrates reveals that compounds 5aec, with the nitro
group in the 7-position, have the electron withdrawing quinone-
corresponding halogenated compounds 6aec.
These observations correlate well with Kitamura’s experi-
ments28 and help to explain the absence of visible reduction for
substrates 6aec, while substrates 5aec and 7a,b were reduced to
some extent.
3. Conclusions
Although some substrate selectivity has been previously ob-
served with the nitroreductase from the food and gastrointestinal
bacterium, Lactobacillus plantarum,29 the differences in the

8460
A.F. Bedernjak et al. / Tetrahedron 69 (2013) 8456e8462
reduction of the substrates, particularly by the bacteria used in the
current work, suggest that, rather than detecting a wide range of
Gram-negative and Gram-positive microorganisms, nitroreductase
activity on substituted 7- or 9-nitrophenoxazinones could be used
to distinguish certain pathogenic strains. Furthermore, differences
in the nitroreductase enzymes, or in the nitro group metabolic
pathways, between strains can be inferred by the variations in re-
duction of a particular nitroaromatic substrate.
8-Nitro-1,2,4-trihalogenophenoxazin-3-ones 6aec proved to be
unsuitable for the detection of bacterial nitroreductase; the elec-
tronic factors associated with the position of the 8-nitro group on
the phenoxazin-3-one ring appear to be very unfavourable for
enzymatic reduction. These observations correlate with results
previously published.27,28
7-Nitro-1,2,4-trihalogenophenoxazin-3-ones 5aec and 9-nitro-
1,2,4-trihalogenophenoxazin-3-ones 7a,b showed positive results
for the general detection of Gram-negative bacterial nitroreductase,
excepting B. cepacia, which was distinguished by its lack of reduction
of 7-nitro-1,2,4-trifluorophenoxazin-3-one 5a. Exploration and ex-
ploitation of the differences in substrate structure and properties,
such as replacement of the halogen atoms by other substituents,
could reduce the toxicity of the substrates and improve enzymatic
reduction. Such modifications could also assist in distinguishing
pathogenic strains by conferring even greater selectivity for the
nitroreductase enzymes of particular microorganisms.
and the insoluble residue was washed several times with EtOAc,
giving 12a as a dark violet solid (1.91 g, 4.63 mmol, 48%). A dark
violet microcrystalline solid was obtained from nitrobenzene, mp:
315 ꢁC (dec); (found: (MH)þ, 413.0324. Calcd for C18H7F2N4O6:
(MH)þ, 413.0328); nmax/cmꢀ1 3104 (CeH), 1622 (C]N), 1595 and
1579 (C]C), 1515 and 1332 (NO2). The aqueous layer was dis-
carded and the combined EtOAc filtrates and layers were washed
with water (150 mL) and brine (150 mL), and dried (MgSO4). The
solvent was removed in vacuo and the residue subjected to col-
umn chromatography on silica, eluting with petroleum ether
60e80 ꢁC/EtOAc (80:20). Nitrophenoxazinone 5a was isolated as
a dark purple solid (0.35 g, 1.19 mmol, 12%). Dark red-purple
needle-like crystals were obtained from glacial acetic acid, mp:
218e220 ꢁC; (found: C, 48.5; H, 1.05; N, 9.15%. C12H3F3N2O4 re-
quires C, 48.7; H, 1.0; N, 9.5%); (found: (MH)þ, 297.0118. Calcd for
C
12H4F3N2O4: (MH)þ, 297.0118); nmax/cmꢀ1 3104 (CeH), 1660 (C]
O), 1596 (C]C), 1531 and 1312 (NO2), 1003 (CeF); dH (300 MHz,
THF-d8) 6.41 (1H, d, J¼8.7 Hz, 9-H), 6.58 (1H, dd, J¼8.7, 2.4 Hz, 8-
H), 6.74 (1H, d, J¼2.4 Hz, 6-H); dC (75 MHz, THF-d8) 110.3 (CH, 6-C),
118.6 (CH, 8-C), 129.4 (CH, 9-C), 130.1 (quat, ddd, J¼8.5, 5.2, 1.4 Hz,
10a-C), 134.1 (quat, 9a-C), 136.0 (quat, dd, J¼262.7, 6.1 Hz, 4-C),
141.3 (quat, dt, J¼273.4, 6.9 Hz, 2-C), 142.2 (quat, dd, J¼273.4,
12.2 Hz, 1-C), 141.4 (quat, d, J¼1.5 Hz, 5a-C), 141.7 (quat, ddd,
J¼18.1, 4.8, 1.2 Hz, 4a-C), 147.9 (quat, 7-C), 166.7 (quat, td, J¼21.4,
5.4 Hz, 3-C); dF (282 MHz, THF-d8) ꢀ282.17 (1F, t, J¼2.3 Hz, 4-F),
ꢀ271.89 (1F, dd, J¼9.3, 2.0 Hz, 2-F), ꢀ265.41 (1F, dd, J¼9.3, 2.0 Hz,
1-F); dH (300 MHz, DMSO-d6) 8.23 (1H, d, J¼8.7 Hz, 9-H), 8.31 (1H,
dd, J¼8.7, 2.4 Hz, 8-H), 8.54 (1H, d, J¼2.4 Hz, 6-H); dC (75 MHz,
DMSO-d6) 112.3 (CH, 6-C), 120.8 (CH, 8-C), 131.4 (CH, 9-C), 132.1
(quat, dd, J¼9.4, 3.0 Hz, 10a-C), 135.4 (quat, 9a-C), 136.0 (quat, dd,
J¼262.7, 6.1 Hz, 4-C), 142.6 (quat, dt, J¼268.2, 6.1 Hz, 2-C), 143.6
(quat, dd, J¼270.4, 11.7 Hz, 1-C), 142.5 (quat, 5a-C), 141.7 (quat,
ddd, J¼18.1, 4.8, 1.2 Hz, 4a-C), 149.1 (quat, 7-C), 168.9 (quat, td,
J¼20.3, 5.6 Hz, 3-C); dF (282 MHz, DMSO-d6) ꢀ280.43 (1F, s, 4-F),
ꢀ268.27 (1F, d, J¼11.9 Hz, 2-F), ꢀ263.04 (1F, d, J¼11.3 Hz, 1-F).
4. Experimental section
4.1. General methods
All commercially available reagents and solvents were obtained
from SigmaeAldrich, Fluka or Riedel-de-Haan and were used with-
out further purification. Melting points were recorded on a Reich-
arteKofler hot-stage microscope apparatus and are uncorrected.
Infrared spectra were recorded in the range 4000e600 cmꢀ1 using
a PerkineElmer Spectrum BX FTIR instrument with internal cali-
bration and a Pike sampling system. NMR spectra were obtained
using a Bruker Ultrashield 300 spectrometer at 300 MHz for 1H
spectra, 75 MHz for 13C spectra or at 282 MHz for 19F spectra or
a Bruker Ultrashield 500 spectrometer at 500 MHz for 1H spectra or
at 125 MHz for 13C spectra. High-resolution mass spectra were ob-
tained from the EPSRC National Mass Spectrometry Facility Swansea,
using a Thermofisher LTQ Orbitrap XL. Elemental analyses were
performed using an Exeter Analytical CE-440 Elemental Analyzer.
4.2.2. 7-Nitro-1,2,4-trichloro-3H-phenoxazin-3-one (5b).31 The title
compound was prepared from p-chloranil 11b (3.52 g, 14.32 mmol)
and 2-amino-5-nitrophenol 8 (1.97 g, 12.78 mmol); 5b was isolated
as a purple solid (4.06 g, 11.75 mmol, 90%). Purple lustrous plates of
5b were obtained from glacial acetic acid; mp: 239e241 ꢁC [lit.:
238e240 ꢁC31 (from AcOH)]; (found: C, 41.7; H, 0.9; N, 7.9%.
C
12H3Cl3N2O4 requires: C, 41.7; H, 0.9; N 8.1%); (found: (MH)þ,
35
344.9226. Calcd for C
H
3
Cl3N2O4: (MH)þ, 344.9231; found:
12
(MH)þ, 346.9195. Calcd for C12H335Cl327ClN2O4: (MH)þ, 346.9202;
found: (MH)þ, 348.9162. Calcd for C12H335Cl37Cl2N2O4: (MH)þ,
348.9172); nmax/cmꢀ1 3094 (CeH), 1646 (C]O), 1584 (C]C), 1519
and 1347 (NO2); dH (300 MHz, DMSO-d6) 8.24 (1H, d, J¼8.7 Hz, 9-H),
8.32 (1H, dd, J¼8.7, 2.4 Hz, 8-H), 8.51 (1H, d, J¼2.4 Hz, 6-H); dC
(75 MHz, DMSO-d6) 111.6 (quat, 4-C), 112.7 (CH, 6-C), 121.5 (CH, 8-
C), 132.1 (CH, 9-C), 136.6 (quat, 5a-C), 137.2 (quat, 1-C or 2-C), 138.5
(quat, 1-C or 2-C), 143.5 (quat, 9a-C), 146.4 (quat, 2ꢂ C, 4a-C and
10a-C), 149.8 (quat, 7-C), 171.0 (quat, 3-C).
4.2. Synthesis of nitrohalogenophenoxazin-3-ones 5e7: gen-
eral procedure20,30
2-Aminonitrophenol 8e10 (1 mol equiv), p-tetrahalogeno-
benzoquinone 11aec (1.1 mol equiv) and sodium acetate
(1.2 mol equiv) were suspended in ethanol. The solution was stirred
for 24 h or until no more 2-aminonitrophenol was detected by TLC.
For the brominated and chlorinated derivatives, a red precipitate
progressively appeared as the reaction neared completion. Water
was then added to precipitate completely the red solid out of so-
lution. The solid was recovered by filtration and washed with water
and a little ethanol, then either recrystallised from acetic acid or
purified by column chromatography.
4.2.3. 7-Nitro-1,2,4-tribromo-3H-phenoxazin-3-one (5c). The title
compound was prepared from p-bromanil 11c (1.03 g, 2.42 mmol)
and 2-amino-5-nitrophenol 8 (0.34 g, 2.20 mmol); 5c was isolated
as a bright red solid (1.10 g, 2.30 mmol, 95.0%). A microcrystalline
dark red solid of 5c was obtained from glacial acetic acid, mp:
263e264 ꢁC; (found: C, 30.1; H, 0.7; N, 5.6%. C12H3Br3N2O4 requires:
4.2.1. 7-Nitro-1,2,4-trifluoro-3H-phenoxazin-3-one (5a) and 3,10-
dinitro-6,13-difluorotriphenodioxazine (12a). The title compounds
were prepared from p-fluoranil 11a (1.90 g, 10.57 mmol) and 2-
amino-5-nitrophenol 8 (1.42 g, 9.61 mmol) according to the
general procedure. Water (100 mL) was added to the reaction
mixture and the resulting mixture was extracted with EtOAc
(3ꢂ60 mL). The emulsion was filtered through a sintered funnel
C, 30.1; H, 0.6; N 5.85%); (found: (MH)þ, 476.7714. Calcd for
79
C
12
H
3
Br3N2O4: (MH)þ, 476.7716; found: (MH)þ, 478.7692. Calcd
79
for C
H
3
Br821BrN2O4: (MH)þ, 478.7696; found: (MH)þ, 480.7669.
12
79
Calcd for C
482.7646. Calcd for C
H
Br81Br2N2O4: (MH)þ, 480.7676; found: (MH)þ,
12
3
81
H
3
Br3N2O4: (MH)þ, 482.7658); nmax/cmꢀ1
12
3101 (CeH), 1641 (C]O), 1529 and 1346 (NO2); dH (300 MHz,

A.F. Bedernjak et al. / Tetrahedron 69 (2013) 8456e8462
8461
DMSO-d6) 8.21 (1H, d, J¼9.0 Hz, 9-H), 8.29 (1H, dd, J¼8.7, 2.4 Hz, 8-
H), 8.44 (1H, d, J¼2.4 Hz, 6-H); dC (75 MHz, DMSO-d6) 103.2 (quat,
4-C),112.5 (CH, 6-C), 121.4 (CH, 8-C), 131.9 (CH, 9-C), 134.9 (quat,1-C
or 2-C),137.0 (quat,1-C or 2-C),137.1 (quat, 5a-C),143.6 (quat, 9a-C),
147.2 (quat, 4a-C or 10a-C), 148.3 (quat, 4a-C or 10a-C), 149.7 (7-C),
171.5 (quat, 3-C).
(from acetone)]; found: C, 30.1; H, 0.6; N, 5.6%. C12H3Br3N2O4 re-
quires: C, 30.1; H, 0.6; N 5.85%; found: (MH)þ, 476.7716. Calcd for
79
C
H
Br3N2O4: (MH)þ, 476.7716; found: (MH)þ, 478.7694. Calcd
12
3
79
for C
H
3
Br821BrN2O4: (MH)þ, 478.7696; found: (MH)þ, 480.7670.
12
79
Calcd for C
H
3
Br81Br2N2O4: (MH)þ, 480.7676; found: (MH)þ,
12
81
482.7646. Calcd for C
H
3
Br3N2O4: (MH)þ, 482.7658); nmax/cmꢀ1
12
3078 (CeH), 1638 (C]O), 1581 (C]C), 1508 and 1328 (NO2); dH
(300 MHz, DMSO-d6) 7.82 (1H, d, J¼9.0 Hz, 6-H), 8.47 (1H, dd, J¼9.3,
2.7 Hz, 7-H), 8.60 (1H, d, J¼2.7 Hz, 9-H); dC (75 MHz, DMSO-d6)
103.4 (quat, 4-C), 118.1 (CH, 6-C), 125.6 (CH, 9-C), 129.0 (CH, 7-C),
133.0 (quat, 9a-C), 134.8 (quat, 1-C or 2-C), 136.5 (quat, 1-C or 2-C),
145.3 (quat, 8-C), 146.5 (quat, 4a-C or 10a-C), 147.9 (quat, 4a-C or
10a-C), 148.2 (quat, 5a-C), 171.6 (quat, 3-C).
4.2.4. 8-Nitro-1,2,4-trifluoro-3H-phenoxazin-3-one (6a). The title
compound was prepared from p-fluoranil 11a (1.51 g, 8.39 mmol)
and 2-amino-4-nitrophenol 9 (1.27 g, 8.22 mmol) according to the
general procedure. The precipitate was taken into EtOAc (100 mL)
and the resulting mixture filtered through a sintered funnel. The
insoluble residue was washed several times with EtOAc and dis-
carded. The combined organic filtrates were washed with 10%
aq NaOH solution (4ꢂ100 mL) and water (1ꢂ100 mL), and
dried (MgSO4). The solvent was removed under reduced pressure
and the residue subjected to column chromatography, eluting
with a gradient mixture of petroleum ether 60e80 ꢁC/Et2O
(50:50e0:100). Compound 6a was isolated as a red solid (0.94 g,
3.17 mmol, 38.5%). An orange crystalline solid of 6a was obtained
from glacial acetic acid, mp: 212e214 ꢁC; (found: C, 48.6; H, 1.05; N,
9.1%. C12H3F3N2O4 requires C, 48.7; H, 1.0; N, 9.5%); nmax/cmꢀ1 3093
(CeH), 1648 (C]O), 1608 (C]C), 1518 and 1308 (NO2), 1004 (CeF);
dH (300 MHz, THF-d8) 6.03 (1H, d, J¼9.3 Hz, 6-H), 6.80 (1H, dd,
J¼9.0, 2.7 Hz, 7-H), 7.06 (1H, d, J¼2.7 Hz, 9-H); dC (75 MHz, THF-d8)
115.4 (CH, 6-C), 123.8 (CH, 9-C), 126.2 (CH, 7-C), 129.9 (quat, ddd,
J¼9.6, 5.1, 1.3 Hz, 10a-C), 130.2 (quat, 9a-C), 136.0 (quat, dd, J¼262.4,
6.5 Hz, 4-C), 141.3 (quat, dt, J¼272.7, 6.9 Hz, 2-C), 142.2 (quat, dd,
J¼273.9, 12.0 Hz, 1-C), 140.8 (quat, ddd, J¼18.3, 5.5, 1.1 Hz, 4a-C),
143.4 (quat, 8-C), 145.3 (quat, d, J¼1.6 Hz, 5a-C), 166.8 (quat, td,
J¼21.0, 5.6 Hz, 3-C); dF (282 MHz, THF-d8) ꢀ282.05 (1F, t, J¼2.8 Hz,
4-F), ꢀ271.73 (1F, dd, J¼9.6, 1.7 Hz, 2-F), ꢀ266.07 (1F, dd, J¼9.3,
2.5 Hz, 1-F); dH (300 MHz, DMSO-d6) 7.93 (1H, d, J¼9.0 Hz, 6-H),
8.56 (1H, dd, J¼9.0, 2.4 Hz, 7-H), 8.76 (1H, d, J¼2.4 Hz, 9-H); dC
(75 MHz, DMSO-d6) 118.4 (CH, 6-C), 125.9 (CH, 9-C), 129.0 (CH, 7-C),
132.1 (quat, 9a-C), 132.5 (quat, dd, J¼8.8, 5.2 Hz, 10a-C), 137.8 (quat,
dd, J¼256.3, 6.45 Hz, 4-C), 143.2 (quat, dt, J¼267.75, 6.8 Hz, 2-C),
144.2 (quat, dd, J¼270.4, 11.9 Hz, 1-C), 142.8 (quat, d, J¼14.1 Hz, 4a-
C), 145.2 (quat, 8-C), 147.3 (quat, 5a-C), 169.5 (quat, td, J¼20.0,
4.9 Hz, 3-C); dF (282 MHz, DMSO-d6) ꢀ279.65 (1F, s, 4-F), ꢀ268.13
(1F, d, J¼11.3 Hz, 2-F), ꢀ264.22 (1F, d, J¼11.8 Hz, 1-F).
4.2.7. 9-Nitro-1,2,4-trifluoro-3H-phenoxazin-3-one (7a). The title
compound was prepared from p-fluoranil 11c (0.90 g, 5.00 mmol)
and 2-amino-3-nitrophenol 10 (0.74 g, 4.82 mmol) according to
the general procedure. The precipitate was taken into EtOAc
(100 mL) and the resulting mixture filtered through a sintered
funnel. The insoluble residue was washed several time with
EtOAc and discarded. The combined organic filtrates were washed
with water (1ꢂ100 mL), brine (1ꢂ100 mL) and dried (MgSO4). The
solvent was removed under reduced pressure and the residue
subjected to column chromatography, eluting with petroleum
ether 60e80 ꢁC/EtOAc (80:20). Compound 7a was isolated as
a dark red solid (0.22 g, 0.73 mmol, 15%). Ruby red prisms of 7a
with a metallic lustre were obtained from glacial acetic acid, mp:
216e218 ꢁC; (found: C, 48.5; H, 1.0; N, 9.2%. C12H3F3N2O4 requires
C, 48.7; H, 1.0; N, 9.5%); (found: (MH)þ, 297.0118. Calcd
for C12H4F3N2O4: (MH)þ, 297.0118); nmax/cmꢀ1 3084 (CeH), 1649
(C]O), 1603 (C]C), 1530 and 1313 (NO2), 1004 (CeF); dH
(300 MHz, THF-d8) 6.04e6.13 (m, 3H, 6-H, 7-H and 8-H); dC
(75 MHz, THF-d8) 117.6 (CH, 8-C), 117.9 (CH, 6-C), 123.0 (quat, 9a-
C), 130.0 (quat, ddd, J¼10.0, 5.0, 1.2 Hz, 10a-C), 131.5 (CH, 7-C),
135.8 (quat, dd, J¼261.9, 6.3 Hz, 4-C), 140.7 (quat, ddd, J¼18.2, 4.8,
1.2 Hz, 4a-C), 141.4 (quat, dt, J¼273.2, 6.8 Hz, 2-C), 141.5 (quat, d,
J¼1.7 Hz, 5a-C), 142.1 (quat, dd, J¼274.7, 12.2 Hz, 1-C), 146.4 (quat,
9-C), 166.7 (quat, td, J¼21.1, 5.5 Hz, 3-C); dF (282 MHz, THF-d8)
ꢀ282.53 (1F, t, J¼2.3 Hz, 4-F), ꢀ271.43 (1F, dd, J¼9.0, 2.3 Hz, 1-F),
ꢀ265.36 (1F, dd, J¼9.3, 2.3 Hz, 2-F); dH (300 MHz, DMSO-d6) 7.95
(1H, t, J¼8.4 Hz, 7-H), 8.01 (1H, dd, J¼8.4, 1.8 Hz, 8-H), 8.08 (1H,
dd, J¼7.5, 1.8 Hz, 6-H); dC (75 MHz, DMSO-d6) 120.5 (CH, 8-C),
120.7 (CH, 6-C), 124.8 (quat, 9a-C), 132.8 (quat, dd, J¼9.0, 4.5 Hz,
10a-C), 134.6 (CH, 7-C), 137.5 (quat, dd, J¼255.0, 6.0 Hz, 4-C),
142.7 (quat, ddd, J¼17.3, 4.5, 1.5 Hz, 4a-C), 143.2 (quat, d, J¼0.8 Hz,
5a-C), 143.3 (quat, dt, J¼267.8, 7.5 Hz, 2-C), 144.0 (quat, dd,
J¼270.0, 10.5 Hz, 1-C), 147.8 (quat, 9-C), 169.4 (quat, td, 21.0,
6.0 Hz, 3-C); dF (282 MHz, DMSO-d6) ꢀ280.98 (1F, t, J¼2.3 Hz, 4-
F), ꢀ268.59 (1F, dd, J¼11.0, 2.3 Hz, 1-F), ꢀ262.77 (1F, dd, J¼11.0,
2.3 Hz, 2-F).
4.2.5. 8-Nitro-1,2,4-trichloro-3H-phenoxazin-3-one (6b).21 The title
compound was prepared from p-chloranil 11b (1.74 g, 7.07 mmol)
and 2-amino-4-nitrophenol 9 (0.991 g, 6.43 mmol); 6b was isolated
as a red solid (2.19 g, 6.34 mmol, 99%). Red plates of 6b were ob-
tained from glacial acetic acid; mp: 261e263 ꢁC [lit.: 248e250 ꢁC21
(from acetone)]; (found: C, 41.7; H, 0.9; N, 7.9%. C12H3Cl3N2O4 re-
quires: C, 41.7; H, 0.9; N 8.1%); found: (MH)þ, 344.9229. Calcd for
C
H
Cl3N2O4: (MH)þ, 344.9231; found: (MH)þ, 346.9197. Calcd
35
12
3
35
for C
H
3
Cl327ClN2O4: (MH)þ, 346.9202; found: (MH)þ, 348.9164.
12
35
Calcd for C
H
3
Cl37Cl2N2O4: (MH)þ, 348.9172); nmax/cmꢀ1 3088
4.2.8. 9-Nitro-1,2,4-trichloro-3H-phenoxazin-3-one (7b). The title
compound was prepared from p-chloranil 11c (1.17 g, 7.61 mmol)
and 2-amino-3-nitrophenol 10 (1.07 g, 6.92 mmol); 7b was isolated
as a dark purple solid (1.08 g, 3.13 mmol, 45%). Lustrous deep purple
needles of 7b were obtained from glacial acetic acid, mp:
254e258 ꢁC; (found: C, 41.6; H, 0.9; N, 7.8%. C12H3Cl3N2O4 requires
12
(CeH), 1649 (C]O), 1597 (C]C), 1510 and 1331 (NO2); dH
(300 MHz, DMSO-d6) 7.95 (d, 1H, J¼9.0 Hz, 6-H), 8.57 (dd, 1H, J¼9.0,
2.7 Hz, 7-H), 8.73 (d,1H, J¼2.7 Hz, 9-H); dC (75 MHz, DMSO-d6) 111.8
(quat, 4-C), 118.3 (CH, 6-C), 125.9 (CH, 9-C), 129.1 (CH, 7-C), 132.6
(quat, 9a-C), 137.2 (quat, 1-C or 2-C), 138.2 (quat, 1-C or 2-C), 145.3
(quat, 8-C), 145.6 (quat, 4a-C or 10a-C), 146.2 (quat, 4a-C or 10a-C),
147.7 (quat, 5a-C), 171.1 quat, 3-C).
C, 41.7; H, 0.9; N, 8.1%); (found: (MH)þ, 344.9227. Calcd for
35
C
H
Cl3N2O4: (MH)þ, 344.9231; found: (MH)þ, 346.9197. Calcd
12
3
35
for C
H
Cl327ClN2O4: (MH)þ, 346.9202; found: (MH)þ, 348.9165.
3
12
35
4.2.6. 8-Nitro-1,2,4-tribromo-3H-phenoxazin-3-one (6c).21 The title
compound was prepared from p-bromanil 11c (4.05 g, 9.57 mmol)
and 2-amino-4-nitrophenol 9 (1.34 g, 8.70 mmol). Elution with
petroleum ether 60e80 ꢁC/EtOAc (60:40) gave 6c as a red solid
(3.01 g, 6.29 mmol, 72%). Lustrous cardinal red crystals of 6c were
obtained from glacial acetic acid, mp: 285e287 ꢁC [lit.: 287 ꢁC21
Calcd for C H
Cl37Cl2N2O4: (MH)þ, 348.9172); nmax/cmꢀ1 3100
12 3
(CeH), 1647 (C]O), 1590 (C]C), 1532 and 1331 (NO2); dH
(300 MHz, DMSO-d6) 7.97 (1H, t, J¼8.4 Hz, 7-H), 8.02 (1H, dd, J¼8.4,
2.1 Hz, 8-H), 8.09 (1H, dd, J¼7.5, 2.1 Hz, 6-H); dC (75 MHz, DMSO-d6)
111.2 (quat, 4-C), 120.6 (CH, 8-C), 120.8 (CH, 6-C), 125.3 (quat, 9a-C),
134.6 (CH, 7-C), 136.9 (quat, 1-C or 2-C), 138.6 (quat, 1-C or 2-C),

8462
A.F. Bedernjak et al. / Tetrahedron 69 (2013) 8456e8462
143.7 (quat, 5a-C), 145.5 (quat, 4a-C or 10a-C), 146.3 (quat, 4a-C or
10a-C), 147.8 (quat, 9-C), 171.0 (quat, 3-C).
Conflict of interest
No conflicts of interest are noted. The synthetic chemistry and
characterisation were conducted at the University of Sunderland;
the microbiological evaluation was carried out (by Alexandre
Bedernjak) at Freeman Hospital (Newcastle upon Tyne, UK); the
analysis and rationalisation of data were the responsibility of the
team at University of Sunderland; the final decision to submit these
data and the rationalisation for publication were taken by
4.3. Catalytic hydrogenation of 8-nitrohalogenophenoxazin-
3-ones 6aec: general procedure
Nitro-1,2,3-trihalogeno-3H-phenoxazin-3-ones 6aec were dis-
solved in a solvent mixture of EtOAc/MeOH (1:1) and either Pd/C 10%
or PtO2 (10% of the reactant mass) was added to the solution. The
reaction mixture was then hydrogenated for 2e20 h, the initial hy-
drogen pressure being set at 2.4 bars. Hydrogenation was continued
until the hydrogen pressure was steady, after which the catalyst was
removed from the reaction mixture by filtration through Celite 451.
The Celite cake was washed several times with methanol. MnO2
(3 mol equiv) was added to the clear solution and the resulting
mixture stirred until no more amino-3-hydroxyphenoxazine was
observed by TLC. The reaction mixture changed to a deep colour,
ranging from blue to blue-violet, and was again filtered through
Celite 451 to remove inorganic insoluble materials and the cake was
washed several times with THF, until the filtrate was clear of colour.
The solvent mixture was evaporated in vacuo and the residue was
subjected to column chromatography on silica.
ꢀ
bioMerieux.
Acknowledgements
ꢀ
We are grateful to bioMerieux and the University of Sunderland
for their generous financial support of this project (Alexandre
Bedernjak). We also thank the EPSRC UK National Mass Spec-
trometry Facility, Swansea University, for providing all accurate
mass measurements.
References and notes
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4.3.1. 8-Amino-1,2,4-trifluoro-3H-phenoxazin-3-one (17a). The title
compound was prepared from 8-nitro-1,2,3-trifluoro-3H-phenox-
azin-3-one 6a (0.38 g, 1.29 mmol) using Pd/C 10%. Elution used
a
gradient mixture of petroleum ether (60e80 ꢁC)/EtOAc
(30:70e10:90). 8-Amino-1,2,4-trifluoro-3H-phenoxazin-3-one 17a
was isolated as a dark violet solid (0.34 g, 1.27 mmol, 98.0%); mp:
277 ꢁC (dec); (found: C, 54.2; H, 2.1; N, 10.1%. C12H5F3N2O2 requires
C, 54.15; H, 1.9; N, 10.5%); (found: (MH)þ, 267.0373. Calcd for
C
12H6F3N2O2: (MH)þ, 267.0376); nmax/cmꢀ1 3503, 3358 and 3230
(NH2), 1638 (C]O), 1610, 1575 and 1513 (C]C), 1002 (CeF); dH
(500 MHz, TFA-d1) 8.17 (1H, d, J¼9.0 Hz, 6-H), 8.35 (1H, dd, J¼9.0,
2.0 Hz, 7-H), 8.74 (1H, d, J¼1.5 Hz, 9-H); dC (125 MHz, TFA-d1) 119.1
(CH, 6-C), 125.7 (CH, 9-C), 127.1 (quat, 9a-C), 129.4 (CH, 7-C), 132.5
(quat, dd, J¼16.5, 3.9 Hz, 10a-C), 132.8 (quat, 8-C), 138.2 (quat, dd,
J¼263.5, 5.4 Hz, 4-C), 141.6 (quat, dd, J¼18.0, 7.8 Hz, 4a-C), 143.9
(quat, dt, J¼275.0, 5.4 Hz, 2-C), 144.2 (quat, 5a-C), 144.4 (quat, dd,
J¼279.2, 13.2 Hz, 1-C), 172.3 (quat, td, J¼19.0, 6.6 Hz, 3-C).
4.4. Microbiological methods
Test compounds 5e7 were dissolved in a small volume of
N-methylpyrrolidone (100
mL) and Tween 20 (100 mL) and in-
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corporated into molten Columbia agar (Oxoid, Basingstoke) to give
a final concentration of 50 mg/L; the use of a surfactant (Tween 20)
was necessary to avoid precipitation of the substrates during in-
corporation into the agar medium. The resulting chromogenic
media were poured into Petri dishes and each solution dried to
a gel. The resultant agar plates were each inoculated with 10 Gram-
negative, 8 Gram-positive bacteria and 2 yeasts (from the National
Collection of Type Cultures, London, UK): colonies of each strain
were suspended in sterile deionised water to generate a suspension
with a turbidity equivalent to 0.5 McFarland units (approximately
1.5ꢂ108 colony forming units per mL), as confirmed with a densi-
tometer. One microlitre of this suspension was inoculated onto the
agar plates containing the various substrates using an automated
multipoint inoculator. All inoculated plates were incubated at 37 ꢁC
for 24 h and then examined visually for the presence of growth and
colouration of bacterial colonies.
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€
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~
26. Sousa, S. A.; Ramos, C. G.; Leitao, J. H. Int. J. Microbiol. 2011, 2011, 1.
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ꢀ
~
29. Guillen, H.; Curiel, J. A.; Landete, J. M.; Munoz, R.; Herraiz, T. J. Agric. Food Chem.
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30. Although some of the compounds described herein have been previously
synthesized and reported in the literature, few analytical data are available
(NMR characterization was not reported) and, for that reason, a full charac-
terization of each compound is provided.
31. Tschlenowa, V. Zh. Obshch. Khim. 1958, 28, 2977.