Synthesis and testing of chromogenic phenoxazinone substrates for β-alanyl aminopeptidase

Article abstract of DOI:10.1039/b716978g

Novel 7-N-(β-alanyl)aminophenoxazin-3-one salts 27a-d have been synthesized and tested as chromogenic substrates for β-alanyl aminopeptidase, which is present in Pseudomonas aeruginosa, the most common respiratory pathogen in patients with cystic fibrosis. The biological results show that 7-N-(β-alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate salt 27a is a chromogenic substrate for this bacterium, with a low degree of diffusion in nutrient media for growing bacterial cultures and a bright red colour, making it easily distinguishable from the agar background. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b716978g

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Volume 6 | Number 4 | 21 February 2008 | Pages 613–800
ISSN 1477-0520
FULL PAPER
2WT\XRP[ꢀBRXT]RT
Paul Groundwater et al.
Synthesis and testing of chromogenic
phenoxazinone substrates for β-alanyl
aminopeptidase
In this issue...
1477-0520(2008)6:4;1-D

PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and testing of chromogenic phenoxazinone substrates for b-alanyl
aminopeptidase†
Andrey V. Zaytsev,a Rosaleen J. Anderson,*a Alexandre Bedernjak,a Paul W. Groundwater,*a Yongxue Huang,a
John D. Perry,b Sylvain Orenga,c Celine Roger-Dalbertc and Arthur Jamesd
Received 2nd November 2007, Accepted 6th December 2007
First published as an Advance Article on the web 4th January 2008
DOI: 10.1039/b716978g
Novel 7-N-(b-alanyl)aminophenoxazin-3-one salts 27a–d have been synthesized and tested as
chromogenic substrates for b-alanyl aminopeptidase, which is present in Pseudomonas aeruginosa, the
most common respiratory pathogen in patients with cystic fibrosis. The biological results show that
7-N-(b-alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate salt 27a is a chromogenic substrate for
this bacterium, with a low degree of diffusion in nutrient media for growing bacterial cultures and a
bright red colour, making it easily distinguishable from the agar background.
Techniques for the detection and differentiation of different species
of bacteria often utilize the action of bacterial enzymes on
chromogenic or fluorogenic substrates1—liberating coloured or
fluorescent products, respectively. Such substrates are of particular
interest as they allow the rapid and accurate detection of bacteria,
since these tests may be performed on the primary isolation media,
thus avoiding time-consuming isolation procedures prior to the
bacterial identification step.
reported and compared to a monoclonal antibody detection
method.12
Phenoxazinones are the chromophores in a number of synthetic
dyestuffs and natural coloured matters and the phenoxazinone
chromophore has been found in a number of mould metabolites
and other natural products. The actinomycins13 are a group of
antibiotics produced by Streptomyces species, which are very
toxic but some of which show chemotherapeutic activity in
certain neoplastic diseases. The actinomycins contain a 2-amino-
4,6-dimethylphenoxazin-3(3H)one-1,9-dicarboxylic acid nucleus
1 bound via peptide links at the carboxyl groups to pentapeptide
side chains. Compounds with different amino acid sequences in
the peptide chains have been isolated, e.g. actinomycin A, and
synthesised, as have their analogues.13
We wish to report here the preparation and testing of chro-
mogenic phenoxazinone substrates for b-alanyl aminopeptidase.
b-Alanyl aminopeptidase has been detected in Pseudomonas sp.,2
while alanyl aminopeptidase is a periplasmic aminopeptidase
present in significant quantities in Pseudomonas aeruginosa,3
the most common respiratory pathogen in patients with cystic
fibrosis.4 P. aeruginosa is also responsible for 28% of the cases
of bacteremia in patients receiving organ transplants5 and causes
urinary tract infections and a variety of systemic infections in
patients with severe burns,6 and in cancer and AIDS patients
who are immunosuppressed.7,8 It has been shown that in such
patients as few as 10–100 cells of Pseudomonas aeruginosa can
lead to gut colonization by this Gram-negative aerobic bacterium,9
which is naturally resistant to many antibiotics.10,11 Although
classical microbiological techniques for the detection and iden-
tification of P. aeruginosa are satisfactory in many situations,
methods for the rapid detection of this bacterium are still under
investigation. For example, P. aeruginosa identification using
a disk of phenanthroline and 9-chloro-9-[4-(dimethylamino)]-
9,10-dihydro-10-phenylacridine hydrochloride (PC disk) has been
Results and discussion
7-Amino-1-pentylphenoxazin-3-one (1-pentylresorufamine [PRF])
6a can be prepared by nitrosation of 3-acetamidophenol 2,
condensation of the resulting 2-nitroso-5-acetamidophenol 3
with olivetol 4 and acidic hydrolysis of amide 5 using 85%
H2SO4, Scheme 1 (steps a–c),14 or by the reaction of 1,4-
dichlorobenzoquinonediimine 8, generated in situ by the oxidative
chlorination of p-phenylenediamine 7,15 with olivetol 4,14 Scheme 1
(step d). The overall yield and purity of the resorufamines 6
prepared via these routes is poor, so that these methods are not
suitable for multi-gram syntheses or the preparation of a range
of derivatives, and an alternative route to these phenoxazinones
is thus required. In particular, the route from the diimine leads
to the production of chlorinated derivatives, e.g. the 2-chloro-1-
pentyl derivative 6b, which are difficult to separate from the desired
1-pentylresorufamine 6a.
aPharmacy, Chemistry and Biomedical Sciences, University of Sunderland,
Sunderland, SR1 3SD, U.K.. E-mail: paul.groundwater@sunderland.ac.uk;
Fax: +44(0)191 515 3405; Tel: +44(0)191 515 2600
bDepartment of Microbiology, Freeman Hospital, Newcastle upon Tyne,
NE7 7DN U.K. E-mail: john.perry@nuth.northy.nhs.uk; Fax: +44(0)191
223 1224; Tel: +44(0)191 284 3111 × 26691
cR & D Microbiology, bioMerieux, 3 Route del Port Michaud, 28 390
La Balme-les-Grottes, France. E-mail: sylvain.orenga@eu.biomerieux.com;
Fax: +33 (0)474 952 632; Tel: +33 (0)474 952 543
dSchool of Applied Sciences, Ellison Place, Northumbria University, New-
castle upon Tyne, NE1 8ST, U.K.
† Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b716978g
682 | Org. Biomol. Chem., 2008, 6, 682–692
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The Royal Society of Chemistry 2008
©

Scheme 1 Reagents and conditions: [a] ‘HNO2’; [b] nBuOH, conc. H2SO4; [c] 85% H2SO4, ethanol; [d] MeOH, CO(NH2)2, Cl2, then 4, reflux.
Similar heterocyclic systems 10 have previously been synthesised
by Bird and Latif16 via the reductive cyclisation of 3-hydroxy-
2ꢀ-nitrodiphenyl ethers 9, Scheme 2, but the preparation of
resorufamines by this method was unsuccessful. An alternative
route was therefore investigated, involving the reduction of a
dinitrodihydroxydiaryl ether 14, which can be prepared via the
coupling of 2,5-dinitrofluorobenzene 11 with suitably substituted
phenols 12, Scheme 3, prepared via a sequence of reactions in-
volving; methylation of a hydroquinone, formylation and Baeyer–
Villiger oxidation.
Scheme 2 Reagents and conditions: [a] Zn, NH4Cl, H2O, DME, 40 ◦C.
Alkylation of hydroquinones 15a–d and monomethyl ether
15e with methyl iodide was carried out using an adaptation
of the method of Michman et al.17 and the yields of the
corresponding dimethyl ethers 16a–e were excellent (82–99%),
Scheme 4. The introduction of the hydroxyl group onto the
aromatic ring, to form the phenols 12, was then accomplished
via the Baeyer–Villiger oxidation of the corresponding benzalde-
hydes 17, which were prepared using the Duff reaction,18 with
Scheme 3 Reagents and conditions: [a] NaH, DMF, 40 ◦C; [b] BBr3, DCM,
−78 ◦C; [c] 5% Pd-on-C, MeOH then air/silica.
hexamine (hexamethylenetetramine) as the formylating agent, in
refluxing trifluoroacetic acid. No product was isolated from the
attempted formylation of the tert-butyl substituted hydroquinone
ethers 16a,d,e, Scheme 3, presumably due to the cumulative
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Scheme 5 Reagents and conditions: [a] nBuLi, THF, −78 ◦C then B(OPri)3;
[b] 10% NH4Cl; [c] 27% H2O2, THF.
dinitrofluorobenzene 11 and this proceeded smoothly to give the
ethers 13a–c in good yields, as shown in Scheme 3. Cleavage
of the methoxyaryl ethers 13 was carried out under standard
conditions, with BBr3 in DCM at −78 ◦C, and the resulting
hydroquinones 14a,b were subjected to further transformation
without purification, Scheme 3.
The final stage in the preparation of aminophenoxazinones
6c and 6d, involved the reduction of the nitro groups in ethers
14a,b with palladium-on-carbon under hydrogen (1 atm.), fol-
lowed by aerial oxidation and subsequent cyclization on work-
up/chromatography on silica.
The reduction of the nitro groups in the ethers 14 leads to the
formation of the diamine 20a, which can exist as a zwitterion
20b. This zwitterion undergoes oxidation by molecular oxygen,19
involving the autoxidation of the dianion, Scheme 6, in a process
similar to the autoxidation of duroquinone dianion 24, Scheme 7.
By analogy, this process would be expected to be autocatalytic,
Scheme 4 Reagents and conditions: [a] NaH, MeI, DMF, 40 ◦C, 1 h; [b]
hexamine, TFA, reflux; [c] i, MMPP, MeOH; ii, NaOH then HCl.
steric hindrance of the bulky tert-butyl and methoxy groups.
Baeyer–Villiger oxidation of aldehydes 17a,b with magnesium
monoperoxyphthalate (MMPP), followed by the hydrolysis of the
formate esters, gave the corresponding phenols 12a,b, Scheme 4.
A similar steric effect was observed during ortho-lithiation of
ether 16a with n-BuLi in THF at −78 ◦C. The anion initially
formed reacts with triisopropyl borate to give the corresponding
arylboronic ester 18, hydrolysis of which, under acidic conditions,
afforded boronic acid 19, which was oxidised with 27% H2O2 to
form phenol 12c in a total yield of 41%, Scheme 5.
The next step in the preparation of the diaryl ethers is
the nucleophilic aromatic substitution of the fluorine in 2,5-
Scheme 6
684 | Org. Biomol. Chem., 2008, 6, 682–692
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©

Scheme 7
to be accelerated by the quinone, and to proceed at a rate which
Compounds 27a–d are weakly coloured as the amino lone-
pair is no longer available for conjugation through the system
to the carbonyl group at the 3-position. These compounds
were tested for their activity to act as a substrate for the
enzyme, b-alanyl aminopeptidase, which is present in significant
amounts in Pseudomonas aeruginosa. b-Alanyl aminopeptidase
cleaves these chromatogenic substances to release the coloured 7-
aminophenoxazinones 6 and one of the advantages of this test is
the simple visual interpretation of the results, which can be carried
out without the use of any specialized instrumentation.
is independent of oxygen pressure. The addition of an oxygen-
transfer catalyst, salcomine 25, did not influence the rate, thus
confirming that this is an autocatalytic oxidation. According to
this mechanism, the semiquinone 21 formed in the first step
undergoes attack by a hydroxyperoxide radical, resulting in the
formation of a biradical 22. Such biradicals readily self-terminate
through the formation of a quinone 23. In a slightly acidic medium,
in this case due to the presence of the silica, this quinone undergoes
intramolecular cyclization to the resorufamine 6, Scheme 6.
7-Amino-1-pentyl-3H-phenoxazin-3-one (1-pentylresorufamine
[PRF]) 6a, which is produced by the b-alanyl aminopeptidase
hydrolysis of the 7-N-(b-alanyl)amino derivative 27a (b-Ala-
PRF), was shown to be a very good chromophore, having a
low degree of diffusion in nutrient media for growing bacterial
cultures and a bright red colour, making it easily distinguishable
from a pale yellow agar background. Fig. 1 shows the results
obtained after 24 hours incubation using 7-N-(b-alanyl)amino-1-
pentylphenoxazin-3-one (b-Ala-PRF) 27a, 7-N-(b-alanyl)amino-
1,2-dimethyl-(b-Ala-diMRF) 27c and 7-N-(b-alanyl)amino-1,2,4-
trimethyl-3H-phenoxazin-3-one (b-Ala-triMRF) 27d trifluoroac-
etate salts at a concentration of 50 mg l−1 in the presence of
Pseudomonas aeruginosa, Burkholderia cepacia, and Escherichia
coli. The selectivity of these substrates for P. aeruginosa is clearly
shown by the absence of the red colour in the case of Escherichia
coli, Fig. 1, and Pseudomonas fluorescens, Fig. 2, which do not have
b-alanyl aminopeptidase and so cannot release the chromogen.
Burkholderia cepacia strains may exhibit b-alanyl aminopeptidase
For the biological tests, the aminophenoxazinones 6a–d were
coupled to Boc-b-Ala-OH using N,Nꢀ-diisopropylcarbodiimide
as the coupling reagent, affording the protected monopeptides
26, Scheme 8. The Boc group was then removed under standard
conditions (TFA in DCM) to give the corresponding trifluoroac-
etate salts 27. It should be noted that the nucleophilicity of the
amino group in phenoxazinones 6 is very low and coupling by
this method is impossible without reduction prior to the coupling,
Scheme 8. In a similar manner, 7-N-(L-Ala)aminophenoxazinones
29a,b were prepared by the coupling of the aminophenaxozinones
6a–d with Boc-L-Ala-OH to give the carbamates 28a,b, Scheme 9,
followed by deprotection.
Scheme 8 Reagents and conditions: [a] 5% Pd-on-C, DMF, N-tBoc-b-alanine, HOBt, DIC, DCM; [b] TFA.
Scheme 9 Reagents and conditions: [a] 5% Pd-on-C, DMF, N-tBoc-L-alanine, HOBt, DIC, DCM; [b] TFA.
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©

Table 2 Detection of L-alanyl aminopeptidase activity via the hydrolysis
of L-Ala-pentylresorufamine 29a (50 mg l−1). Positive results are indicated
by a purple colour, and + and − signify the growth or inhibition of growth
of the bacteria, respectively
Species
Growth
Colour
Escherichia coli NCTC 10418
Klebsiella pneumoniae NCTC 10896
Salmonella hadar
Proteus mirabilis NCTC 10975
Providencia rettgeri NCTC 7475
Pseudomonas aeruginosa NCTC 10662
Staphylococcus aureus NCTC 6571
Listeria monocytogenes NCTC 11994
Streptococcus pyogenes NCTC 8306
+
+
+
+
+
+
−
−
−
Purple
Purple
Purple
Purple
Purple
Purple
No colour
No colour
No colour
Fig. 1 Incubation of 7-N-(b-alanyl)amino-1-pentylphenoxazin-3-one
(b-Ala-PRF) 27a, 7-N-(b-alanyl)amino-1,2-dimethyl-(b-Ala-diMRF)
27c and 7-N-(b-alanyl)amino-1,2,4-trimethyl-3H-phenoxazin-3-one
(b-Ala-triMRF) 27d trifluoroacetate salts, at a concentration of 50 mg
l−1, for 24 hours, with Pseudomonas aeruginosa, Burkholderia cepacia,
and Escherichia coli. In each photograph, the Pseudomonas aeruginosa
plates are top left and bottom left, Burkholderia cepacia top right, and
Escherichia coli bottom right.
chromogen for good localisation. The dimethyl substitution in 6c
(generated by the hydrolysis of 27c) is not sufficient to localise
this chromogen and the colour is seen to be diffused across
the medium. The trimethyl substituted 7-aminophenoxazin-3-one
6d (from 27d) is visibly localised, although not as efficiently as
the pentyl-substituted derivative (b-Ala-PRF) 6a. The increased
lipophilicity of the substituted chromogens aids in the retention of
the colour in the cell wall of the bacterium and also results in an
increase in the hydrophobic properties, which prevents the spread
of chromogen into the medium. The same is true with an increase
in length of the alkyl chain (as in the case of b-Ala-PRF 6a), Fig. 1.
The data in Table 1 shows that b-Ala-PRF 27a can be used
to distinguish between P. aeruginosa and other non-fermenting
bacteria, since b-alanyl aminopeptidase activity is present in all P.
aeruginosa strains but not in Burkholderia gladioli, Acinetobacter
sp., Stenotrophomonas maltophilia, Brevindumonas sp. or Chry-
seobacterium meningosepticum. The detection of 99% of strains
using the pentyl derivative 27a as a test substrate for the detection
of P. aeruginosa is vitally important as a recent study of 1225
isolates from cystic fibrosis patients from 31 centres in the U.K.
showed that, although cross-infection by transmissable strains
does occur, at least 72% of patients harboured strains with unique
genotypes.20
activity (see data in Table 1) and the presence of a weak red
colouration indicates some enzymatic activity for this strain.
In contrast, the L-Ala derivative 29a is hydrolyzed by all the
Gram-negative bacteria tested, Table 2, including Escherichia coli
(NCTC 10418), thus suggesting that b-alanyl aminopeptidase is
responsible for the specific hydrolysis of the b-Ala analogue 27a.
Another requirement of a test substrate is that it should localise
to the bacterial cells, aiding the visualisation of colonies. Fig. 1
also highlights the importance of lipophilic substituents on the
Table 1 Assessment of b-Ala-pentylresorufamines 27a,b (50 mg l−1) as
substrates for non-fermenting bacteria (% of strains with positive response
due to b-alanyl aminopeptidase activity)
Species
No. of strains
27a
27b
Burkholderia cepacia
Burkholderia gladioli
Other Burkholderia sp.
Pseudomonas aeruginosa
Other Pseudomonas sp.
Pandorea sp.
34
6
9
74
17
8
4
16
1
2
7
47
0
26
83
44
73
24
25
50
13
0
0
14
0
100
33
0
33
99
41
25
50
19
0
0
0
0
0
It has therefore been established that compounds 27a,c,d are
sensitive, effective and specific for the detection of Pseudomonas
aeruginosa, but in the case of 27c, spreading of the colour of
the chromogen 6c from the colonies into the medium prevents
it from being used as a diagnostic tool on agar plates (it
could, however, be used in solution-based tests). In addition, 7-
N-(b-alanyl)amino-1-pentylphenoxazin-3-one (b-Ala-PRF) 27a,
like the L-Ala derivative 29a, inhibited the growth of Gram-
positive bacteria, e.g. Staphylococcus aureus, Enterococcus faecalis,
Ralstonia picketti
Other Ralstonia sp.
Sphingobacterium spiritivorum
Stenotrophomonas maltophilia
Acinetobacter sp.
Brevindumonas sp.
2
1
3
1
Chryseobacterium meningosepticum
Moraxella sp.
67
100
Oligella sp.
Fig. 2 Incubation of 7-N-(b-alanyl)amino-1,2-dimethylpentylphenoxazin-3-one (b-Ala-diMRF) 27c trifluoroacetate salt, at a concentration of 50 mg
l−1, for 18 hours, with Pseudomonas aeruginosa and Pseudomonas fluorescens. From left, dishes 1–4 Pseudomonas aeruginosa and dish 5 Pseudomonas
fluorescens.
686 | Org. Biomol. Chem., 2008, 6, 682–692
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Table 3 Impact of 7-N-(b-alanyl)amino-1-pentylphenoxazin-3-one 27a
(50 mg l−1) in Columbia agar medium on growth and colour of colonies of
different strains of bacteria after 24 hours of incubation. Positive results are
indicated by a purple colour, and + and − signify the growth or inhibition
of growth of the bacteria, respectively
portions. After the base had been added and the evolution of
H2 had ceased, methyl iodide (4 equiv.) was added dropwise over
15–20 min. When the addition was finished, the reaction mixture
was stirred at 40 ◦C for 2 hours. Brine (200 ml) was added to the
flask and the resulting mixture was extracted with diethyl ether
(3 × 50 ml). The combined organic layers were washed with water
(2 × 50 ml) and dried over MgSO4. The solvent was evaporated
under reduced pressure and the residue was subjected to column
chromatography.
Species
Growth
Colour
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilis
+
+
+
+
+
+
−
−
−
−
No colour
No colour
No colour
Red
No colour
Red
No colour
No colour
No colour
No colour
No colour
Pseudomonas aeruginosa
Pseudomonas fluorescens
Burkholderia cepacia
Staphylococcus aureus
Streptococcus pyogenes
Enterococcus faecalis
Listeria monocytogenes
Candida albicans
2-tert-Butyl-1,4-dimethoxybenzene 16a
Prepared from 2-tert-butylhydroquinone 15a (2.90 g, 17.5 mmol)
and purified by column chromatography using petroleum ether
(60–80 ◦C)–diethyl ◦ether (95 : 5) as eluent; yellow oil (3.35 g,
99%) (lit.17 bp 240 C/50 mm Hg) (found: M+, 194.1301. Calc.
for C12H18O2: M, 194.1316); dH (300 MHz; CDCl3) 1.40 (9H, s,
C(CH3)3), 3.80 (3H, s, OCH3), 3.83 (3H, s, OCH3), 6.72 (1H, dd,
J = 8.8 Hz and J = 3.1 Hz, H-5), 6.84 (1H, d, J = 8.8 Hz, H-6),
6.93 (1H, d, J = 3.1 Hz, H-3); dC (75 MHz; CDCl3) 30.1 (CH3,
C(CH3)3), 35.3 (quat., C(CH3)3), 56.0 (CH3, OCH3), 56.0 (CH3,
OCH3), 110.3 (CH, C-5), 112.8 (CH, C-6), 114.7 (CH, C-3), 140.3
(quat., C-2), 153.4 (quat., C-1 or C-4), 153.7 (quat., C-4 or C-1).
Listeria monocytogenes, and Streptococcus pyogenes, Table 3, when
incorporated into Columbia agar at 50 mg l−1, and this suppression
of the growth of Gram-positive bacteria in the presence of Gram-
negative bacteria is a useful additional property when developing
a selective means for the detection of Pseudomonas aeruginosa.
These b-Ala-resorufamines 27 represent some of the first
chromogenic substrates which can be used directly in culture media
to differentiate bacterial colonies based upon the detection of b-
alanyl aminopeptidase activity.
1,4-Dimethoxy-2,3-dimethylbenzene 16b
Prepared from 2,3-dimethylhydroquinone 15b (1.96 g, 14.2 mmol)
and purified by column chromatography using petroleum ether
(60–80 ◦C)–diethyl ether (95 : 5) as eluent; white solid (2.27 g,
80%); mp 75–76 C (lit.21 mp 73–74 C); dH (300 MHz; CDCl3)
2.09 (6H, s, 2 × CH3), 3.70 (6H, s, 2 × OCH3), 6.58 (2H, s, 2 ×
ArH); dC (75 MHz; CDCl3) 12.4 (CH3, 2 × ArCH3), 56.5 (CH3,
2 × OCH3), 108.4 (2 × CH), 127.1 (2 × quat.), 152.4 (2 × quat.).
Experimental
General
◦
◦
Melting points were determined on a Gallenkamp apparatus
and are uncorrected. Elemental analyses were performed on an
Exeter Analytical CE-440 Elemental Analyzer. IR spectra were
recorded on a Perkin-Elmer 1600 series FTIR spectrophotometer.
1H NMR spectra were acquired on a Bruker AVANCE 300 at
300 MHz or AVANCE 500 at 500 MHz. Coupling constants are
given in Hz and all chemical shifts are relative to the chemical
shift of the residual non-deuterated solvent. 13C NMR spectra
were obtained on the Bruker AVANCE 300 at 75 MHz. Low
resolution electrospray mass spectra were obtained on a Bruker
Esquire 3000+ and high resolution spectra on a Bruker APEX II
FT mass spectrometer. Thin layer chromatography was performed
on Merck silica gel 60F254. All solvents were purified according
to standard procedures. Diethyl ether and tetrahydrofuran were
freshly distilled over sodium wire with a trace of benzophenone.
Fisons silica gel 60 (35–70 micron) was used for wet flash
chromatography. The samples were applied in liquid form or were
pre-adsorbed onto silica 60 (35–70 micron). Experimental and
spectroscopic data are given for one example of each synthetic
method; data for the other compounds included in this work are
reported in the ESI accompanying this paper.†
Formylation of dimethoxybenzenes via the Duff reaction
The dimethoxybenzene (1 equiv.) was dissolved in TFA (20 ml) and
hexamine (1.05 equiv.) was added to the resulting solution. The
reaction mixture was refluxed under dry conditions for 2 hours.
The TFA was evaporated under reduced pressure, the residue was
dissolved in ether (100 ml) and the organic solution was washed
with water (3 × 50 ml) and then dried over MgSO4. The solvent was
evaporated and the residue subjected to column chromatography,
eluting with petroleum ether (60–80 ◦C)–diethyl ether (80 : 20).
2,5-Dimethoxy-3,4-dimethylbenzaldehyde 17a
Prepared from 1,4-dimethoxy-2,3-dimethylbenzene 16b (2.270 g,
13.7 mmol). 2,5-Dimethoxy-3,4-dimethylbenzaldehyde ◦17a was
isolated as a white solid (1.18 g, 44%); mp 61–62 C (lit.22
◦
mp 67.5–68.5 C) (found: MH+, 195.1019. Calc. for C11H15O3:
MH, 195.1016); mmax (KBr)/cm−1 1685 (C O), 1595 (C C); dH
(300 MHz; CDCl3) 2.26 (3H, s, CH3), 2.29 (3H, s, CH3), 3.83
(3H, s, OCH3), 3.85 (3H, s, OCH3), 7.16 (1H, s, H-6), 10.38 (1H, s,
CHO); dC (75 MHz; CDCl3) 12.5 (CH3), 13.3 (CH3), 56.1 (OCH3),
64.2 (OCH3), 105.4 (CH, C-6), 127.0 (quat., C-1), 132.3 (quat., C-
3), 135.8 (quat., C-4), 154.7 (quat., C-2), 156.9 (quat., C-5), 190.4
(CHO).
=
=
General procedure for the preparation of dimethoxybenzenes
16a–e17
In a dry 2-necked round bottom flask equipped with a condenser,
a magnetic stirring bar and a calcium chloride guard tube, the
hydroquinone 15 (1 equiv.) was dissolved in dry DMF (50 ml)
and NaH (2.2 equiv., 60% dispersion in oil) was added in small
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2,5-Dimethoxy-3,4,6-trimethylbenzaldehyde 17b
diethyl ether (3 × 50 ml), and dried over MgSO4. The solvent was
removed under vacuum and the residue was purified by column
chromatography using light petroleum (60–80 ◦C)–ethyl acetate
(80 : 20) as eluent to give the title compound◦12c as a white solid
(1.010 g, 41%), mp 46–47 ◦C (lit.24 mp 54–55 C); mmax(film)/cm−1
Prepared from 1,4-dimethoxy-2,3,5-trimethylbenzene 16c
(2.274 g, 12.6 mmol). 2,5-Dimethoxy-3,4,6-trimethylbenzaldehyde
17b was isolated as a yellow solid (1.21 g, 46%); mp 65–66 ◦C
◦
(lit.23 mp 80 C) (found: MH+, 209.1176. Calc. for C12H17O3:
=
3542, 3433 (OH), 1600 (C C); dH (300 MHz; CDCl3) 1.22 (9H, s,
MH, 209.1172); mmax (KBr)/cm−1 1685 (C O), 1586 (C C), 1255
(C–O); dH (500 MHz; CDCl3) 2.23 (3H, s, CH3), 2.31 (3H, s, CH3),
2.53 (3H, s, CH3), 3.70 (3H, s, OCH3), 3.82 (3H, s, OCH3), 10.47
(1H, s, CHO); dC (125 MHz; CDCl3) 12.5 (CH3), 13.3 (CH3),
14.2 (CH3), 60.7 (OCH3), 63.8 (OCH3), 126.0 (quat., C-1), 129.7
(quat., C-3), 132.1 (quat., C-6), 140.2 (quat., C-4), 153.6 (quat.,
C-2), 160.0 (quat., C-5), 194.9 (CHO).
=
=
C(CH3)3), 3.61 (3H, s, OCH3), 3.71 (3H, s, OCH3), 6.43 (1H, s,
H-6), 6.70 (1H, s, H-3); dC (75 MHz; CDCl3) 30.6 (CH3, C(CH3)3),
34.9 (quat., C(CH3)3), 56.1 (OCH3), 57.3 (OCH3), 100.9 (CH, C-
6), 111.2 (CH, C-3), 130.1 (quat., C-4), 139.9 (quat.), 144.6 (quat.),
153.6 (quat.).
General preparation of biaryl ethers 13a–c
2,5-Dimethoxy-3,4-dimethylphenol 12a
The appropriate phenol (1 equiv.) was dissolved in dry DMF (10
ml) and NaH (60% dispersion in oil; 1.1 equiv.) was added in small
portions. After the evolution of gas was complete, the resulting
solution of the sodium phenolate was stirred at room temperature
for 15 min. A solution of 2,5-dinitrofluorobenzene 11 (1 equiv.)
in dry THF (5 ml) was then added dropwise to the flask and the
reaction mixture was stirred for 2 hours. Finally, the contents of
the flask were poured into water (50 ml), extracted with ether (3 ×
20 ml) and the combined organic layers were dried over MgSO4.
The solvent was removed under reduced pressure and the residue
was subjected to column chromatography on silica.
Prepared from 3,4-dimethyl-2,5-dimethoxybenzaldehyde 17a
(1.126 g, 5.8 mmol). Using light petroleum (60–80 ◦C)–diethyl
ether (60 : 40) as eluent, 2,5-dimethoxy-3,4-dimethylphenol 12a
was isolated as a yellow solid (0.239 g, 23%), mp 69–71 ◦C (lit.21
mp 70–71 ◦C) (found: C, 65.9; H, 7.7. C10H14O3 requires C, 65.9;
H, 7.7%); mmax (film)/cm−1 3263 (OH), 1598 (C C), 1261 (C–O);
=
dH (300 MHz; CDCl3) 2.09 (3H, s, CH3), 2.22 (3H, s, CH3), 3.74
(3H, s, OCH3), 3.78 (3H, s, OCH3), 4.74 (1H, br s, OH), 6.44
(1H, s, H-6); dC (75 MHz; CDCl3) 11.7 (CH3), 13.0 (CH3), 56.1
(OCH3), 61.4 (OCH3), 97.0 (CH, C-6), 117.4 (quat., C-4), 130.6
(quat., C-3), 139.5 (quat., C-2), 147.1 (quat., C-1), 154.7 (quat.,
C-5); m/z 183 (MH+).
1-(2ꢀ,5ꢀ-Dinitrophenoxy)-3,4-dimethyl-2,5-dimethoxybenzene 13a
2,5-Dimethoxy-3,4,6-trimethylphenol 12b
Prepared from 2,5-dinitrofluorobenzene 11 (0.293 g, 1.6 mmol)
and 2,5-dimethoxy-3,4-dimethylphenol 12a (0.287 g, 1.6 mmol).
Purified using light petroleum (60–80 ◦C)–ethyl acetate (85 :
15) as eluent to give 1-(2ꢀ,5ꢀ-dinitrophenoxy)-3,4-dimethyl-2,5-
dimethoxybenzene 13a as an orange solid (0.415 g, 76%); mp
135–136 ◦C; (found: MH+, 349.1030. Calc. for C16H17N2O7: MH,
349.1017); mmax (KBr)/cm−1 1551, 1346 (NO2), 1246 (C–O); dH
(300 MHz; CDCl3) 2.10 (3H, s, CH3), 2.17 (3H, s, CH3), 3.58
(3H, s, OCH3), 3.71 (3H, s, OCH3), 6.49 (1H, s, H-6), 7.52 (1H, d,
J = 2.2 Hz, H-6ꢀ), 7.84 (1H, dd, J = 8.8 Hz and 2.2 Hz, H-4ꢀ), 7.93
(1H, d, J = 8.8 Hz, H-3ꢀ); dC (75 MHz; CDCl3) 12.4 (CH3), 13.0
(CH3), 56.3 (OCH3), 61.7 (OCH3), 102.6 (CH, C-6), 112.8 (CH,
C-6ꢀ), 116.7 (CH, C-4ꢀ), 125.5 (quat.), 126.4 (CH, C-3ꢀ), 133.7
(quat.), 143.1 (quat.), 143.6 (quat.), 150.8 (quat.), 152.6 (quat.),
154.8 (quat.).
Prepared from 2,5-dimethoxy-3,4,6-trimethylbenzaldehyde 17b
(1.205 g, 5.8 mmol). Using light petroleum (60–80 ◦C)–diethyl
ether (75 : 25) as eluent, 2,5-dimethoxy-3,4,6-trimethylphenol 12b
was isolated as a white solid (0.856 g, 75%) mp 105–106 ◦C,
(found: C, 67.4; H, 8.2. C11H16O3 requires C, 67.3; H, 8.2%); mmax
(KBr)/cm−1 3401 (OH), 1265 (C–O); dH (300 MHz; CDCl3) 2.18
(3H, s, CH3), 2.23 (6H, s, 2 × CH3), 3.71 (3H, s, OCH3), 3.77 (3H, s,
OCH3), 5.71 (1H, br s, OH); dC (75 MHz; CDCl3) 9.1 (CH3), 12.0
(CH3), 12.5 (CH3), 60.2 (OCH3), 60.9 (OCH3), 115.0 (quat.), 121.0
(quat.), 126.8 (quat.), 141.7 (quat.), 145.3 (quat.), 153.4 (quat.).
Preparation of 4-tert-butyl-2,5-dimethoxyphenol 12c
In a flame-dried flask, 2-tert-butyl-1,4 dimethoxybenzene 16a
(2.27 g, 11.7 mmol) was dissol◦ved in dry THF (30 ml), the resulting
solution was cooled to −78 C and a solution of BuLi (2.82 M
in hexanes, 5.0 ml, 14.0 mmol) was added dropwise. After the
addition was complete, the reaction mixture was allowed to warm
to room temperature, stirred for 15 min at this temperature and
cooled again to −78 ◦C. Triisopropyl borate (3.2 ml, 0.01402 mol)
was introduced dropwise and the reaction mixture was left to stir
at room temperature. After 12 hours the reaction mixture was
quenched with 10% NH4Cl solution (50 ml) and water (50 ml) and
extracted with diethyl ether (3 × 50 ml). The combined organic
layers were washed with brine (50 ml) and dried over MgSO4. The
solvent was evaporated under reduced pressure, the residue was
dissolved in THF (10 ml) and aqueous H2O2 (27% w/v, 5 ml) was
added. The emulsion was stirred at room temperature for 30 min.
Water (100 ml) was added and the mixture was extracted with
General procedure for preparation of 7-aminophenoxazin-3-ones
6c,d
The dihydroxydiaryl ether (1.3 mmol) was dissolved in methanol
(5 ml) and 5% Pd/C (10% w/w) was added to the solution. The
reaction mixture was stirred at room temperature in a hydrogena-
tor under a hydrogen atmosphere for 4 hours. Sufficient silica to
adsorb the residue for the subsequent column chromatography
was added to the flask, and the mixture was stirred vigorously
for a further 4 hours in air. When the oxidation was complete,
the solvent was removed and the residue was subjected to column
chromatography on silica eluting with light petroleum (60–80 ◦C)–
ethyl acetate (50 : 50 to 0 : 100) then with ethyl acetate–methanol
(90 : 10).
688 | Org. Biomol. Chem., 2008, 6, 682–692
This journal is
The Royal Society of Chemistry 2008
©

7-Amino-1,2-dimethylphenoxazin-3-one 6c
N-methylmorpholine (0.408 g, 4.0 mmol) were dissolved in dry
THF (10 ml), the solution was cooled to −20 ◦C and isobutyl
chloroformate (0.56 ml, 4.0◦mmol) was added with stirring. The
mixture was stirred at −20 C for a further 30 min, after which
time the mixture was introduced into the reduced resorufamine
Prepared from 1-(2ꢀ,5ꢀ-dinitrophenoxy)-3,4-dimethyl-2,5-dime-
thoxybenzene 13a (0.266 g, 0.76 mmol) in a one-pot reaction.
7-Amino-1,2-dimethylphenoxazin-3-one 6c was obtained as a
brown-red solid (0.125 g, 68%); mp > 270 ◦C; mmax (KBr)/cm−1
◦
solution at −10 C with the continued passage of hydrogen gas.
=
=
3315, 3201 (NH2), 1600 (C O), 1545 (C C); dH (300 MHz;
DMSO-d6) 2.02 (3H, s, CH3), 2.33 (3H, s, CH3), 6.10 (1H, s,
H-4), 6.47 (1H, d, J = 2.3 Hz, H-6), 6.67 (1H, dd, J = 8.8 and
Hz, H-8), 6.73 (2H, s, NH2), 7.48 (1H, d, J = 8.8 Hz, H-9); dC
(75 MHz; DMSO-d6) 13.3 (CH3), 13.5 (CH3), 98.0 (CH, C-6),
104.4 (CH, C-4), 113.8 (CH, C-8), 125.9 (quat., C-7), 132.6 (CH,
C-9), 136.7 (quat.), 138.1 (quat.), 141.0 (quat.), 147.0 (quat., C-9a),
150.3 (quat., C-4a), 155.3 (quat., C-5a), 184.4 (quat., C-3).
After 15 min, hydrogen was no longer admitted, the system
was sealed and the reaction mixture was stirred overnight at
room temperature. The reaction mixture was filtered and solvent
was evaporated under reduced pressure, the residual solid was
dissolved in DCM (50 ml), filtered, and the DCM solution
washed with NaHCO3 (5%, 2 × 50 ml) and water (50 ml). The
organic phase was dried (MgSO4), filtered and concentrated to
afford a residue consisting of two products, which was purified
by column chromatography on silica, eluting with petrol–ethyl
acetate (6 : 4), to give 7-N-(N-tBoc-b-alanyl)amino-2-chloro-
1-pentylphenoxazin-3-one 26b (as the first spot) as an orange
7-Amino-1,2,4-trimethylphenoxazin-3-one 6d
1-(2ꢀ,5ꢀ -Dinitrophenoxy)-3,4,6-trimethyl-2,5-dihydroxybenzene
14b was prepared from 1-(2ꢀ,5ꢀ-dinitrophenoxy)-3,4,6-trimethyl-
2,5-dimethoxybenzene 13b (0.626 g, 1.73 mmol), using light
petroleum (60–80 ◦C)–diethyl ether (70 : 30) as eluent, and isolated
as an orange solid (0.435 g, 75%); mp 180–181 ◦C; (found: MH+,
255.1129. Calc. for C15H15N2O2: MH, 255.1128); mmax (KBr)/cm−1
◦
solid (0.12 g) mp 226–227 C; (found: MH+, 488.1942. Calc. for
C25H31ClN3O5 MH, 488.1945); mmax (KBr)/cm−1 3388 (NH), 3269
=
=
=
(NH), 1699 (C O), 1603 (C O), 1577 (C C); dH (300 MHz,
DMSO-d6) 0.89 (3H, t, J = 6.8 Hz, 5ꢀ-CH3), 1.38 (13H, m, 3ꢀ-CH2,
4ꢀ-CH2, C(CH3)), 1.59 (2H, m, 2ꢀ-CH2), 2.56 (2H, t, J = 6.8 Hz,
NHCH2CH2CO), 3.00 (2H, m, 1ꢀ-CH2), 3.25 (2H, q, J = 6.8 Hz,
NHCH2CH2CO), 6.40 (1H, s, H-4), 6.93 (1H, d, J = 4.95 Hz,
NH), 7.52 (1H, d, J = 8.4 Hz, H-8), 7.79 (1H, d, J = 8.2 Hz,
H-9), 7.93 (1H, s, H-6), 10.60 (1H, br, ArNH); dC (75.5 MHz,
DMSO-d6) 14.6 (CH3, C-5ꢀ), 22.6 (CH2, C-4ꢀ), 28.4 (CH2, C-2ꢀ),
28.5 (CH2, C-1ꢀ), 29.1 (3 × CH3), 32.0 (CH2, C-3ꢀ), 37.1 (CH2),
37.9 (CH2), 78.55 (quat.), 105.3 (CH, C-4), 108.2 (CH, C-6), 117.5
(CH, C-8), 129.6 (quat., C-7), 131.9 (CH, C-9), 136.7 (quat., C-2),
142.9 (quat., C-1), 143.65 (quat., C-9a), 144.5 (quat., C-5a), 145.1
=
=
3490 (NH2 and OH), 1606 (C O), 1591 (C C); dH (300 MHz;
DMSO-d6) 1.98 (3H, s, CH3), 2.11 (3H, s, CH3), 2.14 (3H, s, CH3),
7.23 (1H, d, J = 2.3 Hz, H-6ꢀ), 7.94 (1H, br s, OH), 7.99 (1H,
dd, J = 8.9 and 2.3 Hz, H-4ꢀ), 8.28 (1H, d, J = 8.9 Hz, H-3ꢀ),
8.38 (1H, br s, OH); dC (75 MHz; DMSO-d6) 10.6 (CH3), 13.3
(CH3), 13.7 (CH3), 111.0 (CH), 116.4 (quat.), 117.4 (quat.), 124.1
(quat.), 124.2 (quat.), 127.7 (CH), 137.6 (quat.), 140.7 (quat.),
143.3 (quat.), 146.9 (quat.), 150.9 (quat.), 152.0 (quat.).
1-(2ꢀ,5ꢀ-Dinitrophenoxy)-3,4,6-trimethyl-2,5-dihydroxybenzene
14b (0.435 g, 1.30 mmol) was then treated as described above to
give 7-amino-1,2,4-trimethylphenoxazin-3-one 6d as a brown-red
=
=
(quat., C-10a), 149.6 (quat., C-4a), 156.4 (C O), 171.4 (C O),
=
187.8 (C O, C-3).
◦
7-N-(N-tBoc-b-alanyl)amino-1-pentylphenoxazin-3-one 26a
solid (0.237 g, 72%); mp > 270 C; mmax (KBr)/cm−1 3320, 3211
(second spot) was obtained as an orange solid (0.11 g) mp
=
(NH2), 1610 (C C); dH (300 MHz; DMSO-d6) 1.96 (3H, s, CH3),
◦
212.5–214.0 C; (found: MH+, 454.2330. Calc. for C25H32N3O5:
2.04 (3H, s, CH3), 2.31 (3H, s, CH3), 6.49 (1H, d, J = 2.3 Hz,
H-6), 6.60 (2H, br s, NH2), 6.63 (1H, dd, J = 8.7 and 2.3 Hz,
H-8), 7.44 (1H, d, J = 8.7 Hz, H-9); dC (75 MHz; DMSO-d6)
8.5 (CH3), 13.3 (CH3), 13.6 (CH3), 98.2 (CH, C-6), 111.9 (quat.,
C-4), 113.3 (CH, C-8), 125.4 (quat., C-7), 132.3 (CH, C-9), 136.0
(quat.), 137.1 (quat.), 141.3 (quat.), 146.4 (quat., C-9a), 147.2
(quat., C-4a), 154.8 (quat., C-5a), 184.1 (quat., C-3).
MH, 454.2334); mmax (KBr)/cm−1 3379 (NH), 3265 (NH), 1703
=
=
=
=
(C O), 1647 (C O), 1612 (C O), 1591 (C C); dH (300 MHz,
CD3OD) 0.96 (3H, t, J = 7.0 Hz, 5ꢀ-CH3), 1.29–1.44 (15H,
m, 2ꢀ-CH2, 3ꢀ-CH2, 4ꢀ-CH2, C(CH3)3), 2.63 (2H, t, J = 6.6 Hz,
NHCH2CH2CO), 2.91 (2H, t, J = 8.0 Hz, 1ꢀ-CH2), 3.43 (2H, t, J
= 6.7 Hz, NHCH2CH2CO), 6.26 (1H, d, J = 2.1 Hz, H-4), 6.68
(1H, d, J = 2.1 Hz, H-2), 7.50 (1H, dd, J = 8.7 and 2.3 Hz, H-8),
7.81 (1H, d, J = 8.8 Hz, H-9), 8.01 (1H, d, J = 2.1 Hz, H-6); dC
(75.5 MHz, CD3OD) 14.4 (CH3, C-5ꢀ), 22.85 (CH2, C-4ꢀ), 28.8
(C(CH3)3), 29.1 (CH2, C-2ꢀ), 30.0 (CH2, C-1ꢀ), 32.0 (CH2, C-3ꢀ),
37.1 (CH2), 37.9 (CH2), 78.55 (quat.), 106.25 (CH, C-4), 106.4
(CH, C-6), 116.7 (CH, C-8), 129.75 (quat., C-7), 131.3 (CH, C-9),
131.75 (CH, C-2), 142.4 (quat., C-1), 144.9 (quat., C-9a), 146.8
(quat., C-5a), 147.65 (quat., C-10a), 150.4 (quat., C-4a), 157.3
7-N-(N-tBoc-b-alanyl)amino-1-pentylphenoxazin-3-one 26a and
7-N-(N-tBoc-b-alanyl)amino-2-chloro-1-pentylphenoxazin-3-one
26b
Acetic acid (30%, 200 ml) was added dropwise, with stirring,
to a solution in which sodium borohydride (4–6 g) and sodium
hydroxide (0.2 g) were dissolved in water (200 ml). The hydrogen
gas produced was passed into a three-necked flask in which a
mixture of 7-amino-1-pentylphenoxazin-3-one 6a and 7-amino-
2-chloro-1-pentylphenoxazin-3-one 6b14 (0.564 g) was dissolved
in dry DMF (15 ml), and the solution was diluted with dry
THF (15 ml). 5% Pd/C (0.2 g) was added and hydrogen gas was
bubbled slowly through the solution for 1 hour after the reduction
appeared to be complete, as evidenced by the replacement of
the purple colour of the solution by a weak grey-green colour.
In a separate flask, N-tBoc-b-alanine (0.756 g, 4.0 mmol) and
=
=
=
(carbamate C O), 172.25 (amide C O), 184.7 (C O, C-3).
General procedure for the peptide coupling of
7-aminophenoxazin-3-ones 6c,d
The 7-aminophenoxazin-3-one 6c,d (0.4 mmol) was dissolved in
dry DMF (5 ml) and 5% Pd/C (0.010 g) was added to the solution.
The flask was placed in a hydrogenator at room temperature and
an atmosphere of hydrogen was maintained while the reaction
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 682–692 | 689
©

mixture was stirred for 1 hour. The completion of the reduction
was indicated by the replacement of the deep purple colour of
the solution by a greyish-green colour. In a separate flask, N-
tBoc-b-alanine (0.089 g, 0.47 mmol), HOBt (0.072 g, 0.47 mmol),
and DIC (0.07 ml, 0.47 mmol) were dissolved in dry DCM
(5 ml) and the resulting mixture was stirred at room temperature
for 1 hour. After this period, the contents of the second flask
were introduced into the first flask (which contained the reduced
form of 7-aminophenoxazin-3-one) via syringe, under an inert
atmosphere. The mixture was stirred for a further 20 hours at
room temperature then filtered through celite and the solvent
evaporated under reduced pressure. The residue was redissolved
in ethyl acetate (20 ml), the organic layer was washed with 1 M
HCl (20 ml), 10% Na2CO3 (20 ml) and water (20 ml). The organic
solution was dried over MgSO4, filtered and evaporated under
reduced pressure to give a residue, which was purified by column
chromatography using light petroleum (60–80 ◦C)–ethyl acetate
(30 : 70) as eluent.
(quat., C-5a), 146.0 (quat., C-4a), 146.8 (quat., C-4a), 156.2 (quat.,
=
=
carbamate C O), 171.1 (quat., amide C O), 184.7 (quat., C-3).
Deprotection of N-tbutoxycarbonyl group
The corresponding N-tbutoxycarbonyl protected compound 26
(0.2 mmol) was dissolved in dry DCM (3 ml) and TFA (1 ml) or
neat TFA (2 ml) was added to the solution. The reaction mixture
was stirred at room temperature until completion of reaction (as
indicated by TLC). The solvent and excess of TFA were evaporated
under reduced pressure and the residue was purified by column
chromatography on silica, using a gradient eluent starting with
light petroleum (60–80 ◦C)–ethyl acetate (50 : 50 to 0 : 100) and,
finally, ethyl acetate–methanol (90 : 10).
7-N-(b-Alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate
salt 27a
Prepared from 7-N-(N-tBoc-b-alanyl)amino-1-pentylphenoxazin-
3-one 26a (80 mg, 0.18 mmol) and TFA (2 ml). After work-up, 7-N-
(b-alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate salt
27a was obtained as a brown solid (80 mg, 97%) mp 215–216 ◦C;
(found: M+, 354.1798. Calc. for C20H24N3O3: M, 354.1812); mmax
7-N-(N-tButoxycarbonyl-b-alanyl)amino-1,2-dimethylphenoxazin-
3-one 26c
Prepared from 7-amino-1,2-dimethylphenoxazin-3-one 6c
(0.110 g, 0.4578 mmol). 7-N-(N-tButoxycarbonyl-b-alanyl)amino-
1,2-dimethylphenoxazin-3-one 26c was obtained as a brown-red
(KBr)/cm−1 3448, 3259 (NH), 1678 (C O), 1647 (C O), 1612
=
=
◦
solid (0.104 g, 55%); mp 222–223 C (decomp.); (found: MH+,
=
(C O), 1250 (C–O); dH (300 MHz, DMSO-d6) 0.89 (3H, t, J =
6.9 Hz, 5ꢀ-CH3), 1.16 (4H, m, 3ꢀ-CH2, 4ꢀ-CH2), 1.62 (2H, m, 2ꢀ-
CH2), 2.77 (4H, m, 1ꢀ-CH2,CH2), 3.13 (2H, m, CH2), 6.19 (1H, d,
J = 1.4 Hz, H-4), 6.59 (1H, d, J = 1.4 Hz, H-2), 7.50 (1H, d, J =
412.1871. Calc. for C22H26N3O5: MH, 412.1867); mmax (KBr)/cm−1
=
=
3341, 3272 (NH), 1705, 1689 (C O), 1616 (C C), 1253 (C–O);
dH (300 MHz; DMSO-d6) 1.38 (9H, s, C(CH3)3), 2.06 (3H, s,
CH3), 2.36 (3H, s, CH3), 2.53–2.55 (2H, m, H-2ꢀ), 3.22–3.28 (2H,
m, H-3ꢀ), 6.22 (1H, s, H-4), 6.88 (1H, br s, NH), 7.48 (1H, dd, J =
8.7 and 2.0 Hz, H-8), 7.75 (1H, d, J = 8.7 Hz, H-9), 7.87 (1H, d,
J = 2.0 Hz, H-6), 10.47 (1H, s, ArNH); dC (75 MHz; DMSO-d6)
13.4 (CH3), 13.5 (CH3), 29.1 (CH3, C(CH3)), 37.3 (CH2, C-3ꢀ),
37.9 (CH2, C-2ꢀ), 78.5 (quat., C(CH3)3), 103.8, (CH), 105.5 (CH,
C-4), 117.0 (CH, C-8), 129.4 (quat., C-7), 131.3 (CH, C-9), 138.4
(quat.), 139.0 (quat.), 143.7 (quat.), 144.9 (quat.), 147.0 (quat.,
+
8.6 Hz, H-8), 7.8 (1H, d, J = 8.7 Hz, H-9), 7.9 (4H, br, H-6, NH3 ),
10.82 (1H, br, ArNH); dC (75.5 MHz, DMSO-d6) 14.7 (CH3, C-5ꢀ),
22.7 (CH2, C-4ꢀ), 29.1 (CH2, C-2ꢀ), 29.8 (CH2, C-1ꢀ), 31.8 (CH2, C-
3ꢀ), 34.5 (CH2), 35.5 (CH2), 105.6 (4-CH), 106.0 (CH, C-6), 117.1
(CH, C-8), 122.6 (CH, C-9), 129.4 (quat., C-7), 131.7 (CH, C-2),
143.8 (quat., C-1), 145.2 (quat., C-9a), 146.7 (quat., C-5a), 147.4
=
=
(quat., C-10a), 150.85 (quat., C-4a), 170.2 (C O), 185.9 (C O,
C-3).
=
C-4a), 150.0 (quat.), 156.3 (quat., carbamate C O), 171.2 (quat.,
=
amide C O), 185.1 (quat., C-3).
7-N-(b-Alanyl)amino-2-chloro-1-pentylphenoxazin-3-one
trifluoroacetate salt 27b
7-N-(N-tButoxycarbonyl-b-alanyl)amino-1,2,4-
Prepared from 7-N-(N-tBoc-b-alanyl)amino-2-chloro-1-pentyl-
phenoxazin-3-one 26b (80 mg, 0.18 mmol) and TFA (2 ml). Af-
ter work-up, 7-N-(b-alanyl)amino-2-chloro-1-pentylphenoxazin-
3-one trifluoroacetate salt 27b was obtained as a brown solid
trimethylphenoxazin-3-one 26d
Prepared from 7-amino-1,2,4-trimethylphenoxazin-3-one 6d
(0.100 g, 0.39 mmol). 7-N-(N-tButoxycarbonyl-b-alanyl)amino-
1,2,4-trimethylphenoxazin-3-one 26d was obtained as an orange
solid (0.113 g, 68%); mp 215–216 ◦C; (found: MH+, 426.2025. Calc.
for C23H28O5N3: MH, 426.2023); mmax (KBr)/cm−1 3341 (NH), 1704
◦
(80 mg, 97%) mp 220–221 C; (found: M+, 388.1422. Calc. for
C20H23ClN3O3: M, 388.1422); mmax (KBr)/cm−1 3454, 3265 (NH),
=
=
=
1678 (C O), 1601 (C O), 1577 (C C), 1252 (C–O); dH (300 MHz,
DMSO-d6) 0.89 (3H, m, 5ꢀ-CH3), 1.38 (4H, m, 3ꢀ-CH2, 4ꢀ-CH2),
1.59 (2H, m, 2ꢀ-CH2), 2.81 (2H, m, CH2), 3.00 (1ꢀ-CH2), 3.14 (2H,
m, CH2), 6.42 (1H, s, H-4), 7.55 (1H, d, J = 7.9 Hz, H-8), 7.82
=
=
=
(C O), 1686 (C O), 1616 (C C), 1250 (C–O); dH (500 MHz;
DMSO-d6) 1.39 (9H, s, C(CH3)3), 1.98 (3H, s, CH3), 2.05 (3H, s,
CH3), 2.31 (3H, s, CH3), 2.55 (2H, t, J = 7.0 Hz, H-2ꢀ), 3.24–3.28
(2H, m, H-3ꢀ), 6.92 (1H, t, J = 5.2 Hz, NH), 7.37 (1H, dd, J =
8.7 and 2.1 Hz, H-8), 7.68 (1H, d, J = 8.7 Hz, H-9), 7.91 (1H, d,
J = 2.1 Hz, H-6), 10.43 (1H, s, ArNH); dC (125 MHz; DMSO-
d6) 8.5 (CH3), 13.3 (CH3), 13.7 (CH3), 29.1 (CH3, C(CH3)3), 37.2
(CH2, C-3ꢀ), 37.9 (CH2, C-2ꢀ), 78.5 (quat., C(CH3)3), 105.6 (CH,
C-6), 113.1 (quat., C-4), 116.6 (CH, C-8), 129.0 (quat., C-7), 131.0
(CH, C-9), 137.4 (quat.), 138.3 (quat.), 143.3 (quat., C-9a), 145.2
+
(1H, d, J = 8.4 Hz, H-9), 7.93 (4H, br, H-6, NH3 ), 10.85 (1H, br,
ArNH); dC (75.5 MHz, DMSO-d6) 14.6 (CH3, C-5ꢀ), 22.6 (CH2,
C-4ꢀ), 28.4 (CH2, C-2ꢀ), 29.8 (CH2, C-1ꢀ), 32.0 (CH2, C-3ꢀ), 34.6
(CH2), 35.5 (CH2), 105.4 (CH, C-4), 105.5 (CH, C-6), 117.5 (CH,
C-8), 129.7 (quat., C-7), 132.0 (CH, C-9), 136.8 (quat., C-2), 143.7
(quat., C-1), 144.4 (quat., C-9a), 144.85 (quat., C-5a), 145.1 (quat.,
=
=
C-10a), 150.8 (quat., C-4a), 169.8 (C O), 177.8 (C O, C-3).
690 | Org. Biomol. Chem., 2008, 6, 682–692
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7-N-(b-Alanyl)amino-1,2-dimethylphenoxazin-3-one
trifluoroacetate salt 27c
146.7 (quat., C-5a), 148.5 (quat., C-10a), 151.05 (quat., C-4a),
=
=
168.9 (C O), 187.3 (C O, C-3).
Prepared from 7-N-(N-tBoc-b-alanyl)amino-1,2-dimethylpheno-
xazin-3-one 26c (0.047 g, 0.1138 mmol), dry DCM (3 ml) and
TFA (1 ml). 7-N-(b-Alanyl)amino-1,2-dimethylphenoxazin-3-one
trifluoroacetate salt 27c was isolated as a red solid (0.046 g, 95%)
mp 191–192 ◦C; (found: M+, 312.1338. Calc. for C17H18N3O3: M,
7-N-(L-Alanyl)amino-2-chloro-1-pentylphenoxazin-3-one
trifluoroacetate salt 29b
Prepared from 7-N-(N-tBoc-L-alanyl)amino-2-chloro-1-pentyl-
phenoxazin-3-one 28b (0.10 g, 0.21 mmol) and TFA (2 cm3). Af-
ter work-up, 7-N-(L-alanyl)amino-2-chloro-1-pentylphenoxazin-
3-one trifluoroacetate salt 29b was obtained as a brown solid
(0.095 g, 92%) mp >290 ◦C; (HRMS found: M+, 388.1422. Calc.
for C20H23ClN3O3: M, 388.1422), [a]2D0 +100◦ (c 0.07, MeOH); mmax
312.1343); mmax (KBr)/cm−1 3274, 3192, 3111 (NH), 1676 (C O),
=
=
1592 (C C), 1254 (C–O); dH (300 MHz, CD3OD) 2.01 (3H, s,
CH3), 2.29 (3H, s, CH3), 2.78 (2H, t, J = 6.2 Hz, H-2ꢀ), 3.20–3.22
(2H, m, H-3ꢀ), 6.03 (1H, s, H-4), 7.27 (1H, dd, J = 8.7 and 2.2 Hz,
H-8), 7.55 (1H, d, J = 8.7 Hz, H-9), 7.80 (1H, d, J = 2.2 Hz,
H-6); dC (125 MHz; CD3OD) 11.8 (CH3), 12.0 (CH3), 33.1 (CH2),
35.6 (CH2), 104.8 (CH, C-4), 105.8 (CH, C-6), 116.6 (CH, C-8),
129.8 (quat., C-7), 130.8 (CH, C-9), 139.0 (quat.), 139.3 (quat.),
142.8 (quat.), 144.7 (quat.), 146.6 (quat., C-4a), 150.0 (quat.), 169.9
(KBr)/cm−1 3452 (NH), 3276 (NH), 1682 (C O), 1645 (C O),
=
=
=
1583 (C C), 1250 (C–O); dH (300 MHz, CD3OD) 0.85 (3H, t,
J = 6.8 Hz, 5ꢀ-CH3), 1.31 (4H, m, 3ꢀ-CH2, 4ꢀ-CH2), 1.47 (2H,
m, 2ꢀ-CH2), 1.56 (3H, d, J = 6.8 Hz, ala-CH3), 2.98 (2H, m, 1ꢀ-
CH2), 4.05 (1H, q, J = 6.9 Hz, CHa), 6.17 (1H, s, H-4), 7.36
(1H, d, J = 7.6 Hz, H-8), 7.64 (1H, d, J = 8.7 Hz, H-9), 7.89
(1H, s, H-6); dC (75.5 MHz, CD3OD) 13.3 (5ꢀ-CH3), 16.4 (ala-
CH3), 22.4 (4ꢀ-CH2), 28.0 (2ꢀ-CH2), 28.2 (1ꢀ-CH2), 32.0 (3ꢀ-CH2),
50.2 (CH), 104.85 (CH, C-4), 106.0 (CH, C-6), 117.3 (CH, C-
8), 130.1 (quat., C-7), 131.4 (CH, C-9), 136.8 (quat., C-2), 143.3
(quat., C-1), 144.0 (quat., C-9a), 144.8 (quat., C-5a), 144.9 (quat.,
=
(quat., amide C O), 186.4 (quat., C-3).
7-N-(b-Alanyl)amino-1,2,4-trimethylphenoxazin-3-one
trifluoroacetate salt 27d
Prepared from 7-N-(N-tBoc-b-alanyl)amino-1,2,4-trimethylphe-
noxazin-3-one 26d (0.081 g, 0.1897 mmol). 7-N-(b-Alanyl)amino-
1,2,4-trimethylphenoxazin-3-one trifluoroacetate salt 27d was iso-
lated as a red solid (0.080 g, 96%) mp 217–219 ◦C (decomp.);
(found: M+, 326.1506. Calc. for C18H20N3O3: M, 326.1499); mmax
=
=
C-10a), 150.2 (quat., C-4a), 169.0 (C O), 178.8 (C O, C-3).
Columbia agar solution preparation
(KBr)/cm−1 3328, 3108 (NH), 1701 (C O), 1686 (C O), 1578
=
=
Gram-positive and Gram-negative bacteria were cultured on
Columbia agar. 1 Litre of Columbia agar was prepared as follows;
Columbia agar (41 g) was dissolved by boiling in distilled water
(1 l). The solution was then autoclaved at 116 ◦C for 10 min and
left to cool at 50 ◦C.
=
(C C), 1207 (C–O); dH (300 MHz; DMSO-d6) 1.96 (3H, s, CH3),
2.04 (3H, s, CH3), 2.30 (3H, s, CH3), 2.76–2.81 (2H, m, H-2ꢀ),
3.11–3.16 (2H, m, H-3ꢀ), 7.38 (1H, dd, J = 8.7 and 2.2 Hz, H-8),
+
7.69 (1H, d, J = 8.7 Hz, H-9), 7.86 (3H, br s, NH3 ), 7.88 (1H, d,
J = 2.2 Hz, H-6), 10.69 (1H, s, ArNH); dC (75 MHz; DMSO-d6)
7.6 (CH3), 12.4 (CH3), 12.8 (CH3), 33.5 (CH2), 34.7 (CH2), 104.9
(CH, C-6), 112.3 (quat., C-4), 115.7 (CH, C-8), 128.3 (quat., C-7),
130.2 (CH, C-9), 136.5 (quat.), 137.5 (quat.), 142.0 (quat.), 144.3
(quat.), 145.1 (quat., C-4a), 146.2 (quat., C-10a), 169.1 (quat.,
Media preparation
The substrates to be tested were initially dissolved in DMSO or
distilled water to give solutions of 10 mg ml−1. The substrate
solutions were incorporated into Columbia agar solution (200 ml)
and added to sterile plates to give final concentrations of 50 mg
l−1. Columbia agar alone was used as a growth control. Solidified
plates were surface dried in a warm air cabinet for 5 min.
=
amide C O), 183.9 (quat., C-3).
7-N-(L-Alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate
salt 29a
Prepared from 7-N-(N-tBoc-L-alanyl)amino-1-pentylphenoxazin-
3-one 28a (80 mg, 0.18 mmol) and TFA (2 cm3). After work-
up, 7-N-(L-alanyl)amino-1-pentylphenoxazin-3-one trifluoroac-
etate salt 29a was obtained as a brown solid (80 mg, 97%) mp
168–170 ◦C; (found: C, 56.5; H, 5.15; N, 9.0. C22H24F3N3O5
requires C, 56.5; H, 5.2; N, 9.0%) (found: M+, 354.1809. Calc.
Bacterial suspension preparation
Bacterial strains were obtained from the National Collection of
Type Cultures (NCTC), Colindale, U.K., the American Type Cul-
ture Collection (ATCC), Cockeysville, USA, or were isolated from
clinical samples (wild strains) at the Microbiology Department of
the Freeman Hospital, Newcastle-upon-Tyne, U.K.
McFarland tubes were labelled with numbers corresponding to
the bacterial code on the plates. Sterile distilled water (2 ml) was
added to each tube. Each bacterium was inoculated into the tube
using a sterile loop. A densitometer was used to adjust the turbidity
to 0.5 McFarland units (1.5 × 108 organisms per ml).
for C20H24N3O3: M, 354.1812); [a]20 +145◦ (c 0.06, MeOH); mmax
D
(KBr)/cm−1 3452 (NH), 3276 (NH), 1682 (C O), 1645 (C O),
=
=
=
1585 (C C), 1250 (C–O); dH (300 MHz, CD3OD) 0.84 (3H, t, J
= 6.9 Hz, 5ꢀ-CH3), 1.31 (4H, m, 3ꢀ-CH2, 4ꢀ-CH2), 1.55 (3H, d, J
= 7.05 Hz, ala-CH3), 1.58 (2H, m, 2ꢀ-CH2), 2.76 (2H, m, 1ꢀ-CH2),
4.05 (1H, m, CHa), 6.09 (1H, d, J = 2.1 Hz, H-4), 6.53 (1H, d, J
= 2.1 Hz, H-3), 7.41 (1H, dd, J = 8.7 and 2.2 Hz, H-8), 7.68 (1H,
d, J = 8.7 Hz, H-9), 7.83 (1H, d, J = 2.2 Hz, H-6); dC (75.5 MHz,
CD3OD) 13.2 (5ꢀ-CH3), 16.4 (ala-CH3), 22.35 (4ꢀ-CH2), 29.1 (2ꢀ-
CH2), 29.7 (1ꢀ-CH2), 31.7 (3ꢀ-CH2), 50.2 (CH), 105.4 (CH, C-4),
106.4 (CH, C-6), 117.2 (CH, C-8), 130.2 (quat., C-7), 131.1 (CH,
C-9), 131.3 (CH, C-2), 142.75 (quat., C-1), 145.1 (quat., C-9a),
Multipoint inoculation
Each bacterial suspension (200 ll) was pipetted into the corre-
sponding tubes of a multipoint inoculator. Each set of plates
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The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 682–692 | 691
©

received 1 ll of bacterial suspension, giving 1.5 × 105 organisms
per spot on each inoculation. Twenty strains were inoculated per
plate and the plates were incubated for 24 and 48 hours at 30 ◦C,
and 24 and 48 hours at 37 ◦C.
5 R. G. Brooks and J. S. Remington, Transplant-related infections, in
Hospital infections, ed. J. V. Bennett and P. S. Brachman, Little, Brown
and Co., Boston, 2nd edn, 1986, pp. 581–618.
6 J. C. Valdez, M. C. Peral, M. Rachid, M. Santana and G. Perdigon,
Clin. Microbiol. Infect., 2005, 11, 472.
7 I. Gustafsson and J. L. Martinez, Rev. Med. Microbiol., 2005, 16, 155.
8 V. Nagappan and P. Kazanjian, HIV Clinical Trials, 2005, 6, 213.
9 D. von der Waaij, J. Antimicrob. Chemother., 1982, 10, 263.
10 H. Nikaido, Science, 1994, 264, 382.
Activity determination
The activity of the test substrates was determined by the develop-
ment of red, pink, purple or orange colonies after incubation. The
control plate was first taken for each substrate tested and examined
for growth and colour. Each test plate was then compared to the
control and the presence of red, pink, purple or orange colour
was considered as positive evidence for the hydrolysis of the
substrate by alanyl aminopeptidase; no colour or a pale yellow
was considered as negative.
11 P. Richard, J. Infect. Dis., 1994, 170, 377.
12 S. Fujita, A. Tonohata, T. Matsuoka, N. Okado and T. Hashimoto,
J. Clin. Microbiol., 1992, 30, 2728.
13 H. Hollstein, Chem. Rev., 1974, 74, 625; H. Brockmann, Angew. Chem.,
1960, 72, 939.
14 R. J. Anderson, P. W. Groundwater, A. James, D. Monget and A. V.
Zaytsev, PCT Patent WO 2006/030119 A1.
15 R. Willsta¨tter and E. Mayer, Ber. Dtsch. Chem. Ges., 1904, 1498.
16 C. W. Bird and M. Latif, Tetrahedron, 1980, 36, 1813; C. W. Bird and
M. Latif, Tetrahedron, 1980, 36, 529.
17 M. Michman, M. Oron and H. J. Schaefer, Collect. Czech. Chem.
Commun., 2000, 65, 924.
18 L. N. Ferguson, Chem. Rev., 1946, 38, 230.
References
19 T. H. James and A. Weissberger, J. Am. Chem. Soc., 1938, 60, 98.
20 F. W. Scott and T. L. Pitt, J. Med. Microbiol., 2004, 53, 609.
21 B. M. Trost, H. C. Shen, L. Dong, J.-P. Surivet and C. Sylvain, J. Am.
Chem. Soc., 2004, 126, 11966.
1 A. Carricajo, S. Boiste, J. Thore, G. Aubert, Y. Gille and A. M.
Freydiere, Eur. J. Clin. Microbiol. Infect. Dis., 1999, 18, 796; F. Fenollar
and D. Raoult, Eur. J. Clin. Microbiol. Infect. Dis., 2000, 19, 33.
2 H. Komeda and Y. Asano, FEBS J., 2005, 272, 3075.
3 B. Fricke and H. Aurich, J. Basic Microbiol., 1993, 33, 291.
4 M. Laurans, A. Arion, M. Fines-Guyon, A. Regeasse, J. Brouard, R.
Leclercq and J. H. Duhamel, Arch. Pediatr., 2006, 13, S22.
22 T. Smith, J. Am. Chem. Soc., 1944, 66, 1523.
23 L. Giraud and A. Giraud, Synthesis, 1998, 1153.
24 C. J. K. Adderley and F. R. Hewgill, J. Chem. Soc. C, 1968, 1438.
692 | Org. Biomol. Chem., 2008, 6, 682–692
This journal is
The Royal Society of Chemistry 2008
©