2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase activity in clinically important bacteria

Article abstract of DOI:10.1016/j.bmc.2011.03.043

A series of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitrophenyl)benzoxazole 10 and 2-(2-nitrophenyl)benzimidazole 13 derivatives have been synthesised and assessed as indicators of nitroreductase activity across a range of clinically important Gram negativ

Full text of DOI:10.1016/j.bmc.2011.03.043

Bioorganic & Medicinal Chemistry 19 (2011) 2903–2910
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives
as fluorogenic substrates for the detection of nitroreductase
and aminopeptidase activity in clinically important bacteria
Marie Cellier a, Olivier J. Fabrega b, Elizabeth Fazackerley b, Arthur L. James b, Sylvain Orenga a,
John D. Perry c, Vindhya L. Salwatura b, Stephen P. Stanforth b,
⇑
a Research & Development Microbiology, bioMérieux SA, 3 route de Port Michaud, 38 390 La-Balme-les-Grottes, France
b School of Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK
c 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:
A
series of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitrophenyl)benzoxazole 10 and 2-(2-nitro-
Received 12 October 2010
Revised 11 March 2011
Accepted 18 March 2011
Available online 8 April 2011
phenyl)benzimidazole 13 derivatives have been synthesised and assessed as indicators of nitroreductase
activity across a range of clinically important Gram negative and Gram positive bacteria. The majority of
Gram negative bacteria produced strongly fluorescent colonies with substrates 7 and 10 whereas fluores-
cence production in Gram positive bacteria was less widespread. The L-alanine 16 and 19 and b-alanine
21 and 23 derivatives have been prepared from 2-(2-aminophenyl)benzothiazole 14 and 2-(2-aminophe-
nyl)benzoxazole 17. These four compounds have been evaluated as indicators of aminopeptidase activity.
The growth of Gram positive bacteria was generally inhibited by these substrates but fluorescent colonies
were produced with the majority of Gram negative bacteria tested.
Keywords:
Fluorgenic substrates
Nitroreductase
Aminopeptidase
Benzothiazoles
Benzoxazoles
Ó 2011 Elsevier Ltd. All rights reserved.
Benzimidazoles
Bacteria detection
1. Introduction
distinct microbial genera, including a wide range of clinically
important species.4,5 Other coumarin-derived nitroreductase sub-
Enzymatic substrates have emerged as important and powerful
tools for the detection and identification of bacteria1,2 and in this
paper we describe the synthesis of a number of new fluorogenic
nitroreductase and aminopeptidase substrates for this purpose.
These substrates have subsequently been evaluated for their ability
to detect a range of Gram negative and Gram positive bacteria of
clinical importance.
strates have also been prepared by us.6
Aminopeptidase activity in bacteria has frequently been de-
tected by observing the change in fluorescence when the amide
bond in a weakly fluorescent substrate 3 is hydrolysed by an ami-
nopeptidase producing a highly fluorescent amine product 2
(Scheme 1). The substrate 3 is designed so that the amide compo-
nent is derived from an amino acid, for example, the
L-alanine
Nitroreductase activity in bacteria can often be detected by
observing the change in fluorescence when the nitro-group of a
weakly fluorescent substrate 1 is reduced by a nitroreductase
enzyme giving a strongly fluorescent amine (or hydroxylamine
product) 2 (Scheme 1). Many bacteria can utilise aromatic nitro-
compounds as substrates and hence the detection of nitroreduc-
tase activity by observing a substantial change in fluorescence
intensity can offer a useful method for analysing biological and
environmental samples for the presence of bacteria.3 A series
of several 7-nitrocoumarin derivatives has been prepared by one
of us (ALJ) and assessed against 30 strains encompassing 24
derivative 3 (R = Me). -Alanine aminopeptidase activity has previ-
L
ously been used to differentiate between Gram negative and Gram
positive bacteria because Gram negative organisms demonstrate
the presence of this enzyme but in contrast, Gram positive bacteria
do not. b-Alanyl aminopeptidase has been detected in Pseudomonas
sp. and new chromogenic substrates have recently been described
for identifying Pseudomonas aeruginosa, a common respiratory
pathogen in cystic fibrosis patients.7 Colormetric methods for
detecting aminopeptidase activity have also been reported
recently.7,8
Fluorogenic enzyme substrates derived from O-substituted
2-(2-hydroxyphenyl)benzothiazoles, 2-(2-hydroxyphenyl)benzox-
azoles and 2-(2-hydroxyphenyl)benzimidazoles have been pre-
pared for the detection of phosphatase and other enzymatic
⇑
Corresponding author. Tel.: +44 191 227 4784; fax: + 44 191 227 3519.
E-mail address: steven.stanforth@northumbria.ac.uk (S.P. Stanforth).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.043

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M. Cellier et al. / Bioorg. Med. Chem. 19 (2011) 2903–2910
Scheme 1. Generation of fluorescence from nitroreductase and aminopeptidase activity.
activities (Fig. 1).9,10 The core 2-(2-hydroxyphenyl)heterocycles are
highly fluorescent in comparison to their O-substituted derivatives
which has been attributed to excited-state intramolecular proton
transfer (ESIPT). However, there was no indication of whether sub-
strates based upon the corresponding 2-(2-aminophenyl)heterocy-
clic core, that is, structure 2 in Scheme 1 could also furnish useful
fluorogenic enzyme substrates.
cases, the isolatable dihydrobenzothiazoles 6 which were subse-
quently dehydrogenated by treatment with p-chloroanil producing
the required products 7. Reaction of 2-aminophenol 8 with 2-nitro-
benzaldehydes 5 afforded Schiff bases 9 which underwent cyclisa-
tion and dehydrogenation giving the required substrates 10 when
treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).
N-Methyl-1,2-diphenylamine 11 and aldehydes 5 in the presence
of potassium peroxymonosulfate (‘OxoneÒ’) afforded the benzimi-
dazoles 13 directly without isolation of the intermediate dihydro-
benzimidazoles 12.
2. Synthesis of nitroreductase substrates
A selection of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitro-
phenyl)benzoxazole 10 and 2-(2-nitrophenyl)benzimidazole 13
derivatives have been prepared as potential nitroreductase sub-
strates as part of this work (Scheme 2). The benzothiazole deriva-
3. Evaluation of nitroreductase substrates
The nitroreductase substrates 7, 10 and 13 were evaluated
against the clinically important bacteria listed in Table 1.11 The
benzothiazole substrates 7a–d generally gave strongly blue
tives were prepared by reacting 2-aminothiophenol
4 with
appropriately substituted 2-nitrobenzaldehydes 5, yielding in most
Figure 1. Fluorogenic substrates based upon the 2-(2-hydroxyphenyl)heterocyclic core system.
Scheme 2. Synthesis of nitroreductase substrates 7, 10 and 13. Reagents and conditions: (i) EtOH, rt or reflux (6a, 6c and 6d); (ii), 2,3,5,6-tetrachloro-1,4-benzoquinone (p-
chloranil), CH2Cl2, reflux (7a, 7c and 7d); (iii), EtOH, reflux; (iv), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), CH2Cl2, rt; (v), ‘OxoneÒ’, DMF, rt.

M. Cellier et al. / Bioorg. Med. Chem. 19 (2011) 2903–2910
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Table 1
Fluorescent colonies produced by various organisms in the presence of the substrates 7, 10 and 13
Substrate
Fluorescencea
7a
7b
7c
7d
10a
10b
10c
10d
13a
13b
Purpled
Gram negative organismsb
Escherichia coli NCTC 10418
++ Blue
++ Blue
NF
++ Blue
++ Blued
++ Blue
++ Blue
++ Blue
++ Blue
Blue
++ Blue
+ Blued
+ Blue
+ Blue
++ Blue
++ Blue
NF
++ Blue
++ Blue
++ Blue
++ Blue
++ Blue
++ Blue
++ Blue
++ Blue
++ Blued
++ Blue
++ Blue
++ Purple
++ Purple
++ Purple
++ Purple
++ Purple
++ Purple
++ Purple
++ Blue
+ Blue
Blue
+ Blue
++ Blue
+ Blue
++ Blue
++ Blued
++ Blued
NF
+ Blue
+ Blue
NF
NF
Blue
NF
NF
NF
NF
Blue
NF
NF
Serratia marcescens NCTC 10211
Pseudomonas aeruginosa NCTC 10662
Salmonella typhimurium NCTC 74
Morganella morganii WS 462403
Enterobacter cloacae NCTC 11936
Providencia rettgeri NCTC 7475
Purpled
Purpled
Purpled
Purpled
+ Purple
Purple
++ Blued
++ Blued
++ Blued
++ Blued
+ Blue
+ Blue
Gram positive organismsb,c
Staphylococcus epidermidis NCTC 11047
Staphylococcus aureus NCTC 6571
Staphylococcus aureus (MRSA) NCTC 11939
Trace blue
++ Blue
++ Blue
NF
++ Blue
++ Blue
NF
++ Blue
++ Blue
Trace blue
++ Blue
++ Blue
+ Purple
++ Purple
++ Purple
NF
+ Blue
+ Blue
NF
NF
+ Blue
+ Blue
NF
NF
NF
NF
+ Purple
+ Purple
++ Blued
++ Blued
a
b
c
++ strong fluorescence, + moderate fluorescence, weak fluorescence, NF no significant fluorescence. Substrate concentration = 100 mg LÀ1
All Gram negative bacteria exhibited good growth in the presence of the substrate. Gram positive organisms generally exhibited moderate growth.
The following Gram positive organisms did not give fluorescent colonies with any of the substrates: Enterococcus faecalis NCTC 775, Enterococcus faecium NCTC 7171,
.
Streptococcus pyogenes NCTC 8306, Listeria monocytogenes NCTC 11994.
d
There was noticeable diffusion of the fluorophore into the medium.
fluorescent colonies with the majority of the seven Gram negative
organisms shown in Table 1. With the exception of P. aeruginosa,
most of these Gram negative organisms produced blue or purple
fluorescent colonies with the benzoxazole substrates 10a–c but
in contrast substrate 10d only moderately or weakly fluorescent
colonies were produced with the seven Gram negative bacteria.
Figure 2 illustrates the fluorescent colonies produced when sub-
strate 10c was incubated with Serratia marcescens. The benzimid-
azole derivatives 13a and 13b were poor substrates in
comparison with their benzothiazole and benzoxazole analogues.
No significant fluorescence was observed for substrate 13a and
most Gram negative bacteria and substrate 13b gave only weakly
fluorescent purple colonies with Gram negative bacteria. Some
undesirable diffusion of the fluorescence into the surrounding
media was noted particularly with substrates 10c and 13b.
In contrast to the Gram negative bacteria, the Gram positive
bacteria Enterococcus faecalis, Enterococcus faecium, Streptococcus
pyogenes and Listeria monocytogenes did not produce fluorescent
colonies with any of the substrates and Staphylococcus epidermidis
gave weakly fluorescent colonies with the benzothiazole sub-
strates 7a and 7d. Staphylococcus aureus NCTC 6571 and S. aureus
NCTC 11939 (MRSA) however gave strongly fluorescent colonies
with all of the benzothiazole substrates 7a–d. Similarly, the
benzoxazole substrates 10a and 10c gave strongly fluorescent col-
onies with S. aureus NCTC 6571 and S. aureus NCTC 11939 (MRSA)
whereas the other benzoxazole substrates produced inferior
results with these two bacteria. Substrate 10a however was supe-
rior to substrate 10c for P. aeruginosa and S. epidermidis detection.
The benzimidazole derivatives 13a and 13b were generally poor
substrates for the Gram positive bacteria.
In order to confirm that enzyme activity was responsible for
the production of fluorescence Escherichia coli BL21 was grown
and the cell culture was subsequently sonicated and centrifuged.
The resulting cell-free extract was incubated with the substrate
10c overnight producing a fluorescent solution (Fig. 3). Addition-
ally, when E. coli was grown in a broth in the presence of substrate
7a, the corresponding amine 14 was the only product detected by
HPLC. The identity of the amine 14 was confirmed by comparison
with an authentic sample, thus confirming that the anticipated
reduction of the nitro-group had taken place with this organism.
4. Synthesis of aminopeptidase substrates
In view of the superior results obtained with the benzothiazole
and benzoxazole nitroreductase substrates described above, L-ala-
nine and b-alanine aminopeptidase substrates were prepared from
Figure 3. Incubation of substrate 10c with a Escherichia coli BL21 cell-free extract in
a Tris buffer solution (pH 7.4) viewed under UV light (365 nm). Left: buffer solution
and substrate 10c; Centre: buffer solution, substrate 10c and cell-free extract;
Right: buffer solution and cell-free extract.
Figure 2. Fluorescent colonies produced from substrate 10c and Serratia marcescens
(NCTC 10211) viewed under UV light at 365 nm.

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M. Cellier et al. / Bioorg. Med. Chem. 19 (2011) 2903–2910
Scheme 3. Synthesis of aminopeptidase substrates 16, 19, 21 and 23. Reagents and conditions: (i) Boc-L-alanine, N-methylmorpholine (NMM), isobutylchloroformate (IBCF),
À10 to À15 °C, then add 14 or 17; (ii), Boc-b-alanine, NMM, IBCF, À10 to À15 °C, then add 14 or 17; (iii), trifluoroacetic acid, rt.
Table 2
Fluorescent colonies produced by various bacteria in the presence of the substrates 16, 19, 21 and 23
Substrate
16
Fluorescenceb
19
Fluorescenceb
21
Fluorescenceb
23
Fluorescenceb
Growtha
Growtha
Growtha
Growtha
Escherichia coli NCTC 10418
++
++
++
NG
++
++
++
+ Blue
+ Blue
+ Yellow
—
+ Blue
+ Blue
+ Blue
++
++
++
++
++
++
++
++ Bluec
++ Bluec
++ Bluec
++ Bluec
++ Bluec
++ Bluec
++ Bluec
+
+
++ Yellow
++ Yellow
++ Yellow
—
++ Yellow
—
++
++
++
+
++
++
+ Green
+ Green
+ Green
—
+ Green
+ Green
Green
Serratia marcescens NCTC 10211
Pseudomonas aeruginosa NCTC 10662
Salmonella typhimurium NCTC 74
Morganella morganii WS 462403
Enterobacter cloacae NCTC 11936
Providencia rettgeri NCTC 7475
++
NG
++
NG
NG
—
a
b
c
Growth in absence of substrate: ++ strong growth, + moderate growth, poor growth, NG no growth. Substrate concentration = 100 mg LÀ1
++ strong fluorescence, + moderate fluorescence, weak fluorescence, NF no significant fluorescence.
There was noticeable diffusion of the fluorophore into the medium.
.
these two heterocyclic cores as shown in Scheme 3. Reduction of
the nitro-derivative 7a using tin(II) chloride gave the amine 14
which was condensed with Boc-L-alanine using the mixed anhy-
dride method giving compound 15. Removal of the Boc group by
treatment with trifluoroacetic acid afforded the required substrate
16 as its TFA salt. Similarly, the nitro-derivative 10a afforded the
substrate 19 via the amine 17 and Boc-protected intermediate
cens, P. aeruginosa and Morganella morganii but this substrate
inhibited the growth of the other Gram negative bacteria. It is
interesting to note the contrast in fluorescent colours between
substrates 16 and 21 that possess the same benzothiazole core
structure. The L-alanyl substrate 16 generally produced blue fluo-
rescent colonies following hydrolysis whereas in contrast the b-
alanyl substrate 21 gave yellow fluorescent colonies. The origin
of this effect has not yet been established but may be a conse-
quence of either the localisation of the fluorophore in different re-
gions of the organism or the result of further metabolic
transformation of the initially formed fluorophore. Compound 23
was not a particularly effective substrate producing only moder-
ately or weakly fluorescent colonies with most Gram negative bac-
teria. We have previously demonstrated that aminopeptidase
activity is linked to enzyme activity.8
18. By replacing Boc-L-alanine with Boc-b-alanine, amines 14 and
17 gave the corresponding Boc-protected derivatives 20 and 22
from which the b-alanine aminopeptidase substrates 21 and 23
were obtained.
5. Evaluation of aminopeptidase substrates
The four substrates 16, 19, 21 and 23 were evaluated for amino-
peptidase activity12 against the seven Gram negative and seven
Gram positive bacteria listed in Table 1. The results for the Gram
negative bacteria are only shown in Table 2 because all of these
substrates were generally inhibitory to the Gram positive organ-
isms and only occasionally weakly fluorescent colonies were
produced (data not shown). These substrates would therefore have
potential in the differentiation between Gram negative and Gram
positive bacteria. With the exception of Salmonella typhimurium,
6. Conclusions
Nitroreductase activity was efficiently detected by the benzox-
azole substrate 10a across all of the Gram negative bacteria and
S. aureus strains examined. In general, the full range of benzothi-
azole substrates 7, enabled detection of most Gram negative
bacteria but the benzimidazole substrates 13a and 13b were inef-
fective. The benzoxazole substrate 19 was effective for determin-
the L-alanine aminopeptidase benzothiazole substrate 16 gave
moderately fluorescent colonies with Gram negative bacteria. In
contrast, benzoxazole substrate 19 gave strongly blue fluorescent
colonies but diffusion of the fluorophore into the surrounding
medium was observed. The b-alanine aminopeptidase substrate
21 gave strongly yellow fluorescent colonies with E. coli, S. marces-
ing L-alanyl aminopeptidase activity although there was diffusion
of the fluorophore into the surrounding medium. The benzothia-
zole substrate 21 allowed detection of b-alanyl aminopeptidase
activity in cases where microorganism growth was not inhibited
by this substrate.

M. Cellier et al. / Bioorg. Med. Chem. 19 (2011) 2903–2910
2907
(200 mL) at reflux. Vivid red needles (7.23 g, 65%), mp 156–
158 °C (EtOH); mmax cmÀ1: 3376, 1584, 1516, 1342, 1326, 1257,
1214, 1046; dY (270 MHz; CDCl3) 4.56 (1H, broad s, >NH), 6.82–
6.91 (3H, m, 3 Â Ar-H or 2 Â Ar-H and >CH-Ar), 6.99–7.14 (3H, m,
3 Â Ar-H or 2 Â Ar-H and >CH-Ar), 7.69 (1H, dd, J = 9.7 and
2.7 Hz, Ar-H), 8.18 (1H, dd, J = 9.2 and 5.2 Hz, Ar-H); dC (68 MHz;
CDCl3) 64.1, 112.2, 115.7 (d, J = 25.8 Hz), 115.8 (d, J = 23.1 Hz),
122.0, 122.4, 125.8, 127.3, 128.5 (d, J = 10.2 Hz), 141.8, 143.8 (d,
J = 7.5 Hz), 145.5, 165.8 (d, J = 258.4 Hz). Compound 7c was pre-
pared from compound 6c (2.23 g, 8.07 mmol) and p-chloranil
(1.98 g, 8.07 mmol) in CH2Cl2 (125 mL). Fluffy yellow solid
(1.42 g, 64%), mp 114–116 °C (EtOH); (found: MH+, 275.0283. Calcd
for C13H8FN2O2S: MH, 275.0285); mmax cmÀ1 3082, 1510, 1348,
1313, 1269, 1093, 1223; dY (270 MHz; CDCl3) 7.33 (1H, ddd,
J = 9.0, 7.2 and 2.7 Hz, Ar-H), 7.45–7.59 (3H, m, Ar-H), 7.96 (1H,
ddd, J = 7.8, 1.5 and 1.0 Hz, Ar-H), 8.03 (1H, dd, J = 9.0 and 5.0 Hz,
Ar-H), 8.10 (1H, ddd, J = 7.8, 1.5 and 1.0 Hz, Ar-H); dC (68 MHz;
CDCl3) 117.8 (d, J = 23.8 Hz), 119.2 (d, J = 25.2 Hz), 121.8, 124.2,
126.3, 126.9, 127.4 (d, J = 9.5 Hz), 131.2 (d, J = 8.8 Hz), 135.9,
153.4, 161.1, 163.9 (d, J = 257.9 Hz).
7. Experimental
7.1. Synthetic work
1H NMR spectra (270 MHz) and 13C NMR spectra (68 MHz) were
recorded on a Jeol EX270 instrument. High resolution mass spec-
trometry (HRMS) (electrospray) was performed by the EPSRC mass
spectrometry service. Infra-red spectra were obtained via a dia-
mond anvil sample cell using a Perkin Elmer 1000 spectrometer.
Melting points are reported uncorrected as determined on a Stuart
SMP 1 melting point apparatus. Thin layer chromatography was
performed on Merck plastic foil plates pre-coated with silica gel
60 F254. Merck silica gel 60 was used for column chromatography.
7.1.1. General procedure for the preparation of the
dihydrobenzothiazoles 6a–d and benzothiazoles 7a–d
A mixture of 2-aminothiophenol 4 (1 equiv) and the appropriate
nitrobenzaldehyde derivative 5 (1 equiv) in EtOH was heated either
at reflux for 2–3 h or stirred at room temperature. After cooling to
room temperature (if necessary), the solution was evaporated and
the residue was partitioned between water and ethyl acetate. The
organic layer was separated and the aqueous layer was extracted
with ethyl acetate. The combined organic layers were dried
(MgSO4) and evaporated giving the crude product 6. To a solution
of compound 6 (1 equiv) in CH2Cl2 at room temperature was added
p-chloranil (1 equiv) and the resultingmixture was stirred overnight.
The mixture was washed with a dilute aqueous NaOH solution, the
organic phase was separated, dried (MgSO4) and evaporated. The
crude product 7 thus obtained was recrystallised from ethanol.
7.1.1.4.
thiazole 6d and 2-(3,4-dimethoxy-6-nitrophenyl)benzothiazole
7d. Compound 6d was prepared from 2-aminothiophenol
2-(3,4-Dimethoxy-6-nitrophenyl)-2,3-dihydrobenzo-
4
(1.88 g, 15 mmol) and 6-nitroveratraldehyde (3.17 g, 15 mmol) in
EtOH (50 mL) at reflux for 2 h. After cooling at room temperature,
the solution was partially concentrated giving compound 6d as a
yellow solid (1.98 g, 41%); dY (270 MHz; CDCl3) 3.82 (3H, s, –
OCH3), 3.88 (3H, s, –OCH3), 4.51 (1H, br d, J = 5.0 Hz, >NH), 6.72–
6.78 (2H, m, Ar-H), 6.88–7.02 (2H, m, Ar-H), 6.92 (1H, d,
J = 5.0 Hz, >CH–), 7.47 (1H, s, Ar-H), 7.59 (1H, s, Ar-H). Compound
7d was prepared from compound 6d (1.59 g, 5 mmol) and
p-chloranil (1.23 g, 5 mmol) in CH2Cl2 (80 mL). Yellow solid
(1.40 g, 89%), mp 153–154 °C; (found: MH+, 317.0584. Calcd for
7.1.1.1. 2-(2-Nitrophenyl)-2,3-dihydrobenzothiazole 6a and 2-
(2-nitrophenyl)benzothiazole 7a. Compound 6a13 was prepared
from 2-nitrobenzaldehyde (1.00 g, 6.6 mmol), and 2-aminothiophe-
nol (0.83 g, 6.6 mmol) in EtOH (50 mL) under nitrogen at room tem-
perature for 1 h. Red needles (1.01 g, 59%); mmax cmÀ1 3387, 1587,
1518, 1344, 1326, 1271, 1058; dY (270 MHz; CDCl3) 4.57 (1H, broad
s, >NH), 6.75–6.82 (3H, m, 2 Â Ar-H and >CH-Ar), 6.93–7.06 (2H, m,
Ar-H), 7.45 (1H, dd, J = 1.5 and 7.8 Hz, Ar-H), 7.63 (1H, ddd, J = 0.6,
0.6 and 7.7 Hz, Ar-H), 7.95–8.05 (2H, m, Ar-H). Compound 7a was
prepared from compound 6a (1.01 g, 3.91 mmol) and p-chloroanil
(0.96 g, 3.91 mmol) in CH2Cl2 (75 mL). Yellow solid (0.78 g, 77%),
mp 117 °C (lit.14 122–123 °C); mmax cmÀ11531, 1362, 1305, 1232;
dY (270 MHz; CDCl3) 7.40 (1H, ddd, J = 1.3, 7.6 and 7.6 Hz, Ar-H),
7.48 (1H, ddd, J = 1.3, 7.6 and 7.6 Hz, Ar-H), 7.54–7.68 (2H, m, Ar-
H); 7.74 (1H, dd, J = 1.6 and 7.6 Hz, Ar-H), 7.84–7.91 (2H, m, Ar-H),
8.02 (1H, dd, J = 1.3 and 8.2 Hz, Ar-H).
C
15H13N2O4S: MH, 317.0596); mmax cmÀ1 3008, 2937, 2845, 1496,
1330, 1272, 1230, 1088; dY (270 MHz; CDCl3) 3.94 (3H, s,
–OCH3), 3.96 (3H, s, –OCH3), 7.07 (1H, s, Ar-H), 7.35–7.42 (1H, m,
Ar-H), 7.44–7.51 (1H, m, Ar-H), 7.56 (1H, s, Ar-H), 7.84–7.89 (1H,
m, Ar-H), 8.00–8.05 (1H, m, Ar-H); dC (68 MHz; CDCl3) 56.7, 56.8,
108.0, 113.3, 121.7, 123.0, 123.7, 125.8, 126.5, 136.1, 141.5,
150.1, 152.4, 153.2, 163.6.
7.1.2. General procedure for the preparation of the Schiff bases
9a–d and benzoxazoles 10a–d
A mixture of 2-hydroxyaniline 8 (1 equiv) and the appropriate
2-nitrobenzaldehyde derivative 5 (1 equiv) in ethanol (30 mL)
was heated at reflux for 3–4 h. After cooling to room temperature
the resulting precipitate 9a–d was collected and dried. To a stirred
solution of the Schiff’s base 9a–d (1 equiv) in CH2Cl2 (30 mL) at
room temperature, DDQ (1 equiv) was added over a 1 min. The
reaction mixture was stirred at room temperature for 1–2 days,
filtered and evaporated giving the crude product 10a–d.
7.1.1.2. 2-(3-Chloro-6-nitrophenyl)benzothiazole 7b. Compound
7b was prepared directly from 2-aminothiophenol
4 (1.35 g,
0.011 mol) and 3-chloro-6-nitrobenzaldehyde (2.0 g, 0.011 mol) in
EtOH (20 mL) at reflux. The intermediate dihydro-derivative 6b
was not isolated and was oxidised to compound 7b under the reac-
tion conditions. Yellow-brown solid (0.66 g, 24%), mp 126–128 °C;
(found: MH+, 290.9992. Calcd for C13H835ClN2O2S: MH, 290.9990).
mmax cmÀ1 2962, 1526, 1340, 1259, 753; dY (270 MHz; CDCl3)
7.36–7.48 (2H, m, Ar-H), 7.52 (1H, dd, J = 8.7 and 2.2 Hz, Ar-H),
7.70 (1H, d, J = 2.2 Hz, Ar-H), 7.83 (1H, d, J = 8.7 Hz, Ar-H), 7.87
(1H, dd, J = 7.7 and 1.5 Hz, Ar-H), 8.01 (1H, dd, J = 7.7 and 1.5 Hz,
Ar-H); dC (68 MHz; CDCl3) 121.8, 124.2, 126.1, 126.3, 126.9, 130.0,
130.9, 131.9, 135.9, 138.9, 147.2, 153.4, 161.0.
7.1.2.1. 2-[(2-Nitrobenzylidene)amino]phenol 9a and 2-(2-
nitrophenyl)benzoxazole 10a. Compound 9a was synthesised
from 2-hydroxyaniline 8 (4.03 g, 0.037 mol) and 2-nitrobenzalde-
hyde (3.73 g, 0.02 mol). Yellow needles (6.36 g, 70%), mp
105–107 °C (lit.15 mp 104 °C); dY (270 MHz; CDCl3) 6.91 (1H, td,
J = 7.4 and 1.5 Hz, Ar-H), 7.01 (1H, dd, J = 9.4 and 1.2 Hz, Ar-H),
7.15 (1H, s, –OH), 7.23 (1H, t, J = 8.7 Hz, Ar-H), 7.33 (1H, dd,
J = 7.9 and 1.5 Hz, Ar-H), 7.61 (1H, td, J = 8.9 and 1.5 Hz, Ar-H),
7.72 (1H, t, J = 8.4 Hz, Ar-H), 8.03 (1H, dd, J = 7.9 and 1.5 Hz,
Ar-H), 8.24 (1H, dd, J = 7.7 and 1.7 Hz, Ar-H), 9.13 (1H, s, =CH–);
dC (68 MHz; CDCl3) 115.5, 116.5, 120.5, 124.8, 129.6, 130.6,
131.5, 131.5, 133.5, 134.8, 149.4, 152.1, 152.9. Compound 10a
was prepared from compound 9a (6.36 g, 0.026 mol) and DDQ
7.1.1.3. 2-(3-Fluoro-6-nitrophenyl)-2,3-dihydrobenzothiazole
6c and 2-(3-fluoro-6-nitrophenyl)benzothiazole 7c. Compound
6c was prepared from 2-aminothiophenol 4 (5.00 g, 0.04 mol)
and 3-fluoro-6-nitrobenzaldehyde (6.75 g, 0.04 mol) in EtOH

2908
M. Cellier et al. / Bioorg. Med. Chem. 19 (2011) 2903–2910
(5.90 g, 0.026 mol). Pale orange solid (4.41 g, 69%), mp 104–106 °C
(lit.15 mp 104–106 °C); dY (270 MHz; CDCl3) 7.36–7.45 (2H, m, Ar-
H), 7.54–7.62 (1H, m, Ar-H), 7.66–7.79 (2H, m, Ar-H), 7.79–7.84
(1H, m, Ar-H), 7.89 (1H, dd, J = 7.7 and 1.7 Hz, Ar-H), 8.14 (1H,
dd, J = 7.4 and 1.7 Hz, Ar-H); dC (68 MHz; CDCl3) 108.7, 110.3,
116.3, 116.8, 119.4, 124.4, 124.8, 128.8, 132.5, 141.9, 147.9,
149.3, 163.2.
dd, J = 7.9 and 1.5 Hz, Ar-H), 7.62 (1H, s, Ar-H), 7.69 (1H, s, Ar-H),
9.25 (1H, s, =CH–); dC (68 MHz; CDCl3) 56.7, 56.8, 107.6, 109.6,
115.4, 116.7, 120.5, 125.5, 129.9, 135.3, 142.8, 151.0, 152.5,
153.1, 153.2. Compound 10d was synthesised from compound 9d
(1.52 g, 0.005 mol) and DDQ (1.13 g, 0.005 mol). Orange-brown
powder (1.35 g, 90%), mp 148–150 °C (EtOH); (found: MH+,
301.0819. Calcd for C15H13N2O5: MH, 301.0819); mmax cmÀ1 1519,
1582, 1448, 1378, 1189, 1177; dY (270 MHz; CDCl3) 4.00 (3H, s, –
OCH3), 4.01 (3H, s, –OCH3), 7.37–7.41 (2H, m, Ar-H), 7.43 (1H, s,
Ar-H), 7.52 (1H, s, Ar-H), 7.53–7.57 (1H, m, Ar-H), 7.78–7.82 (1H,
m, Ar-H); dC (68 MHz; CDCl3) 56.1, 56.2, 107.8, 111.0, 112.9,
115.8, 120.4, 124.9, 125.8, 141.5, 142.4, 150.9, 151.1, 152.2, 159.8.
7.1.2.2. 2-[(3-Chloro-6-nitrobenzylidene)amino]phenol 9b and
2-(3-chloro-6-nitrophenyl)benzoxazole 10b. Compound 9b was
synthesised from 2-hydroxyaniline 8 (2.18 g, 0.02 mol) and 5-
chloro-2-nitrobenzaldehyde (5.59 g, 0.037 mol). Orange needles
(4.17 g, 80%), mp 144–146 °C (EtOH); (found: MH+, 277.0373. Calcd
for C13H1035ClN2O3: MH, 277.0374); mmax cmÀ1 3442, 1558, 1376,
1521, 1461, 1341, 1301, 1176, 1143, 756; dY (270 MHz; CDCl3)
6.95 (1H, t, J = 8.2 Hz, Ar-H), 7.05 (1H, dd, J = 8.2 and 1.2 Hz, Ar-
H), 7.10 (1H, broad s, –OH), 7.28 (1H, t, J = 8.2 Hz, Ar-H), 7.33
(1H, dd, J = 8.2 and 1.5 Hz, Ar-H), 7.60 (1H, dd, J = 8.7 and 2.5 Hz,
Ar-H), 8.05 (1H, d, J = 8.7 Hz, Ar-H), 8.23 (1H, d, J = 2.5 Hz, Ar-H),
9.27 (1H, s, =CH–); dC (68 MHz; CDCl3) 115.8, 116.6, 120.5, 126.5,
129.3, 130.8, 131.3, 132.3, 134.4, 140.4, 147.4, 150.8, 153.0. Com-
pound 10b was prepared from compound 9b (1.38 g, 0.005 mol)
and DDQ (1.14 g, 0.005 mol). Greenish brown powder (0.97 g,
70%), mp 130–132 °C (EtOH); (found: MH+, 274.0141. Calcd for
7.1.3. Preparation of the benzimidazoles 13a and 13b
7.1.3.1. 1-Methyl-2-(2-nitrophenyl)benzimidazole 13a. Com-
pound 13a (80%) was prepared using a similar procedure to that
described below for compound 13b, mp 134–136 °C (EtOH) (lit.14
135–137 °C); dY (270 MHz; CDCl3) 3.65 (3H, s, –NCH3), 7.30–7.50
(3H, m, Ar-H), 7.56–7.90 (4H, m, Ar-H), 8.35 (1H, dd, J = 8.0 and
2.0 Hz, Ar-H); dC (68 MHz; CDCl3) 30.7, 109.8, 120.2, 122.6, 123.3,
124.9, 126.1, 131.2, 133.0, 133.7, 135.8, 143.0, 148.8, 149.9.
7.1.3.2. 1-Methyl-2-(3-chloro-6-nitrophenyl)benzimidazole 13b.
To a stirred solution of N-methyl-1,2-phenylenediamine 11 (0.5 g,
0.004 mol) and 5-chloro-2-nitrobenzaldehyde (0.76 g, 0.004 mol)
in DMF (25 mL) and H2O (1 mL) at room temperature, OxoneÒ
(1.47 g, 0.0024 mol) was added in portions over 15 min. The mix-
ture was stirred overnight at room temperature. Water (10 mL)
was added and the reaction mixture was extracted twice with
CH2Cl2 (20 mL). The combined CH2Cl2 layers were washed several
times with water, dried (MgSO4) and evaporated giving compound
13b (0.94 g, 83%) as yellow-brown needles, mp 148–150 °C (EtOH)
(lit.14 144–145 °C); dY (270 MHz; CDCl3) 3.65 (3H, s, –CH3),
7.30–7.45 (2H, m, Ar-H), 7.70 (2H, m, Ar-H), 7.85 (1H, m, Ar-H),
7.80 (1H, m, Ar-H), 8.20 (1H, d, J = 9.4 Hz, Ar-H); dC (68 MHz;
CDCl3) 30.8, 109.8, 120.3, 122.8, 123.7, 126.4, 127.9, 131.2, 133.2,
135.7, 140.4, 142.9, 147.1, 148.5.
C
13H8Cl35N2O3: MH, 274.0140); mmax cmÀ1 1616, 1572, 1534,
1461, 1371, 1236, 1182, 1153, 743; dY (270 MHz; CDCl3) 7.40–
7.45 (2H, m, Ar-H), 7.56–7.61 (1H, m, Ar-H), 7.65 (1H, dd, J = 8.7
and 2.2 Hz, Ar-H), 7.80–7.85 (1H, m, Ar-H), 7.87 (1H, d, J = 8.7 Hz,
Ar-H), 8.16 (1H, d, J = 2.2 Hz, Ar-H); dC (68 MHz; CDCl3) 111.1,
120.9, 123.3, 125.3, 125.7, 126.5, 131.4, 131.8, 138.8, 141.4,
147.3, 151.6, 157.5.
7.1.2.3. 2-[(3-Fluoro-6-nitrobenzylidene)amino]phenol 9c and
2-(3-fluoro-6-nitrophenyl)benzoxazole 10c. Compound 9c was
synthesised from 2-hydroxyaniline 4 (10.91 g, 0.100 mol) and 3-
fluoro-6-nitrobenzaldehyde (16.91 g, 0.100 mol). Orange solid,
(24.09 g, 93%) mp 167–168 °C; dY (270 MHz; CDCl3) 6.92–6.99
(1H, m, Ar-H), 7.03–7.08 (1H, m, Ar-H), 7.09 (1H, broad s, –OH),
7.27–7.35 (2H, m, Ar-H), 7.35–7.40 (1H, m, Ar-H), 7.97 (1H, dd,
J = 9.2 and 2.7 Hz, Ar-H), 8.18 (1H, dd, J = 9.2 and 5.0 Hz, Ar-H),
9.23 (1H, d, J = 2.0 Hz, =CH–); dC (68 MHz; CDCl3) 115.8, 116.1 (d,
J = 25.2 Hz), 116.6, 118.4 (d, J = 22.4 Hz), 120.6, 128.0 (d,
J = 9.5 Hz), 130.8, 134.0 (d, J = 8.8 Hz), 145.4, 150.9, 153.0, 165.0
(d, J = 255.0 Hz). The compound was used for the next step without
further characterisation. Compound 10c was synthesised from
compound 9c (24.09 g, 0.093 mol) and DDQ (21.04 g, 0.093 mol).
Beige solid (14.49 g, 61%), mp 112–114 °C; (found: MH+,
259.0513. Calcd for C13H8FN2O3: MH, 259.0513); mmax cmÀ1 3084,
1589, 1527, 1357, 1298, 1219, 1056; dY (270 MHz; CDCl3) 7.34–
7.47 (3H, m, Ar-H), 7.57–7.62 (1H, m, Ar-H), 7.80–7.87 (1H, m,
Ar-H), 7.84 (1H, dd, J = 8.2 and 2.9 Hz, Ar-H), 7.97 (1H, dd, J = 8.2
and 4.7 Hz, Ar-H); dC (68 MHz; CDCl3) 111.1, 118.7 (d,
J = 21.8 Hz), 118.7 (d, J = 26.5 Hz), 121.0, 124.4 (d, J = 9.9 Hz),
125.3, 126.5, 127.0 (d, J = 9.4 Hz), 141.4, 145.4, 151.1, 157.7,
157.7, 164.0 (d, J = 257.0 Hz).
7.1.4. General procedure for the preparation of t-Boc-L-alanyl
derivatives 15, 18, 20 and 22
2-(2-Aminophenyl)benzothiazole 14 was prepared by
SnCl2.2H2O reduction of compound 7a.16 2-(2-Aminophenyl)benz-
oxazole 1717 was prepared by a similar procedure. t-Boc-
L-alanine
or t-Boc-b-alanine (1.33 equiv) was dissolved in dry THF (10 mL).
The solution was cooled down to À15 °C and N-methylmorpholine
(NMM) (1.66 equiv) was added drop-wise over 1 min, followed by
isobutyl chloroformate (IBCF) (1.33 equiv). The mixture was stirred
for 5–10 min and then a solution of either 2-(2-aminophenyl)ben-
zothiazole 14 (1 equiv) or 2-(2-aminophenyl)benzoxazole 17
(1 equiv) dissolved in minimum amount of dry THF (ca 5 mL)
was added drop-wise over 2 min at À15 °C. The reaction mixture
was stirred in an ice-bath for 2–3 h, allowed to warm to room tem-
perature and stirred at room temperature overnight. The reaction
mixture was filtered and the resulting filtrate was reduced in vol-
ume and poured into a cooled aqueous solution of sodium carbon-
ate. The resulting precipitate was collected and recrystallised from
either methanol or ethanol.
7.1.2.4.
2-[(3,4-Dimethoxy-6-nitrobenzylidene)amino]phenol
9d and 2-(3,4-dimethoxy-6-nitrophenyl)benzoxazole 10d.
Compound 9d was synthesised from 2-hydroxyaniline 8 (3.27 g,
0.03 mol) and 6-nitroveratraldehyde (6.33 g, 0.03 mol). Orange
needles (8.20 g, 90%), mp 154–156 °C (EtOH); (found: MH+,
303.0973. Calcd for C15H15N2O5: MH, 303.0975); mmax cmÀ1 3416,
2838, 1589, 1566, 1461, 1384, 1344, 1317, 1181, 1149; dY
(270 MHz; CDCl3) 4.01 (3H, s, –OCH3), 4.05 (3H, s, –OCH3), 6.92
(1H, t, J = 7.9 Hz, Ar-H), 7.01 (1H, dd, J = 7.9 and 1.5 Hz, Ar-H),
7.12 (1H, broad s, –OH), 7.22 (1H, t, J = 7.9 Hz, Ar-H), 7.32 (1H,
7.1.4.1. 2-[(t -Boc-
Compound 15 was synthesised from t-Boc-
L
-alanylamino)phenyl]benzothiazole 15.
-alanine (1.51 g,
L
8 mmol), NMM (1.01 g, 10 mmol), IBCF (1.09 g, 8 mmol) and 2-
(2-aminophenyl)benzothiazole 14 (1.36 g, 6 mmol). Yellow solid
(0.87 g, 36%), mp 202–204 °C (MeOH); (found: MNa+, 420.1347.
Calcd for C21H23NaN3O3S: MNa, 420.1352); mmax cmÀ1 3336,
1707, 1681, 1514, 1250, 1158, 1062; dY (270 MHz; CDCl3) 1.43
(9H, s, 3 Â –CH3), 1.58 (3H, d, J = 7.4 Hz, –CH3), 4.51 (1H, quintet,

M. Cellier et al. / Bioorg. Med. Chem. 19 (2011) 2903–2910
2909
J = 7.4 Hz, >CH–), 5.33 (1H, d, J = 5.9 Hz, >NH), 7.19 (1H, td, J = 7.7
and 1.2 Hz, Ar-H), 7.41–7.56 (3H, m, Ar-H), 7.88 (1H, dd, J = 7.9
and 1.5 Hz, Ar-H), 7.93 (1H, broad d, J = 7.7 Hz, Ar-H), 8.14 (1H,
broad d, J = 7.4 Hz, Ar-H), 8.83 (1H, broad d, J = 8.4 Hz, Ar-H) (one
>NH was not located); dC (68 MHz; CDCl3) 19.5, 28.4, 52.1, 80.3,
119.5, 121.0, 121.5, 123.1, 123.5, 125.9, 126.6, 129.9, 132.0,
133.3, 137.7, 152.9, 155.4, 168.7, 172.1.
1704 , 1141; dY (270 MHz; DMSO-d6) 1.63 (3H, d, J = 7.2 Hz, –
CH3), 4.30–4.40 (1H, m, >CH–), 7.34–7.41 (1H, m, Ar-H), 7.49–
7.57 (1H, m, Ar-H), 7.58–7.66 (2H, m, Ar-H), 8.00–8.05 (1H, m,
Ar-H), 8.11–8.16 (1H, m, Ar-H), 8.18–8.23 (1H, m, Ar-H), 8.29–
8.37 (1H, m, Ar-H), 8.33 (3H, broad s, –NH3+), 11.97 (1H, broad s,
>NH); dC (68 MHz; DMSO-d6) 17.1, 50.0, 122.2, 122.8, 123.3 (2C),
125.7, 126.7, 127.5, 130.9, 132.5, 134.0, 136.4, 152.9, 167.6, 169.2.
7.1.4.2. 2-[(t -Boc-
pound 18 was synthesised from t-Boc-
L
-alanylamino)phenyl]benzoxazole 18. Com-
-alanine (5.03 g,
7.1.5.2. 2-[(2-L-alanylamino)phenyl]benzoxazole 19. Compound
L
18 (6.10 g, 0.016 mol) was dissolved in a minimum amount of dry
ethyl acetate. The resulting solution was added drop-wise to dry
ethyl acetate that had been saturated with HCl (15 mL). The mix-
ture was stirred overnight and saturated aqueous Na2CO3 solution
(20 mL) and CH2Cl2 (30 mL) was added. After stirring for 3 h organ-
ic layer was separated, washed with water, dried (MgSO4) and
evaporated giving compound 19 (3.59 g, 81%) as a cream powder,
mp 222–224 °C (EtOH–H2O); (found: MH+, 282.1233. Calcd for
0.0266 mol), NMM (3.36 g, 0.033 mol), IBCF (3.63 g, 0.0266 mol)
and 2-(2-aminophenyl)benzoxazole 17 (4.20 g, 0.02 mol). Cream
crystals (8.00 g, 80%), mp 152–154 °C (EtOH); (found: MH+,
382.1760. Calcd for C21H24N3O4: MH, 382.1761); mmax cmÀ1 3365,
3210, 3105, 1675, 1624, 1585, 1556, 1496, 1183, 1165; dY
(270 MHz; CDCl3) 1.44 (9H, s, 3 Â –CH3), 1.59 (3H, d, J = 7.30 Hz,
–CH3), 4.54 (1H, quintet, J = 7.3 Hz, >CH–), 5.39 (1H, d, J = 7.3 Hz,
>NH), 7.23 (1H, t, J = 7.1 Hz, Ar-H), 7.40 (1H, t, J = 1.5 Hz, Ar-H),
7.54 (1H, m, Ar-H), 7.61 (2H, m, Ar-H), 7.81 (1H, t, J = 6.4 Hz, Ar-
H), 8.24 (1H, dd, J = 7.9 and 1.7 Hz, Ar-H), 8.84 (1H, d, J = 8.7 Hz,
Ar-H), 12.35 (1H, s broad, >NH); dC (68 MHz; CDCl3) 19.6, 28.4,
52.0, 80.0, 110.5, 113.5, 120.0, 120.5, 123.3, 124.9, 125.8, 128.4,
132.9, 138.8, 141.0, 149.3, 155.5, 162.0, 172.4.
C
16H16N3O2: MH, 282.1237); mmax cmÀ1 3414, 3352, 3093, 2921,
1667, 1619, 1525, 1581, 1437, 1245, 1194, 1139; dY (270 MHz;
CDCl3) 1.53 (3H, d, J = 6.9 Hz, –CH3), 1.74 (2H, broad s, –NH2),
3.76 (1H, q, J = 6.9 Hz, >CH–), 7.20 (1H, m, Ar-H), 7.38 (2H, m, Ar-
H), 7.52 (1H, m, Ar-H), 7.60 (1H, m, Ar-H), 7.73 (1H, m, Ar-H),
8.21 (1H, dd, J = 8.2 and 1.5 Hz, Ar-H), 8.84 (1H, dd, J = 8.7 and
1.5 Hz, Ar-H) (one >NH was not located); dC (68 MHz; CDCl3)
21.9, 52.7, 110.7, 113.6, 119.7, 120.7, 123.2, 124.9, 125.7, 128.5,
132.8, 138.9, 141.1, 149.4, 162.0, 175.9.
7.1.4.3.
2-[(t-Boc-b-alanylamino)phenyl]benzothiazole
20.
Compound 20 was synthesised from t-Boc-b-alanine (1.51 g,
8 mmol), NMM (1.01 g, 10 mmol), IBCF (1.09 g, 8 mmol) and com-
pound 14 (1.36 g, 6 mmol). Yellow-green solid (1.60 g, 67%), mp
114–116 °C (MeOH); (found: MNa+, 420.1357. Calcd for C21H23Na-
N3O3S: MNa, 420.1352); mmax cmÀ1 3365, 1706, 1679, 1624, 1247,
1162, 974; dY (270 MHz; CDCl3) 1.40 (9H, s, 3 Â –CH3), 2.79 (2H, t,
J = 6.0 Hz, >CH2), 3.60 (2H, q, J = 6.0 Hz, >CH2), 5.27 (1H, broad s,
>NH), 7.17 (1H, td, J = 7.7 and 1.2 Hz, Ar-H), 7.41–7.57 (3H, m,
Ar-H), 7.86 (1H, dd, J = 7.9 and 1.5 Hz, Ar-H), 7.92 (1H, dd, J = 7.9
and 1.0 Hz, Ar-H), 8.05 (1H, d, J = 7.9 Hz, Ar-H), 8.77 (1H, dd,
J = 8.4 and 1.2 Hz, Ar-H) (one >NH was not located); dC (68 MHz;
CDCl3) 28.5, 36.6, 38.2, 79.3, 119.1, 120.8, 121.5, 123.0, 123.4,
126.0, 126.8, 129.9, 132.0, 133.2, 137.8, 152.8, 156.0, 168.7, 170.9.
7.1.5.3. 2-[(2-b-alanylamino)phenyl]benzothiazole 21 bis TFA
salt. Compound 21 was prepared as its bis TFA salt from com-
pound 20 (0.25 g, 0.629 mmol) and TFA (7 mL). Yellow-green solid
(0.24 g, 73%), mp 170–174 °C; (found: M+, 298.1008. Calcd for
C
16H16N3OS: M+, 298.1009); dY (270 MHz; DMSO-d6) 2.91 (2H, t,
J = 6.7 Hz, >CH2), 3.19 (2H, sextet, J = 6.7 Hz, >CH2), 7.30–7.38
(1H, m, Ar-H), 7.51–7.66 (3H, m, Ar-H), 7.88 (3H, broad s, –NH3+),
8.03–8.08 (1H, m, Ar-H), 8.12–8.17 (1H, m, Ar-H), 8.19–8.24 (1H,
m, Ar-H), 8.38–8.45 (1H, m, Ar-H), 11.94 (1H, br s, >NH); dC
(68 MHz; DMSO-d6) 35.0, 35.5, 121.4, 122.6, 122.8, 123.3, 125.1,
126.7, 127.4, 130.6, 132.5, 133.9, 137.3, 152.8, 167.8, 169.5.
7.1.4.4. 2-[(t-Boc-b-alanylamino)phenyl]benzoxazole 22. Com-
pound 22 was synthesised from t-Boc-b-alanine (0.75 g, 4 mmol),
NMM (0.50 g, 5 mmol), IBCF (0.54 g, 4 mmol) and compound 17
(0.63 g, 3 mmol). Cream crystals (0.40 g, 33%), mp 143–145 °C
(MeOH); (found: MH+, 382.1760. Calcd for C21H24N3O4: MH,
382.1761); mmax cmÀ1 3365, 1679, 1619, 1244, 1168, 1040; dY
(270 MHz; CDCl3) 1.42 (9H, s, 3 Â –CH3), 2.84 (2H, t, J = 5.9 Hz,
>CH2), 3.60 (2H, q, J = 5.9 Hz, >CH2), 5.27 (1H, broad s, >NH),
7.19–7.25 (1H, m, Ar-H), 7.38–7.45 (2H, m, Ar-H), 7.51–7.58 (1H,
m, Ar-H), 7.60–7.66 (1H, m, Ar-H), 7.76–7.83 (1H, m, Ar-H), 8.22–
8.27 (1H, m, Ar-H), 8.77–8.82 (1H, m, Ar-H), 12.01 (1H, broad s,
>NH).
7.1.5.4. 2-[(2-b-alanylamino)phenyl]benzoxazole 23 TFA salt.
Compound 23 was prepared as its TFA salt from compound 22
(0.17 g, 0.45 mmol) and TFA (5 mL). Beige solid (0.19 g, 100%),
mp 188–191 °C; (found: M+, 282.1236. Calcd for C16H16N3O2: M+,
282.1237); mmax cmÀ1 3146, 1679, 1623, 1594, 1116, 1180, 1040;
dY (270 MHz; DMSO-d6) 2.93 (2H, t, J = 6.7 Hz, >CH2), 3.19 (2H, sex-
tet, J = 6.7 Hz, >CH2), 7.33–7.39 (1H, m, Ar-H), 7.46–7.56 (2H, m, Ar-
H), 7.62–7.69 (1H, m, Ar-H), 7.84 (3H, broad s, –NH3+), 7.85–7.89
(2H, m, Ar-H), 8.21–8.26 (1H, m, Ar-H), 8.50–8.55 (1H, m, Ar-H),
11.52 (1H, broad s, >NH); dC (68 MHz; DMSO-d6) 35.0, 35.5,
111.7, 114.4, 120.2, 121.5, 124.5, 125.8, 126.8, 129.3, 133.4,
138.4, 140.9, 149.5, 161.9, 169.3.
7.1.5. General procedure for the preparation of 2-[(2-alanylami-
no)phenyl]benzothiazoles 2-[(2-alanylamino)phenyl]benzoxaz-
oles 16, 19, 21 and 23
7.2. Microbiological work
7.2.1. Agar plate preparation
A solution of the t-Boc protected derivative 15, 20 or 22 in TFA
was stirred at room temperature overnight and then evaporated
yielding the products 16, 21 and 23, respectively, as their TFA
salts.18 Compound 18 was deprotected using HCl/EtOAc as detailed
below.
Each substrate (10 mg) was dissolved in 1-methyl-2-pyrroli-
done (0.2 mL) and added to molten Columbia agar (100 mL) (Ox-
oid, Basingstoke) at 50 °C to a final concentration of 100 mg/L.
Agar plates were then prepared and dried. Bacterial strains (ob-
tained from the National Collection of Type Cultures, London, UK)
were sub-cultured onto Columbia agar. Colonies of each strain
were sampled using a loop and suspended in 0.85 % sterile physi-
ological saline to generate a suspension equivalent to 108 colony
forming units per mL using a densitometer. Each agar plate was
7.1.5.1. 2-[(2-L-alanylamino)phenyl]benzothiazole 16 bis TFA
salt. Compound 16 was prepared as its bis TFA salt from
compound 15 (0.30 g, 0.755 mmol) and TFA (7 mL). Yellow-green
solid (0.35 g, 88%), mp 150–154 °C; (found: M+, 298.1008. Calcd
for C16H16N3OS: M+, 298.1009); mmax cmÀ1: 2940, 1653, 1596,
then inoculated with 10
lL of this suspension and spread to obtain
single colonies. Plates were incubated at 37 °C in air for 24 h.

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7.2.2. Cell-free extract preparation
References and notes
E. coli BL21 was grown in a nutrient broth for 24 h, centrifuged
to form a pellet which was subsequently suspended in the mini-
mum amount of Tris pH 7.4 buffer. The mixture was then sonicated
and centrifuged again. The supernatant liquid was separated and
used directly.
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Acknowledgements
We thank bioMérieux SA for generous financial support and the
EPSRC Mass Spectrometry Centre, Swansea, for high resolution
mass spectra. We thank Dr S. Reed for preparing the Escherichia coli
BL21cell-free extract and Mr E. Ludkin for assistance with the HPLC
work.
18. We thank
a referee for pointing out that the pKa values of the parent
heterocycles suggests that the benzothiazoles will give bis TFA salts whereas
the benzoxazoles will produce mono TFA salts.
Supplementary data
Supplementarydata (HPLCtraces)associatedwith thisarticlecan
be found, in the online version, at doi:10.1016/j.bmc.2011.03.043.