Synthesis of 2-arylbenzothiazole derivatives and their application in bacterial detection

Article abstract of DOI:10.1016/j.bmc.2014.01.013

A series of 2-arylbenzothiazole derivatives have been prepared as fluorogenic enzyme substrates in order to detect aminopeptidase, esterase, phosphatase and β-galactosidase activity in clinically important Gram-negative and Gram-positive bacteria. Substrates were incorporated into an agar-based culture medium and this allowed growth of intensely fluorescent bacterial colonies based on hydrolysis by specific enzymes. Substrate 20 targeted l-alanine aminopeptidase activity and was hydrolysed exclusively by a range of Gram-negative bacteria and inhibited the growth of a range of Gram-positive bacteria. Substrate 19a targeted β-alanyl aminopeptidase activity and generated fluorescent colonies of selected Gram-negative species including Pseudomonas aeruginosa. Substrate 21b targeted C8-esterase activity and resulted in strongly fluorescent colonies of selected species known to harbour such enzyme activity (e.g., Salmonella and Pseudomonas). Most Gram-negative species produced colonies with an intense blue fluorescence due to hydrolysis of phosphatase substrates 24a-c and substrate 24c was also hydrolysed by strains of Staphylococcus aureus. Compounds 26b and 26c targeted β-galactosidase activity and generated strongly fluorescent colonies with coliform bacteria that produced this enzyme (e.g., Escherichia coli).

Full text of DOI:10.1016/j.bmc.2014.01.013

Bioorganic & Medicinal Chemistry 22 (2014) 1250–1261
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 2-arylbenzothiazole derivatives and their application
in bacterial detection
Marie Cellier a, Elizabeth Fazackerley b, Arthur L. James b, Sylvain Orenga a, John D. Perry c,
Graeme Turnbull b, Stephen P. Stanforth b,
⇑
a Research & Development Microbiology, bioMérieux SA, 3 route de Port Michaud, 38 390 La-Balme-les-Grottes, France
b Department of Applied 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-arylbenzothiazole derivatives have been prepared as fluorogenic enzyme substrates in order
to detect aminopeptidase, esterase, phosphatase and b-galactosidase activity in clinically important
Gram-negative and Gram-positive bacteria. Substrates were incorporated into an agar-based culture
medium and this allowed growth of intensely fluorescent bacterial colonies based on hydrolysis by spe-
Received 21 November 2013
Revised 6 January 2014
Accepted 8 January 2014
Available online 15 January 2014
cific enzymes. Substrate 20 targeted L-alanine aminopeptidase activity and was hydrolysed exclusively by
a range of Gram-negative bacteria and inhibited the growth of a range of Gram-positive bacteria. Sub-
strate 19a targeted b-alanyl aminopeptidase activity and generated fluorescent colonies of selected
Gram-negative species including Pseudomonas aeruginosa. Substrate 21b targeted C8-esterase activity
and resulted in strongly fluorescent colonies of selected species known to harbour such enzyme activity
(e.g., Salmonella and Pseudomonas). Most Gram-negative species produced colonies with an intense blue
fluorescence due to hydrolysis of phosphatase substrates 24a–c and substrate 24c was also hydrolysed by
strains of Staphylococcus aureus. Compounds 26b and 26c targeted b-galactosidase activity and generated
strongly fluorescent colonies with coliform bacteria that produced this enzyme (e.g., Escherichia coli).
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Fluorogenic substrates
Aminopeptidase
Esterase
Phosphatase
b-Galactosidase
Bacteria detection
1. Introduction
heterocycles 7 are highly fluorescent in comparison to their O-
substituted derivatives and this has been rationalised by an ex-
The use and application of enzymatic substrates as tools for the
detection and identification of bacteria is a subject of intense aca-
demic and commercial interest.1,2 One strategy for detecting and
identifying bacteria is to allow the bacteria to grow in the presence
of an enzymatic substrate which is transformed by bacterial en-
zymes into a product that can readily be detected. The formation
of fluorescent heterocycles from bacterial transformations of het-
erocyclic substrates has been frequently used for this purpose as
outlined in Scheme 1. Thus, enzyme induced hydrolysis of weakly
fluorescent amino acid 3, ester 4, phosphate 5 and sugar 6 deriva-
tives by aminopeptidase, esterase, phosphatase and glycosidase
enzymes, respectively liberates either fluorescent amines 1 or phe-
nols 2. The generation of fluorescence (and also colour) from the
hydrolytic enzyme activities depicted in Scheme 1 has found use
in numerous commercially available protocols for bacterial
detection.1
cited-state intramolecular proton transfer (ESIPT) process. In our
recent work in this area,5,6 we prepared a series of
L-alanyl and
b-alanyl aminopeptidase substrates from heterocycles 8 and ob-
served that these substrates were hydrolysed by some bacteria lib-
erating the corresponding fluorescent amines 8. During the course
of this work we also observed that the 2-(4-hydroxyphenyl)- and
2-(4-aminophenyl)benzothiazoles 9a and 10a, respectively ap-
peared to be relatively good fluorophores. We were therefore inter-
ested in discovering whether enzyme substrates based upon these
structures might also be useful for the detection of bacterial en-
zyme activity. In this paper we report the synthesis and evaluation
of aminopeptidase substrates derived from the amines 9a and 9b
and also esterase, phosphatase and glycosidase substrates derived
from the phenols 10a–10c.
2. Synthesis of substrates
Enzyme substrates derived from O-substituted 2-(2-hydroxy-
phenyl)heterocycles 7 have been reported and these compounds
have found application in the detection of phosphatase and other
enzymatic activities (Fig. 1).3,4 The core 2-(2-hydroxyphenyl)
The parent amine 9a7 and phenol 10a8 have been reported pre-
viously and the synthetic routes used for the preparation of the
other amine 9b and phenols 10b and 10c are shown in Schemes
2 and 3, respectively. Thus, reaction of 2-amino-4-(trifluoro-
methyl)benzenethiol hydrochloride with 4-nitrobenzaldehyde
afforded the dihydro derivative 11 which was subsequently
⇑
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 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.013

M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
1251
O
O
esterase
RCO2H
aminopeptidase
O
NH2
R
Het
N
H
Het XH
O
Het
R
1
, X = NH
4
NH2
HO
3
2, X = O
weakly fluorescent
weakly fluorescent
highly fluorescent
R
_
O
_
O
O
phosphatase
glycosidase
sugar-OH
sugar
P
O
Het
O
Het
2-
HPO4
5
6
weakly fluorescent
weakly fluorescent
Scheme 1. Generation of fluorescence from hydrolytic enzymatic activity (Het = heterocycle).
X
N
X
ESIPT
X
N
N
H
HO
O
H2N
7
, X = S, O, NR
8
, X = S, O
R2
R3
S
S
N
NH2
OH
R1
N
R1
R1 R2 R3
, R1 = H
9a
9b
10a
10b
10c
H
H
H
F
H
F
F
, R1 = CF3
H
Cl
Figure 1. The structures of fluorescent heterocycles relevant to this paper.
SH
H
S
(i)
+
OHC
NO2
NO2
91%
N
H
F3C
NH2 .HCl
F3C
11
S
N
S
(iii)
(ii)
NH2
NO2
69%
N
95%
F3C
F3C
12
9b
Scheme 2. Synthesis of 2-(4-aminophenyl)-5-(trifluoromethyl)benzothiazole 9b. Reagents and conditions: (i) NaHCO3, EtOH, rt, 2 h; (ii) 2,3,5,6-tetrachloro-1,4-
benzoquinone (p-chloranil), CH2Cl2, rt, 20 h; (iii) SnCl2Á2H2O, EtOH, reflux, 2 h.
R2
R3
R2
SH
+
H
S
(i)
OHC
OMe
OMe
OH
R1
NH2
38-46%
R1
N
H
R3
R2
13
R2
R3
S
N
(ii)
73-91%
S
N
OMe
R1
R1
R3
R1 R2 R3
R1 R2 R3
14b
14c
H
Cl
F
F
H
F
10b
10c
H
Cl
F
F
H
F
Scheme 3. Synthesis of the phenols 10b and 10c. Reagents and conditions: (i) EtOH, rt then p-chloranil, CH2Cl2, rt; (ii) pyridinium hydrobromide, melt.

1252
M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
oxidised using p-chloranil giving the nitro-compound 12. Reduc-
tion of compound 12 with tin(II) chloride dihydrate gave the re-
quired amine 9b.
phenols 10a–c, respectively following a literature method.10 Deb-
enzylation of these compounds giving the required substrates
24a–c was achieved by treatment with trifluoroacetic acid. Phenols
3-Fluoro-4-methoxybenzaldehyde9 was reacted with 2-amino-
thiophenol and the resulting dihydro-intermediate 13 was not
isolated but was oxidised with p-chloranil giving the
methoxy-derivative 14b (Scheme 3). Heating this compound with
pyridinium hydrobromide in the melt afforded the required phenol
10b. Compounds 14c and 10c were prepared using a similar
procedure.
10a–c were reacted with 2,3,4,6-tetra-O-acetyl-a-D-galactosyl
bromide using Koenigs–Knorr methodology giving the acylated
b-galactoside derivatives 25a–c, respectively. Deacylation of these
compounds was achieved by treatment with methanolic sodium
methoxide solution which afforded the b-galactoside substrates
26a–c.
Amine 9a was coupled to t-Boc-protected
L-alanine, b-alanine
3. Evaluation of substrates
and di- -alanine giving the amino acid derivatives 15a, 16a and
L
17, respectively as shown in Scheme 4. Removal of the t-Boc-
groups from these compounds yielding the required substrates
18a, 19a and 20 as their bis-trifluoroacetate salts was achieved
using trifluoroacetic acid. Similarly prepared from amine 9b were
the t-Boc-protected trifluoromethylated derivatives 15b and 16b
from which the substrates 18b and 19b were obtained.
A series of esterase, phosphatase and glycosidase substrates
have been prepared from phenols 10a–c as outlined in Scheme 5.
Thus, treatment of the phenols 10a–c with octanoyl chloride in
the presence of triethylamine afforded the corresponding octano-
ate derivatives 21a–c, respectively. The nonate derivative, com-
pound 22b, was also prepared from phenol 10b using a similar
procedure. The benzyl phosphates 23a–c were synthesised from
Bacterial colonies were grown on Columbia agar plates in the
presence of a substrate. Plates were incubated (18 h) at 37 °C be-
fore being viewed in order to assess colony growth and fluores-
cence in comparison with control plates which did not contain
the substrate. Ideally, when fluorescent colonies are generated by
enzymatic hydrolysis of the substrate, the fluorescence should be
localised within the bacterial colonies. However, in some cases
the fluorophore does diffuse noticeably into the media.
L
-Alanyl aminopeptidase activity has been used to differentiate
between Gram-negative bacteria and Gram-positive bacteria and
b-alanyl aminopeptidase activity has been used for specific detec-
tion of Pseudomonas spp., a common respiratory pathogen associ-
ated with cystic fibrosis.11,12 The five substrates listed in Table 1
O
O
S
N
S
N
(ii)
(i)
NH2
NHBoc
2 TFA
N
H
9a, 9b
N
H
R1
Me
O
43-94%
83-97%
R1
Me
O
R1 amino-acid
R1 amino-acid
18a
18b
19a
H
L-alanine
CF3 L-alanine
β-alanine
N
H
NH2
Me
15a
15b
16a
H
L-alanine
CF3 L-alanine
β-alanine
N
H
NHBoc
H
H
O
O
Me
19b CF3 β-alanine
20 di-L-alanine
16b CF3 β-alanine
17 di-L-alanine
H
H
N
N
H
H
N
NH2
N
NHBoc
H
H
Me
O
Me
O
Scheme 4. Synthesis of aminopeptidase substrates. Reagents and conditions: t-Boc-amino acid, 1-ethyl-3-(3-diethylamino)propylcarbodiimide hydrochloride (EDAC), 1-
hydroxybenzotriazole, CH2Cl2, rt; (ii) CF3CO2H, rt.
R2
R2
R3
O
S
N
S
N
(i)
O
R4
OH
81-95%
R1
R1
R3
4 = C7H15
4 = C8H17
10
21
22
R
R
10
21 26
- :
In structures
and
R2
R2
O
P
O
P
R1 R2 R3
H
H
Cl
S
S
N
OCH2Ph
OCH2Ph
OH
OH
(ii)
(iii)
O
a
b
c
H
F
F
H
H
F
O
52-84%
85-94%
R1
R1
N
R3
R3
24
23
OH
OAc
OH
OH
OAc
R2
R3
R2
R3
O
O
S
N
S
(v)
(iv)
OAc
O
O
OH
R1
83-99%
27-76%
OAc
R1
N
26
25
Scheme 5. Synthesis of esterase, phosphatase and glycosidase substrates. Reagents and conditions: (i) RCOCl, Et3N, CH2Cl2, rt; (ii) HP(O)(OCH2Ph)2, CCl4, CH3CN, i-Pr2NEt, 4-
dimethylaminopyridine (cat.), -10 °C; (iii) trifluoroacetic acid, rt; (iv) CH2Cl2, 2,6-lutidine, Ag2CO3, 2,3,4,6-tetra-O-acetyl- -galactosyl bromide, rt; (v), NaOMe, MeOH, rt.
a-D

M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
1253
were evaluated for aminopeptidase activity against six Gram-neg-
ative and six Gram-positive bacteria. The substrates caused growth
inhibition of all Gram-positive species. It is evident from the data
presented in Table 1 that the trifluoromethylated substrates 18b
and 19b are also inhibitory towards most of the Gram-negative
bacteria. In cases where bacterial growth had occurred, the gener-
ation of fluorescence was generally weak, or at best, moderate.
Salmonella typhimurium and Enterobacter cloacae colonies showing
particularly intense fluorescence. With the exception of Escherichia
coli and Salmonella typhimurium, the other Gram-negative bacteria
listed in Table 1 all grew well in the presence of the b-alanyl sub-
strate 19a but only two bacteria, Serratia marcescens and Pseudo-
monas aeruginosa, produced fluorescent colonies, as expected.11
The b-alanyl substrate derived from heterocycle 8 (X = S) was more
inhibitory than substrate 19a but gave more intensely fluorescent
colonies.5
Bacterial growth was generally good in the presence of the L-alanyl
aminopeptidase substrate 18a (with the exception of Escherichia
coli and Salmonella typhimurium) and moderately fluorescent
colonies were produced. These results are broadly similar in terms
of organism growth and the intensity of fluorescence to those of
As a consequence of the trifluoromethyl-substituent in sub-
strates 18b and 19b inhibiting bacterial growth, this group was re-
placed by
a chlorine atom located at the 5-postion of the
the
previously by us.5 Based on previous work,13 the di-
substrate 20 was expected to be less inhibitory than the mono-
alanyl substrate 18a, but in fact growth was only moderate. Blue
fluorescent colonies were produced with substrate 20, with
L
-alanyl substrate derived from heterocycle 8 (X = S) reported
benzothiazole ring in some esterase, phosphatase and b-galactosi-
dase substrates. Additionally, up to two fluorine atoms were lo-
cated ortho to the phenolic hydroxyl group in order to produce a
series of substrates with increasingly acidic hydroxyl groups. Thus,
it was anticipated that the fluorinated substrates might give
L-alanyl
L
-
Table 1
Fluorescent colonies produced by various bacteria in the presence of the aminopeptidase substrates
Substrate
18a
18b
19a
19b
20
Growtha Fluorescenceb,c Growtha Fluorescenceb Growtha Fluorescenceb Growtha Fluorescenceb Growtha Fluorescenceb,c
Gram-negative organismsd
Escherichia coli NCTC
10418
Serratia marcescens
NCTC 10211
Pseudomonas aeruginosa
NCTC 10662
Salmonella typhimurium
NCTC 74
Morganella morganii WS
462403
Enterobacter cloacae
NCTC 11936
NG
++
NF
NG
+
NF
NG
++
++
NG
++
++
++
NF
NG
NG
+/À
NG
+
NF
NF
NG
+
NF
+Blue
+Blue
+Blue
+Blue
+Blue
+Blue
+Purple/Blue
+Purple/Blue
NF
+Blue
+Blue
NF
+Blue
+/À Blue
++Blue
+Blue
++Blue
+Blue
+
+
+/À Purple/
Blue
NF
+/À
+
Trace
++
NG
+
+Purple/Blue
+Purple/Blue
NF
NF
+/À Purple/
Blue
NF
+
++
+
NF
NG
+
+
Providencia rettgeri NCTC ++
NG
NF
NF
+
7475
a
b
c
++ strong growth, + moderate growth, +/À weak growth, NG no significant growth.
++ strong fluorescence, + moderate fluorescence, +/À weak fluorescence, NF no significant fluorescence. Substrate concentration = 100 mg LÀ1
.
There was noticeable diffusion of the fluorophore into the medium.
The following Gram-positive bacteria did not grow on the media and failed to give fluorescent colonies with any of the substrates: Staphylococcus epidermidis NCTC 11047,
d
Staphylococcus aureus NCTC 6571, Staphylococcus aureus (MRSA) NCTC 11939, Enterococcus faecalis NCTC 775, Enterococcus faecium NCTC 7171, Streptococcus pyogenes NCTC
8306, Listeria monocytogenes NCTC 11994.
Table 2
Fluorescent colonies produced by various bacteria in the presence of the esterase substrates
Substrate
21a
21b
21c
22b
Growtha
Fluorescenceb,c
Growtha
Fluorescenceb,c
Growtha
Fluorescence
Growtha
Fluorescenceb,c
Gram-negative organisms
Escherichia coli NCTC 10418
++
++
++
++
++
++
NF
++
++
++
++
++
++
NF
++
++
++
++
++
++
See footnote d
++
++
++
++
++
++
NF
Serratia marcescens NCTC 10211
Pseudomonas aeruginosa NCTC 10662
Salmonella typhimurium NCTC 74
Enterobacter cloacae NCTC 11936
Providencia rettgeri NCTC 7475
+Blue
+ Blue
+/À Blue
NF
++ Purple
++ Purple
++ Purple
NF
+Purple
+Purple
+Purple
NF
NF
NF
NF
Gram-positive organismse
Staphylococcus epidermidis NCTC 11047
Staphylococcus aureus NCTC 6571
Staphylococcus aureus (MRSA) NCTC 11939
+
+
+
+/À Blue
+ Blue
+ Blue
+
+
+
++ Purple
++ Purple
++ Purple
+
+
+
+
+
+
+Purple
+ Purple
+Purple
a
b
c
++ strong growth, + moderate growth, +/À weak growth, NG no significant growth.
++ strong fluorescence, + moderate fluorescence, +/À weak fluorescence, NF no significant fluorescence. Substrate concentration = 100 mg LÀ1
There was noticeable diffusion of the fluorophore into the medium.
.
d
e
A strong blue fluorescent background was produced in all plates.
The following Gram-positive bacteria 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. Growth of these bacteria was moderate.

1254
M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
improved fluorescent characteristics as they were expected to be
more ionised in agar media than their non-fluorinated
counterparts.
Phosphatase substrates are typically targeted at the detection of
Staphylococcus spp.15 Particularly important is the detection of S.
aureus, the most common cause of wound infections. Several chro-
mogenic media are commercially available that utilise a phospha-
tase substrate, usually in combination with complementary
glycosidase substrates.16 All three phosphatase substrates 24a–c
gave good growth and strongly fluorescent colonies with Gram-
negative bacteria (Table 3). With Gram-positive bacteria, growth
of all bacteria was moderate with all substrates. Substrate 24a gave
only weakly fluorescent colonies with S. aureus and no fluores-
cence was observed with other Gram-positive bacteria. In contrast,
substrates 24b and 24c both produced fluorescent colonies with all
the Gram-positive bacteria listed in Table 3 and particularly strik-
ing was the production of very intense blue-coloured colonies with
S. aureus from substrate 24c.
Salmonella is frequently detected in culture media by utilising a
C8 esterase substrate (usually in combination with other sub-
strates, inhibitors of other bacteria and other additives) because
few other species of Enterobacteriaceae produce a C8 esterase.14
The C8 esterase substrates 21a–c and the C9 substrate 22b were
therefore examined (Table 2). With this series of substrates, strong
growth was evident for all of the Gram-negative bacteria and mod-
erate growth was apparent for the Gram-positive bacteria. Com-
pound 21c was ineffective as a substrate because an intense blue
background fluorescence was produced. This was attributed to a
facile non-enzymatic hydrolysis of this substrate as a consequence
of the phenolic moiety being a relatively good leaving group due to
the presence of the two fluorine atoms. In cases where fluores-
cence was generated from the substrate, compound 21b produced
strongly fluorescent purple colonies (including Salmonella typhimu-
rium) whereas the intensity of fluorescence for the other two
substrates 21a and 22b was moderate at best.
In order to evaluate the effectiveness of the b-galactosidase sub-
strates 26a–c, these substrates were incubated in the presence of
isopropyl-b-D-1-thiogalactopyranoside, a promoter of b-galactosi-
dase activity.17 b-Galactosidase activity is typically used for the
identification of coliforms (e.g. E. coli, E. cloacae, S. marcesens) that
Table 3
Fluorescent colonies produced by various bacteria in the presence of the phosphatase substrates
Substrate
24a
24b
Fluorescenceb,c
24c
Fluorescenceb,c
Growtha
Fluorescenceb
Growtha
Growtha
Gram-negative organisms
Escherichia coli NCTC 10418
++
++
++
++
++
++
++ Blue
++ Blue
+ Blue
++ Blue
++ Blue
++ Blue
++
++
++
++
++
++
++ Blue
++ Blue
+ Blue
++ Blue
++ Blue
++ Blue
++
++
++
++
++
++
++ Blue
++ Blue
++ Blue
++ Blue
++ Blue
++ Blue
Serratia marcescens NCTC 10211
Pseudomonas aeruginosa NCTC 10662
Salmonella typhimurium NCTC 74
Enterobacter cloacae NCTC 11936
Providencia rettgeri NCTC 7475
Gram-positive organisms
Staphylococcus epidermidis NCTC 11047
Staphylococcus aureus NCTC 6571
Staphylococcus aureus (MRSA) NCTC 11939
Enterococcus faecalis NCTC 775
Enterococcus faecium NCTC 7171
Streptococcus pyogenes NCTC 8306
Listeria monocytogenes NCTC 11994
+
+
+
+
NF
+
+
+
+
+
+
+
+/À Blue
+/À Blue
+ Blue
+/À Blue
+/À Blue
+ Blue
+
+
+
+
+
+
+
+/À Blue
++ Blue
++ Blue
+/À Blue
+/À Blue
+/À Blue
+/À Blue
+/À Blue
+/À Blue
NF
NF
NF
+
+/À
+
NF
+/À Blue
a
b
c
++ strong growth, + moderate growth, +/À weak growth, NG no significant growth.
++ strong fluorescence, + moderate fluorescence, +/À weak fluorescence, NF no significant fluorescence. Substrate concentration = 100 mg LÀ1
.
There was noticeable diffusion of the fluorophore into the medium.
Table 4
Fluorescent colonies produced by various bacteria in the presence of the b-galactosidase substrates
Substrate
26a
Fluorescenceb,c
26b
Fluorescenceb,c
26c
Growtha
Growtha
Growtha
Fluorescenceb,c
Gram-negative organisms
Escherichia coli NCTC 10418
+
+
+
+
+
+
+ Purple
+Purple
NF
NF
+Purple
NF
+
+
+
+
+
+
++ Purple
++ Purple
NF
NF
++ Purple
NF
++
++
++
++
++
++
++ Blue
++ Blue
+/À Blue
+/À Blue
++ Blue
+/À Blue
Serratia marcescens NCTC 10211
Pseudomonas aeruginosa NCTC 10662
Salmonella typhimurium NCTC 74
Enterobacter cloacae NCTC 11936
Providencia rettgeri NCTC 7475
Gram-positive organisms
Staphylococcus epidermidis NCTC 11047
Staphylococcus aureus NCTC 6571
Staphylococcus aureus (MRSA) NCTC 11939
Enterococcus faecalis NCTC 775
Enterococcus faecium NCTC 7171
Streptococcus pyogenes NCTC 8306
Listeria monocytogenes NCTC 11994
+
+
+
+
+
+
+
NF
NF
NF
+/À Purple
+/À Purple
NF
+
+
+
+
+
+
+
+/À Purple
+/À Purple
+/À Purple
+Purple
+
+
+
+
+
+
+
Trace Blue
+Blue
+Blue
Trace Blue
Trace Blue
Trace Blue
Trace Blue
+Purple
+/À Purple
NF
NF
a
b
c
++ strong growth, + moderate growth, +/À weak growth, NG no significant growth. IPTG (30 mg LÀ1) was added to the media to promote b-galactosidase activity.
++ strong fluorescence, + moderate fluorescence, +/À weak fluorescence, NF no significant fluorescence. Substrate concentration = 100 mg LÀ1
.
There was noticeable diffusion of the fluorophore into the medium.

M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
1255
are often found as a result of faecal contamination of water sup-
plies.1 There are numerous commercial products which contain a
b-galactosidase substrate indicating the importance of such com-
pounds in diagnostic microbiology.1 Moderate growth was ob-
served for all of the bacteria listed in Table 4 in the presence of
substrates 26a and 26b. Only five of the bacteria produced
weakly-moderately intense purple-coloured fluorescent colonies
with substrate 26a. However, substrate 26b showed a broader
spectrum of activity and three coliform species, E. coli, E. cloacae
and S. marcesens, produced strongly fluorescent colonies. Similarly,
substrate 26c gave strongly fluorescent blue-coloured colonies
with these three species and bacterial growth was stronger with
this substrate compared to substrate 26b.
The heterocycles 21b, 24c and 26b, which have emerged as
promising substrates for the detection of esterase, phosphatase
and b-galactosidase activities, respectively in bacteria, have been
compared with their corresponding 4-methylumbelliferone (4-
MU) substrates (Figs. 2–4). All three substrates produced a deeper
blue fluorescence than their 4-MU analogues. Additionally,
significantly less diffusion of the substrates into the media was
O
C7H15
O
O
O
C7H15
S
N
O
O
F
Me
21b
Figure 2. Substrate 21b and its 4-MU analogue for Salmonella typhimurium detection (concentration of each substrate was 0.05 mmol LÀ1).
F
F
O
P
OH
OH
S
N
HO
HO
O
O
O
O
P
Cl
O
24c
Me
Figure 3. Substrate 24c and its 4-MU analogue for S. aureus detection (concentration of each substrate was 0.05 mmol LÀ1).

1256
M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
OH
OH
OH
OH
O
O
O
O
O
S
N
O
HO
OH
OH
O
OH
Me
F
26b
Figure 4. Substrate 26b and its 4-MU analogue for E. coli detection (concentration of each substrate was 0.05 mmol LÀ1).
noticeable for the substrates 21b, 24c and 26b compared with their
4-MU counter-parts.
bright yellow crystals, mp 121–122 °C; dH (270 MHz; CDCl3) 4.70
(1H, d, J = 2.7 Hz, NH), 6.52 (1H, d, J = 2.7 Hz, CH), 6.87 (1H, d,
J = 1.2 Hz, Ar-H), 6.99–7.10 (2H, m, Ar-H), 7.65 (2H, d, J = 8.7 Hz,
Ar-H), 8.19 (2H, d, J = 8.7 Hz, Ar-H); dC (68 MHz; CDCl3) 68.7,
106.0 (q, J = 3.6 Hz), 118.1 (q, J = 4.2 Hz), 121.5, 124.3 (q,
J = 272.0 Hz), 124.3, 127.4, 128.5 (q, J = 32.7 Hz), 130.4, 146.3,
4. Conclusions
A series of novel fluorogenic enzymatic substrates have been
successfully prepared for the purpose of detecting bacterial activ-
148.1, 148.7; IR
1451, 1514, 1581, 1604, 3076, 3347.
m
max cmÀ1: 840, 859, 870, 1161, 1238, 1324,
ity. The
Gram-negative and Gram-positive bacteria but these compounds
offer no significant advantage over existing -alanyl substrates. Of
L-alanyl substrates 18a and 20 can differentiate between
5.1.2. 2-(4-Nitrophenyl)-5-(trifluoromethyl)benzothiazole 12
A mixture of the dihydrobenzothiazole derivative 11 (1.31 g,
4.0 mmol) and p-chloranil (0.98 g, 4.0 mmol) in CH2Cl2 (200 mL)
was stirred (20 h) at room temperature. The resulting mixture
was filtered and the filtrate was washed with 10% aqueous NaOH
solution, dried (Na2SO4) and evaporated. The crude product was
purified by recrystallisation from MeOH giving compound 12
(0.89 g, 69%) as orange needles, mp 167–168 °C; HRMS (+NSI)
found: MH+, 325.0255. Calcd for C14H8F3N2O2S: MH, 325.0253; dH
(270 MHz; CDCl3) 7.67 (1H, dd, J = 1.7, 8.4 Hz, Ar-H), 8.06 (1H, d,
J = 8.4 Hz, Ar-H), 8.28 (2H, d, J = 8.9 Hz, Ar-H), 8.33–8.38 (3H, m,
Ar-H); dC (68 MHz; CDCl3) 121.2 (q, J = 4.2 Hz), 122.6 (q,
J = 3.6 Hz), 124.1 (q, J = 272.0 Hz), 124.5, 128.5, 129.7 (q,
L
particular interest are substrates for the detection of Salmonella
typhimurium, Staphylococcus aureus and coliforms for which com-
pounds 21b, 24c and 26b/c, respectively are well suited.
5. Experimental
1H NMR spectra (270 or 400 MHz) and 13C NMR spectra (68 or
100 MHz) were recorded on a Jeol EX270 or Jeol ECS400 instru-
ment. High resolution mass spectrometry (HRMS) was performed
by the EPSRC mass spectrometry service. Infrared spectra were ob-
tained via a diamond anvil sample cell using a Perkin Elmer 1000
spectrometer. Melting points are reported uncorrected as deter-
mined on a Stuart SMP 1 melting point apparatus. Thin layer chro-
matography was performed on Merck plastic foil plates pre-coated
with silica gel 60 F254. Merck silica gel 60 was used for column
chromatography.
J = 33.2 Hz), 130.6, 138.5, 138.8, 149.4, 153.7, 167.0; IR m :
max cmÀ1
685, 817, 851, 1144, 1264, 1335, 1526, 1597.
5.1.3. 2-(4-Aminophenyl)-5-(trifluoromethyl)benzothiazole 9b
A stirred solution of nitro-compound 12 (0.50 g, 1.5 mmol) and
tin(II) chloride dihydrate (1.74 g, 7.7 mmol) in EtOH (50 mL) was
heated (2 h) at reflux. The mixture was allowed to cool to room
temperature and poured onto ice. The pH was made slightly basic
by the addition of 5% aqueous sodium hydrogen carbonate solu-
tion. The mixture was extracted with ethyl acetate (3 Â 50 mL)
and the combined organic layers were washed with brine, dried
(Na2SO4) and evaporated giving compound 9b (0.42 g, 95%) as an
orange solid, mp 185–186 °C; HRMS (+NSI) found: MH+,
295.0515. Calcd for C14H10F3N2S: MH, 295.0511; dH (270 MHz;
CDCl3) 4.00 (2H, br s, NH2), 6.68 (2H, d, J = 8.7 Hz, Ar-H), 7.48
(1H, dd, J = 1.5, 8.2 Hz, Ar-H), 7.83 (2H, d, J = 8.7 Hz, Ar-H), 7.89
5.1. Synthetic work
5.1.1. 2-(4-Nitrophenyl)-5-(trifluoromethyl)-1,2-
dihydrobenzothiazole 11
A mixture of 4-nitrobenzaldehyde (0.66 g, 4.40 mmol), 2-ami-
no-4-(trifluoromethyl)benzenethiol
4.40 mmol) and NaHCO3 (0.74 g, 8.80 mmol) in EtOH (50 mL)
was stirred (2 h) at room temperature under a nitrogen atmo-
sphere. The mixture was filtered and water was slowly added to
the filtrate until crystals began to form. The solid was collected
and dried under vacuum giving compound 11 (1.31 g, 91%) as
hydrochloride
(1.00 g,

M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
1257
(1H, d, J = 8.2 Hz, Ar-H), 8.17 (1H, d, J = 1.5 Hz, Ar-H); dC (68 MHz;
CDCl3) 114.8, 119.6 (q, J = 4.2 Hz), 120.8 (q, J = 3.6 Hz), 122.0,
123.3, 124.4 (q, J = 272.0 Hz), 128.6 (q, J = 32.7 Hz), 129.5, 138.1,
149.9, 154.0, 170.6; IR
1470, 1603, 3226, 3330.
(1H, ddd, J = 1.4, 7.8, 8.0 Hz, Ar-H), 7.69 (1H, dd, J = 1.6, 8.5 Hz,
Ar-H), 7.81 (1H, dd, J = 1.6, 11.9 Hz, Ar-H), 7.95 (1H, d, J = 8.0 Hz,
Ar-H), 8.03 (1H, d, J = 8.0 Hz, Ar-H), 10.69 (1H, s, OH); dC
(100 MHz; DMSO-d6) 115.2 (d, J = 20.1 Hz), 118.9 (d, J = 2.9 Hz),
122.7, 123.0, 124.9 (d, J = 2.9 Hz), 125.0 (d, J = 6.7 Hz), 125.7,
127.1, 134.8, 148.7 (d, J = 12.5 Hz), 151.6 (d, J = 243.5 Hz), 154.1,
m
max cmÀ1: 700, 710, 735, 1108, 125, 1412,
5.1.4. 2-(3-Fluoro-4-methoxyphenyl)benzothiazole 14b
166.8 (d, J = 2.4 Hz); IR m
max/cmÀ1 754, 1209, 1263, 1301, 1434,
A mixture of 2-aminothiophenol (0.69 mL, 6.45 mmol) and 3-
fluoro-4-methoxybenzaldehyde (0.99 g, 6.42 mmol) were stirred
in EtOH (15 mL) at room temperature (2 h) before cooling in an
ice-water bath. The resulting light coloured precipitate was filtered
and washed with ice-cold EtOH. The filtered material was dis-
solved in CH2Cl2 (10 mL) and p-chloranil (1.58 g, 6.43 mmol) was
added with stirring at room temperature. After 19 h the mixture
was filtered through a shallow bed of Celite and the residue rinsed
with CH2Cl2 (20 mL). The filtrate was washed with aqueous NaOH
(1 M, 20 mL) and then brine (20 mL), dried (MgSO4) and evapo-
rated yielding compound 14b (0.76 g, 46%) as a light brown pow-
der. The compound was used without further purification. An
analytical portion was recrystallised from aqueous EtOH giving li-
lac-coloured needles, mp 115 °C; HRMS (+NSI) found: MH+,
260.0543. Calcd for C14H11FNOS: MH, 260.0540; dH (400 MHz;
CDCl3) 3.95 (3H, s, OCH3), 7.03 (1H, t, J = 8.2 Hz, Ar-H), 7.36 (1H,
ddd, J = 0.9, 7.6, 8.2 Hz, Ar-H), 7.47 (1H, ddd, J = 0.9, 7.6, 8.2 Hz,
Ar-H), 7.77–7.80 (1H, m, Ar-H), 7.83–7.88 (2H, m, Ar-H), 8.02
(1H, d, J = 8.2 Hz, Ar-H); dC (100 MHz; CDCl3) 56.4, 113.3, 115.2
(d, J = 21.1 Hz), 121.7, 123.1, 124.0 (d, J = 2.9 Hz), 125.2, 126.5,
126.9 (d, J = 6.9 Hz), 135.0, 150.2 (d, J = 11.5 Hz), 152.5 (d,
2200–3100 (broad).
5.1.7. 2-(3,5-Difluoro-4-hydroxyphenyl)-5-chlorobenzothiazole
10c
Using a similar procedure to that described above for the syn-
thesis of compound 10b, compound 14c (3.83 g, 12.29 mmol) gave
compound 10c (2.67 g, 73%) as a light brown powder, mp 240–
242 °C; HRMS (+NSI) found: MH+, 297.9895. Calcd for C13H735ClF2-
NOS: MH, 297.9899; dH (400 MHz; DMSO-d6) 7.39 (1H, dd, J = 1.8,
8.7 Hz, Ar-H), 7.63 (2H, dd, J = 1.4, 7.3 Hz, Ar-H), 7.96 (1H, d,
J = 1.8 Hz, Ar-H), 8.05 (1H, d, J = 8.7 Hz, Ar-H), 11.13 (1H, br s,
OH); dC (100 MHz; DMSO-d6) 111.4 (dd, J = 16.3, 8.2 Hz), 122.5,
123.3 (t, J = 9.1 Hz), 124.2, 126.0, 131.9, 133.8, 137.7 (t,
J = 16.8 Hz), 152.8 (dd, J = 7.7, 243.5 Hz), 154.7, 167.9; IR mmax
/
cmÀ1 748, 754, 797, 856, 1017, 1261, 1333, 1434, 1485, 2200–
3200 (broad).
5.1.8. General procedure for the preparation of the Boc-prote-
cted derivatives 15–17
To a stirred solution of the amine 9a or 9b (1 equiv) in CH2Cl2
was added 1-ethyl-3-(3-dimethylamino)propylcarbodiimideÁHCl
(EDAC) (0.6 equiv), 1-hydroxybenzotriazole (HOBt) (0.6 equiv)
and the relevant t-Boc protected amino acid (0.6 equiv). The mix-
ture was allowed to stir at room temperature (24 h) and a further
0.6 equiv of each reagent was added. The mixture was then al-
lowed to stir at room temperature (4 days) and the solvent was
evaporated giving the crude product.
J = 246.8 Hz), 154.2, 166.6; IR
1116, 1267, 1278, 1493, 1616.
m
max/cmÀ1 719, 748, 860, 1000,
5.1.5. 2-(3,5-Difluoro-4-methoxyphenyl)-5-chlorobenzothiazole
14c
3,5-Difluoro-4-methoxybenzaldehyde9 (2.38 g, 13.83 mmol)
was dissolved in EtOH (40 mL) and 2-amino-4-chlorothiophenol
(2.24 g, 14.03 mmol) was added with stirring at room temperature.
After 18 h solvent was evaporated and replaced by CH2Cl2 (40 mL).
p-chloranil (3.41 g, 13.87 mmol) was added and the mixture was
stirred (20 h) at room temperature. The mixture was filtered
through a bed of Celite and the residue rinsed by CH2Cl2 (50 mL).
The filtrate was washed sequentially with aqueous NaOH (1 M,
2 Â 50 mL), water (50 mL) and brine (50 mL), dried (MgSO4) and
evaporated. The crude product was purified by column chromatog-
raphy over silica gel (eluent: heptane/EtOAc, 10:1) giving com-
pound 14c (1.65 g, 38%). An analytical portion was recrystallised
from aqueous EtOH giving white crystals, mp 161–162 °C; HRMS
(+NSI) found: MH+, 312.0055. Calcd for C14H935ClF2NOS: MH,
312.0056; dH (400 MHz; CDCl3) 4.10 (3H, s, CH3), 7.38 (1H, dd,
J = 2.1, 8.7 Hz, Ar-H), 7.63 (2H, d, J = 9.2 Hz, Ar-H), 7.80 (1H, d,
J = 8.7 Hz, Ar-H), 8.03 (1H, d, J = 2.1 Hz, Ar-H); dC (100 MHz; CDCl3)
61.9, 111.7 (dd, J = 17.3, 7.7 Hz), 122.4, 123.3, 126.2, 127.7 (t,
J = 9.6 Hz), 132.7, 133.4, 139.1 (t, J = 13.4 Hz), 154.8, 155.6 (dd,
5.1.8.1.
15a.
2-[4-(t-Boc-
L-alanylamino)phenyl]benzothiazole
Compound 15a was synthesized from amine 9a (0.50 g,
2.21 mmol), EDACÁHCl (0.46 g, 2.44 mmol), HOBt (0.32 g,
2.44 mmol), and t-Boc- -alanine (0.42 g, 2.22 mmol) in CH2Cl2
L
(50 mL) following the general procedure described above. The
crude product was purified by column chromatography [eluent:
CH2Cl2 changing to CH2Cl2/MeOH (97:3)] giving compound 15a
(75%) as pale green crystals, mp 197–198 °C; HRMS (+NSI) found:
MH+, 398.1530. Calcd for C21H24N3O3S: MH, 398.1533; dH
(270 MHz; DMSO-d6) 1.27 (3H, d, J = 7.2 Hz, CH3), 1.37 (9H, s, t-
Bu), 4.12 (1H, td, J = 7.2, 7.2 Hz, CH), 7.20 (1H, d, J = 7.2 Hz, NH),
7.40–7.45 (1H, m, Ar-H), 7.49–7.55 (1H, m, Ar-H), 7.80 (2H, d,
J = 8.7 Hz, Ar-H), 8.00–8.03 (1H, m, Ar-H), 8.05 (2H, d, J = 8.7 Hz,
Ar-H), 8.11 (1H, ddd, J = 0.7, 1.5, 7.9 Hz, Ar-H), 10.29 (1H, s, NH);
dC (68 MHz; DMSO-d6) 18.5, 28.8, 51.2, 78.7, 119.9, 122.8, 123.1,
125.8, 127.1, 128.1, 128.6, 134.9, 142.5, 154.2, 155.8, 167.5,
173.0; IR: mmax cmÀ1: 724, 752, 834, 1155, 1248, 1314, 1367,
1455, 1515, 1672, 2988, 3327.
J = 6.7, 250.2 Hz), 166.9; IR m
max/cmÀ1 750, 804, 871, 944, 1032,
1281, 1343, 1426, 1440, 1486, 2951, 3086.
5.1.8.2. 2-[4-(t-Boc-L-alanylamino)phenyl]-5-trifluoromethyl-
5.1.6. 2-(3-Fluoro-4-hydroxyphenyl)benzothiazole 10b
benzothiazole 15b.
Compound 15b was synthesized from
Compound 14b (0.75 g, 2.89 mmol) was mixed with pyridine
hydrobromide (3.70 g, 23.12 mmol) and heated under an argon
atmosphere until melting had occurred. After 1.5 h the mixture
was cooled and ice (50 g) was added with stirring. The resulting
slurry was extracted into EtOAc (3 Â 50 mL) and the combined or-
ganic layers washed with brine (50 mL), dried (MgSO4) and evapo-
rated giving compound 10b (0.65 g, 91%) as light brown crystals,
mp 222–223 °C; HRMS (+NSI) found: MH+, 246.0387. Calcd for
amine 9b (0.50 g, 1.70 mmol), EDACÁHCl (0.40 g, 2.04 mmol), HOBt
(0.28 g, 2.04 mmol), and t-Boc- -alanine (0.38 g, 2.04 mmol) in
L
CH2Cl2 (50 mL) following the general procedure described above.
The crude product was purified by recrystallisation from MeOH/
H2O giving compound 15b (43%) as pale green crystals, mp 217–
219 °C; HRMS (+NSI) found: MH+, 466.1402. Calcd for C22H23F3N3-
O3S: MH, 466.1407; dH (270 MHz; DMSO-d6) 1.27 (3H, d, J = 7.2 Hz,
CH3), 1.37 (9H, s, t-Bu), 4.13 (1H, td, J = 7.2, 7.2 Hz, CH), 7.19 (1H, d,
J = 7.2 Hz, NH), 7.75 (1H, d, J = 8.7 Hz, Ar-H), 7.83 (2H, d, J = 8.7 Hz,
Ar-H), 8.08 (2H, d, J = 8.7 Hz, Ar-H), 8.45–8.39 (2H, m, Ar-H), 10.34
C13H9FNOS: MH, 246.0383; dH (400 MHz; DMSO-d6) 7.08 (1H, t,
J = 8.5 Hz, Ar-H), 7.36 (1H, ddd, J = 1.2, 7.8, 8.0 Hz, Ar-H), 7.46

1258
M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
(1H, s, NH); IR mmax cmÀ1: 669, 819, 834, 1119, 1147, 1250, 1318,
5.1.9.1. 2-[4-(L-Alanylamino)phenyl]benzothiazole bis TFA salt
1338, 1406, 1515, 1673, 2985, 3334.
18a. Compound 15a (0.50 g, 1.30 mmol) in TFA (5 mL) gave
compound 18a (0.66 g, 97%) as a pale green solid, mp 112 °C;
HRMS (ESI) found: MH+, 298.1005. Calcd for C16H16N3OS: MH,
298.1009; dH (270 MHz; DMSO-d6) 1.47 (3H, d, J = 6.9 Hz, CH3),
4.02–4.08 (1H, m, CH), 7.44 (1H, ddd, J = 1.2, 6.9, 7.4 Hz, Ar-H),
7.53 (1H, ddd, J = 1.2, 6.9, 7.7 Hz, Ar-H), 7.80 (2H, d, J = 8.7 Hz,
Ar-H), 8.02 (1H, ddd, J = 0.5, 1.2, 7.4 Hz, Ar-H), 8.10 (2H, d,
J = 8.7 Hz, Ar-H), 8.11–8.12 (1H, m, Ar-H), 8.25 (3H, d, J = 5.7 Hz,
NH3+), 10.77 (1H, s, NH); dC (68 MHz; DMSO-d6) 17.6, 49.7, 120.3,
122.8, 123.2, 125.9, 127.1, 128.7, 128.9, 134.9, 141.5, 154.2,
5.1.8.3.
16a.
2-[4-(t-Boc-b-alanylamino)phenyl]benzothiazole
Compound 16a was synthesized from amine 9a (0.39 g,
1.72 mmol), EDACÁHCl (0.40 g, 2.06 mmol), HOBt (0.28 g,
2.06 mmol) and t-Boc-b-alanine (0.38 g, 2.06 mmol) in CH2Cl2
(50 mL) following the general procedure described above. The
crude product was purified by column chromatography (eluent;
CH2Cl2/MeOH, 9:1) giving compound 16a (94%) as pale green crys-
tals, mp 171–173 °C; HRMS (+NSI) found: MH+, 398.1535. Calcd for
C21H24N3O3S: MH, 398.1533; dH (270 MHz, DMSO-d6) 1.31 (9H, s,
167.3, 169.3; IR
1524, 1667, 2400–3000, 2920, 3358.
m
max cmÀ1: 702, 721, 760, 1127, 1314, 1445,
t-Bu), 2.44–2.49 (2H, m, CH2), 3.18 (2H, td, J = 5.4, 6.7 Hz, CH2),
6.87 (1H, t, J = 5.4 Hz, NH), 7.34–7.39 (1H, m, Ar-H), 7.43–7.49
(1H, m, Ar-H), 7.74 (2H, d, J = 8.7 Hz, Ar-H), 7.94–7.96 (1H, m, Ar-
H), 7.98 (2H, d, J = 8.7 Hz, Ar-H), 8.05 (1H, ddd, J = 0.5, 0.7, 7.9 Hz,
Ar-H), 10.24 (1H, br s, NH); dC (68 MHz; DMSO-d6) 28.8, 37.0,
37.5, 78.2, 119.8, 122.8, 123.1, 125.8, 127.1, 128.0, 128.6, 134.8,
5.1.9.2. 2-[4-(
thiazole bis TFA salt 18b.
L
-Alanylamino)phenyl]-5-trifluoromethylbenzo-
Compound 15b (0.30 g, 0.65 mmol)
in TFA (5 mL) gave compound 18b as a pale orange solid, (0.37 g,
96%), mp 205–207 °C; HRMS (+NSI) found: MH+, 366.0888. Calcd
for C17H15F3N3OS: MH, 366.0882; dH (270 MHz; DMSO-d6) 1.48
(3H, d, J = 6.7 Hz, CH3), 4.09 (1H, q, J = 6.7 Hz, CH), 7.75 (1H, d,
J = 8.4 Hz, Ar-H), 7.84 (2H, d, J = 8.7 Hz, Ar-H), 8.13 (2H, d,
J = 8.7 Hz, Ar-H), 8.31–8.39 (5H, m, 2Â Ar-H, NH3+), 10.83 (1H, s,
NH); dC (68 MHz; DMSO-d6) 17.6, 49.7, 119.9 (q, J = 4.2 Hz),
120.3, 121.9 (q, J = 3.6 Hz), 124.3, 124.9 (q, J = 272.5 Hz), 128.1 (q,
J = 32.2 Hz), 128.3, 129.0, 139.0, 142.1, 153.8, 169.4, 170.0; IR mmax
cmÀ1: 673, 724, 802, 1122, 1313, 1333, 1485, 1544, 1672, 2700–
3200, 2917, 3305.
142.6, 154.2, 156.1, 167.6, 170.5; IR
1163, 1227, 1247, 1406, 1480, 1590, 1683, 2985, 3360.
m
max cmÀ1: 724, 751, 832,
5.1.8.4. 2-[4-(t-Boc-b-alanylamino)phenyl]-5-trifluoromethyl-
benzothiazole 16b.
Compound 16b was synthesized from
amine 9b (0.5 g, 1.70 mmol), EDACÁHCl (0.40 g, 2.04 mmol), HOBt
(0.28 g, 2.04 mmol) and t-Boc-b-alanine (0.38 g, 2.04 mmol) in
CH2Cl2 (50 mL) following the general procedure described above.
The crude product was purified by recrystallisation from MeOH/
CH2Cl2 giving compound 16b (40%) as pale green crystals, mp
197–198 °C; HRMS (+NSI) found: MH+, 466.1391. Calcd for C22H23-
F3N4O4S: MH, 466.1407; dH (270 MHz; DMSO-d6) 1.32 (9H, s, t-Bu),
2.49–2.53 (2H, m, CH2), 3.23 (2H, td, J = 5.7, 6.6, 6.3 Hz, CH2), 6.92
(1H, t, J = 5.7 Hz, NH), 7.73 (1H, dd, J = 1.2, 8.7 Hz, Ar-H), 7.81 (2H,
d, J = 8.7 Hz, Ar-H), 8.05 (2H, d, J = 8.7 Hz, Ar-H), 8.34–8.35 (1H, m,
Ar-H), 8.37 (1H, d, J = 8.7 Hz, Ar-H), 10.34 (1H, s, NH); dC (68 MHz;
DMSO-d6) 28.8, 37.0, 37.5, 78.2, 119.7 (q, J = 4.2 Hz), 119.8, 121.7
(q, J = 4.2 Hz), 124.3, 124.9 (q, J = 272.0 Hz), 127.3, 128.0 (q,
J = 31.7 Hz), 128.9, 139.0, 143.2, 153.8, 156.1, 170.3, 170.6; IR
5.1.9.3. 2-[4-(b-Alanylamino)phenyl]benzothiazole bis TFA salt
19a.
Compound 16a (0.50 g, 1.30 mmol) in TFA (5 mL) gave
compound 19a as a pale green solid (0.63 g, 92%), mp 137 °C;
HRMS (+NSI) found: MH+, 298.1008. Calcd for C16H16N3OS: MH,
298.1009; dH (270 MHz, DMSO-d6) 2.76 (2H, t, J = 6.7 Hz, CH2),
3.06–3.18 (2H, m, CH2), 7.39–7.46 (1H, m, Ar-H), 7.49–7.55 (1H,
m, Ar-H), 7.80 (2H, d, J = 8.7 Hz, Ar-H), 8.01 (1H, ddd, J = 0.5, 1.2,
7.9 Hz, Ar-H), 8.06 (2H, d, J = 8.7 Hz, Ar-H), 8.11 (1H, ddd, J = 0.5,
1.0, 7.7 Hz, Ar-H), 10.52 (1H, s, NH); dC (68 MHz, DMSO-d6) 34.0,
35.4, 119.9, 122.8, 123.1, 125.8, 127.2, 128.3, 128.6, 134.9, 142.3,
m
max cmÀ1: 669, 819, 831, 1119, 1146, 1248, 1334, 1406, 1517,
1681, 2926, 3358.
154.2, 167.5, 169.4; IR m
max cmÀ1: 725, 753, 827, 1073, 1311,
1481, 1538, 1661, 2700–3100, 3053, 3342.
5.1.8.5. 2-[4-(t-Boc-L-alanyl-L-alanylamino)phenyl]benzothia-
zole 17.
(0.5 g, 2.21 mmol), EDACÁHCl (0.50 g, 2.64 mmol), HOBt (0.36 g,
2.64 mmol), and t-Boc- -alanine- -alanine (0.68 g, 2.64 mmol) in
Compound 17 was synthesized from amine 9a
5.1.9.4. 2-[4-(b-Alanylamino)phenyl]-5-trifluoromethylbenzo-
thiazole bis TFA salt 19b.
Compound 16b (0.30 g, 0.58 mmol)
L
L
in TFA (5 mL) gave compound 19b as cream crystals (0.33 g, 96%),
mp 229–230 °C; HRMS (+NSI) found: MH+, 366.0883. Calcd for C17-
H15F3ON3S: MH, 366.0882; dH (270 MHz; DMSO-d6) 2.78 (2H, t,
J = 6.4 Hz, CH2), 3.13 (2H, t, J = 6.4 Hz, CH2), 7.71 (1H, dd, J = 1.2,
8.4 Hz, Ar-H), 7.82 (2H, d, J = 8.7 Hz, Ar-H), 7.99 (3H, br s, NH3+),
8.06 (2H, d, J = 8.7 Hz, Ar-H), 8.30 (1H, d, J = 1.2 Hz, Ar-H), 8.33
(1H, d, J = 8.4 Hz, Ar-H), 10.61 (1H, s, NH); dC (68 MHz; DMSO-d6)
34.0, 35.4, 119.7 (q, J = 4.2 Hz), 119.9, 121.7 (q, J = 4.2 Hz), 124.2,
124.8 (q, J = 272.0 Hz), 127.8, 128.0 (q, J = 31.7 Hz), 128.8, 139.0,
142.8, 153.8, 169.5, 170.1; IR: mmax cmÀ1: 654, 723, 796, 1135,
1329, 1409, 1520, 1669, 2600–3000, 2922, 3323.
CH2Cl2 (50 mL) following the general procedure described above.
The crude product was purified by recrystallisation from MeOH/
CH2Cl2 (with charcoal) giving compound 17 (66%) as white crys-
tals, mp 265–266 °C; HRMS (+NSI) found: MH+, 469.1893. Calcd
for C24H29N4O4S: MH, 469.1904; dH (270 MHz; DMSO-d6) 1.19
(3H, d, J = 7.2 Hz, CH3), 1.33 (3H, d, J = 6.9 Hz, CH3), 1.38 (9H,
s, t-Bu), 3.95–4.06 (1H, m, CH), 4.37–4.48 (1H, m, CH), 6.95
(1H, d, J = 7.2 Hz, NH); 7.43 (1H, ddd, J = 1.2, 7.2, 7.9 Hz, Ar-H),
7.53 (1H, ddd, J = 1.2, 7.2, 8.3 Hz, Ar-H), 7.80 (2H, d, J = 8.7 Hz,
Ar-H), 8.00–8.07 (4H, m, 3Â Ar-H, 1Â NH), 8.12 (1H, ddd,
J = 0.5, 1.2, 7.9 Hz, Ar-H), 10.27 (1H, s, NH); IR mmax cmÀ1: 724,
752, 834, 1162, 1227, 1251, 1366, 1480, 1520, 1652, 2988, 3295.
5.1.9.5.
20.
2-[4-(
L-Alanyl-L-alanylamino)phenyl]benzothiazole
Compound 17 (0.50 g, 1.07 mmol) in TFA (5 mL) gave
5.1.9. General procedure for the preparation of the amino acid
derivatives 18–20
compound 20 as off-white crystals (0.53 g, 83%), mp 241–242 °C;
HRMS (+NSI) found: MH+, 369.1383. Calcd for C19H21N4O2S: MH,
369.1380; dH (270 MHz; DMSO-d6) 1.37 (3H, d, J = 7.2 Hz, CH3),
1.38 (3H, d, J = 6.7 Hz, CH3), 3.91 (1H, q, J = 6.7 Hz, CH), 4.50 (1H,
dq, J = 6.9, 7.2 Hz, CH), 7.42 (1H, ddd, J = 1.0, 7.2, 8.0 Hz, Ar-H),
7.52 (1H, ddd, J = 1.0, 7.0, 8.0 Hz, Ar-H), 7.81 (2H, d, J = 8.7 Hz,
Ar-H), 8.01 (1H, ddd, J = 0.5, 1.0, 7.2 Hz, Ar-H), 8.05 (2H, d,
A mixture of the appropriate t-Boc-protected derivative in tri-
fluoroacetic acid (TFA) was stirred at room temperature for 24 h.
The excess trifluoroacetic acid was removed under reduced pres-
sure and bis-TFA salt obtained was dried under vacuum. No further
purification was required.

M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
1259
J = 8.7 Hz, Ar-H), 8.10 (1H, ddd, J = 0.5, 1.0, 7.0 Hz, Ar-H), 8.19 (3H,
br s, NH3+), 8.81 (1H, d, J = 6.9 Hz, NH), 10.49 (1H, s, NH); dC
(68 MHz; DMSO-d6) 17.7, 18.4, 48.6, 50.0, 120.0, 122.8, 123.2,
125.9, 127.2, 128.3, 128.6, 134.8, 142.3, 154.2, 167.5, 170.0,
noyl chloride and was obtained as a waxy cream solid, mp 67–
68 °C; HRMS (+NSI) found: MH+, 386.1586. Calcd for C22H25FNO2S:
MH, 386.1585; dH (400 MHz; CDCl3) 0.89 (3H, t, J = 7.3 Hz, CH3),
1.46–1.24 (10H, m, 5Â CH2), 1.78 (2H, quintet, J = 7.3 Hz, bCH2),
171.8; IR
m
max cmÀ1: 699, 722, 756, 1117, 1382, 1484, 1538,
2.62 (2H, td, J = 7.3 Hz, aCH2), 7.22–7.25 (1H, m, 1H, Ar-H), 7.39
1651, 2700–3100, 2986, 3282.
(1H, ddd, J = 0.9, 6.9, 8.2 Hz, Ar-H), 7.49 (1H, ddd, J = 1.4, 6.9,
8.5 Hz, Ar-H), 7.82–7.84 (1H, m, Ar-H), 7.89 (1H, d, J = 8.5 Hz, Ar-
H), 7.95 (1H, dd, J = 2.3, 10.5 Hz, Ar-H), 8.06 (1H, d, J = 8.2 Hz, Ar-
H); dC (100 MHz; CDCl3) 14.2, 22.8, 25.0, 29.1, 29.2, 29.3, 31.9,
34.0, 115.8 (d, J = 21.1 Hz), 121.8, 123.5, 123.8 (d, J = 3.8 Hz),
124.5, 125.6, 126.6, 132.9 (d, J = 7.7 Hz), 135.2, 140.4 (d,
5.1.10. General procedure for the preparation of the esters 21
and 22
The procedure described below for the synthesis of compound
21a is typical. The other esters were prepared similarly.
J = 13.4 Hz), 154.1, 154.4 (d, J = 251.1 Hz), 165.7, 171.0; IR mmax
/
5.1.10.1. 4-(Benzothiazol-2-yl)-2-phenyl octanoate 21a.
A
cmÀ1 730, 755, 878, 994, 1118, 1134, 1270, 1432, 1775, 2847, 2914.
solution of octanoyl chloride (0.21 g, 1.32 mmol) in CH2Cl2
(10 mL) was added drop-wise to a stirred solution of compound
10a (0.30 g, 1.32 mmol) and Et3N (0.27 g, 2.64 mmol) in CH2Cl2
(10 mL). The mixture was allowed to stir (1 h) at room tempera-
ture. The mixture was neutralized by the addition of dilute aque-
ous HCl solution and then water (10 mL) was added. The mixture
was extracted with CH2Cl2 (2 Â 10 mL) and the combined organic
extracts were dried (MgSO4) and evaporated yielding the crude
product. The crude product was purified by recrystallisation from
CH2Cl2/MeOH giving compound 21a as a cream solid, (0.38 g,
81%), mp 88–89 °C; HRMS (+NSI) found: MH+, 354.1518. Calcd
for C21H23NO2S: MH, 354.1522; dH (270 MHz; CDCl3) 0.89 (3H, t,
J = 6.4 Hz, CH3), 1.30–1.36 (8H, m, 4Â CH2), 1.76 (2H, tt, J = 6.9,
7.3 Hz, CH2), 2.57 (2H, t, J = 7.30 Hz, CH2), 7.22 (2H, d, J = 8.7 Hz,
Ar-H), 7.33–7.39 (1H, m, Ar-H), 7.44–7.50 (1H, m, Ar-H), 7.87
(1H, dd, J = 1.2, 7.9 Hz, Ar-H), 8.05 (1H, dd, J = 1.2, 8.2 Hz, Ar-H),
8.09 (2H, d, J = 8.7 Hz, Ar-H); dC (68 MHz; CDCl3) 14.2, 22.7, 25.0,
29.0, 29.1, 31.7, 34.5, 121.7, 122.4, 123.3, 125.3, 126.5, 128.8,
5.1.11. General procedure for the synthesis of the dibenzyl
phosphates 23
To a stirred solution of the appropriate phenol (1 equiv) in
anhydrous CH3CN, at À10 °C, was added CCl4 (5 equiv), N,N-diiso-
propylethylamine (DIPEA) (2.1 equiv), and N,N-dimethylaminopyr-
idine (DMAP) (0.1 equiv). The mixture was allowed to stir (1 min)
before dibenzyl phosphite (1.45 equiv) was added drop-wise while
keeping the temperature of the reaction mixture below À10 °C. The
reaction was allowed to stir (1.5 h) and 0.5 M aq KH2PO4 (32 mL
per 100 mL CH3CN) was then added. The mixture allowed to warm
to room temperature and was extracted with EtOAc (3 Â 20 mL).
The combined organic extracts were washed successively with
water (30 mL) and brine (30 mL), dried (NaSO4) and evaporated.
5.1.11.1.
23a.
4-(Benzothiazol-2-yl)phenyl-1-dibenzylphosphate
Compound 23a was synthesized from compound 10a
(0.30 g, 1.32 mmol), CCl4 (1.02 g, 6.6 mmol), DIPEA (0.36 g,
2.77 mmol), DMAP (0.16 g, 0.13 mmol), and dibenzyl phosphite
(0.50 g, 1.91 mmol) in acetonitrile (20 mL) following the general
procedure described above. Yield (0.54 g, 84%), mp 110–112 °C;
HRMS (+NSI) found: MH+, 488.1085. Calcd for C27H23NO4PS: MH,
488.1080; dH (270 MHz; DMSO-d6) 5.08 (4H, d, J = 8.7 Hz, 2Â
CH2), 7.15 (2H, d, J = 8.4 Hz, Ar-H), 7.21–7.26 (10H, m, Ar-H),
7.27–7.32 (1H, m, Ar-H), 7.40 (1H, ddd, J = 1.2, 7.2, 8.2 Hz, Ar-H),
7.80 (1H, ddd, J = 0.5, 1.2, 7.9 Hz, Ar-H), 7.92 (2H, d, J = 8.4 Hz, Ar-
H), 7.97 (1H, ddd, J = 0.5, 1.2, 8.2 Hz, Ar-H); dC (68 MHz; DMSO-d6)
70.3 (d, J = 5.7 Hz), 120.8 (d, J = 5.2 Hz), 122.0, 123.2, 125.6, 126.7,
128.3, 128.8, 129.0, 129.1, 130.6, 135.1, 135.4 (d, J = 6.8 Hz),
131.3, 135.2, 152.9, 154.2, 167.1, 172.0; IR
755, 1112, 1164, 1410, 1465, 1478, 1606, 1749, 2922.
m
max cmÀ1: 690, 724,
5.1.10.2.
21b.
4-(Benzothiazol-2-yl)-2-fluorophenyl
octanoate
Compound 10b (277 mg, 90%) was obtained as a waxy
yellow solid, mp 77–78 °C; HRMS (+NSI) found: MH+, 372.1424.
Calcd for C21H23FNO2S: MH, 372.1428; dH (400 MHz; CDCl3) 0.90
(3H, t, J = 6.9 Hz, CH3), 1.24–1.46 (8H, m, 4Â CH2), 1.78 (2H, quin-
tet, J = 7.8 Hz, bCH2), 2.62 (2H, t, J = 7.8 Hz,
aCH2), 7.22–7.26 (1H,
m, Ar-H), 7.39 (1H, ddd, J = 0.9, 7.7, 8.2 Hz, Ar-H), 7.49 (1H, ddd,
J = 0.9, 7.7, 8.2 Hz, Ar-H), 7.82–7.84 (1H, m, Ar-H), 7.89 (1H, d,
J = 8.2 Hz Ar-H), 7.93 (1H, dd, J = 2.3, 11.0 Hz, Ar-H), 8.06 (1H, d,
J = 8.2 Hz, Ar-H); dC (100 MHz; CDCl3) 14.2, 22.7, 25.0, 29.0, 29.1,
31.7, 34.0, 115.8 (d, J = 20.1 Hz), 121.8, 123.5, 123.8 (d,
J = 2.9 Hz), 124.5, 125.6, 126.6, 132.9 (d, J = 7.7 Hz), 135.2, 140.4
152.7 (d, J = 6.8 Hz), 154.1, 166.6; IR
m
max cmÀ1: 696, 731, 744,
1013, 1168, 1208, 1274, 1436, 1479, 1606, 3059.
5.1.11.2. 4-(Benzothiazol-2-yl)-2-fluorophenyl-1-dibenzylphos-
(d, J = 13.4 Hz), 154.1, 154.4 (d, J = 250.2 Hz), 165.1, 171.0; IR mmax
/
phate 23b.
10b (490 mg, 2.00 mmol), CCl4 (969
(418 L, 2.40 mmol), DMAP (a few crystals), and dibenzyl phos-
phite (944 L, 4.27 mmol) in CH3CN (25 mL) following the general
procedure described above. The crude product was purified by col-
umn chromatography (eluent: CH2Cl2 changing to EtOAc/CH2Cl2,
1:10) yielding compound 23b (674 mg, 67%) as a pale yellow solid,
mp 96–97 °C; HRMS (+NSI) found: MH+, 506.0980. Calcd for
Compound 23b was synthesized from compound
cmÀ1 726, 760, 890, 1108, 1125, 1269, 1430, 1770, 2847, 2918.
lL, 10.04 mmol), DIPEA
l
5.1.10.3. 4-(5-Chlorobenzo[d]thiazol-2-yl)-2,6-difluorophenyl
octanoate 21c. Compound 21c (137 mg, 95%) was obtained
l
as a waxy light-brown solid, mp 66–67 °C; HRMS (+NSI) found:
MH+, 424.0946. Calcd for C21H2135ClF2NO2S: MH, 424.0944; dH
(400 MHz; CDCl3) 0.84–0.91 (3H, t, CH3), 1.25–1.46 (8H, m, 4Â
CH2), 1.79 (2H, quintet, J = 7.6 Hz, CH), 2.66 (2H, t, J = 7.6 Hz,
CH2), 7.37 (1H, dd, J = 2.3, 8.7 Hz, Ar-H), 7.67 (2H, d, J = 8.2 Hz,
Ar-H), 7.79 (1H, d, J = 8.7 Hz, Ar-H), 8.02 (1H, d, J = 2.3 Hz, Ar-H);
C27H21FNO4PS: MH, 506.0986; dH (400 MHz; CDCl3) 5.18 (4H, d,
J = 8.7 Hz, 2Â CH2), 7.37 (1H, d, J = 9.2 Hz, Ar-H), 7.33–7.40 (10H,
m, Ar-H), 7.40 (1H, m, Ar-H), 7.50 (1H, ddd, J = 1.4, 7.8, 8.2 Hz,
Ar-H), 7.72 (1H, d, J = 8.2 Hz, Ar-H), 7.87–7.90 (2H, m, Ar-H), 8.06
(1H, d, J = 8.2 Hz, Ar-H); dC (100 MHz; CDCl3) 70.6 (d, J = 5.8 Hz),
116.0 (d, J = 20.1 Hz), 121.8, 122.9 (d, J = 2.9 Hz), 123.5, 123.8 (d,
J = 1.9 Hz), 125.7, 126.7, 128.2, 128.7, 128.9, 131.9 (d, J = 7.2 Hz),
135.2 (d, J = 6.7 Hz), 135.3, 140.3 (dd, J = 6.7, 12.5 Hz), 154.1,
IR m
max/cmÀ1 805, 846, 879, 906, 1027, 1043, 1092, 1431, 1488,
1777, 2856, 2925, 3027. The 1H-NMR spectrum indicated that a
small quantity of an aliphatic impurity was present.
5.1.10.4.
22b.
4-(Benzothiazol-2-yl)-2-fluorophenyl
Compound 22b (297 mg, 92%) was prepared using nona-
nonanoate

1260
M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
153.7 (dd, J = 5.8, 251.1 Hz), 165.6; IR
894, 930, 994, 1021, 1270, 1490.
m
max/cmÀ1 694, 732, 868,
134.3, 154.6, 155.8 (dd, J = 4.8, 255.0 Hz), 167.3;
906, 962, 1037, 1346, 1436, 1434, 2200–3200.
m
max/cmÀ1 808,
5.1.11.3. 4-(5-Chlorobenzothiazol-2-yl)-2,6-difluorophenyl-1-
dibenzyl phosphate 23c. Compound 23c was prepared in a
5.1.13. General procedure for the preparation of the acylated
sugars 25
similar manner to compound 23b. The crude product was purified
was by column chromatography (eluent: CH2Cl2) yielding com-
pound 23c (583 mg, 52%) as a cream solid, mp 106–107 °C; HRMS
(+NSI) found: MH+, 558.0497. Calcd 558.0502 for C27H2035ClF2NO4-
PS: MH, 558.0502; dH (400 MHz; CDCl3) 5.26 (4H, dd, J = 1.8, 7.3 Hz,
2Â CH2), 7.34–7.40 (11H, m, Ar-H), 7.65 (2H, d, J = 8.2 Hz, Ar-H),
7.77 (1H, d, J = 8.7 Hz, Ar-H), 8.02 (1H, d, J = 1.8 Hz, Ar-H); dC
(100 MHz; CDCl3) 70.7 (d, J = 5.8 Hz), 111.6 (dd, J = 5.8, 18.2 Hz),
122.6, 123.5, 126.5, 128.2, 128.7, 128.9, 130.0 (td, J = 7.7,
16.3 Hz), 131.0 (t, J = 8.6 Hz), 132.9, 133.4, 135.2 (d, J = 6.7 Hz),
The procedure described below for the synthesis of compound
25a is typical. Compounds 25b and 25c were prepared similarly.
5.1.13.1.
4-(Benzothiazol-2-yl)-1-(tetra-O-acetyl-b-
D-galacto-
syl)phenol 25a.
A
mixture of compound 10a (0.46 g,
2.02 mmol) and 2,6-lutidine (0.23 mL, 1.97 mmol) in dry CH2Cl2
(10 mL) was stirred at room temperature (CaCl2 guard tube).
After 10 min Ag2CO3 (0.55 g, 1.99 mmol) and additional 2,6-luti-
dine (0.23 mL, 1.97 mmol) were added and the vessel was
covered to exclude light. After a further 15 min 2,3,4,6-tetra-O-
154.7, 155.2 (dt, J = 3.4, 252.1 Hz), 166.1;
m
max/cmÀ1 696, 734,
acetyl-a-D-galactosyl bromide (0.82 g, 1.99 mmol) was added
912, 1016, 1283, 1432, 1487.
and stirring continued (60 h) in the dark. The reaction mixture
was filtered through a shallow bed of silica and the residue
rinsed with CH2Cl2 (10 mL). The filtrate was dried (MgSO4) and
evaporated. The residue was recrystallised from aqueous EtOH
yielding compound 25a (0.62 g, 56%) as pale pink needles, mp
147–149 °C; HRMS (+NSI) found: MH+, 558.1419. Calcd for C27-
H28NO10S: MH, 558.1428; dH (400 MHz; DMSO-d6) 1.92 (3H, s,
COCH3), 2.00 (3H, s, COCH3), 2.02 (3H, s, COCH3), 2.12 (3H, s,
COCH3), 4.09 (2H, d, J = 6.2 Hz, CH), 4.46 (1H, dd, J = 6.2, 6.2 Hz,
CH), 5.30–5.20 (2H, m, 2Â CH), 5.34 (1H, d, J = 7.3 Hz, CH), 5.61
(1H, d, J = 7.3 Hz, anomeric CH), 7.14 (2H, d, J = 9.2 Hz, Ar-H),
7.41 (1H, ddd, J = 1.4, 6.9, 7.9 Hz, Ar-H), 7.50 (1H, ddd, J = 1.4,
6.9, 7.9 Hz, Ar-H), 8.00 (1H, d, J = 7.9 Hz, Ar-H), 8.05 (2H, d,
J = 9.2 Hz, Ar-H), 8.09 (1H, d, J = 7.9 Hz, Ar-H); dC (100 MHz;
DMSO-d6) 20.9, 21.0, 21.1, 61.9, 67.7, 68.8, 70.6, 71.1, 97.7,
117.5, 122.8, 123.2, 125.9, 127.2, 128.1, 129.5, 134.9, 154.1,
5.1.12. General procedure for preparing the phosphate
derivatives 24
TFA was added drop-wise to the appropriate compound 23 and
the mixture was stirred (4–24 h) at room temperature. The mix-
ture was evaporated and the resulting TFA salt was dried under
vacuum in a desiccator. No further purification was required.
5.1.12.1. 4-(Benzothiazol-2-yl)phenyl-1-dihydrogen phosphate
24a.
Following the general procedure described above, com-
pound 23a (0.50 g, 0.62 mmol) in TFA (5 mL) (24 h) gave com-
pound 24a as an off-white solid (0.18 g, 94%), mp 235–236 °C;
HRMS (+NSI) found: MH+, 308.0144. Calcd for C13H11NO4PS: MH,
308.0141; dH (270 MHz; DMSO-d6) 7.36 (2H, d, J = 8.7 Hz, Ar-H),
7.44 (1H, ddd, J = 1.2, 7.2, 7.9 Hz, Ar-H), 7.53 (1H, ddd, J = 1.2, 7.2,
7.9 Hz, Ar-H), 8.04 (1H, ddd, J = 0.5, 1.2, 7.9 Hz, Ar-H), 8.08 (2H, d,
J = 8.7 Hz, Ar-H), 8.11–8.14 (1H, m, Ar-H); dC (68 MHz; DMSO-d6)
121.4 (d, J = 5.2 Hz), 122.9, 123.3, 126.0, 127.2, 129.1, 129.3,
159.2, 167.2, 169.8, 170.1, 170.4, 170.5;
1044, 1210, 1740.
m
max/cmÀ1 758, 832,
135.0, 154.2, 154.6 (d, J = 6.2 Hz), 167.2; IR
m
max cmÀ1: 689, 726,
5.1.13.2. 4-(Benzothiazol-2-yl)-2-fluoro-1-(tetra-O-acetyl-b-
D-
755, 963, 1186, 1253, 1444, 1505, 1604, 2100–2700.
galactosyl)phenol 25b. Compound 25b (880 mg, 76%) was
obtained as pink needles, 139–140 °C after recrystallisation form
from aqueous EtOH. HRMS (+NSI) found: MH+, 576.1334. Calcd
for C27H27FNO10S: MH, 576.1331; dH (400 MHz; DMSO-d6) 1.90
(3H, s, COCH3), 1.97 (3H, s, COCH3), 2.00 (3H, s, COCH3), 2.11
(3H, s, COCH3), 4.07 (2H, d, J = 6.0 Hz, CH), 4.42 (1H, dd, J = 6.0,
6.0 Hz, C), 5.22–5.28 (2H, m, 2Â CH), 7.34–7.40 (2H, m, Ar-H),
7.47 (1H, ddd, J = 1.4, 7.8, 8.1 Hz, Ar-H), 7.82 (1H, d, J = 9.2 Hz,
Ar-H), 7.87 (1H, dd, J = 1.8, 11.5 Hz, Ar-H), 7.96 (1H, d, J = 8.1 Hz,
Ar-H), 8.02 (1H, d, J = 8.1 Hz, Ar-H); dC (100 MHz; DMSO-d6)
20.8, 20.9, 21.0, 61.8, 67.6, 68.6, 70.5, 71.3, 98.9, 115.6 (d,
J = 21.1 Hz), 119.1, 122.9, 123.4, 124.7, 126.2, 127.3, 129.1 (d,
J = 6.7 Hz), 135.1, 146.9 (d, J = 10.5 Hz), 152.6 (d, J = 247.3 Hz),
5.1.12.2.
phosphate 24b.
4-(Benzothiazol-2-yl)-2-fluorophenyl-1-dihydrogen
Following the general procedure described
above, compound 23b (193 mg, 0.38 mmol) in TFA (5 mL) (4 h)
yielded compound 24b (121 mg, 97%) as a pale yellow powder
after evaporation of the solvent and trituration of the residue with
ether, mp 189–190 °C; HRMS found: MH+, 326.0051. Calcd for C13-
H10FNO4PS: MH, 326.0047; dH (400 MHz; DMSO-d6) 7.43 (1H, m,
Ar-H), 7.52 (1H, ddd, J = 0.9, 7.4, 8.2 Hz, Ar-H), 7.56 (1H, dd,
J = 8.2, 8.2 Hz, Ar-H), 7.88 (1H, d, J = 8.7 Hz, Ar-H), 7.96 (1H, dd,
J = 1.4, 11.5 Hz, Ar-H), 8.02 (1H, d, J = 8.0 Hz, Ar-H), 8.12 (1H, d,
J = 8.0 Hz, Ar-H); dC (100 MHz; DMSO-d6) 115.6 (d, J = 20.1 Hz),
123.0, 123.5, 123.4 (d, J = 3.8 Hz), 124.4 (d, J = 1.9 Hz), 126.2,
127.3, 130.1 (d, J = 5.8 Hz), 135.2, 142.2 (dd, J = 5.8, 11.5 Hz),
153.9, 166.0, 169.7, 170.1, 170.4, 170.5;
1047, 1210, 1738.
m
max/cmÀ1 758, 1014,
153.8 (dd, J = 7.2, 247.8 Hz), 153.9, 166.0;
m
max/cmÀ1 712, 751,
824, 881, 913, 966, 1135, 1293, 1448, 1510, 1613, 2500–3200.
5.1.13.3. 4-(5-Chlorobenzothiazol-2-yl)-2,6-difluoro-1-(tetra-O-
acetyl-b- -galactosyl)phenol 25c. Compound 25c (340 mg,
D
5.1.12.3. 4-(5-Chlorobenzothiazol-2-yl)-2,6-difluorophenyl-1-
27%) was obtained as light orange crystals, 179–181 °C; HRMS
(+NSI) found: MH+, 628.0851. Calcd for C27H2535ClF2NO10S: MH,
628.0850; dH (400 MHz; DMSO-d6) 1.90 (3H, s, Ac-CH3), 1.92 (3H,
s, COCH3), 2.06 (3H, s, COCH3), 2.14 (3H, s, 3H, COCH3), 3.98–4.11
(2H, m, 2Â CH), 4.28 (1H, dd, J = 6.4, 6.4 Hz, CH), 5.19–5.31 (3H,
m, 3Â CH), 5.37 (1H, d, J = 7.8 Hz, anomeric CH), 7.50 (1H, dd,
J = 1.8, 8.7 Hz, Ar-H), 7.88 (2H, d, J = 8.2 Hz, 2H, Ar-H), 8.12 (1H,
d, J = 1.8 Hz, Ar-H), 8.15 (1H, d, J = 8.7 Hz, Ar-H); dC (100 MHz;
DMSO-d6) 20.9, 21.0, 61.1, 67.5, 69.2, 70.4, 71.2, 102.4, 112.1 (dd,
J = 7.7, 18.2 Hz), 123.0, 124.6, 126.7, 130.2 (t, J = 9.6 Hz), 132.2,
134.3, 135.3 (t, J = 14.9 Hz), 154.6, 155.9 (dd, J = 4.8, 250.2 Hz),
dihydrogen phosphate 24c.
Following the general procedure
described above, compound 23c (192 mg, 0.34 mmol) in TFA (5 mL)
(4 h) yielded compound 24c (111 mg, 85%) as a pale yellow powder
after evaporation of the solvent and trituration of the residue with
ether, mp 197 °C; HRMS (+NSI) found: MH+, 377.9568. Calcd for C13-
H735ClF2NO4PS: MH, 377.9563; dH (400 MHz; DMSO-d6) 6.05 (br s,
2Â OH), 7.51 (1H, dd, J = 1.8, 8.5 Hz, Ar-H), 7.87 (2H, d, J = 8.2 Hz,
Ar-H), 8.11 (1H, d, J = 1.8 Hz, Ar-H), 8.18 (1H, d, J = 8.5 Hz, Ar-H);
dC (100 MHz; DMSO-d6) 111.9 (dd, J = 6.7, 18.2 Hz), 123.0, 124.6,
126.6, 129.7 (t, J = 10.5 Hz), 131.4 (td, J = 7.7, 16.3 Hz), 132.2,

M. Cellier et al. / Bioorg. Med. Chem. 22 (2014) 1250–1261
1261
167.2, 169.8, 170.0, 170.3, 170.5;
1368, 1434, 1486, 1745.
m
max/cmÀ1 923, 1027, 1065, 1212,
248.7 Hz), 167.4;
3100–3500.
m
max/cmÀ1 878, 1027, 1078, 1246, 1347, 1426,
5.1.14. General procedure for preparation of the galactose
derivatives 26
5.2. Microbiological work
The procedure described below for the synthesis of compound
26a is typical. Compounds 26b and 26c were prepared similarly.
5.2.1. Agar plate preparation
Each substrate (10 mg) was dissolved in 1-methyl-2-pyrroli-
done (0.2 mL) and added to molten Columbia agar (100 mL) (Ox-
5.1.14.1.
26a.
4-(Benzothiazol-2-yl)-1-(b-
Compound 26a (0.33 g, 0.59 mmol) and sodium methox-
D
-galactosyl)phenol
oid, Basingstoke) at 50 °C to a final concentration of 100 mg LÀ1
.
Agar plates were then prepared and dried to remove excess mois-
ture. Bacterial strains (obtained 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 physiological saline to generate a suspension
equivalent to 108 colony forming units per mL using a densitome-
ide (0.64 g, 11.85 mmol) were added to LC-grade methanol (15 mL)
and stirred (70 h) at room temperature. The mixture was cooled in
an ice-water bath the precipitate was collected yielding compound
26a (0.19 g, 83%) as a white powder, >180 °C (decomp.); HRMS
(+NSI) found: MH+, 390.1009. Calcd for C19H20NO6S: MH,
390.1011; dH (400 MHz; DMSO-d6) 3.28–3.61 (5H, m, 5Â CH),
3.69 (1H, d, J = 2.8 Hz, CH), 4.61 (1H, br s, OH), 4.72 (1H, br s,
OH), 4.93 (1H, d, J = 7.8 Hz, anomeric CH), 4.99 (1H, br s, OH),
5.28 (1H, br s, OH), 7.16 (2H, d, J = 8.7 Hz, Ar-H), 7.40 (1H, ddd,
J = 1.4, 6.9, 8.0 Hz, Ar-H), 7.49 (1H, ddd, J = 1.4, 6.9, 8.1 Hz, Ar-H),
7.97–8.01 (3H, m, Ar-H), 8.08 (1H, d, J = 8.0 Hz, Ar-H); dC
(100 MHz; DMSO-d6) 60.8, 68.6, 70.7, 73.8, 76.2, 101.1, 117.4,
122.8, 123.1, 125.7, 127.0, 127.1, 129.3, 134.8, 154.2, 160.4,
ter. Each agar plate was then inoculated with 10 lL of this suspen-
sion and spread to obtain single colonies. Plates were incubated at
37 °C in air for 24 h. For the evaluation of the b-galactosidase sub-
strates, IPTG (3 mg) in sterile deionised water (1 mL) was also
added to the molten agar.
Acknowlegements
167.5;
m
max/cmÀ1 757, 825, 1035, 1064, 1078, 3000–3500.
We thank bioMérieux SA for generous financial support and the
EPSRC Mass Spectrometry Centre, Swansea, for high resolution
mass spectra.
5.1.14.2. 4-(Benzothiazol-2-yl)-2-fluoro-1-(b-
D-galactosyl)phe-
nol 26b. Compound 26b (0.35 g, 99%) was obtained as a white
References and notes
powder, >195 °C (decomp.); HRMS (+NSI) found: MH+, 408.0912.
Calcd for C19H19FNO6S: MH, 408.0912; dH (400 MHz; DMSO-d6)
3.40–3.55 (2H, m, 2Â CH), 3.60–3.64 (3H, m, 3Â CH), 3.70 (1H, d,
J = 2.8 Hz, CH), 4.62 (1H, br s, –OH), 4.71 (1H, br s, OH), 4.98 (1H,
br s, OH), 5.04 (1H, d, J = 7.8 Hz, anomeric CH), 5.33 (1H, br s,
OH), 7.38–7.43 (2H, m, Ar-H), 7.50 (1H, ddd, J = 1.4, 7.8, 8.1 Hz,
Ar-H), 7.79–7.82 (1H, m, Ar-H), 7.90 (1H, dd, J = 1.8, 11.5 Hz, Ar-
H), 8.00 (1H, d, J = 8.1 Hz, Ar-H), 8.09 (1H, dd, J = 1.4, 8.1 Hz, Ar-
H); dC (100 MHz; DMSO-d6) 60.8, 68.6, 70.6, 73.8, 76.3, 101.3,
115.1 (d, J = 21.1 Hz), 118.1, 122.9, 123.3, 124.6, 126.0, 127.3,
127.5 (d, J = 6.7 Hz), 135.0, 148.0 (d, J = 10.5 Hz), 152.3 (d,
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m
max/cmÀ1 751, 1027, 1052, 1072,
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galactosyl)phenol 26c.
4-(5-Chlorobenzothiazol-2-yl)-2,6-difluoro-1-(b-
D-
Compound 26c (89 mg, 97%) was ob-
tained as a white powder, >200 °C (decomp.); HRMS (+NSI) Found
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Ar-H), 7.81 (2H, d, J = 8.7 Hz, Ar-H), 8.10 (1H, d, J = 2.1 Hz, Ar-H),
8.18 (1H, d, J = 8.5 Hz, Ar-H); dC (100 MHz; DMSO-d6) 60.5,
68.3, 71.5, 73.5, 76.5, 104.8, 112.0, 122.9, 124.6, 126.5, 128.6,
132.2, 134.2, 135.9 (t, J = 12.5 Hz), 154.7, 155.8 (dd, J = 5.8,
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