The synthesis and evaluation of some coumarin derivatives as fluorescent indicators of nitroreductase activity

Article abstract of DOI:10.1002/jhet.5570430241

The carbamate 8 was reacted with appropriate β-keto esters in a Pechmann reaction giving the coumarin derivatives 9-11 bearing 4-nitrobenzylcarbamoyl triggers. These compounds were assessed as potential indicators of nitroreductase activity.

Full text of DOI:10.1002/jhet.5570430241

Mar-Apr 2006
The Synthesis and Evaluation of Some Coumarin Derivatives as
Fluorescent Indicators of Nitroreductase Activity
515
a
b
a †
Arthur L. James , John D. Perry and Stephen P. Stanforth *
a
School of Applied Sciences, University of Northumbria, Newcastle upon Tyne, NE1 8ST, UK.
b
Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, NE7 7DN, UK.
Received July 27, 2005
The carbamate 8 was reacted with appropriate β-keto esters in a Pechmann reaction giving the coumarin
derivatives 9-11 bearing 4-nitrobenzylcarbamoyl triggers. These compounds were assessed as potential indi-
cators of nitroreductase activity.
J. Heterocyclic Chem., 43, 515 (2006).
Many bacteria have the ability to reduce aromatic nitro
ger’ mechanism in anti-cancer pro-drug design [2-7].
Reduction of the nitro-group in these inactive pro-drugs 4
gave the corresponding hydroxylamine derivatives 5 that
underwent spontaneous fragmentation as shown in
compounds and the detection of nitroreductase activity
may offer a useful means of analysing biological and envi-
ronmental samples for the presence of microorganisms.
The reduction of a series of seven non-fluorogenic nitro-
coumarins 1 to their corresponding fluorogenic hydroxy-
lamine derivatives 2 or amine derivatives 3 has been
recently investigated for 30 strains encompassing 24 dis-
tinct microbial genera, including a wide range of clinically
important species [1] (Scheme 1). The coumarin deriva-
tives 2/3 are highly fluorogenic thus giving a sensitive
method for the detection of nitroreductase activity.
Scheme 2 releasing the active drug (RNH ). In this note
2
we report on our preliminary studies using a 4-nitrobenzyl-
carbamate trigger as a method for releasing fluorescent 7-
hydroxylamine derivatives of coumarin thus giving a
method for detecting nitroreductase activity. Substrates
bearing triggers based upon 7-aminocoumarin derivatives
and 7-hydroxycoumarin derivatives have recently found
application in enzymatic profiling [8], image contrast
agents [9], and probes for the detection of hydrogen perox-
ide [10], phosphatases [11] and Baeyer-Villigerases [12].
The 7-aminocoumarin derivatives 9-11 possessing 4-
nitrobenzylcarbamate triggers were prepared by the method
shown in Scheme 3. This route was chosen because the
intermediate carbamate 8 could act as a precursor to a wide
range of different coumarin derivatives in a Pechmann reac-
tion [13]. Additionally, the majority of the nitro-compounds
shown in Scheme 1 were prepared from 4-nitrosalicyalde-
hyde using a Knoevenagel condensation reaction to con-
struct the coumarin ring system and the Pechmann route
would therefore enable the preparation of an extended range
The enzymatic reduction of nitro-groups in 4-nitroben-
zylcarbamate derivatives 4 has also been used as a ‘trig-

5
16
A. L. James, J. D. Perry and S. P. Stanforth
Vol. 43
of potential nitroreductase substrates from readily available
starting materials. Thus, 3-aminophenol 6 was reacted with
spectra were determined at 270 MHz using a Jeol GX270 instru-
ment. Elemental analyses were performed by the Department of
Chemistry, University of Newcastle upon Tyne, UK. High resolu-
tion mass spectra were performed at the EPSRC national mass
spectrometry service centre, Swansea, UK.
4
-nitrobenzyl chloroformate 7 giving the carbamate deriva-
tive 8 in 82 % yield which has been previously obtained in
5 % yield [14]. Reaction of this carbamate 8 with the
3
(
3-Hydroxyphenyl)carbamic Acid 4-Nitrobenzyl Ester 8.
appropriate β-keto esters in the presence of 75 % sulphuric
acid at room temperature afforded the coumarin derivatives
To a boiling solution of 3-aminophenol 6 (1.0 g, 9.17 mmol) in
9
(73 %), 10 (86 %) and 11 (64 %). The carbamate 8 and all
ethyl acetate (10 mL) was added 4-nitrobenzyl chloroformate 7
(1.0 g, 4.65 mmol) in portions over 5 minutes. A white precipitate
(3-aminophenol hydrochloride) formed as the 4-nitrobenzyl
chloroformate was added. The mixture was stirred at reflux (5
of the coumarin derivatives 9-11 had proton NMR spectral
data, microanalytical and high resolution mass spectra
consistent with their proposed structures.
The nitroreductase substrates 9-11 (10 mg) were dis-
solved in N-methylpyrrolidinone (500 µL) and the resulting
solutions added to Columbia agar and then assessed against
five organisms, Escherichia coli, Klebsiella pneumoniae,
Serratia marcescens, Pseudomonas aeruginosa and
Staphylococcus aureus (MRSA). Substrates 9 and 11 did
not produce any fluorescence in the presence of these
organisms but compound 10 produced blue fluorescent
colonies in the presence of Serratia marcescens and
Pseudomonas aeruginosa. One of the consistent difficulties
in working with these substrates is their inherent insolubil-
ity. Although they can be solubilized in N-methylpyrrolidi-
none, subsequent addition to the aqueous bacteriological
media resulted in precipitation. Thus, we have been unable
to produce at this stage quantitative data that demonstrates
significant levels of fluorescence over and above those pro-
duced by substrate-free controls. Thus, the synthetic route
to the coumarins described in this communication is now
being applied to the production of coumarin derivatives that
are expected to be more soluble in aqueous media.
min), filtered while hot and the filtrate was allowed to cool to
room temperature and then evaporated giving the carbamate 8
(
1.1 g, 82 %) as a pale yellow solid, m.p. 170-172 °C (from
methanol); lit. m.p. 168 °C [14]. Compound 8 had ir: 3354, 1706,
-1 1
1
600, 1558, 1511, 1439, 1346, 1238, 1212 and 1070 cm ; H-
nmr: (DMSO-d ): δ 9.76 (1H, broad s, >NH), 9.38 (1H, s, -OH),
6
8
.24 (2H, d, J 8 Hz, Ar-H), 7.68 (2H, d, J 8 Hz, Ar-H), 7.02 (1H,
t, J 8 Hz, Ar-H), 7.00 (1H, s, Ar-H), 6.84 (1H, d, J 8 Hz, Ar-H),
6
.39 (1H, d, J 8 Hz, Ar-H), 5.28 (2H, s, >CH2).
Anal. Calcd. for C H N O : C, 58.4; H, 4.1; N, 9.7. Found:
1
4 12 2 5
C, 58.3; H, 4.2; N, 9.7 %.
Coumarins 9-11: General Procedure.
In a typical procedure a mixture of the carbamate 8 (1.0
mmol), the β-keto ester (1 mL) and 75 % sulphuric acid (1 mL)
were stirred at room temperature for 2 days. The mixture was
diluted with water and the solid was collected and washed several
times with water and then with ether giving the products 9-11.
(
4-Methyl-2-oxo-2H-chromen-7-yl)-carbamic Acid 4-Nitro-
benzyl Ester 9.
Compound 9 (73 %) had: m.p. >250 °C (from warm ca. 100 °C
-1
DMF); ir: 3266, 1703, 1583, 1520, 1348, 1232, 1203 and 1064 cm ;
1
H-nmr: (DMSO-d ): δ 10.42 (1H, broad s, >NH), 8.29 (2H, d, J 8
6
EXPERIMENTAL
Hz, Ar-H), 7.72 (3H, 2 x overlapping d, J 8 Hz, Ar-H), 7.56 (1H, d, J
1
Hz, Ar-H), 7.42 (1H, dd, J 8 and 1 Hz, Ar-H), 6.26 (1H, s, Ar-H),
Infra-red spectra were recorded on a Perkin Elmer Paragon
5.36 (2H, s, >CH ) and 2.39 (3H, s, -CH ); ms: m/z (electrospray pos-
2
3
1
000 spectrophotometer using a diamond anvil. Proton NMR
itive) calcd. for C H N O (M+H): 355.0925. Found: 355.0931.
18 15 2 6

Mar-Apr 2006
The Synthesis and Evaluation of Some Coumarin Derivatives
517
Anal. Calcd. for C H N O : C, 55.6; H, 3.4; N, 7.2. Found:
C, 55.3; H, 3.5; N, 7.1 %.
lution mass spectra. We also thank Mr A. M. Lawrie for assis-
tance in part of this project.
1
8 14 2 6
(
4-Chloromethyl-2-oxo-2H-chromen-7-yl)-carbamic Acid 4-
REFRERENCES AND NOTES
Nitrobenzyl Ester 10.
Compound 10 (86 %) had: m.p. >250 °C (from warm ca. 100
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1 1
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6
(
2H, d, J 8 Hz, Ar-H), 7.78 (1H, d, J 8 Hz, Ar-H), 7.71 (2H, d, J 8
Hz, Ar-H), 7.59 (1H, d, J 1 Hz, Ar-H), 7.43 (1H, dd, J 8 and 1 Hz,
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2
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2
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35
C H
ClN O (M+NH ): 406.0804. Found: 406.0800.
3 6 4
1
8
17
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6
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-
1 1
°C DMF); ir: 3280, 1701, 1625, 1516, 1342 and 1238 cm ; H-
nmr: (DMSO-d ): δ 10.40 (1H, broad s, >NH), 8.29 (2H, d, J 8
Hz, Ar-H), 7.71 (2H, d, J 8 Hz, Ar-H), 7.61 (1H, d, J 1 Hz, Ar-H),
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6
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7
5
.56 (1H, d, J 8 Hz, Ar-H), 7.42 (1H, dd, J 8 and 1 Hz, Ar-H),
.35 (2H, s, >CH ), 3.05 (2H, t, J 7 Hz, -CH CH CH -), 2.73
2
2
2
2
(
(
2H, t, J 7 Hz, -CH CH CH -) and 2.10 (2H, m, -CH CH CH -).
2 2 2 2 2 2
ms: m/z (electrospray positive) calcd. for C H N O (M+H):
2
0 17 2 6
3
81.1081. Found: 381.1081.
Anal. Calcd. for C H N O : C, 63.2; H, 4.2; N, 7.4. Found:
[
2
0 16 2 6
C, 63.1; H, 4.3; N, 7.5 %.
[
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Acknowledgement.
We thank the EPSRC mass spectrometry service for high reso-