7-Chloro-4-methyl-6-nitro-2H-chromen-2-one: A novel type of reagent for fluorescence analysis

Article abstract of DOI:10.1016/S0040-4039(02)02638-2

Starting from 7-chloro-4-methyl-2H-chromen-2-one 1 we have obtained a new reagent for fluorescence analysis. The synthesised key compound 7-chloro-4-methyl-6-nitro-2H-chromen-2-one 2 forms highly fluorescent derivatives with different amines, which can be introduced by nucleophilic substitution of the chloro atom. Two examples are given which describe the preparation of these derivatives. Additionally, we demonstrate that an increase of fluorescence can be achieved, if a reduction step is performed after the substitution.

Full text of DOI:10.1016/S0040-4039(02)02638-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 845–848
7-Chloro-4-methyl-6-nitro-2H-chromen-2-one: a novel type of
reagent for fluorescence analysis
Christian R. Noe,a,* Spyridon Korniliosb and Bodo Lachmanna
aInstitute of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria
bInstitute of Organic Chemistry, Technical University of Vienna, Getreidemarkt 9, A-1060 Vienna, Austria
Received 25 May 2002; revised 19 November 2002; accepted 20 November 2002
Abstract—Starting from 7-chloro-4-methyl-2H-chromen-2-one 1 we have obtained a new reagent for fluorescence analysis. The
synthesised key compound 7-chloro-4-methyl-6-nitro-2H-chromen-2-one 2 forms highly fluorescent derivatives with different
amines, which can be introduced by nucleophilic substitution of the chloro atom. Two examples are given which describe the
preparation of these derivatives. Additionally, we demonstrate that an increase of fluorescence can be achieved, if a reduction step
is performed after the substitution. © 2003 Elsevier Science Ltd. All rights reserved.
It has been shown that—depending on their substitu-
tion pattern—derivatives of 2H-chromen-2-one may
exhibit high fluorescence activity and therefore are use-
ful candidates for dyes, fluorescent substrates in enzy-
matic assays or derivatisation reagents in analytical
assays.1 With the latter application, derivatisation has
been hitherto carried out via amidation, transesterifica-
tion, esterification, reaction with isothiocyanates or by
condensation.
expected. Due to its character as an aromatic second
order substituent, the nitro group is required to achieve
nucleophilic aromatic substitution. It may be reduced
after the reaction to enhance fluorescence. The resulting
amino group does not only have a positive impact on
fluorescence properties of the compound, but also pro-
vides, if intended, an additional position for attachment
of analytes.
Although 2H-chromen-2-ones (coumarins) are com-
pounds which are known for more than 100 years,3 the
methods reported for their synthesis require major
modifications to obtain compound 2 in acceptable
yields. Therefore an efficient synthesis of compound 2
will be reported in the present paper together with one
example to illustrate the synthetic procedure for nucle-
ophilic substitution and reduction of the nitro group
and a second example, by which the impact of the
introduction of the reagent on the optical rotation of
In the present paper we present 7-chloro-4-methyl-6-
nitro-2H-chromen-2-one 2,
a compound, which—
although itself exhibiting no fluorescence—nevertheless
is a versatile derivatisation reagent for analysis using
fluorescence detection. Nucleophilic aromatic substitu-
tion has been-since Sanger2—an established reaction for
derivatisation of analytes. It allows reagent attachment
at nitrogen, oxygen and sulfur atoms. The spatial dis-
tance between reagent and analyte is minimised, when
compared to other methods of attachment (e.g. via
carboxyl groups: esters, amides, carbamates, ureas).
Due to the fact that by this reaction the analyte is
linked to the chromophore of the reagent via a het-
eroatom bearing a free electron pair, it may be expected
that specific changes in the spectroscopic properties
might occur. Apart from an impact on absorption
wavelengths or extinction coefficients of the reagent,
also effects on chiroptical properties (e.g. enhancement
of optical rotation values) of the analyte are to be
L
-prolinol is demonstrated.
Synthesis of compound 2
A significant portion of published 2H-chromen-2-ones
bear a 4-methyl group. Such compounds with halogen
or nitro groups at the aromatic nucleus may be synthe-
sised according to the procedure published by Clayton.4
He describes a modification of the Pechmann coumarin
synthesis, by which a phenolic compound reacts with a
b-keto ester derivative. In this reaction the substituent
at the meta-position of the phenol used in the reaction
will become the substituent at position 7 of the cou-
marin. It has also been reported that an amino group
* Corresponding author. Tel.: +43-1-427755103; fax: +43-1-42779551;
e-mail: christian.noe@univie.ac.at
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02638-2

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C. R. Noe et al. / Tetrahedron Letters 44 (2003) 845–848
increases fluorescence of coumarins. Amino groups
have been introduced by nitration followed by a reduc-
tion step. Following this synthetic approach several
derivatives of aminoumbelliferones5 have been synthe-
sised. However, the 7-chloro-6-nitro derivative 2 has
not yet been described.
carried out using concentrated sulphuric acid as solvent
(50 ml for 10 g 1), adding a mixture of concentrated
sulphuric (5.2 ml) acid and nitric acid (4.3 ml) below
0°C, yielding 7-chloro-6-nitro-4-methyl-2H-chromen-2-
one in a yield of more than 90% after 1 h of stirring.
The removal of side products, particularly traces of
7-chloro-3,6-dinitro-4-methyl-2H-chromen-2-one
2b,
We obtained compound 2 by selective nitration of
7-chloro-4-methyl-2H-chromen-2-one 1, which was syn-
thesised according to the procedure of Brubaker et al.,
who describes a modification of the Pechmann conden-
sation leading to improved, but still moderate yields
and increased purity of the product.6 This protocol
could be reproduced, leading to compound 1 in 30%
yield.
which had been observed by TLC, could be achieved by
recrystallisation from toluene.10
Nucleophilic substitution of 2 and reduction of the nitro
group
Nucleophilic substitution was carried out using the
secondary amines pyrrolidine and
L-prolinol as sub-
strates. Thus, compound 3a was synthesised by dissolv-
ing 1 g (4.2 mmol) of 2 in 20 ml of DMF, adding 0.65
g (9.2 mmol) of pyrrolidine and heating the reaction
mixture to reflux temperature for 1 h under an argon
atmosphere. After cooling to room temperature, the
mixture was treated with crushed ice, the precipitate
was filtered off and recrystallised from methanol to
yield 0.72 g (63%) of 3a.11,12 Applying the same condi-
tions, 1 g (4.2 mmol) of 2 and 0.94 g (9.2 mmol) of
Different methods have been reported describing the
nitration of closely related compounds of the coumarin
type.7 In general, mixtures of mono and dinitro prod-
ucts resulted in almost equal amounts and were sepa-
rated
by
chromatographic
methods
or
recrystallisation.7,8 In order to avoid these impurities
and to achieve higher yields, reaction conditions were
modified as follows: first, we applied concentrated nitric
acid both as solvent and reagent for nitration. Com-
pound 1 (1.33 g, 6.8 mmol) was dissolved in 30 ml of
concentrated nitric acid keeping the solution constantly
below −5°C for 2 h. After that time the starting com-
pound had reacted quantitatively and only one product
was detected by TLC. The mixture was treated with
crushed ice and the precipitated product isolated by
suction filtration. Recrystallisation from ethanol yielded
2.5 g of pure product. NMR spectroscopy showed that
the isolated product was neither the desired mononitro
compound 2 nor the 6,8-dinitro compound 2a, a substi-
tution pattern which has been described in literature for
related coumarins,5,7,8 but was 7-chloro-3,6-dinitro-4-
methyl-2H-chromen-2-one 2b (Fig. 1).
L
-prolinol yielded 0.9 g (78.9%) of 3b. Considering the
fact that one molar equivalent of -prolinol was con-
L
sumed in the reaction as basic scavenger, we decided to
attempt the use of triethylamine for this purpose. Thus,
6 g (25.2 mmol) of 2 were dissolved in 120 ml DMF
and 7.8 g of triethylamine (77.5 mmol). After adding
2.82 g (27.6 mmol) of L-prolinol the mixture was heated
to reflux for 3 h under an argon atmosphere and
worked up as described for compound 3a. The modifi-
cation afforded 6.28 g (81.7%) of 3b.13
Therefore, addition of triethylamine seems to be recom-
mendable, particularly in cases where the amines are
not readily available (Scheme 1).
Structure and substitution patterns of this compound
Three different methods for reduction of the nitro
1
were confirmed by 13C and H NMR analyses, mass
group were evaluated: SnCl2/HCl,14 sodium dithionite/
spectroscopy and elemental analyses.9 Monitoring of
the reaction mixture by TLC showed that during the
reaction other products could be observed only in
traceable amounts. The formation of the undesired
dinitro product was probably caused by the excess of
nitric acid applied. To avoid this, the reaction was
NH3 and SnCl2/ethanol.16 The method using SnCl2 in
15
ethanol without adding acid proved to be the most
Figure 1.
Scheme 1. Nucleophilic substitution.

C. R. Noe et al. / Tetrahedron Letters 44 (2003) 845–848
847
suitable. Compound 4a was prepared using SnCl2/HCl
in ethanol for the reduction of 3a. This procedure gave
72% of a crude product, which after purification by
VFC17 afforded 36% of 4a18 as pure product. Reacting
compound 3b with SnCl2 in ethanol at 60°C without
addition of acid yielded the desired compound 4b,19
which was recrystallised from methanol and isolated in
nearly quantitative yield. At this point it seems worth
mentioning that compounds 4a and 4b are the first
6,7-diamino substituted coumarins hitherto described
(Scheme 2).
higher optical rotation values were obtained after the
nucleophilic substitution reaction.
In conclusion, the coumarin 2 has been described for
the first time. A method for its synthesis has been
presented. Nucleophilic aromatic substitution allows
attachment of an amine. Reduction of the nitro group
leads to hitherto not described 6,7-diamino substituted
4-methyl-coumarins, which are highly fluorescent com-
pounds useful in fluorescence analysis. The loss in
fluorescence was remarkably less pronounced, com-
pared to other dyes, like NBD,22 if water was used as
solvent. Even though the fluorescence does not come up
to that of fluorescamine, the limit of detection is almost
similar to the one, which is obtained with dyes of the
naphtholsulfonic acid-type.23
The resulting purified amines were dissolved and
fluorescence spectra were measured. In Figure 2 the
fluorescence spectrum of 4b is shown. The substance
shows high fluorescence, its excitation maxima depend
on both pH and type of solvent. Compared to the
unreduced nitro-derivative, an approximately 100-fold
increase of fluorescence was observed.20
References
Effect of the ‘coumarinisation’ on optical rotation values
1. Haugland, R. P. Handbook of Fluorescent Probes and
Research Chemicals; Molecular Probes Inc: Eugene, Ore-
gon, 1996.
2. Sanger, F. J. Biochem. 1949, 45, 563–574.
3. Pechmann, H.; Duisberg, C. Berichte 1884, 17, 929–932.
4. Clayton, A. J. Chem. Soc. 1908, 93, 2016–2022.
5. Kozlova, I. K. Heterocycl. Compounds Engl. Transl. 1985,
21, 750–753.
of L-prolinol
Comparing the values of optical rotation of
L
-prolinol21
with the ‘coumarinised’ nitro compound 3b and the
corresponding amino compound 4b revealed that much
6. Brubaker, A. B.; DeRuiter, J.; Whitmer, W. L. J. Med.
Chem. 1986, 29, 1094–1099.
7. Shah, N. M.; Mehta, D. H. J. Indian Chem. Soc. 1954,
31, 784–786.
8. Clayton, A. J. Chem. Soc. 1910, 97, 1397–1400.
9. Analytical data for compound 2b: yellow crystals; mp
205–207°C; 1H NMR (300 MHz, DMSO-d6): l 8.74 [s,
1H]; 8.14 [s, 1H], 2.53 [s, 3H]; 13C NMR (50 MHz,
DMSO-d6):
l
153.3 [C2ꢀO], 152.1 [C9(Ar)], 144.9
[C6(Ar)], 144.4 [C4], 137.8 [C3], 130 [C7(Ar)], 124.9
[C5(Ar)], 120.1 [C8(Ar)], 117.9 [C10(Ar)], 14.1 [CH3]; IR
(KBr): 3113, 3051, 1742, 1555, 1347, 1041 cm−1; MS: m/z
(ES-): 282.9. Anal. calcd for C10H5ClN2O6: (284.6): C,
42.2; H, 1.77; N, 9.84. Found: C, 42.18; H, 1.85; N,
9.48%.
10. Analytical data for compound 2: white crystals; mp
1
255°C; H NMR (300 MHz, DMSO-d6): l 8.48 [s, 1H];
7.94 [s, 1H], 6.6 [s, 1H], 2.46 [s, 3H]; 13C NMR (50 MHz,
DMSO-d6): l 158.3 [C2ꢀO], 154.7 [C9(Ar)], 151.9 [C4],
143.6 [C6(Ar)], 128.0 [C7(Ar)], 123.1 [C5(Ar)], 119.5
[C8(Ar)], 119.3 [C10(Ar)], 116.1 [C3], 17.9 [C11H3]; MS:
m/z (ES-): 239.0. Anal. calcd for C10H6ClNO4: (239.62):
C, 50.13; H, 2.52; N, 5.85. Found: C, 50.07; H, 2.72; N,
5.88%.
Scheme 2. Reagents and conditions: (i) SnCl2/conc. HCl/
EtOH, 6 h, rt; (ii) SnCl2×2H2O/EtOH, 1 h, reflux.
11. Analytical data for compound 3a: yellow needles; mp
1
167–168°C; H NMR (90 MHz, CDCl3): l 7.94 [s, 1H];
6.67 [s, 1H], 6.05 [s, 1H], 3.39–3.14 [t, 4H], 2.36 [s, 3H],
2.11–1.96 [m, 4H]; IR (KBr): 1740, 1530, 1390, 1290
cm−1. Anal. calcd for C14H14N2O4: (274.28): C, 61.31; H,
5.14; N, 10.21. Found: C, 61.15; H, 5.16; N, 10.32%.
12. Kornilios, S. Diploma Thesis, Technical University of
Vienna, 1986.
13. Analytical data for compound 3b: golden crystals; mp
172–173°C; 1H NMR (300 MHz, DMSO-d6): l 8.1 [s,
1H]; 7.0 [s, 1H], 6.18 [s, 1H], 4.86–4.82 [t, 1H], 4.0–3.9 [m,
Figure 2. Fluorescence spectra of 4b in dichloromethane (10
mg/ml), EM: 250–700 nm, EX: 250–700 nm.

848
C. R. Noe et al. / Tetrahedron Letters 44 (2003) 845–848
68.83; H, 6.6; N, 11.47. Found: C, 68.72; H, 6.62; N,
1H], 3.5 [m, 1H], 3.36 [m, 2H], 2.8 [m, 1H], 2.3 [s, 3H],
1.9–1.6 [m, 4H]; 13C NMR (50 MHz, DMSO-d6): l 159.8
[CꢀO], 155.7 [C9(Ar)], 153.1 [C4], 144.3 [C6(Ar)], 135.0
[C7(Ar)], 124.3 [C5(Ar)], 110.9 [C3], 109.4 [C10(Ar)], 102.9
[C8(Ar)], 61.7 [C6%], 60.9 [C2%], 52.6 [C5%], 28.6 [C3%], 24.4
[C4%], 17.9 [C11H3]; MS: m/z (ES+): 305.1. Anal. calcd for
C15H16N2O5: (304.31): C, 59.21; H, 5.3; N, 9.21. Found:
11.41%.
19. Analytical data for compound 4b: green–yellow crystals;
1
mp 256–259°C; H NMR (300 MHz, DMSO-d6): l 6.87
[s, 1H]; 6.8 [s, 1H], 6.0 [s, 1H], 4.75 [s, 2H], 4.46–4.42 [t,
1H], 3.8 [bs, 1H], 3.5 [m, 1H], 3.26–3.11 [m, 2H], 2.77 [m,
1H], 2.26 [s, 3H], 2.0–1.7 [m, 4H]; 13C NMR (50 MHz,
DMSO-d6): l 160.8 [CꢀO], 152.8 [C4], 146.5 [C9(Ar)],
141.4 [C6(Ar)], 139.0 [C7(Ar)], 113.5 [C10(Ar)], 110.7 [C3],
108.2 [C5(Ar)], 105.3 [C8(Ar)], 62.4 [C6%], 60.0 [C2%], 51.3
[C5%], 27.9 [C3%], 23.6 [C4%], 18.1 [CH3]; MS: m/z (ES+):
20
20
C, 58.97; H, 5.36; N, 9.11%. [h] : −1995; [h] : −1354 (c
546
589
1.0, DCM).
14. (a) Adams, R.; Mecorney, J. W. J. Am. Chem. Soc. 1944,
66, 802–805; (b) Xing, W. K.; Ogata, Y. J. Org. Chem.
1982, 47, 3577–3581.
15. See: Ref. 3 and references cited therein.
16. Bellamy, F. D.; Ou, K. Tetrahedron Lett. 1984, 25,
839–842.
275.1. Anal. calcd for C10H7NO5: (274.32): C, 64.41; H,
20
546
6.70; N, 10.01. Found: C, 64.13; H, 6.82; N, 9.36%. [h]
:
+169; [h]25089: +125 (c 1.0, DCM).
20. Fluorescence was measured and quantified at three differ-
ent wavelengths both for excitation and emission: EX
350/EM 500 nm; EX 350/EM 580 nm; EX 325/EM 520
17. Vacuum-flash-chromatography.
18. Analytical data for compound 4a: bright yellow crystals;
mp 164–166°C; 1H NMR (90 MHz, CDCl3): l 6.81
[s, 2H]; 6.08 [s, 1H], 3.66 [s, 2H], 3.26–3.07 [t, 4H],
2.33 [s, 3H], 2.08–1.82 [m, 4H]; IR (KBr): 3380, 3300,
1690 cm−1. Anal. calcd for C14H16N2O2: (244.29): C,
nm.
20
546
21.
L-Prolinol [h] : +37 (c 1.0, toluene).
22. 7-Nitrobenz-2-oxa-1,3-diazole.
23. The compounds 2, 3b and 4b are commercially available
from Prochem KFT (prochem@chello.at).