On the development of NAD(P)H-sensitive fluorescent probes

Article abstract of DOI:10.1039/b001296n

In contrast to current, multi-reagent assay systems, the development of a single reagent that can be used to assay NAD(P)H is described. The reagent eliminates the fluorophore 4-methylumbelliferone from a quinoxalinium adduct upon reduction and the chemistry of this process is described. The Royal Society of Chemistry.

Full text of DOI:10.1039/b001296n

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On the development of NAD(P)H-sensitive fluorescent probes
Nicola L. Maidwell, M. Reza Rezai, Carl A. Roeschlaub and Peter G. Sammes*
Molecular Probes Unit, Department of Chemistry, University of Surrey, Guildford, Surrey,
UK GU2 5XH
1
Received (in Cambridge, UK) 16th February 2000, Accepted 17th March 2000
In contrast to current, multi-reagent assay systems, the development of a single reagent that can be used to assay
NAD(P)H is described. The reagent eliminates the fluorophore 4-methylumbelliferone from a quinoxalinium adduct
upon reduction and the chemistry of this process is described.
1
,4-Dihydronicotinamide adenine dinucleotide (NADH) and
which is extremely unstable and rapidly undergoes further
redox reactions. This instability is probably because the inter-
mediate dihydrospecies 4 is formally anti-aromatic.
In order to avoid the need for a two-component system,
involving a reducible fluorophore and a catalyst, we considered
the possibility of using a reducible substrate that could release
a masked fluorophore after reduction with NAD(P)H. The
general scheme was as depicted in Scheme 1. The first step was
its phosphate ester, NADPH, are ubiquitous reducing agents in
Nature, and measurement of intracellular turnover of these
vectors is often used to indicate cellular viability and activity.
In rapidly proliferating cervical cancer cells the turnover of
NAD(P)H is greater than in normal cells and thus it can be
used as a biological marker for the detection of cancer. One
1
1
therefore requires a means for the detection and estimation of
NAD(P)H levels in cells.
2
NAD(P)H concentrations can be monitored by direct
measurement of their incipient fluorescence but, since it is
ϩ
rapidly turned over [reoxidized to NAD(P) ], absolute levels do
not necessarily reflect the level of enzymic and metabolic activ-
ity occurring within the cell. Furthermore, interference from
background signals from other biochemical entities in cells can
also obscure any reliable estimation.
As a consequence of these problems there is considerable
interest in using reagents that themselves are selectively reduced
by NAD(P)H with the formation of either a coloured or fluor-
escent signal. Few dye precursors react directly with NAD(P)H
and, in order to overcome this problem, a catalyst usually has to
Scheme 1 Reduction of a protected fluorophore A should lead to an
intermediate dihydro-compound B that collapses spontaneously with
liberation of the free fluorophore.
to generate masked fluorophore substrates, such as A, that
would interact with NAD(P)H to produce a reduced species,
such as B, that would spontaneously collapse with the liber-
ation of the fluorophore.
3
be used. The catalyst is either an enzyme, such as diaphorase,
or a known electron carrier, such as methylphenazinium methyl
4
sulfate 1. Thus NADH reduction of the colourless or pale
yellow tetrazolium salts, such as iodonitrotetrazolium blue,
does not occur by direct interaction but can be catalysed by the
enzyme diaphorase to form the intensely coloured formazan
dye. The product formazans are generally highly insoluble
Apart from work with methylphenazinium methyl sulfate,
surprisingly little work has been reported on the use of NADH
as a direct reductant of other organic substrates, i.e. not involv-
ing an enzyme-mediated process. Substrate requirements are for
a system that is sufficiently oxidizing to drive the equilibrium
5
and non-fluorescent. Use has also been made of the weakly
fluorescent dye resazurin 2, which is reduced by NADH in
ϩ
with NADH over to the NAD state, preferably without being
6
7
the presence of either diaphorase or the phenazinium salt 1
too strong an oxidant so as not to interfere with other com-
ponents in any assay. Also, since NADH reductions generally
involve a face-to-face interaction with the substrate, thus
encouraging a net hydride delivery, planar systems should
initially be investigated.
to give, in this case, the strongly fluorescent dye resorufin 3.
The mechanism of the catalysis with the phenazinium salt 1
proceeds with formation of the dihydrophenazine species 4,
Results and discussion
In this work two systems have been explored, the phenazinium
and quinoxalinium systems. Phenazine derivatives of the type 6,
upon N-methylation, should give the phenazinium system,
4
related to 1 and known to be rapidly reduced with NADH. 4-
Methylcatechol was oxidized with sodium periodate to produce
the ortho-quinone 5 which was immediately treated with 1,2-
diaminobenzene to produce 2-methylphenazine 6 in good yield
(
see Scheme 2). The methyl group could be brominated, using
N-bromosuccinimide (NBS), to give the monobromide 7,
characterized as its phenyl ether 8. Reaction of the mono-
bromide with 4-methylumbelliferone produced the required
DOI: 10.1039/b001296n
J. Chem. Soc., Perkin Trans. 1, 2000, 1541–1546
This journal is © The Royal Society of Chemistry 2000
1541

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the colour. Examination of the products again showed a
complex mixture, amongst which were small quantities of the
fluorescent 4-methylumbelliferone. A fluorescence study also
showed that some of the fluorophore was freed during reduc-
tion but the yields obtained were small (<10%). A possible
mechanism for this elimination is shown in Scheme 3; the
Scheme 3
reduced intermediate (14a, or its regioisomer, 14b) would be
expected to eliminate the fluorophore to produce the intermedi-
ate 15 and hence re-form the phenazinium species, i.e. 10a(b),
before further reduction with NADH.
Scheme 2 Reagents: i, 1,2-diaminobenzene; ii, NBS; iii, sodium
phenoxide; iv, methyl triflate; v, 4-methylumbelliferone, base; vi,
dimethyl sulfate.
It is clear that, for the phenazinium series, the intermediate
dihydro-species, such as 4 and 13, were too sensitive to com-
peting aerial oxidation and redox reactions to be of value in
estimating NADH. Encouraged by the appearance of small
quantities of the free fluorophore, our attention was turned
to exploring other heterocyclic substrates. Of these, the
quinoxalinium series was considered, since, it was argued, the
corresponding reduced system should be less susceptible to
these oxidative side reactions.
conjugate 9. Reaction of the parent phenazine 6 with methyl
triflate gave mixtures of the 5- and 10-methyl regioisomers, 10a
and 10b, not easily separable, and means to overcome this
problem could not be found. As a consequence the qualitative
behaviour of these mixtures towards NADH was studied.The
conjugate 9 was only sparingly soluble in organic solvents and
attempts to make its triflate derivative failed. Instead this con-
jugate was methylated with dimethyl sulfate to produce the
regioisomeric mixture 11.
7
Condensation of 1,2-diaminobenzene with pyruvaldehyde
13
A solution of the yellow salts 10 in 4-(2-hydroxyethyl)-
piperazine-1-ethanesulfonic acid (HEPES) buffer at pH 7.5 was
treated with an excess of a freshly prepared solution of NADH,
gave 2-methylquinoxaline 16 in good yields.
In contrast to the phenazine system, the quinoxaline was
found to react regioselectively with methylating agents, such as
methyl iodide or methyl triflate, to afford the corresponding 1,3-
Ϫ8
Ϫ3
Ϫ7
using salt concentrations in the range 10 mol dm to 10
Ϫ3
1
mol dm . Reaction was monitored by disappearance of the
yellow colour (λmax 390 nm), the solutions going colourless
within minutes. When only a slight excess of the NADH
reductant was used, the initial bleaching of the colour was fol-
lowed by its reappearance. This observation is in agreement
with the observations of Davis and Thornalley, Zaugg, and
Chew and Bolton on the parent phenazinium methyl sulfate.
They suggested that aerial reoxidation of the reduced inter-
mediate 13 (cf. 4) occurs. Attempts to isolate the dihydro-
species 13 (X = H) were unsuccessful, the compound being
readily oxidized in air, only small samples of the demethylated
phenazine 6 being obtained from the complex reaction mixture.
Aerial photo-oxidation of the phenazinium system is well
dimethylquinoxalinium salt 17. H NMR spectral examination
showed that a less than 5% yield of the 1,2-dimethyl regiosomer
18 was formed, and the major isomer was readily purified by
recrystallization. Reduction of these salts with NADH at pH
7.5 rapidly produced a dihydro-compound (see Scheme 4).
Whilst mechanistic studies with NADH predicted that this
7
8
9
10
14
would be the 1,4-dihydro-isomer 19, this tautomer was not
isolated since it rapidly rearranges to the 1,2-dihydro-isomer
20, isolated in high yield (>90%). Again, in contrast to the
dihydrophenazine intermediates, the dihydroquinoxaline 20
is relatively stable, although, upon exposure to air it is oxid-
ized to give a variety of highly coloured products. It is to be
noted that compound 19, like 4, is also formally anti-aromatic
and this undoubtedly contributes to its chemical behaviour.
Whereas the dihydroquinoxaline 19 has the potential to
tautomerize to the more stable isomer 20, the dihydrophenazine
system cannot undergo a similar tautomerism to a more stable
form.
11
12
recorded and results in formation of products such as
pyocyanin and 12.
NADH reduction of the yellow-green conjugate 11 also
resulted in a rapid decoloration followed by reappearance of
Reduction of the salt can also be effected with NaBH4,
although with an excess of this reagent further reduction to the
tetrahydroquinoxaline system 21 was observed. None of the
tetrahydro-product was observed upon the reduction of 17 with
NADH.
Because of its bimolecular nature, the rate of reduction of
the quinoxalinium salt 17 is concentration dependent. At
concentrations in the micromolar range the reduction takes
1
542
J. Chem. Soc., Perkin Trans. 1, 2000, 1541–1546

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Fig. 1 Increase in umbelliferone fluorescence by addition of NADH
Ϫ3
Ϫ3
Ϫ4
to the probe 26. NADH, 1 × 10 mol dm mixed with probe, 1 × 10
Ϫ3
mol dm , in TRIS buffer at pH 7.6 sampled at 2 min intervals, aliquots
diluted 100-fold before measurement; λex 360 nm; λmax reached within
2
4 min.
The fate of the quinoxalinium moiety has also been explored
and the dihydro-species 20 was also isolated from the NADH
reduction mixture. Formation of the latter is explained as in
Scheme 5. Initial reduction by NADH is assumed to give the
Scheme 4 Reagents: i, CH I; ii, NBS; iii, NADH; iv, NaBH ; v,
3
4
4
-methylumbelliferone, base.
several hours to complete but, at millimolar concentrations, the
reaction is complete within seconds.
In order to generate a useful fluorescent probe, 2-methyl-
quinoxaline 16 was brominated at the methyl group using NBS.
Selective monobromination proved difficult to control and
samples of the dibromo- and tribromo-compounds 23 and 24
always accompanied the required monobromide 22. The
monobromide was unstable to heat, general decomposition
occurring to form a range of dark green/blue polar materials.
The monobromide was conjugated with 4-methylumbelli-
ferone to form the derivative 25, which was a stable compound.
Methylation of this conjugate with methyl triflate afforded the
triflate salt 26. Neither compound 25 nor 26 showed any signifi-
cant fluorescence. An aqueous solution of the probe, main-
tained at pH 7.6 and kept in the dark at room temperature, was
stable over several days, no free methylumbelliferone being
released.
Reduction of the salt 26 with an excess of NADH was
studied under a range of conditions. In a quantitative experi-
ment at pH 7.5, reduction to the (unstable) intermediate 27
occurred and an emission spectrum characteristic of the
free umbelliferone species 29 immediately started to form.
After reduction was complete, the free umbelliferone could
be isolated and a recovery of >85% was obtained. In the
absence of NADH no liberation of 4-methylumbelliferone was
observed, confirming that the liberation was a direct result of
reduction by NADH. The rate of the reaction is concentration
dependent. At concentrations of both probe and reductant in
the micromolar range, reaction was extremely slow. However,
in the lower millimolar concentration range an acceptable rate
of reduction was observed. Fig. 1 shows the emission results of
a typical reduction. The increase in 4-methylumbelliferone 29
fluorescence is obtained by mixing a solution of the salt 26
Scheme 5 Reagent: i, NADH.
1,4-dihydro-intermediate 27. We speculate that rather than
tautomerizing to the isomer 28, the intermediate 27 ejects
4-methylumbelliferone forming the species 30, which can
tautomerize back to the parent quinoxalinium species 17, itself
a substrate for further reduction with NADH to give the
dihydro-species 20. In principle, the initial reduction product 27
could be in an equilibrium with its tautomer 28, the reaction
ultimately leading to the same products. However, by monitor-
Ϫ4
Ϫ3
Ϫ3
Ϫ3
at 1 × 10 mol dm with NADH at 1 × 10 mol dm in
tris(hydroxymethyl)methylamine (TRIS) buffer at pH 7.5,
taking aliquots at 2 min intervals and diluting 100-fold in
ing reactions in D O, no evidence for the formation of
2
Ϫ6
Ϫ3
buffer (to give a probe concentration of 1 × 10 mol dm )
before measuring the fluorescence emission spectrum (λex 350
nm), monitoring the methylumbelliferone signal at λem 450
nm. Reaction takes less than 24 min under these conditions
and, whilst not fast, is adequate for assaying NADH concen-
trations. Using an excess of the quinoxalinium salt, a linear
relationship between emission intensities and NADH concen-
substantial amounts of the tautomer 28 could be obtained.
From the reduction process it was noted that the strength of
the fluorescence emission signal at 450 nm was not as high as
that obtained for a control sample of 4-methylumbelliferone 29
at the same concentration, indicating some quenching process.
This was assumed to be by the co-product dihydroquinoxaline
20.
Ϫ7
Ϫ4
trations could be obtained in the range 5 × 10 to 10 mol
A Stern–Volmer quenching experiment confirmed this
assumption (Fig. 2). At the concentrations used in fluorescence
Ϫ3
dm range.
J. Chem. Soc., Perkin Trans. 1, 2000, 1541–1546
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3
cm ) with stirring. To the solution was then added, dropwise
3
over a period of 10 min, glacial acetic acid (20 cm ) and the
mixture was stirred for a further 30 min at room temperature
before being heated to reflux for 4 h. The solution was cooled,
3
and washed successively with water (100 cm ), aq. sodium
3
3
hydrogen carbonate (2 × 100 cm ) and finally brine (100 cm ).
The organic layer was dried, filtered, and evaporated to dryness
to afford an orange solid. Filtration through a plug of alumina,
using methylene dichloride as solvent, afforded the product as
17
a yellow solid (3.08 g, 60%), mp 117 ЊC (lit., 117 ЊC); δH 2.66
3 H, s), 7.64 (1 H, dd, J 7, 2 Hz), 7.83 (2 H, m), 7.99 (1 H, s),
.13 (1 H, d, J 7 Hz), 8.22 (2 H, m).
(
8
Fig. 2 Stern–Volmer quenching of 4-methylumbelliferone 29 fluor-
escence by dihydroquinoxaline 20 in TRIS buffer at pH 7.6. [29] =
2-(Bromomethyl)phenazine 7
-Methylphenazine (1.63 g, 8.3 mmol) was dissolved in CCl (70
Ϫ6
Ϫ3
1
× 10 mol dm ; λex 360 nm; λem 450 nm.
2
4
3
cm ) and NBS (1.48 g, 8.3 mmol, freshly recrystallized from
water) and benzoyl peroxide (50 mg) were added. The mixture
was heated to a gentle reflux for 2 days, with addition of two
further portions of benzoyl peroxide (50 mg) after 20 and 30 h.
The mixture was cooled to room temperature, filtered, and
measurements for the NADH-reduction experiment, this
quenching accounts for almost all of the observed reduction in
fluorophore signal intensity.
Little is known about the factors responsible for increasing
the kinetic rate of direct reduction of heterocyclic substrates
with NAD(P)H. For example, it was noted that the observed
rate of reduction of the quinoxalinium derivative 17 was much
slower compared with that for methylphenazinium methyl
sulfate 1. Presumably the increased aromatic footprint of the
latter enhances the initial interactions between the two species.
Studies on such factors, and also to define the scope of such
reductions, are in progress.
3
the filtrate washed successively with water (50 cm ) and brine
3
(
50 cm ), before drying and removal of solvent under reduced
pressure. The brown solid was purified by chromatography
through silica gel (1:5 ethyl acetate–light petroleum) to afford
the title compound as a yellow solid (0.43 g, 80%), mp 156–
1
62 ЊC; δ 4.74 (2 H, s, CH Br), 7.80–7.88 and 8.21–8.28 (7 H,
H
2
m, ArH); δC 143.8, 143.7, 143.05, 140.0, 131.1, 130.7, 130.5,
29.65, 129.6, 129.5, 128.85, 32.75 (2 overlapping signals); m/z
1
ϩ
(
EI) 274 (M Ϫ Br), 193.
Experimental
Mps were determined on a Kofler hot-stage apparatus and are
2
-(Phenoxymethyl)phenazine 8
uncorrected. IR specta were recorded on a Perkin-Elmer System
To a solution of phenol (0.846 g, 9.1 mmol) in 50:1 aceto-
nitrile–water (51 cm ) was added potassium hydroxide (0.512 g,
3
2
000 FTIR spectrometer, for samples either as films, Nujol
mulls or solutions in chloroform. UV spectra were determined
on a Perkin-Elmer Lambda 9 spectrophotometer for sample
solutions in 10 mm cuvettes. H NMR spectra were recorded on
9.1 mmol) and the solution was heated to reflux for 30 min
and then cooled slightly before addition of (bromomethyl)-
phenazine 7 (0.5 g, 1.82 mmol) and heating of the solution for a
further 4 h. The reaction mixture was diluted with ethyl acetate
1
either a Bruker 300 MHz spectrometer or a JEOL FX270 MHz
instrument, for samples as solutions in deuteriochloroform,
unless otherwise stated, using tetramethylsilane as internal
reference. Mass spectra were obtained from the EPSRC
National Mass Spectrometry Service Centre, Swansea, and
microanalytical determinations were by MEDAC Ltd,
Englefield Green, Surrey. All fluorescence spectra were
obtained on a Perkin-Elmer LS50B spectrofluorimeter loaded
with FLwinlab software.
3
3
(100 cm ) and washed successively with water (2 × 70 cm ), 5%
3
3
w/v aq. sodium hydroxide (70 cm ) and brine (50 cm ) before
separation of the organic layer, drying, filtering and removal of
solvent to give the product as a brown solid. The material was
purified by column chromatography (5:1 light petroleum–ethyl
acetate) to afford the title compound (0.43 g, 80%), mp 118–
119 ЊC; δ 5.37 (2 H, s, CH O), 7.00 (1 H, t, J 7 Hz, ArH), 7.06
H
2
(2 H, d, J 8.4 Hz, ArH), 7.32 (2 H, dd, J 7, 8.4 Hz, ArH), 7.87–
7.84 (2 H, m, ArH), 7.91 (1 H, dd, J 1.8, 8.9 Hz, ArH), 8.24–
TLC was carried out on glass plates precoated with 0.25 mm
Kieselgel 60 GF254 and column chromatography was through
Merck Kieselgel 60G. Solvents were distilled before use and
solvent ratios are in volumes before mixing. Light petroleum
refers to the fraction of boiling range 60–80 ЊC and ether refers
to diethyl ether. Brine is a saturated aqueous solution of
sodium chloride. Reagents and chemicals obtained from
external sources were checked by TLC and NMR prior to use.
ϩ
8.29 (3 H, m, ArH), 8.32 (1 H, s, ArH); m/z (EI) 286 (M Ϫ Ph),
ϩ
210 (M Ϫ OPh) (Found: C, 79.6; H, 4.9; N, 9.7. C H N O
1
9
14
2
requires C, 79.7; H, 4.9; N, 9.8%).
2-(4-Methylcoumarin-7-yloxymethyl)phenazine 9
Freshly recrystallized 4-methylumbelliferone 29 (0.58 g, 3.3
mmol) and sodium hydroxide (0.132 g, 3.3 mmol) were dis-
15
Solvents were purified by the methods described in Perrin.
3
solved in 50:1 acetonitrile–water (51 cm ) and the solution was
Solutions were dried over anhydrous sodium sulfate unless
otherwise specified.
heated to reflux for 10 min before cooling, and addition of
(
bromomethyl)phenazine 7 (0.45 g, 1.65 mmol). The mixture
was heated to reflux for 4 h before cooling, and refiltering off of
the light green solid (0.45 g, 40%). A sample was recrystallized
from ethanol to give pale green crystals of the title compound,
mp 255–257 ЊC; δ 2.39 (3 H, s, CH ), 5.42 (2 H, s, CH O), 6.12
Methylphenazinium methyl sulfate 1
16
This was prepared according to the literature method, mp
1
58–160 ЊC (decomp.).
H
3
2
(
1 H, s, CHCO), 6.95 (1 H, s, ArH), 7.03 (1 H, d, J 8.2 Hz,
1
7
2
-Methylphenazine
6
ArH), 7.53 (1 H, d J 8.1 Hz, ArH), 7.86–7.88 (3 H, m, ArH),
8
3
ϩ
ϩ
.26–8.31 (4 H, m, ArH); m/z 368 (M ), 193 (Found: M ,
68.1161. C H N O requires M, 368.1161).
2
-Methylcatechol (3.72 g, 30 mmol) was dissolved in water (300
cm ) at room temperature and sodium periodate (6.73 g, 33
mmol) added. The mixture was stirred vigorously for 1 min
3
23 16
2
3
2
,5(10)-Dimethylphenazinium trifluoromethanesulfonate 10
3
before extraction with methylene dichloride (2 × 70 cm ). The
organic extract was immediately added to a solution of 1,2-
diaminobenzene (3.24 g, 30 mmol) in methylene dichloride (40
2-Methylphenazine 6 (48.5 mg, 0.25 mmol) was dissolved in
dry toluene (2 cm ) under a dry nitrogen atmosphere in a flask
3
1
544
J. Chem. Soc., Perkin Trans. 1, 2000, 1541–1546

View Article Online
wrapped in aluminium foil. Methyl trifluoromethanesulfo-
nate (41 mg, 0.25 mmol) added dropwise at room temperature
and the mixture was stirred overnight. The crystalline precipi-
tate that formed was collected by filtration, washed with ether,
and dried in the dark under reduced pressure to give a mixture
of the two isomeric title compounds as a bright yellow solid (36
50%), mp 96–99 ЊC; δ 2.13 (3 H, s, CH ), 2.76 (3 H, s, NCH ),
H 3 3
3.73 (2 H, s, NCH ), 6.61 (1 H, d, J 8 Hz, ArH), 6.75 (1 H, t, J 8
Hz, ArH), 7.08 (1 H, t, J 8 Hz, ArH), 7.21 (1 H, d, J 8 Hz, ArH)
(Found: C, 74.9; H, 7.3; N, 17.5. C H N requires C, 75.0; H,
2
10
12
2
7.55; N, 17.5%).
mg, 40%), mp 104–106 ЊC; δ 2.92 and 2.79 (3 H, s, CH ), 5.16
H
3
1,3-Dimethyl-1,2,3,4-tetrahydroquinoxaline 21
and 5.12 (3 H, s, CH ), 8.02–8.88 (7 H, m, ArH) (Found: C,
3
3
The salt 17 (0.286 g, 1 mmol) was dissolved in water (5 cm ) in
5
0.1; H, 3.6; N, 7.7. C H F N O S requires C, 50.3; H, 3.7;
15 13 3 2 3
3
the presence of methylene dichloride (5 cm ) and sodium boro-
N, 7.8%).
hydride (0.076 g, 2 mmol, 8 equiv.) was added. The mixture was
stirred for 1 h before addition of more methylene dichloride (5
5
(10)-Methyl-2-(4-methylcoumarin-7-yloxymethyl)phenazinium
3
cm ), separation of the two layers, washing the organic extract
methyl sulfate 11
3
with water (2 × 5 cm ), drying, filtering and evaporation to dry-
3
The methylphenazine 6 (48.5 mg, 0.25 cm ) was added to freshly
ness to afford a reddish gum (0.117 g, 72%); δH 1.17 (3 H, d,
J 6.3 Hz, CH ), 1.54 (1 H, br s, exch. D O, NH), 2.85 (3 H, s,
3
distilled dimethyl sulfate (2 cm ) and the mixture was heated to
3
2
1
10 ЊC for 10 min. The solution that formed was cooled to
NCH ), 2.92 (1 H, dd, J 8.3, 10.6 Hz, CHHN), 3.15 (1 H, dd,
3
3
room temperature and acetonitrile (3 cm ) was added. To the
brown solution was added ether (10 cm ) to afford a brown
J 2.7, 10.6 Hz, CHHN), 3.60 (1 H, m, NCHCH ), 6.47 (1 H, m,
ArH), 6.56 (2 H, m, ArH), 6.69 (1 H, m, ArH).
3
3
solid precipitate, which was isolated by filtration. The material
was re-dissolved in acetonitrile and re-precipitated with dry
ether to afford the title salt (25 mg, 32%), mp 231–232 ЊC
This was characterized as its N-benzoyl derivative, prepared
by reaction of the isolated amine with benzoyl chloride (0.25
3
3
cm ) in pyridine (0.5 cm ), and work-up in the normal manner.
The product was obtained as a viscous gum; δH 1.21 (3 H, d,
J 6.9 Hz, CH ), 3.0 (1 H, s, NCH ), 3.08 (1 H, dd, J 2, 11.3 Hz,
ϩ
ϩ
(
Found: M Ϫ CH O S 383.1406. C H N O requires m/z,
3 4 24 19 2 3
3
83.1396). This material was used directly in the reactions with
3
3
NADH.
NCHH), 3.67 (1 H, dd, J 4.6, 11.3 Hz, CHH), 5.02 (1 H, br s,
NCHCH ), 6.35 (1H, m, ArH), 6.67 (1 H, m, ArH), 6.98 (1 H,
m, ArH), 7.2–7.5 (6 H, m, ArH) [Found: M (EI-MS) 266.1417.
3
1
3
ϩ
2
-Methylquinoxaline 16
C H N O requires M, 266.1419].
17
18
2
Sodium metabisulfite (23.2 g, 0.122 mol) was added to a solu-
tion of pyruvaldehyde (26 g, 0.36 mol) in water (85 cm ; 23%
3
2
-(Bromomethyl)quinoxaline 22
w/v) and, after 10 min, the solution was added to a vigorously
stirred solution of 1,2-phenylenediamine (10.2 g, 94 mmol) in
A mixture of the quinoxaline 16 (2.16 g, 15 mmol), NBS (fresh-
ly recrystallized from water; 6 g, 33 mmol) and benzoyl peroxide
3
water (52 cm ). After 18 h at room temperature the solution was
adjusted to pH 10 by the addition of sodium carbonate (20 g),
3
(
0.415 g) were added to carbon tetrachloride (75 cm ) and the
3
and the mixture was extracted with ether (3 × 30 cm ). The
mixture was heated to 80 ЊC with stirring. The reaction was
monitored by TLC. After 5 h, when all the starting quinoxaline
had reacted, the mixture was cooled to room temperature and
filtered. After removal of the solvent under reduced pressure,
the residue was immediately chromatographed through silica
gel (methylene dichloride as eluant) to yield, initially, the title
compound (2.10 g, 62%) as a pale blue, crystalline solid, mp 73–
extract was dried, the solvent removed under reduced pressure,
and the residue purified by vacuum distillation to afford the title
quinoxaline as a colourless oil (12.38 g, 91%), bp 125–126 ЊC/11
mmHg (lit., 245–247 ЊC/760 mmHg); δ 2.75 (3 H, s, CH ), 7.65–
H
3
7
.74 (2 H, m, ArH), 7.9–8.07 (2 H, m, ArH), 8.72 (1 H, s, 3-H).
Methylation of 2-methylquinoxaline
-Methylquinoxaline 16 (1.0 g, 7 mmol) was added to neat,
7
4 ЊC; ν (CHCl ) 2990, 1559, 1494, 1216, 1201, 982, 757, 667
max 3
Ϫ1
cm ; δ 4.74 (2 H, s, CH ), 7.77–7.78 (2 H, m, ArH), 8.05–8.14
2
H
2
3
(
2 H, m, ArH), 9.00 (1 H, s, 3-H) (Found: C, 48.6, 3.4; N, 12.7.
freshly distilled methyl iodide (3 cm , 48 mmol) and the solution
was stirred in the dark at room temperature under argon. After
C H BrN requires C, 48.5; H, 3.2; N, 12.55%).
9
7
2
Also isolated, in order of elution, were 2-(dibromomethyl)-
quinoxaline 23 (0.13 g, 3%), mp 101–103 ЊC; δH 6.77 (1 H, s,
1
8 h a green crystalline precipitate had formed. This solid was
collected by filtration, a portion (0.1 g) kept for NMR examin-
CHBr ), 7.85–7.79 (2 H, m, ArH), 8.05–8.17 (2 H, m, ArH),
ation and the rest was dissolved in a minimal amount of dry
2
3
9
1
2
.39 (1 H, s, 3-H); m/z (FAB-MS) 301, 303, 305 (proportions
:2:1, MH ) and 2-(tribromomethyl)quinoxaline 24 (0.11 g,
acetonitrile (≈2 cm ) and then precipitated by the addition of
ϩ
ether, to afford as a yellow powder, 1,3-dimethylquinoxalinium
iodide 17 (1.52 g, 82%), mp 125–127 ЊC (decomp.). The purified
product showed δH 2.92 (3 H, s, ArCH ), 4.67 (3 H, s, NCH ),
%), mp 83–84 ЊC; δ 7.48–7.68 (2 H, m, ArH), 8.09–8.09 (2 H,
H
m, ArH), 9.7 (1 H, s, 3-H); m/z 379, 381, 383, 385 (proportions
3
3
ϩ
1
:3:3:1, MH ).
7
.23–7.27 (2 H, m, ArH), 8.41 (1 H, d, J 8 Hz, ArH), 8.56 (1 H,
d, J 8 Hz, ArH), 9.61 (1 H, s, 3-H) (Found: C, 41.9; H, 3.9;
N, 9.7. C H IN requires C, 42.0; H, 3.9; N, 9.8%).
In its H NMR spectrum the crude methylation product
showed the above signals and small signals (<5%) at δ 3.12 and
2-(4-Methylcoumarin-7-yloxymethyl)quinoxaline 25
1
0
11
2
1
To a stirred solution of 7-hydroxy-4-methylcoumarin 29 (0.8 g,
4
.5 mmol) and sodium hydroxide (0.18 g, 4.5 mmol), in 1:19
4
.52 corresponding to the methyl signals for the 1,2-methylated
water–tetrahydrofuran under an atmosphere of nitrogen, was
added 2-(bromomethyl)quinoxaline 22 (1.01 g, 4.5 mmol) and
the solution was heated to reflux in the dark for 16 h. The bulk
of the solvent was removed under reduced pressure and the
residue extracted into methylene dichloride. The organic extract
was washed successively with 2 M aq. sodium hydroxide (30
regioisomer 18; these were virtually absent in the re-precipitated
salt.
1
,3-Dimethyl-1,2-dihydroquinoxaline 20
A biphasic mixture of a solution of NADH (0.16 g, 0.22 mmol)
3
3
3
3
in water (2 cm ) and chloroform (6 cm ) was stirred vigorously
in the dark, at room temperature, whilst adding, dropwise, over
a period of 5 min, a solution of the salt 17 (60 mg, 0.2 mmol) in
cm ) and brine (2 × 30 cm ) before drying, filtering and concen-
tration under reduced pressure to give a dark red solid, which
was purified by silica gel chromatography (1:49 methanol–
methylene dichloride as eluant) and recrystallization from
methanol to give the title compound as colourless crystals
(0.91 g, 64%), mp 211–213 ЊC; δ 2.40 (3 H, s, CH ), 5.49 (2 H,
3
water (2 cm ). The emulsion was stirred for a further 20 min
before the phases were separated, collection of the organic solu-
tion, drying, filtering and removing the solvent under reduced
pressure to afford the title compound as a red powder (18 mg,
H
3
s, CH ), 6.16 (1 H, s, ArH), 6.98 (1 H, s, ArH), 7.04 (1 H, d, J 9
2
J. Chem. Soc., Perkin Trans. 1, 2000, 1541–1546
1545

View Article Online
Hz, ArH), 7.52 (1 H, d, J 9 Hz, ArH), 7.78–7.82 (2 H, m, ArH),
umbelliferone. A control experiment showed that NADH fluor-
8
4
.10–8.16 (2 H, m, ArH), 9.03 (1 H, s, ArH) (Found: C, 71.8; H,
escence emission under the concentration regimes used is
minimal (<10 units at 1 × 10 mol dm ). Similarly the starting
salts 17 and 26 showed only weak emission signals.
Ϫ5
Ϫ3
.45; N, 8.6. C H N O requires C, 71.7; H, 4.4; N, 8.8%).
19
14
2
3
N-Methyl-2-(4-methylcoumarin-7-yloxymethyl)quinoxalinium
trifluoromethanesulfonate 26
Acknowledgements
We thank the EPSRC for financial support of this work and for
a studentship (to C. A. R.).
To a solution of the quinoxaline 25 (0.25 g, 0.79 mmol) in dry
3
chloroform (5 cm ) under argon, in the dark, was added methyl
3
trifluoromethanesulfonate (0.76 cm , 3.2 mmol, 4 equiv.). The
mixture was stirred at room temperature for 24 h, the solvent
removed under reduced pressure, and a few drops of aceto-
nitrile added to form a mobile solution before addition of
References
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S. K. Jonas, C. Benedetto, A. Flatman, L. Micheletti, C. Riley,
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3
dry ether (20 cm ) to precipitate the title compound as a yellow
powder (0.317 g, 83%), mp 195–198 ЊC; δH 2.42 (3 H, s, CH ),
3
4
.78 (3 H, s, NCH ), 5.77 (2 H, s, CH ), 6.27 (1 H, s, ArH), 7.21
2 For preliminary results, see C. A. Roeschlaub, N. L. Maidwell,
3
2
M. R. Rezai and P. G. Sammes, Chem. Commun., 1999, 1637.
(
1 H, d, J 9 Hz, ArH), 7.25 (1 H, s, ArH), 7.80 (1 H, d, J 9 Hz,
ArH), 8.27–8.35 (2 H, m, ArH), 8.51 (1 H, d, J 8 Hz, ArH), 8.64
1 H, d, J 8 Hz, ArH), 9.85 (1 H, s, ArH) (Found: C, 52.2; H,
3
C. H. Self, J. Immunol. Methods, 1985, 76, 389; A. Johannsson,
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21 17 3 2 3
ϩ ϩ
5.8; S, 6.65%); m/z (FAM-MS) 334 (M ϩ H ).
4 M. M. Nichlas, S. J. Marguiles, G. J. Goldberg and A. M. Seligman,
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971, 226, 269.
Preparative-scale reduction of the quinoxalinium conjugate 26
5
E. Seidler, The Tetrazolium-Formazan System: Design and Histo-
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The conjugate salt (100 mg, 0.207 mmol) was dissolved in
3
acetonitrile (2 cm ) and added to a stirred solution of NADH
Ϫ2
Ϫ3
6 D. B. Cook and C. H. Self, Clin. Chem. (Winston-Salem, N.C.),
993, 39, 965.
(
1.42 g, 2.07 mmol, 10 equiv.) in TRIS buffer (10 mol dm ;
3
1
pH 7.6; 30 cm ). Stirring was continued for 2 h, in the dark,
7
L. P. Candeias, D. P. S. MacFarlane, S. L. W. McWhinnie, N. L.
Maidwell, C. A. Roeschlaub, P. G. Sammes and R. Whittlesey,
J. Chem. Soc., Perkin Trans. 2, 1998, 2333.
before extraction of the reaction mixture with chloroform
3
(
2 × 30 cm ), washing of the organic extracts with water (30
3
cm ), drying and evaporation of the solvent under reduced
pressure. The residue was chromatographed through silica gel
8 G. Davis and P. J. Thornalley, Biochim. Biophys. Acta, 1983, 724,
56. See also ref. 9.
W. S. Zaugg, J. Biol. Chem., 1964, 239, 3964.
0 V. Chew and J. Bolton, J. Phys. Chem., 1980, 84, 1903; 1909.
1 Cf. A. K. Raap and P. van Duijn, Histochem. J., 1983, 15, 881.
2 H. McIlwain, J. Chem. Soc., 1937, 1704; see also G. A. Swan and
D. G. I. Fenton, Phenazines, in The Chemistry of Heterocyclic
Compounds, ed. A. Weissberger, Interscience, New York, 1955,
pp. 20–21 and 174–180.
4
9
(
chloroform, then 1:9 methanol–chloroform as eluant) to
1
1
1
afford methylumbelliferone 29 (32.4 mg, 89%) and the dihydro-
quinoxaline 20 (22 mg, 65%).
Spectroscopic measurements
Ϫ2
Freshly prepared stock solutions of reagents, at 1 × 10 mol
13 R. G. Jones, E. C. Kornfield and K. C. McLaughlin, J. Am. Chem.
Soc., 1950, 72, 3539.
Ϫ3
dm , were 17 made up daily before use. NADH was made up
1
1
1
4 G. Blankenhorn, Pyridine Nucleotide Dependent Dehydrogenases,
ed. H. Sund, Walter de Gruyter, Berlin, 1997, pp. 198–205.
5 D. D. Perrin, W. L. F. Amarego and D. R. Perrin, Purification of
Laboratory Chemicals, Pergamon Press, Oxford, 1986.
in distilled water, and the salts 17 and 26, dihydroquinazoline 20
and 4-methylumbelliferone 29 in acetonitrile. Aliquots were
Ϫ2
Ϫ3
diluted with a solution of TRIS buffer at 1 × 10 mol dm
adjusted to pH 7.6. Excitation was at λ 350 nm and emission
measured at λ 450 nm, the emission maximum for methyl-
6 F. Kehrmann and E. Havas, Ber. Dtsch. Chem. Ges., 1913, 46, 341.
17 G. R. Clemo and H. McIlwain, J. Chem. Soc., 1935, 741.
1
546
J. Chem. Soc., Perkin Trans. 1, 2000, 1541–1546