A novel application of heterocyclic compounds for biosensors based on NAD, FAD, and PQQ dependent oxidoreductases

Article abstract of DOI:10.1007/PL00010188

Various five-, six-, and seven-membered heterocyclic compounds containing two nitrogen atoms as well as quinoid substances were synthesized and investigated. Electrochemical oxidation-polymerization of these heterocycles on carbon and platinum electrodes was performed in aqueous medium. Electrochemically pre-treated carbon electrodes modified with these heterocyclic compounds and were used for the electro-catalytic oxidation of NADH and for the design of mediated glucose biosensors.

Full text of DOI:10.1007/PL00010188

Monatshefte fuÈr Chemie 130, 1269±1281 (1999)
A Novel Application of Heterocyclic Compounds
for Biosensors Based on NAD, FAD, and PQQ
Dependent Oxidoreductases
1
Valdas Laurinavicius , Bogumila Kurtinaitiene , Virginijus Liauksminas1,
Ï
Ç1;Ã
Benedikta Puodziunaite , Regina Janciene , Lidija Kosychova , Rolandas
Ï
Meskys
2
Ï
Ç2
Ï
Ç2
Å
1
1
Laboratory of Bioanalysis, Institute of Biochemistry, 2600 Vilnius, Lithuania
2
Laboratory of Bioorganic Compounds Technology, Institute of Biochemistry, 2600 Vilnius,
Lithuania
Summary. Various ®ve-, six-, and seven-membered heterocyclic compounds containing two nitrogen
atoms as well as quinoid substances were synthesized and investigated. Electrochemical oxidation-
polymerization of these heterocycles on carbon and platinum electrodes was performed in aqueous
medium. Electrochemically pre-treated carbon electrodes modi®ed with these heterocyclic compounds
and were used for the electro-catalytic oxidation of NADH and for the design of mediated glucose
biosensors.
Keywords. Benzimidazoles; 1,5-Benzodiazepines; Biosensors; Electrocatalysis.
Eine neue Anwendung organischer Heterocyklen als Mediatoren fuÈr Biosensoren auf der Basis
È
von NAD-, FAD- und PQQ-abhangigen Oxidoreduktasen
Zusammenfassung. Verschiedene fuÈnf-, sechs- und siebengliedrige heterocyclische Verbindungen
mit zwei Stickstoffatomen sowie Chinone wurden hergestellt und untersucht. Elektrochemische
È
Aktivitat und elektrochemische Polymerisation der Heterocyclen an Platin- und Kohlenstoffelek-
È È
troden wurden in waûriger Losung studiert. Durch die genannten Heterozyclen modi®zierte
È
È
Kohlenstoffelektroden konnen fur die elektrochemische Oxidation von NADH und den Aufbau von
È
Glucosesensoren unter Verwendung von Glucoseoxidase und PQQ-abhangiger Glucosedehydro-
genase verwendet werden.
Introduction
Oxidoreductases are well known enzymes used for biosensor design [1, 2]. The
ef®ciency of biosensor design considerably depends on the ef®ciency of electron
transport from the substrate via the enzyme to the acceptor or/and the electrode.
Ã
Corresponding author

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V. Laurinavicius et al.
1270
From this point of view and based on the cofactor's nature, oxidoreductases can be
divided into three main groups. Flavin dependent oxidases containing tightly
bound FAD or (FMN) in the active center of enzyme belong to the ®rst
group. These enzymes transfer electrons to oxygen. In some cases, one of the
products is H2O2 which can be determined amperometrically using Pt electrodes at
0.5±0.7 V vs. Ag/AgCl as reference electrode. Unfortunately, at such high potential
a variety of electrochemically active components existing in the sample can be also
electrochemically oxidized on the surface of electrode. Arti®cial electron acceptors
that reversibly transfer electrons from the active center of the oxidase to the electrode
surface at low potentials can replace oxygen in this reaction. This replacement
allows to design amperometric enzyme electrodes operating at a lower working
potential and avoiding the in¯uence of interfering substances. 2,6-Dichloropheno-
lindophenol [2], hexacyanoferrite(III) [3], tetrathiafulvalene [4], tetracyanoquino-
dimethane [5], various quinones [6±8], and ferrocene derivatives [9, 10] have been
applied for this purpose. However, there still remain some problems in the long
term operation of such types of biosensors. The ®rst is competition of the mediator
with oxygen for the active center of the enzyme. In the case of uncontrolled
concentration of oxygen, this phenomenon results in unpredictable errors of
measurement. The second is based on fact that reduced forms of the mediators are
not stable in the oxygen environment and can be oxidized not only electro-
chemically, but by oxygen as well. The third is inactivation of enzyme molecules
by H2O2 produced in side reactions.
NAD(P) dependent dehydrogenases belong to the second group. To catalyze
oxidation of the substrates, these enzymes need the free diffusing cofactor
NAD(P) to exert catalytic activity. Electrooxidation of NAD(P)H on the solid
electrodes can be performed only at high potentials (0.7±1 V), and this process
is complicated by side reactions: dimerization of NAD and oxidation of inter-
fering substances. Various redox mediators can be used to shuttle the electrons
from NAD(P)H to the electrode at substantially lower potentials [11]. Quinones
[12, 13], phenazines [14], phenoxazine and phenothiazine derivatives [15±18],
conducting organic salts [19], and inorganic iron salts [20, 21] can act as
catalysts for the electrochemical oxidation of NAD(P)H. Biosensors based on
such type of dehydrogenases, however, show poor stability due to instable
mediators and/or disappearing of NAD(P)H from the surroundings of the
enzyme.
The third group of enzymes consists of PQQ dependent dehydrogenases. These
enzymes contain a tightly bound cofactor (PQQ) in the active center [22].
Cytochromes or ubiquinones are natural acceptors of electrons. Derivatives of
ferrocene and phenazine can also serve as good mediators for PQQ dependent
dehydrogenases [23±26]. Biosensors based on PQQ dependent enzymes are very
promising due to their insensitivity to O2.
We have found that modi®cation of the surface of carbon with some
heterocyclic compounds can considerably improve the electron communication
between the active center of enzymes and the solid surface of the modi®ed carbon
electrode. The goal of our work was to check up a number of organic compounds,
mostly insoluble in water and irreversibly adsorbed on the carbon surface, as
promising agents for this purpose.

Biosensors Based on Heterocycles and Oxidoreductases
1271
Results and Discussion
Various ®ve-, six-, and seven-membered heterocyclic compounds containing two
o-positioned nitrogen atoms or bearing a quinone structure were synthesized and
investigated. The ®rst group consists of benzimidazole and benzimidazolinone
derivatives (1±10). The second group includes quinoxaline, quinoline, and
naphthoquinone derivatives (11±17). 1,5-Benzodiazepine derivatives (18±26)
belong to the third group. Chemical structures and electrochemical parameters of
these compounds are presented in Table 1.
Electrochemical properties of mediators
It was found that all tested materials are completely and irreversibly adsorbed on
carbon electrodes and show irreversible electrochemical behaviour in the potential
range of 0±800 mV vs. saturated Ag/AgCl. A typical pattern of an electrochemical
oxidation is shown in Fig. 1. The benzodiazepine derivative 22 was adsorbed on the
carbon electrode. The ®rst scan resulted in two well-pronounced oxidative peaks at
350 and 600 mV. In the following seven scans, the anodic current of the electrode
constantly decreases. Almost the same electrochemical behaviour was obtained on
Pt electrodes (data not shown).
The electrochemical oxidation of nitrogen containing heterocyclic compounds
is not clear and depends on a number of parameters. Some speculations are based
on the assumption that derivatives of 1,5-benzodiazepine are not completely
aromatic structures; they can be considered as substituted aromatic o-diamines.
Electrochemical oxidation of such compounds may be expressed as a two-electron
transfer from the nitrogen atoms to the electrode (Scheme 1).
The dicationic oxidation product can undergo reorganization to a more stable
system by the elimination of a cationic particle. The process can include reactions
with transfer of protons, especially in the case of compounds 23±26 (Scheme 2).
Following this scheme, a ®rst step of an electrooxidation process of benzo-
diazepine derivatives cannot consist of a two-electron oxidation alone; an additional
one-electron transfer reaction producing a more or less stable cation radical is also
required. This way can become predominant when the possibility to distribute
positive charge on a conjugated electron system exists (Scheme 3).
It seems that both processes can take place depending on the potential of the
electrode and the oxidative state of its surface. In any case, on the surface of
electrode there will be a number of different cation radicals of the heterocyclic
compound.
As mentioned above, the electrochemical oxidation process is completely
irreversible. It seems that cation radicals formed in the ®rst step of electrooxidation
lead to a destruction of the seven-membered ring, and oxidative polymerization or
dismutation polymerization of cations or both takes part (Scheme 4).
A similar coupling reaction occurs during the electrochemical oxidation of 2,5-
dihydro-1H-1-benzazepines in aqueous solutions [27].
Taken into account that the surface of the carbon electrode is covered with a
multi-layer of heterocyclic molecules and cycling of potential has been applied for
a long time (pre-treatment of the electrode), the process of electrochemical

Ï
V. Laurinavicius et al.
1272
Table 1. Parameters of investigated compounds
Compounds
M.p. (ꢀC)
(solvent)
Sensitivity (mA/mM)
Solution in
NADH
Glucose
Benzimidazole derivatives
1
2
R ˆ CH3
R ˆ CH2CH3
1.30
0.80
0.97
0.20
EtOH
EtOH
169±170 (Ref. [34])
(CCl4)
2-Benzimidazolinone derivatives
3
4
R1 ˆ R2 ˆ R3 ˆ H
0.85
2.00
0.21
0.98
EtOH
EtOH
R1 ˆ R2 ˆ H, R3 ˆ NO2
305±306 (Ref. [35])
(EtOH)
5
6
7
8
9
R1 ˆ R3 ˆ H, R2 ˆ CH3
190±192 (Ref. [36])
(EtOH)
4.00
2.00
0.65
0.62
0.63
0.01
0.17
0.28
Acetone
EtOH
R1 ˆ R3 ˆ H,
121±122 (Ref.[37])
(EtOH:H2O ˆ 1:1)
228±230 (Ref. [35])
(AcCN)
R2 ˆ C(CH3)=CH2
R1 ˆ H, R2 ˆ C(CH3)=CH2,
R3 ˆ NO2
DMF:EtOH
(1:1)
R1 ˆ R3 ˆ H,
165±167 (Ref. [38])
(AcCN)
Acetone
R2 ˆ C(Ph)=CH2
R1 ˆ R3 ˆ H, R2 ˆ COCH3
0.43
1.10
0.78
0.38
Acetone
Acetone
10 R1 ˆ COCH3,
108±110 (Ref. [38])
(Et2O)
R2 ˆ C(CH3)=CH2, R3 ˆ H
1,2,3,4-Tetrahydroquinoxalinone-2 derivatives
11 R1 ˆ R2 ˆ H
95±97 (Ref. [39])
(H2O)
1.80
0.83
EtOH
12 R1 ˆ H, R2 ˆ CH2CH3
0.80
0.62
0.71
1.68
EtOH
EtOH
13 R1 ˆ H, R2 ˆ COCH3
165±166 (Ref. [39])
(EtAc)

Biosensors Based on Heterocycles and Oxidoreductases
Table 1 (continued)
Compounds
1273
M.p. (ꢀC)
(solvent)
Sensitivity (mA/mM)
Solution in
NADH
Glucose
1,4-Naphthoquinone derivatives
14 R1 ˆ R2 ˆ Cl
0.4
0.78
3.12
1.14
Acetone
Acetone
Acetone
15 R1 ˆ Phthal, R2 ˆ Cl
16 R1 ˆ R2 ˆ Phthal
>295 dec. (Ref. [40])
no m.p. (Ref. [40])
0.54
0.4
17 3-Acetyl-2-methyl-7,8-dihydroxyquinoline
1.2
1.00
H2O
3H-1,5-Benzodiazepine derivatives
18 R1 ˆ R2 ˆ CH3, R3 ˆ H
132±133 (Ref. [41])
0.93a
1.00a
EtOH
EtOH
EtOH
EtOH
EtOH
(Et2O)
19 R1 ˆ R2 ˆ CH3, R3 ˆ H
201±203 (Ref. [41])
(sublimation)
176±178 (Ref. [42])
(H2O)
(hydrochloric salt)
20 R1 ˆ CH3, R2 ˆ Ph, R3 ˆ H
(hydrogensulfate monohydrate)
21 R1 ˆ R2 ˆ Ph, R3 ˆ H
1.04
a
138±140 (Ref. [42])
(MeOH)
3.75b
1.00c
22 R1 ˆ R2 ˆ R3 ˆ CH3
208±209 (Ref. [41])
(EtOH)
(hydrochloric salt)
23 2,2,4-Trimethyl-2,3-dihydro-1H-1,5-benzodiazepine
125±127 (Ref. [43])
(ligroin)
1.5
EtOH
EtOH
24 4-Methyl-2,3-dihydro-1H-1,5-benzodiazepinone-2
146±148.5 (Ref. [35])
1.10
0.25
(EtOH:H2O ˆ 1:1)

Ï
V. Laurinavicius et al.
1274
Table 1 (continued)
Compounds
M.p. (ꢀC)
(solvent)
Sensitivity (mA/mM)
Solution in
NADH
Glucose
2,3,4,5-Tetrahydro-1H-1,5-benzodiazepinone-2 derivatives
25 R1 ˆ H, R2 ˆ COCH3
26 R1 ˆ R2 ˆ CH3
159±160 (Ref. [34])
(EtAc)
1.4
0.21
0.18
EtOH
EtOH
152±154 (Ref. [44])
(benzene)
0.55
a
Sensitivities were measured using a Pt electrode (ꢁ ˆ 0.2 mm, working potential: 0.4 V vs. Ag/AgCl); bcarbon
c
electrode (ꢁ ˆ 3 mm, 0.4 V vs. Ag/AgCl); Pt electrode (ꢁ ˆ 0.2 mm, 0.4 V vs. Ag/AgCl)
Fig. 1. Cyclic voltammograms of a carbon electrode modi®ed with 22; sweep rate: 50 mV/s;
scanning was started at 0 V; the ®rst seven scans are presented
dimerization and/or polymerization can take part in an extensive range and
probably will lead to the formation of an infra-structure containing semi-oxidized
heterocyclic compounds. This presumption is based on the data of the
electrochemical behavior of a number of heterocyclic compounds during the
electrochemical cycling. The ®rst scan showed different peaks of oxidation for the

Biosensors Based on Heterocycles and Oxidoreductases
1275
Scheme 1
Scheme 2
Scheme 3
Scheme 4
tested heterocyclic compounds. This can be expected to be due to the different
structure of heterocycles and different electrochemical activity of the redox
centers. Further, after several scans the surface of the electrode became very
uniform (from the electrochemical point of view), without any distinctly
pronounced redox peaks and with high background current (compare data in
Fig. 1 and curve 1 in Fig. 2A). This means that the carbon surface is covered with a
number of network-like electrochemically active structures (probably three-
dimensional) possessing different electrochemical activity. However, this network
can facilitate electrochemical communication between the active center of the
enzyme or NADH and accept electrons at much lower potential than an untreated
carbon surface. Thus, the modi®ed carbon surface can act as a mediator between
the active center of the redox enzyme and can mediate electrooxidation of NADH.

Ï
V. Laurinavicius et al.
1276
Fig. 2. A: Cyclic voltammogram of a carbon a electrode modi®ed with 1; sweep rate: 50 mV/s; curve
1: background, curves 2±6 have been obtained in the presence of 0.5, 1.0, 1.5, 2, and 2.5 mM of
NADH, respectively B: catalytic properties of modi®ed carbon electrodes; curve 1: calibration curve
of NADH, Curve 2: calibration curve of glucose biosensor; stationary conditions, potential of the
electrode: ‡200 mV
Catalytic properties of mediators
Electrochemical oxidation of NADH on the modi®ed carbon electrodes showed the
reversible character of the mediator and was observed at potentials near 0.2 V.
Typical cyclic voltammogrames are presented in Fig. 2A. As can be seen, the value
of the catalytic current of electrooxidation of NADH depends on the concentration
of NADH. Curve 1 in Fig. 2B shows the current dependence on NADH concen-
tration under stationary conditions at 200 mV. The calibration curve has a linear
current-concentration dependence up to 1.1 mM of NADH. Almost the same range
of linearity was obtained with all 26 investigated mediators. This means that
modi®ed and electrochemically pre-treated carbon electrodes have similar
electrochemical surfaces with close diffusion parameters. Calculated sensitivity
of the sensor is 1.3 mA/mM. The sensitivities for all investigated compounds are
presented in Table 1 and serv as an index of the electrochemical activity of the
substance as a mediator. The sensitivity of sensors, however, was different and
depends on the nature of the mediator. Molar concentrations of mediators were the
same in all experiments, and differences of sensitivity can be explained by different
sorption parameters of mediators and by differences in NADH oxidation rates. The
best mediator for NADH oxidation was 1-methylbenzimidazolinone-2 (5).
The sensitivity of electrodes to NADH oxidation was measured under
potentiostatic conditions at 200 mV vs. Ag/AgCl (except some determinations
which were carried out at higher potentials as mentioned in Table 1). The
dependence of the catalytic current on the applied potential for some

Biosensors Based on Heterocycles and Oxidoreductases
1277
Fig. 3. Dependence of the current on the potential for different mediators; the numbers of the curves
correspond to the numbers of compounds in Table 1; the data were obtained under stationary
conditions; concentration of NADH: 1 mM
benzimidazolinone derivatives is shown in Fig. 3. Introduction of electron accepting
groups into the molecule of the mediator does not change considerably the
sensitivity of the mediator, whereas electron donor groups such as a methyl or iso-
propenyl signi®cantly increase the sensitivity of NADH oxidation. However, the
replacement of a methyl group with ethyl (compare 1 with 2 and 11 with 12) leads
to a decrease of sensitivity. Quinones are well known mediators of NADH
electrooxidation [28], but the sensitivities of some carbon electrodes modi®ed with
quinone derivatives 14±17 have been found to be not higher than in the case of
other investigated mediators. Probably, the limiting step of electrooxidation
process is not the structure but the diffusion rate of the mediator. Benzodiazepine
derivatives also show catalytic properties in the oxidation of NADH, but
interpretation of this phenomenon is too complicated to ensure a conclusion about
the mechanism of electrochemical oxidation of NADH. The absence of well-
pronounced peaks of electrooxidation of NADH points to a complicated multi-step
process. It is possible that the sensitivity of the modi®ed electrodes is limited by the
structure of polymerized heterocycles rather than by individual electrochemical
parameters of the mediators.
A number of publications and our experience show that the active site of
glucose oxidase, as well as that of PQQ dependent glucose dehydrogenase, does
not communicate directly with the carbon surface of the electrode, since FAD or
PQQ are hidden inside the protein. Only small molecules like ferrocene,
phenoxazines, or quinones can transfer electrons from the active centre to the
surface of the electrode [9, 10, 15±18].
Adsorption of glucose oxidase on the modi®ed carbon electrodes does not
increase the background current of the electrode in the potential range from 0 to
0.5 V. Addition of glucose to the buffer solution leads to an increase of the anodic

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V. Laurinavicius et al.
1278
current of the electrode. This means that the carbon surface modi®ed with the
compounds given in Table 1 and electrochemically pre-treated can communicate
with FAD in the active center of glucose oxidase. This current-concentration
relationship is shown in curve 2 of Fig. 2B. The rate of electrochemical
regeneration of the active center of glucose oxidase is slower (in general) in
comparison with the rate of electro-oxidation of NADH, probably due to high
diffusion restrictions in the globule of the enzyme and worse communication of the
modi®ed surface of the carbon electrode with the active center of the enzyme. This
process is also potential dependent without strong correlation between the
electrochemical parameters of mediators and sensor sensitivity. Increased
sensitivity in the case of quinones was not surprising because quinones are well
known mediators of glucose oxidase [29±31]. Immobilization of PQQ dependent
glucose dehydrogenase on the surface of carbon electrodes modi®ed with these
heterocycles also leads to direct electrochemical communication between the
active center of enzyme and the surface of the modi®ed electrode [32].
It seems that enzymes are incorporated in the three-dimensional semi-oxidized
polymeric structure of heterocycles on the carbon surface. This conclusion is
con®rmed by three facts. The calibration curve of the glucose biosensor (curve 2 of
Fig. 2B) is quite short. If the KM value of glucose (KM ˆ 16 mM) is taken into
account, the shape of the calibration curve con®rms a kinetically controlled action
of the biosensor. Another fact con®rming the tight incorporation of the glucose
oxidase molecule into the polymeric structure is the week dependence of the
sensitivity of the sensor on oxygen concentration. The competition between the
natural acceptor (oxygen) and the surface of the electrode is responsible for the
small lag period in the calibration curve at low concentrations of glucose. The third
argument in favour of tight bonding of the enzyme globule into the polymer matrix
is the stabilization of the enzyme. This phenomenon is well pronounced in the case
Fig. 4. Operational stability of a carbon electrode modi®ed with 15 and PQQ glucose dehydrogenase
at 20ꢀC; inset: same data presented semi-logarithmically

Biosensors Based on Heterocycles and Oxidoreductases
1279
Fig. 5. Calibration curves of carbon electrode modi®ed with 15 and PQQ glucose dehydrogenase vs.
glucose obtained in phosphate buffer solution ( pH ˆ 7.0, 1) and in control human serum contain
about 7 mM of glucose by consecutive addition of equal amounts of glucose (20 mm3) to 0.5 cm3 of
serum buffer solution (1:10) (2)
of immobilization of PQQ glucose dehydrogenase. The activity of the enzyme
incorporated into the biosensor is decreasing very slowly. This inactivation can be
described as a ®rst-order process with a rate constant of kin ˆ 0.0028 hꢂ1 (Fig. 4).
The inactivation of the native PQQ glucose dehydrogenase under the same
conditions is 163 times faster (kin ˆ 0.45 hꢂ1) [32].
High stabilization of the enzyme in the redox matrix and a low potential of
action are essential results of this work. The shape of calibration curves obtained
using buffered glucose solutions and human serum containing added glucose
(Fig. 5) indicates that the surface of the biosensors is not affected by components of
blood serum. The results are promising for the design of new biosensors of
potential use for food industry and medicine.
Experimental
Glucose oxidase from Aspergillus niger and NADH were purchased from Sigma. PQQ glucose
dehydrogenase from Erwinia sp 34-1 (30 U/cm3) was puri®ed in the Laboratory of Bioanalysis of the
Institute of Biochemistry (Vilnius) [33]. Control human serum SERODOS was purchased from
Human GmbH (Germany). Carbon rod, ultra ``F'' purity, 3 mm diameter electrodes were purchased
from Ultra Carbon Company (USA). Compounds 1, 14 (Merck), and 3 (Aldrich) were purchased and
used without further puri®cation. The majority of organic compounds was synthesised according to
previously reported methods (Table 1). Compound 17 was kindly provided by Dr. I. Kovalev
(MOLTECH Corp., Tucson, USA). 9 and 12 gave satisfactory elemental analyses.
All tested compounds were used as 0.1 M solutions in an appropriate organic solvent. For
electrode modi®cation, 3 mm3 solution of organic compound were deposited on the tip of the carbon
rod or Pt electrodes and air-dried. All electrochemical studies were carried out at 25ꢀC using a 1 cm3
cell equipped with a saturated Ag/AgCl reference and Pt auxiliary electrodes. Phosphate buffer

Ï
V. Laurinavicius et al.
1280
(0.05 M, pH ˆ 7.0) containing 0.1 M KCl was used throughout the study. The sensitivity of modi®ed
electrodes to NADH was tested under stationary conditions (working carbon electrode potential 0.2 V
vs. Ag/AgCl. For the preparation of glucose sensors, enzyme solutions (5 U of enzyme per electrode)
were deposited on the modi®ed electrode and cross-linked by glutaraldehyde vapour. The electrodes
were kept over 10% glutaraldehyde at 4ꢀC for 2 h and washed with buffer solution. Coated electrodes
were kept at 4ꢀC until use. A polarograph OH-105 Radelkis (Hungary) was used to run the
electrochemical experiments. Melting points are uncorrected. The 1H NMR spectra were recorded on
a Hitachi R-22 spectrometer operating at 90 MHz. Chemical shifts are reported in ppm from
HMDSO as an internal reference and are given in ꢀ units.
1-Acetyl-2-benzimidazolinone (9; C9H8N2O2)
A mixture of 2.1 g 10 (10 mmol), 3 cm3 H2SO4 (conc.), and 5.2 cm3 water in 40 cm3 ethanol was kept
at room temperature for 30 h. Water (50 cm3) was added, and the reaction mixture was neutralized
(pH ˆ 7.0). The precipitate was collected. Recrystallisation from ethanol afforded 1.2 g (77%) of 9
which sublimates at 301±302ꢀC without melting.
1H NMR (DMSO-d6): 2.53 (s, 3H, CH3) 7.25±6.87 and 8.02±7.87 (m, 4H, aromat. H), 11.26 (br,
1H, NH) ppm.
4-Ethyl-1,2,3,4-tetrahydroquinoxalinone-2 (12; C10H12N2O)
A mixture of 1.48 g 11 (10 mmol), 22 cm3 of ethyl bromide, and 3 g of K2CO3 in 60 cm3 dry
methanol was re¯uxed for 25 h. After ®ltration and evaporation of the solvent in vacuo, the solid was
crystallized from a mixture of ethyl acetate and diethyl ether (2:1) to give 1.1 g (67%) of 12.
M.p: 120±122ꢀC; 1H NMR (CDCl3): 1.17 (t, 3H, CH3, J ˆ 6.5 Hz), 3.19 (q, 2H, NCH2,
J ˆ 6.5 Hz), 3.73 (s, 2H, COCH2), 7.07±6.45 (m, 4H, aromat. H), 9.82 (br, 1H, NH) ppm.
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Received December 14, 1998. Accepted (revised) March 4, 1999