First Synthesis of Highly Chemiluminescent Benzo[b]furan-2(3H)-ones Bearing a Urea Substructure

Article abstract of DOI:10.1002/hlca.201800243

A series of benzo[b]furan-2(3H)-ones (coumaran-2-ones) bearing a urea substructure, namely derivatives of 3-(aminocarbonylamino)benzo[b]furan-2(3H)-one, was prepared for the first time. The accessibility of these compounds through an electrophilic α-amidoalkylation approach of phenols (Tscherniac–Einhorn reaction) in the key step as well as the chemiluminescence (CL) properties of the desired compounds are strongly dependent on the substitution patterns at the urea moiety. Competing reaction pathways are discussed and an improved one pot synthetical approach of also general interest is presented. In conclusion, especially N,N-dialkylaminocarbonylamino-derivatives of benzo[b]furan-2(3H)-ones exhibit a strong flash like blue CL upon treatment with bases such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in the presence of oxygen or hydrogen peroxide. Comparative physico-chemical investigations revealed that novel compounds outperform their urethane-analogues in terms of CL-intensity and the speed of the decay making them potentially useful as new tools for CL-based applications on the short time scale.

Full text of DOI:10.1002/hlca.201800243

HELVETICA
chimica acta
Accepted Article
Title: First synthesis of highly chemiluminescent benzo[b]furan-2-(3H)-
ones bearing a urea substructure
Authors: Reimar Krieg, Birgit Hoffmann, Dieter Weiß, and Christoph
Biskup
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First synthesis of highly chemiluminescent benzo[b]furan-2-
3H)-ones bearing a urea substructure
(
,a
b
c
b
Reimar Krieg * , Birgit Hoffmann , Dieter Weiß , and Christoph Biskup
a
Institute of Anatomy II, University Hospital Jena, Teichgraben 7, 07743 Jena, Germany (e-mail: Reimar.Krieg@med.uni-jena.de)
b
Biomolecular Photonics Group, University Hospital Jena, Nonnenplan 2-4, 07743 Jena, Germany
c
Friedrich-Schiller-University Jena, Institute of Organic and Macromolecular Chemistry, Humboldtstraße 10, 07743 Jena, Germany
Abstract:
A series of benzo[b]furan-2-(3H)-ones (coumaran-2-ones) bearing a urea substructure, namely derivatives of
3
-(aminocarbonylamino)-benzo[b]furan-2-(3H)-one, was prepared for the first time. The accessibility of these compounds via an
electrophilic α-amidoalkylation approach of phenols (Tscherniac-Einhorn reaction) in the key step as well as the chemiluminescence (CL)
properties of the desired compounds are strongly dependent on the substitution patterns at the urea moiety. Competing reaction
pathways are discussed and an improved one pot synthetically approach of also general interest is presented. In conclusion, especially
N,N-dialkylaminocarbonylamino-derivatives of benzo[b]furan-2-(3H)-ones exhibit a strong flash like blue CL upon treatment with bases
such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in presence of oxygen or hydrogen peroxide. Comparative physico-chemical
investigations revealed that novel compounds outperform their urethane-analogues in terms of CL-intensity and the speed of the decay
making them potentially useful as new tools for CL-based applications on the short time scale.
Keywords: Synthesis, benzo[b]furan-2-(3H)-ones, derivatives of urea, Tscherniac-Einhorn-Reaction, chemiluminescence
Introduction
Chemiluminescence (CL) plays an important role in biomedical sciences since many bioanalytical assays are based on this phenomenon.
CL is the emission of light from an excited electronic state as result of a chemical reaction. In this regard it differs from fluorescence or
phosphorescence where the excited electronic state results from the absorption of a photon. Accordingly, no excitation light source, no
monochromator, no beamsplitter and often not even an emission filter are necessary to record chemiluminescence. Just a sensitive light
detector such as a photomultiplier tube suffices to detect the photons emitted by the sample. Enzymes catalyzing a CL reaction can be
detected with an unsurpassed sensitivity since many photons can be generated in the catalyzed reaction. Accordingly, the detection
limits for CL-based methods are often several orders of magnitude lower than for other luminescence based techniques reaching
sensitivities in the attomole and zeptomole range. [1,2] In the absence of other catalysts the reaction is also specific for the enzyme
ensuring at the same time a high specificity for the CL-based assay.
In an analytical setup, usually a substrate and an oxidant react to form an intermediate product in an electronically excited state, which
subsequently relaxes to the ground state by emitting a photon. Often such excited states result from the formation of cyclic peroxide-
(
dioxetane-) intermediates. [3-5] Catalysts and co-factors can reduce the activation energy of this reaction. [2,6-9] In special cases the
irradiation of a fluorescent dye may trigger the light emission from a CL-reagent such as luminol (photo-induced CL). [10] Also, the
transfer of energy from an excited species to a fluorophore that in turn gets excited and relaxes to its ground state by emitting a photon
may occur (sensitized or indirect CL). [6] Today most used CL systems for clinical chemistry labels include luminol, coelenterazine and
analogues, acridinium esters, dioxetanes, systems based on peroxyoxalic acid, and derivatives thereof. [3,4,6,11]
However, there is a great demand for improved tailor-made CL-substrates for bioanalytical applications. Higher quantum yields and
improved bio-compatibility are the main goals to be achieved to facilitate the fast detection of small traces of analytes in vivo and in vitro
in a quantitative manner. [12-15] Also, sophisticated CL-reagents are often expensive due to a high expenditure of preparation.
1
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In 1979 Lofthouse et al. prepared carbamoyl and urethane derivatives of benzo[b]furan-2-(3H)-ones (coumaran-2-ones) and
demonstrated their CL for the first time (Table 1). [16] About two decades later, this promising approach was reprocessed by Matuszczak
et al. reporting on derivatives with an extended substitution pattern (Table 1) but could not clarify the reaction mechanisms in more
detail. [17-20] In a recent publication we showed the preparation of a wide variety of chemiluminescent 2-coumaranone derivatives [21-23]
(
Table 1) and revealed evidence for the underlying CL-mechanism. [3,21,24,25] Among benzo[b]furan-2-(3H)-ones, carbamates (Table 1, Y=
O-alkyl) of low molecular weight represent the most potent chemiluminescent derivatives. They exhibit CL mostly in the blue region of
the visible light after exposure to a base in the presence of oxygen in polar aprotic solvents.
Table 1. Summary of so far synthesized benzo[b]furan-2-(3H)-ones as potential CL-reagents. This compilation does not comprise
corresponding lactone precursors of type 5 (Scheme 2), which in part also exhibit CL.
R1
R2
R3
R4
Z
Y
Lofthouse
et al. 1979
Cl, Br
H
H
H
H
n-BuO
[
16]
Me, n-C
9
H
19, MeO
H
H
H
H
H
H
H
H
H
H
H
n-BuO
EtO, t-BuO
n-BuO
Ph
H
H
H
H
Me
H
t-Bu
H
H
Me
H
H
H
H
H
“dimer”
EtO, t-BuO
n-BuO
n-BuO
Me
Me, Cl
bf *)
H
bf *)
H
“dimer”
H
H
Matuszczak
Cl, Me, MeO, OH, NO2,
H
H
et al. 1996, 1997, 1998
PhCH -
2
*
*) [17-20]
MeO
H
MeO
H
MeO
H
H
Me
MeO
H
H
Me
H
H
OH
Me
H
H
Me
H
H
MeOCOO-
H
Me
Cl
H
H
H
H
Tos
Me, MeO
Me
Me
H
H
PhCH
ClPhS
PhCH
2
S, 4-
S
H
H
H
H
H
2
Me
H, Me, Cl
bf *)
bf *) (1)
Cl, Br
Br
H
H
“dimer”
Me, MeO
bf *)
bf *) (2)
H
H
H
H
H
H
Me
bf *) (1)
bf *) (2)
OMe
Schramm
et al. 2013-2016
H
H
H
H
Ph
[
21-24]
H
4-MePh, 4-BrPh,
4-NO
2
Ph, n-C13
H
27
Cl, Br
F
H
H
H
H
H
H
H
H
Ph, MeO, EtO, n-Bu
MeO, EtO, Ph, 4-MeOPh,
4
-FPh, 4-BrPh, 4-NO Ph,
2
2
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4
-AcNHPh, 2,4,6-Me
CH C(CH , HC≡C-CH
(CH O-
3
Ph,
2
3
)
3
2
O-,
N
3
)
2 6
t-Bu
H
H
H
t-Bu
H
H
Ph
bf *)
bf *)
H
MeO
O
O
*
*
) bf: benzo-fused
*) also reported in [18]:
Me
NH
O
HN
O
Me
O
O
Me
In the present study, the well investigated [21-25] strong chemiluminescent 3-ethoxycarbonylamino-5-fluoro-benzo[b]furan-2-(3H)-one a1
and the newly prepared urethane a2 served as lead for the search for improved CL-agents (Scheme 1). We report here on the synthesis
and the CL-properties of benzo[b]furan-2-(3H)-ones bearing a urea substructure as unique feature (Scheme 1, series b).
Scheme 1. Benzo[b]furan-2-(3H)-ones bearing a urethane moiety (a) and novel urea analogues b. The 5-fluoro substituted urethane a1
[
22]
and the 5,7-di-tert-butyl substituted analog a2 served as reference for comparative physico-chemical investigations.
Results and Discussion
The target compounds were prepared via an electrophilic α-amidoalkylation of phenols (Tscherniac-Einhorn reaction) as outlined in
Scheme 2. Non-commercially available ureas 1 were prepared following published protocols (Supporting Information).
3
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Scheme 2. Benzo[b]furan-2-(3H)-ones 6 - 9 were prepared starting with ureas 1 (substitution patterns a – w, Table 5), glyoxylic acid
monohydrate 2 and phenols 4 via an α-amidoalkylation approach (Tscherniac-Einhorn reaction) in the key step 3 => 5. Substitution
1
2
3
patterns a - w and corresponding substituents R , R , and R flanking the urea moiety as well as the phenol substituents X and Y are
compiled in Table 2.
Condensation of ureas 1 with glyoxylic acid monohydrate 2 to yield α-hydroxyhydantoic acids 3 (glyoxylureas, 2-carbamoylamino-2-
hydroxyacetic acids) was initially conducted in an analogous manner as described for amides and urethanes. [16-22] However, since
diverse side reactions occurred, this step turned out to be one bottleneck of this approach. In the course of our optimization efforts the
reaction was executed in different solvents (acetone, methanol, trifluoroacetic acid) under varying conditions (reflux, room temperature;
2
to 24 hours reaction time) yielding frequently instable mixtures of oily products. This is in agreement with the known instability of the
free α-hydroxyhydantoic acids 3 [26] and the fact, that α-hydroxyhydantoic acids are easily converted to the corresponding α-
alkoxyhydantoic acids in presence of alcohols as solvent. [27] Especially acyl substituted ureas gave only low yields of the desired α-
hydroxyhydantoic acids 3. For example, 3m was isolated in 26 % yield from acetyl urea 1m (60 % recovery of urea 1m; Supplementary
Information). In contrast to this, pyrrolidine N-carboxamide (1c) reacted to the allantoic acid derivate d (Scheme 3) in 70-73 % yield
instead of the desired α-hydroxyhydantoic acid 3c (Supplementary Information). The variety of reactions of ureas with glyoxylic acid is
well reflected by the literature: Attempts from Ben-Ishai et al. to obtain the N,N-dimethyl derivatives of α-methoxyhydantoic acid by
reacting 1,1-dimethyl urea with glyoxylic acid in methanol failed, and the authors obtained a mixture of products. Condensing glyoxylic
acid with two equivalents of methyl urea in aqueous solution or with two equivalents of 1,1-dimethyl urea in acetone gave the allantoic
acid derivatives e and f (Scheme 3) in good yields. [27] Also, favored reactions of ureas with glyoxylic acid are ring closing reactions
leading to cyclic products such as derivatives of hydroxyhydantoin g, allantoin h or uric acid i in strong dependence on the urea
substitution patterns and reaction conditions. [27-29] In case of mono-substituted ureas (substitution patterns e – l herein) condensation
to cyclic derivatives of type g and h is possible. The same applies for 1,3-disubstituted ureas. Thus, reaction of 1,3-dimethylurea and
glyoxal in water in absence of strong acids exclusively yielded 4,5-dihydroxy-1,3-dimethyl-2-imidazolidinone (1,3-dimethyl-4,5-
dihydroxyethyleneurea, a derivative of g). [30] By contrast, following an one-pot approach using trifluoroacetic acid as solvent the desired
5
-fluoro-3-[methylaminocarbonyl-(N-methyl)-amino]-benzo[b]furan-2-(3H)-one 7u is obtained in 61 % yield from 1,3-dimethylurea 1u,
glyoxylic acid monohydrate 2 and 4-fluorophenol.
4
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Scheme 3. Condensation of glyoxylic acid with urea and substituted ureas may lead to a wide range of cyclic and acyclic products as
exemplarily shown by the allantoic acid derivatives d (derived from 1-pyrrolidinecarboxamide), e (derived from methyl urea), f (derived
from 1,1-dimethyl urea), and the cyclic urea derivatives g - i. The occurrence of such side reactions can become dominant and may
explain the diverse experimental results. Compounds of type e - i have been already described. [27-30] During our optimization efforts, the
allanthoic acid d was obtained as main product in 70-73 % yield attempting to prepare the intermediate 2-(1-azolidinylcarbonylamino)-
2
-hydroxy acetic acid 3c (Supplementary Information).
In order to limit the multitude of competing side reactions, it seems promising to replace the glyoxylic acid 2 by its esters, e.g. to
counteract formation of cyclic products. However, this approach would involve the need of subsequent hydrolysis of formed N-
carbamoyl glycinates 5 to make the desired lactones 6 accessible. Although highly critical in view of the pronounced sensitivity of 6
towards bases, this route was already described by Matuszczak for N-acetyl-α-arylglycinate analogues using sodium carbonate/water for
ester cleavage with reasonable success. [19] To address this problem and to take advantage of potentially occurring equilibria between
intermediates (Schemes 3 and 4), we considered a one-pot approach starting with apposite ureas 1, phenols 4, and glyoxylic acid
monohydrate 2.
Scheme 4. Condensation of ureas with glyoxylic acid hydrate 2 may result in a cascade of equilibria as shown here exemplarily for urea.
[
27]
Solely glyoxyl urea j is regarded to be capable to α-amidoalkylate phenols in an electrophilic manner after acid catalyzed elimination
of water (cf. Scheme 2). However, it is also possible that ongoing condensation of allantoin h (and its derivatives herein) with 2 furnish
intermediates also capable to α-amidoalkylate phenols.
According to published protocols, suitable solvents for the Tscherniac-Einhorn reaction are: Acetic acid containing 10 % sulfuric acid as
catalyst, [16,21,22] or, alternatively, formic acid with catalytic amounts of sulfuric acid. [17-20] As result of our optimization efforts,
trifluoroacetic acid emerged as optimal solvent and catalyst, even in context of the preparation of urethane analogues with nearly
duplicated yields (e.g. a1, Supporting Information). Thus, sulfuric acid can be omitted and the work up can be easily performed by
evaporating the solvent in vacuum at 30 °C. This is especially important for the accessibility of sensitive and water soluble reaction
products.
In general, the acid catalyzed electrophilic α-amidoalkylation of phenols (Tscherniac-Einhorn reaction (step 3 => 5) requires lengthy
reaction times of frequently several days at room temperature. [31,32] In our context, reaction times beyond 5 days frequently led to
complex oily product mixtures while after one day of reaction time starting ureas could be partially recovered. Reaction times of about 3
days at room temperature have been proven to be optimal.
5
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Attempts to promote lactonization of the intermediate α-(6-hydrox-phenyl)-glycines 5 by treatment of isolated crude products with
acetic anhydride [16,21,22] (e.g. by heating 30 min at 70 °C) or by addition of acetic anhydride to the reaction mixture caused frequently
1
)
pronounced decomposition processes. This is likely due to the formation of reactive urea-N-acetylation products and subsequent side
reactions. An example referring to that is outlined in Scheme 5. Fortunately, in presence of a 3-fluoro-substituent, α-phenyl glycines 5
are prone to undergo ring closure to desired lactones (series 6 and 7; Scheme 2, Table 2). Interestingly, only 3-fluoro-α-phenyl glycines
can undergo spontaneous lactonization. 2) In case of tert-butyl-substituted phenols (series 8 and 9) lactonization must be enforced by
repeated azeotropic water separation in presence of catalytic amounts of toluene sulfonic acid in the final step.
1
2
Scheme 5. Example for the diverging chemistry of acyl ureas: Aroylation of 1,1-dialkyl ureas furnishes 3,3-diaroyl ureas j (R = R = aroyl,
3
4
R = R = alkyl) in high yields which may rearrange thermically to amids k and highly reactive aroyl isocyanates l. By contrast, mono-
substituted ureas react in a non-uniform manner under these conditions. The same applies for bis-acetylation attempts of asymmetric
ureas whereas urea, mono-substituted and symmetric 1,3-di-substituted ureas yield the mono acetylated products. [33]
In the end, following a one-pot α-amidoalkylation approach of phenols and using the solvent trifluoro acetic acisd,1,1-dialkyl, bulky 1-
alkyl, and 1,3-disubstituted ureas yield corresponding benzo[b]furan-2-(3H)-ones in moderate to high yields. The final outcome of
synthesis is compiled in Table 2. Under the described conditions, the followings attempts failed:
1
. Preparation of thio-urea analogues. Frequently oily mixtures were obtained and virtually no CL of the crude reaction products could
be observed after addition of DBU and hydrogen peroxide as CL-trigger (details are not shown).
2
. The same applies for aryl substituted derivatives (6f and 6g). This is likely attributed to competing electrophilic α-amidoalkylations at
the comparatively electron rich N-aryl moiety.
3
. Derivatives of acetyl urea 8m and 9m: In case of ureas of low reactivity, the competing α-hydroxyalkylation of phenols by glyoxylic
acid becomes dominant leading to 2-aryl-2-hydroxy acetic acid derivatives 10 and corresponding lactones (Scheme 2). For example, the
N-acetyl-derivative 9m (Table 2) could not be obtained. 3) Instead, the α-hydroxyalkylation product, namely 5,7-di-tert-butyl-3-hydroxy-
4
)
benzo[b]furan-2-(3H)-one 11, was isolated in 85 % yield while the urea 1m was recovered in 87 % yield.
Footnotes
1
)
The addition of acetic anhydride causes a pronounced exothermic reaction.
2
)
Interestingly, a higher degree of fluoro-substitution does not promote lactonization. For example, 3,4,5-trifluorophenol, ethyl
urethane and glyoxylic acid monohydrate react readily to N-ethoxycarbonyl-α-(6-hydrox-2,3,4-trifluoro-phenyl)glycine (75 % isolated
yield) instead of yielding the desired lactone (3-ethoxycarbonylamino-4,5,6-trifluoro-benzo[b]furan-2-(3H)-one, details see Supporting
Information) while 3,5-difluorophenol does not react in an analogues manner and gave mixtures of products due to the unsubstituted
phenolic 4-aryl position.
3
)
The intermediate 1-acetyl-3-glyoxyl urea 3m was isolated as described under “Isolated intermediates” in the Supporting
Information in moderate yield.
Details see Supporting Information. The data are in agreement with [39].
4
)
6
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Table 2. Compilation of 3-N-ureyl derivatives synthesized in this work.
Chemi-
luminescence
X
Y
R1
R2
R3
Yield (%)
Remarks
(
visually rated)
6
a
F
F
F
F
F
F
H
H
H
H
H
H
CH
3
CH
3
H
H
H
H
H
H
28
14
29
76
44
strong
strong
strong
no CL
moderate
no CL
-
-
-
-
-
6
b
C
2
H
5
C
2
H
5
6
c
-(CH
2
)
5
-
6d
CH
H
H
3
Ph
6
6
e
f
c-C
6
H
11
+
Ph
(not isolated)
mixture MS/EI: M 286
17)
mixture MS/EI: M 300
(
+
6
6
g
h
F
F
H
H
H
H
tolyl
CH
H
H
(not isolated)
very weak
fairly good
(
100)
-
3
3-13
and isomer 7h:
10-28
6
i
F
H
H
i-C
3
H
7
H
47
no CL
-
(
21 after second
recrystallization)
6
j
F
F
F
F
F
F
H
H
H
H
H
H
H
H
H
H
H
H
n-C
n-C
Ph-CH
CH -CO-
Ph-CO-
4
H
9
H
H
H
H
H
30
9
21
23
63
no CL
no CL
moderate
moderate
virtually no CL
weak
-
-
-
-
-
-
6
k
5
H
11
6
l
2
-
6
m
3
6
6
n
o
44 (crude product)
H
H
6
p
F
H
3
weak
-
-
7
h
q
F
F
H
H
H
H
H
CH
3
10-28 and isomer
h: 3-13
(not isolated)
virtually no CL
weak
6
7
c-C
6
H
11
11
mixture, 45 % of
N(cyclohexyl-
carbamoyl)succinamic
acid isolated
oily mixture,12 %
recovery of urea 1r
7
7
7
7
r
F
H
c-C
6
H
(not isolated)
virtually no CL
s
t
u
v
F
F
F
F
H
H
H
H
H
H
H
Ph
Ph
CH
CH
CH
c-C
3
85
32
61
no CL
no CL
no CL
weak
-
-
-
6
H
11
3
CH
CH
3
7
CH
3
3
3
(not isolated)
mixture containing
predominantly starting
4
-fluorophenol
7
w
F
H
-(CH
2
)
4
-
CH
3
(not isolated)
weak
mixture containing
7
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predominantly starting
4-fluorophenol
8
8
a
c
t-Bu
t-Bu
t-Bu
H
H
H
CH
H
3
CH
CH
3
3
H
H
H
20
22
strong
strong
moderate
-
-
-(CH ) -
2
4
8
h
(not isolated)
complete conversion of
h, but mixture of at
1
least 5 components
containing 8h (MS (ESI):
+
m/z (%)341.2 [M+Na ]
(26); M(100): 531.3)
8
m
t-Bu
H
H
CH
3
-CO-
H
(not isolated)
no CL
mixture of several
products, 50 % recovery
of 1m
9
9
a
c
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
CH
H
3
CH
CH
3
3
H
H
H
24
22
strong
strong
weak
-
-(CH
2
)
4
-
50 % recovery of 1c
complete conversion of
9
h
(not isolated)
1
h, but mixture of at
least 6 components
9
m
t-Bu
t-Bu
H
CH
3
-CO-
H
0
no CL
87 % recovery of 1m, 85
%
of 5,7-di-tert-butyl-3 -
hydroxybenzo-
[
b]furan-2-(3H)-one
*)
isolated
*) TLC-controls revealed that after 5 d reaction at room temperature the starting 2,4-di-tert-butylphenol was completely converted, but
+
about 50 % of the starting urea 1c was recovered: mp 139-142 °C (colorless plates), MS (EI): m/z (%) = 114 [M ] (100); NMR data in
agreement with 1c (data not shown).
Physico-Chemical Investigations
The UV-Vis absorption spectra of reference compounds a1, a2, and of the selected urea-analogues 6a-c, 8a, 8c, 9a, and 9c were
measured in acetonitrile. All maxima are located at 278  2 nm (Table 3; Figure S1, Supporting Information). The emission maxima of the
urea derivatives 6a-c, 8a, 8c, 9a, and 9c are found around 430 nm and are shifted by approximately 10 nm towards shorter wavelengths
as compared to the urethanes a1 and a2 (Table 3). However, there are pronounced differences in the kinetics of the CL-decay. All di-tert-
butyl substituted derivatives (substitution pattern c) feature flash-like CL emission with half times in the 0.5 to 6 s range (Table 3, Figure
1
). In general, this applies for all urea derivatives investigated herein making these compounds potentially interesting as new tools for
bioanalytical CL-based applications.
Spectral measurements in phosphate buffered acetonitrile solution revealed slightly blue shifted absorption maxima and slightly red
shifted emission maxima, each about 10 nm shifted compared to the measurements in acetonitrile (Table 4; Figures S3, Supporting
Information). CL-triggering with horseradish peroxidase (HRP)/hydrogen peroxide gave only in case of mono-tert-butyl substituted
derivatives 8a and 8c a CL-response with CL-half times in the 0.9 to 5.7 s range (Table 4, Figure 2). Interestingly, all di-tert-butyl
substituted derivatives, specifically a2, 9a, and 9c, did not exhibit CL under these conditions while the mono-tert-butyl derivative 8c
gave the best relative performance (Figure 2).
8
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Figure 1. Kinetics of light emission in acetonitrile (10-5 M) after addition of DBU (black line) and in the absence of DBU (control, grey line,
close to the time-axis) at 25 °C. The y-axis denotes the recorded CL-intensity in 10 million counts. Representative intensity-time curves of CL
from independent experiments are shown for the indicated compounds in the time window from 2.0 to 3.0 minutes. The complete CL-
-3
decays are shown in the Supporting Information (Figure S2). The DBU solution (67x10 M in acetonitrile) was added after at least 2 min
baseline recording.
Compound
Absorption
maximum
Extinction
coefficient
Fluorescence
maximum
[nm]
CL-Rise time,
CL-Half time,
CL Quantum yield,
trise  SEM
t1/2  SEM

-1
-1
[
nm]
[M cm ]
2710
1590
4070
2670
4210
2490
1660
2910
1790
[s]
[s]
[%]
a1
a2
280
277
279
280
280
276
277
276
277
436
441
429
428
428
426
427
434
437
2.8  0.6
1.3  0.1
2.2  0.1
2.0  0.4
1.8  0.1
3.2  0.4
3.0  0.5
1.4  0.2
1.7  0.5
64.0  0.4
1.5  0.1
7.0  0.5
4.7  0.4
5.7  0.6
1.4  0.4
0.5  0.2
0.9  0.1
0.6  0.2
4.2
1.4
5.9
5.0
4.5
2.8
1.8
1.7
1.4
6a
6
b
6
c
8a
8
c
9a
9
c
Table 3. Spectroscopic and CL-properties of the indicated compounds dissolved in acetonitrile. CL was induced by DBU. Fluorescence and CL
measurements were performed at 25 °C under stirring. The molar extinction coefficients were determined at the absorption maximum.
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Figure 2. Kinetics of light emission of selected benzofuranones in phosphate buffered acetonitrile solution (2 x 10-4 M) in presence of
HRP/H (black line) and in the absence of HRP/H (control, grey line, close to the time-axis) in the time window from 2.0 to 3.0 minutes.
2
O
2
2
O
2
The entire CL-decays are shown in the Supporting Information (Figure S4). Measurements were performed at 25 °C under stirring. The y-axis
denotes the CL-intensity in 10 000 counts.
Compound
Absorption
maximum
Extinction
coefficient
Fluorescence
maximum
[nm]
CL-Rise time,
CL-Half time,
CL Quantum yield,
trise  SEM
t1/2  SEM

-1
-1
-2
[
nm]
[M cm ]
[s]
[s]
[10 %]
a1
280
266
276
269
274
274
1480
452
443
444
443
437
450
11.8  1.8
3.5  0.2
12.8  0.5
11.8  0.9
6.0  0.4
4.8  0.4
31.0  3.1
114.4  0.9
195.9  1.6
134.7  0.9
5.7  1.9
0.1
0.3
0.2
0.2
0.3
0.3
6a
2590
6
b
1460
6
c
2680
8a
3850
8
c
2490
0.9  0.2
Table 4. Spectroscopic and CL-properties in phosphate buffered acetonitrile solution. CL was induced by HRP/H
2
O
2
. Fluorescence and CL
measurements were performed at 25 °C under stirring. No evaluable CL-signal could be detected for the di-tert-butyl substituted derivatives
a2, 9a, and 9c under the described conditions. The molar extinction coefficients were determined at the absorption maximum.
Mechanistical Consideration
The underlying CL-mechanism of 3-ethoxycarbonylamino-5-fluoro-benzo[b]furan-2-(3H)-one (reference compound a1; Schemes 1 and
6
; a: Z = OEt, X = F, Z = H) was recently investigated in great detail using a combination of experimental and computational methods. [3,25]
Accordingly, the ureides b should follow the same general mechanism (Scheme 6): (i) Initial deprotonation, formation of an electron rich
enolate, which forms after single electron transfer (ii) a 1,2-dioxetanone key intermediate (iii), which decomposes (iv) to the
salicylamide-like emitter in its first excited state relaxing to its ground state (v)under emission of light.
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Scheme 6. Proposed general mechanisms of benzofuranone CL induced by bases (step i): The key step is the formation of the high-
energy 1,2-dioxetanone key-intermediate c which decomposes to an excited transition state TS*. [3,21,24,25] These key intermediates can
be generated via nucleophilic attack of triplet oxygen at the lactone carbon of the deprotonated benzofuranone (subsequently to step
ii) followed by a single electron transfer as recently proposed. [3,25] Alternatively, a mechanism involving an electron acceptor (path viii) is
reasonable first postulated by Lofthouse et al.. [16] Path vi postulates a “dark side reaction” by way of nucleophilic attack of oxygen at the
exocyclic carbonyl function. [25]
A noteworthy part of the reaction mechanism is the nucleophilic attack of oxygen on the amid-carbon (vi) as a possible side reaction.
3
[25]
This is discussed for a1 (R = H, Z = OEt) in detail. This “dark reaction” could lead in this case to final decomposition products identical
to products generated via reaction paths ii and iii including the key intermediate c. [25]
Interestingly, the 5-fluoro derivatives 6a and 6c exhibit high CL-half times and high CL quantum yields in acetonitrile when the reaction
is triggered by DBU. Likewise high CL-half times were found in phosphate buffered acetonitrile solution. 5-tert-butyl and 5,7-di-tert-butyl
derivatives 8a, 8c, 9a, and 9c exhibited both decreased CL-half time and CL quantum yield in acetonitrile. HRP induced CL emission in
series 8 featured CL half-times which were shorter by a factor up to more than 100 compared to analogues from series 6. Thus, typically
compounds 6a and 8a, differing only in respect of the substitution in X, behave considerably different in terms of both CL-half time and
CL-quantum yield. This effect is more pronounced for the reaction in phosphate buffered acetonitrile.
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No evaluable CL-signal could be detected for the di-tert-butyl substituted derivatives a2, 9a, and 9c in HRP induced reactions in
phosphate buffered acetonitrile solution. The same applies for series 7 (R3 ≠ H) and also for compounds with the substitution pattern
R2 = R3 = H (in general: derivatives of mono- and 1,3-disubstituted ureas) including the system acetonitrile/DBU.
In the enzymatically triggerd CL reactions, horseradish peroxidase (HRP) was used to catalyze the oxidation of the substrate by hydrogen
peroxide. The results obtained in these CL experiments might be explained by a radical mechanism proceeding through the known HRP
enzymatic cycle where the reaction of HRP and hydrogen peroxide forms an oxidized HRP-I complex which further reacts with the
substrate to the HRP-II complex producing two molecules of substrate radicals per turn. Usually the conversion of the half-reduced HRP-
II complex to the native enzyme is the rate-determining step as kinetic studies revealed. By varying the reaction conditions (i.e.
decreasing the amount of H
step. [11,34] The often enhanced CL-rise and CL-half times found in our HRP/H
2
O
2
and increasing the amount of substrate), the HRP-I complex formation becomes the rate-controlling
system compared to the results obtained in DBU
2
O
2
triggered CL reactions might be attributed to these rate-determining steps of complex formation and the time-lag phenomenon
described for enhanced HRP enzyme systems where the initiation of the CL reaction and the beginning of the emission of light are
characterized by an interval of time due to free radical quenching mechanisms.[35] Further experiments with varying substrate and
enzyme concentrations could help to get a deeper insight into the reaction mechanism and to elucidate whether reaction rate might be
influenced by benzofuranone and enzyme concentration. On the other hand, such delayed reactions (compounds 6a - 6c: up to
minutes) could be of great benefit regarding specialized biological assays where timing conditions are difficult to control. The low
quantum yields for the HRP/H
2
2
O triggered reactions found for the benzofuranones under study could be further explained by
quenching processes in aqueous solution and high triplet to singlet excited state ratio which might further decrease the amount of
produced light.[11,36]
Recently published calculations using time-dependent-density functional theory and multi reference complete-active-space second-
order perturbation theory showed, that charge transfer-initiated luminescence and chemically induced electron-exchange
luminescence (CIEEL) seem not to adequately describe CL reaction steps. [37] In case of the model compound dioxetanone,
chemiexitation should be rather the result of several processes involving electron/charge transfer, charge annihilation, and efficient
access to the potential energy surface. [37] Improved mechanisms taking into account these findings could help to better describe CL
properties of more complex structures, provided that substitution patterns are taken into account.
Conclusions
In this study, we present the synthesis and characterization of a novel class of chemiluminescent benzo[b]furan-2-(3H)-ones bearing a
urea subunit. Previous reports introduced already a convenient one-pot approach for the synthesis of benzo[b]furan-2-(3H)-ones, which
however gave only good yields for the 5-fluoro-derivatives. In this study, the one-pot approach based on trifluoroacetic acid as solvent
and catalyst was improved to get a diversity of side reactions under control. This approach can be applied in general for the synthesis of
benzo[b]furan-2-(3H)-ones and is especially useful for the preparation of water soluble benzofuranones. The synthetic accessibility
crucially depends on the substitution patterns at the ureic moiety: (a) Reaction of mono-substituted ureas often leads to complex
reaction mixtures. (b) By contrast, 1,1-disubstitution and 1,3-disubstitution of the urea moiety directs the reaction towards the desired
N,N- and N,N’-dialkylaminocarbonylamino-benzo[b]furan-2-(3H)-ones in good to high yields (c) 1,1,3-trisubstituted urea derivatives are
not accessible under the described reaction conditions.
Physico-chemical investigations prove that the novel compounds exhibit a strong flash like CL as compared to a slowly decaying CL
which can be observed in many carbamoyl derivatives. Interestingly, changing the 3-carbamoyl group for the 3-ureyl group, as well as
tiny variations in the substitution pattern, have a strong impact on the kinetics of the CL decay. The kinetics of the light emission is
mainly tunable by the urea moiety and, in a less pronounced manner, by the benzo-substituents at positions 5 and 3: (a) N,N-
disubstitution by short alkyl- or cyclo-alkyl moieties promotes an intense blue light emission upon treatment with strong bases in
presence of oxygen, (b) 1,3-disubstituted analogues virtually do not exhibit any CL. Due to their fast CL-decay in the 0.5 to 6 s range
some of the novel compounds might be suited as tools for the monitoring of dynamic reactions.
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Experimental Section
General Remarks and Materials, the Synthesis of Urea Derivatives 1, Isolated Intermediates, and NMR Spectra are compiled in the
Supporting Information.
Chemical Synthesis
Reference urethanes a1 and a2
3
-Ethoxycarbonylamino-5-fluoro-benzo[b]furan-2-(3H)-one a1 is already described in [22] using acetic acid/sulfuric acid 9:1(v/v as
solvent (6 days reaction time: 24 % yield, mp 156 °C). In an improved manner following the general procedure described herein and
using trifluoroacetic acid as solvent a1 was obtained in 42 % yield after 2 days reaction time: Colorless needles from MeOH, mp 170-
1
70.5 °C, R 0.48 (AcOEt/hexane = 1:2, v/v).
f
3
-Ethoxycarbonylamino-5,7-di-t-butyl-benzo[b]furan-2-(3H)-one a2
(M = 333.43). This compound was obtained following the general procedure described herein. Lacton formation was
C
19
H
27NO
4
promoted by addition of toluene sulfonic acid (30 mg) or alternatively Ac
2
O (7 ml) in the last work up step. After recrystallization from
1
hexane: 36 % yield, colorless powder, R
CH
f
0.38 (AcOEt/hexane = 1:4, v/v), mp 134-135 °C. H-NMR (400 MHz, CDCl
3
): δ (ppm) 1.29 (overl., 3H,
4
3
), 1.33(s, 9H, t-Bu), 1.42 (s, 9H, t-Bu), 4.20 (s, broad, 2H, CH
2
), 5.43 (s, broad, overl., 2H, NH and CH), 7.23 (d, J 1.7 Hz, 1H, arom. H), 7.34
d, J 2.0 Hz, 1H, arom. H) 13C-NMR (101 MHz, CDCl
): δ (ppm) 14.43, 29.59, 31.58, 34.38, 34.88, 51.90, 62.00, 118.96, 124.36, 133.67, 147.65,
4
(
3
+
1
49.42, 156.27, 173.95. MS (EI): m/z (%) 333 [M ] (100), 305 (20), 290 (69), 277 (98), 260 (60), 244 (24), 232 (37), 204 (24), 202 (22). HR-MS -
–
1
calc. for C19
H
27NO : 333.1935, obs. 333.1936. IR (ATR), ν (cm ) 874 (m-s), 884 (m), 1023 (s), 1076 (vs), 1269 (m-s), 1507 (s), 1684 (s), 1815 (s),
4
2958 (m-w, br), 3293 (w, br). EA, calc.: C 68.44, H 8.16, N 4.20; found: C 68.56, H 8.32, N 4.17.
Aminocarbonylamino-benzo[b]furan-2-(3H)-ones 6 - 9
General procedure:
In a round-bottom 250 ml flask fitted with magnetic stirring and a drying tube, 10 mmol of the appropriate urea 1 and 10 mmol (920.5
mg) of glyoxylic acid monohydrate 2 were dissolved in 15 ml of trifluoroacetic acid. After 30 minutes stirring at room temperature, to the
clear solution 10 mmol of the requisite phenol 4 was added. The stirring was kept at room temperature and the reaction progress was
followed by TLC until completion, which typically took 2 days. In case of deviant or incomplete reaction processes the experiment was
terminated after 4 days. After this period, in almost all cases a clear and colorless reaction mixture was obtained. For working up, the
trifluoroacetic acid was slowly removed under reduced pressure at about 50 °C over a period of 30 minutes.
In case of t-butyl mono- and di-t-butyl substituted derivatives (starting phenols 4 with X = t-Bu and Y = H or t-Bu), the formation of
lactones 8 and 9 was promoted by addition of 30 mg of toluene sulfonic acid as catalyst and repeated slow removal of each 20 ml of
toluene at 70 °C over a period of 50 minutes using the a rotating evaporator.
Water soluble by-products were removed by repeated dissolving of the crude product in about 10 ml of methanol followed by addition
of 50 ml of distilled water under vigorous stirring and phase separation. The obtained crude product was purified by crystallization or
chromatography on silica gel (hexane/AcOEt or AcOEt/MeOH gradient) as described below for the individual compounds.
5
-Fluoro-3-(N,N-dimethylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6a)
1
C
11
H
11FN
2
O
3
(M = 238.22), recrystallized from MeOH, yield 28 %; R
f
0.22, mp 212-215 °C (dec., colorless plates and rhombs), H-NMR (250
), 5.15 (d, J 6.8 Hz, 1H, 3-CH), 7.07-7.15 (m, overl., 3H, arom. H), 7.17 (d, J 6.8 Hz, 1H, NH). 13C-
3
3
MHz, DMSO-D
6
): δ (ppm) 2.77 (s, 6H, 2 x CH
3
NMR (63 MHz, DMSO-D
6
): δ (ppm) 36.16, 52.80, 111.24, 111.67, 111.80, 115.28, 1115.67, 130.04, 130.17, 149.89, 149.92, 157.31, 161.08,
+
+
1
74.97.MS (EI): m/z (%) 238 [M ] (100), 193 (69), 137 (21), 72 (96), 46 (31). HR-MS - calc. for C11
H
11FN
2
O
3
Na [M+Na ]: 261.06514, obs.
–
1
2
61.06451. IR (ATR), ν (cm ) 1070 (s), 1085 (s), 1477 (vs), 1527 (m-s, sh), 1635 (m-s, sh), 1811 (s), 2363 (w), 2923 (w), 3317 (w-m, sh).
5
-Fluoro-3-(N,N-diethylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6b)
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1
C
13
H
15FN
2
O
3
(M = 266.27), recrystallized from MeOH, yield 14 %; R
f
0.60, mp 175-178 °C (dec., colorless micro-crystals), H-NMR (400 MHz,
3
3
3
4
DMSO-D
6
): δ (ppm) 1.02 (t, J 7.0 Hz, 6H, 2 x CH
3
), 3.10-3.25 (m, 4H, 2 x CH -N), 5.18 (d, J 6.7 Hz, 1H, 3-CH), 7.09 (dd, J 7.4 Hz J 2.0 Hz, 1H,
2
arom. H), 7.12-7.24 (m, 2H, 2 x arom. H), 7.60 (d, J 6.9 Hz, 1H, NH). 13C-NMR (100 MHz, DMSO-D
3
): δ (ppm) 14.12, 52.77, 111.22, 111.47,
6
+
1
11.75, 111.83, 115.37, 130.27, 130.35, 149.89, 149.91, 156.17, 158.02, 160.40, 175.00. MS (EI): m/z (%) 266 [M ] (100), 193 (32), 100 (68), 74
–
1
(29), 72 (47), 58 (24), 44 (19), 29 (28). HR-MS - calc. for C13
H
15FN
2
O Na: 289.09644, obs.: 289.09660. IR (ATR), ν (cm ) 1059 (s), 1067 (s), 1450
3
(m), 1480 (s), 1517 (s, sh), 1614 (s, sh), 1811 (s, sh), 2361 (w,), 2981 (w), 3327 (w-m).
5
-Fluoro-3-(1-azolidinylcarbonylamino)-benzo[b]furan-2-(3H)-one (6c)
(
N-(5-Fluoro-2-oxo-2,3-dihydrobenzofuran-3-yl)-1-pyrrolidinecarboxamide)
1
C
13
H
13FN
2
O
3
(M = 264.26), recrystallized from MeOH, yield 29 %; R
f
0.24, mp 209-212 °C (dec., colorless columns), H-NMR (250 MHz,
3
3
DMSO-D
6
): δ (ppm) 1.79 (s, broad, 4 H, 2 x CH
2
), 3.19 (t, overl., J 6.3 Hz4H, 2 x CH
2
-N), 5.17 (d, J 7.8 Hz, 1H, 3-CH), 7.08-7.16 (m, overl., 3H,
arom. H), 8.40 (d, J 7.8 Hz, 1H, NH). 13C-NMR (63 MHz, DMSO-D
3
): δ (ppm) 25.40, 45.80, 52.58, 111.34, 111.69, 111.74, 111.82, 115.31,
6
+
1
15.70, 130.04, 130.18, 149.86, 149.89, 155.58, 157.27, 161.08, 174.95. MS (EI): m/z (%) 264 [M ] (100), 197 (35), 98 (85), 72 (38), 55 (35). MS
+
–1
(
ESI): m/z (%) 287.0 [M+Na ] (100). HR-MS - calc. for C13
H
13FN
2
O Na: 287.08079, obs.: 287.08133. IR (ATR), ν (cm ) 1474 (s), 1507 (s), 1631
3
(s, sh), 1807 (s), 2876 (w), 2928 (w), 2976 (w), 3368 (w, br).
5
-Fluoro-3-[(N-methyl-N-phenyl)aminocarbonylamino]-benzo[b]furan-2-(3H)-one (6d)
(M = 300.29), recrystallized from EtOH, yield 76 %; R 0.60, mp 270-274 °C (dec., colorless plates). H-NMR (250 MHz, DMSO-
): δ (ppm) 2.70 (s, 3H, CH ), 5.23 (s, 1H, 3-CH), 6.84 (dd, J 8.8 and 4.6 Hz, 1H, aryl-H), 7.09 (dt, J 8.6 and 3.1 Hz, 1H, aryl-H), 7.25 (dd, J 8.9
and 3.0 Hz, 1H, aryl-H), 7.45-7.55 (m, overl., 5H, aryl-H), 10.06 (s, 1H, NH). 13C-NMR (63 MHz, DMSO-D
): δ (ppm) 27.98, 62.68, 116.98,
17.10, 117.35, 118.42, 118.79, 121.26, 127.05, 128.25, 129.21, 133.11, 152.92, 152.95, 153.72, 155.69, 157.46, 171.13. MS (EI): m/z (%) 300
1
C
16
H
13FN
2
O
3
f
D
6
3
6
1
+
+
–1
[
M ] (100), 152 (47). HR-MS - calc. for C16
H
13FN
2
O
3
Na [M+Na ]: 323.08079, obs. 323.08110.IR (ATR), ν (cm ) 1142 (s), 1190 (s), 1282 (m),
1383 (s), 1440 (vs), 1684 and 1689 (vs), 1759 (m-w), 2361 (w), 3160 (m, br).
5
-Fluoro-3-(cyclohexylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6e)
(M = 292.31), recrystallized from EtOH, yield 44 %; R 0.48, mp 230-232 °C (dec. above 224 °C, small colorless plates), H-NMR
): δ (ppm) 1.02-1.28 (m, overl., 3H, 3 x cyclohexyl-H), 1.16 (d, overl., J 10.8 Hz, 3H, 3 x cyclohexyl-H), 1.76 (d, J 12.5 Hz,
H, 2 x cyclohexyl-H), 1.87-2.16 (m, 2H, 2 x cyclohexyl-H), 3.64-3.89 (m, 1H, 1-cyclohexyl-H), 5.02 (s, 1H, 3-CH), 6.77 (dd, J 9.6 and 4.8 Hz,
H, arom. H), 7.00 (ddq, J 9.9, 6.8 and 3.2 Hz, 2H, arom. H), 8.21 (s, 1H, NH), 9.77 (s, 1H, NH). 13C-NMR (63 MHz, DMSO-D
): δ (ppm) 25.38,
5.83, 25.87, 29.22, 29.34, 50.57, 57.10, 116.08, 116.43, 116.75, 116.88, 117.05, 117.42, 123.99, 124.10, 152.67, 152.70, 153.57, 157.30,
1
C
15
H
17FN
2
O
3
f
(
250 MHz, DMSO-D
6
2
1
2
1
2
6
+
+
57.43, 173.23. MS (EI): m/z (%) 292 [M ] (100), 210 (85), 166 (62), 139 (73), 124 (67). HR-MS - calc. for C15
H
18FN
2
O
3
[M+H ]: 293.13014, obs.
–
1
93.13030. IR (ATR), ν (cm ) 819 (s), 1190 (m-s), 1271 (m), 1420 (s), 1507 (m, sh), 1516 (m), 1663 (vs), 2939 (m), 3297 (m, br).
5
-Fluoro-3-(phenylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6f)
(M = 286.26). Under the described conditions, a complex mixture of several compounds containing 6f was obtained. MS (EI):
C
15
H
11FN
2
O
3
+
+
m/z (%) 286 [M ] (17), 176 [compares to M of tolyl urea] (85), 119 (100).
5
-Fluoro-3-(tolylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6g)
(M = 300.29): Mixture of several compounds. MS (EI): m/z (%) 300 [M ] (100); there are higher mol-peaks that may be
+
C
16
H
13FN
2
O
3
+
attributed to cyclic urea derivatives - for example: MS (EI): m/z (%) 338 [M for bis-tolyl allantoin] (12); cf. h, Scheme 4).
5
-Fluoro-3-(methylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6h)
FN (M = 224.19). After stirring at room temperature for 4 days, the clear and colorless reaction mixture was concentrated at 50 °C
C
10
H
9
2
O
3
in vacuum and kept under these conditions for one hour. The obtained oily crude product was extracted twice with boiling toluene,
then twice with boiling AcOEt. The combined extracts were cooled to room temperature, an excess hexane was added, and the
precipitate filtered off after standing over night at 4 °C. Alternatively, working up was performed by way of adding the crude product
with small amounts of MeOH until precipitation started following by step wise addition of an excess of ice water. The obtained
amorphous white solid (about 1.4-2.0 g from 10 mmol preparations) consists of two isomers (TLC AcOEt/hexane = 2:1, v/v: R 0.45 and
f
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0
.58; dichloromethane/MeOH = 9:1, v/v: R
chromatography. However, repeated slow recrystallization from AcOEt or MeOH/water (1:1, v/v) afforded the polar product (R
AcOEt/hexane = 2:1, v/v, uniform) in the form of an amorphous white powder in 10 to 28 % yield, which was assigned via NMR-
f
0.68 and 0.72; ratio each about 3:2), which could not separated further by column
f
0.45,
investigation (NOESY, TOCSY) to the isomeric 5-Fluoro-3-[N-(aminocarbonyl-)N-methylamino)-benzo[b]furan-2-(3H)-one 7h (data see
there).
The less polar isomer 6h could be enriched by step-wise removal of 7h as described above. Concentration of the mother liquors and
repeated recrystallization from AcOEt/THF (3:1, v/v), MeOH/water (1:4, v/v), and finally AcOEt afforded the enriched compound 6h: R
f
1
0
.58 (AcOEt/hexane = 2 : 1, v/v); TLC: virtually uniform, H-NMR (3-CH and CH
3
-signals): contains 6 % of the polar isomer 7h (see below).
1
Compound 6h: Yield: 3-13 %, amorphous white powder, mp 208-212 °C (dec., above 200 °C sublimation to plates). H-NMR (250 MHz,
DMSO-D ): δ (ppm) 2.87 (s, 3 H, CH ), 5.17 (s, 1H, 3-CH), 6.68-6.85 (m, 1H, arom. H), 6.95-7.10 (m, overl., 2H, 2 x arom. H), 8.32 (s, 1H, NH),
.82 (s, 1H, NH’). 13C-NMR (63 MHz, DMSO-D
6
3
9
1
1
6
): δ (ppm) 25.56, 58.29, 117.08, 117.43, 117.68, 117.72, 117.85, 117.94, 118.05, 124.65, 124.77,
+
53.49, 153.90, 154.58, 158.30, 158.68, 174.41. MS (EI): m/z (%) 224 [M ] (100), 166 (31), 139 (82), 111(27). EA: Calculated: C 53.57, H 4.05, N
–
1
2.50; found: C 53.33, H 4.08, N 12.40. IR (ATR), ν (cm ) 717 (s), 816 (m-s), 1144 (s), 1178 (s), 1262 (s), 1438 (vs), 1507 (s), 1680 (vs, sh), 1768
(
m-s), 3235 (m, br).
5
-Fluoro-3-(i-propylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6i)
(M = 252.25), recrystallized first from MeOH (47 % yield), then from AcOEt/hexane; overall yield 21 %; R
C
12
H
13FN
2
O
3
f
0.43, mp 227-230 °C
1
3
4
(
dec., above 200 °C sublimation; microcrystalline white powder), H-NMR (250 MHz, DMSO-D
6
): δ (ppm) 1.34 (dd, J 6.8 Hz, J 3.3Hz, 6H, 2
3
4
x CH ), 4.08-4.30 (m, 1H, CH), 5.03 (s, 1H, 3-H), 6.80 (dd, J 9.6 Hz, J 4.7 Hz, 1H, arom. H), 6.99-7.05 (m, overl., 2H, arom. H), 8.21 (s, 1H, NH),
3
9
.78 (s, br, 1H, NH). 13C-NMR (63 MHz, DMSO-D
6
): δ (ppm) 20.56, 20.71, 43.77, 58.08, 116.99, 117.35, 117.69, 117.82, 117.97, 118.34, 124.88,
+
1
25.00, 153.59, 153.62, 154.50, 158.23. 158.31, 159.98, 174.8. MS (EI): m/z (%) 252 [M ] (100), 210 (38), 166 (27), 139 (46), 124(15). HR-MS -
–
1
calc. for C12
H
14FN
2
O : 253.09884, obs. 253.09940. IR (ATR), ν (cm ) 753 (s, sh), 757 (s), 822 (s), 1102 (s), 1420 (s), 1507 (m, sh), 1517 (m),
3
1675 (vs), 3201 (m, sh, br), 3274 (m, br).
5
-Fluoro-3-(n-but-1-ylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6j)
(M = 266.27), recrystallized first from MeOH/water (3:2, v/v), then from AcOEt/hexane; yield 30 %; R
C
13
H
15FN
2
O
3
f
0.31, mp 228-231 °C
), 1.16 (td, J 13.8 and 6.6 Hz, 2H, CH ),
), 5.12 (s, 1H, 3-H), 6.81 (dd, J 8.0, J 4.7 Hz; 1H, arom. H), 7.00-7.15 (m, overl., 2H, arom.
1
3
(
dec., amorphous white powder), H-NMR (400 MHz, DMSO-D
.21-1.37 (m, 2H, CH ), 3.28-3.35 (m, overl., 2H, CH
H), 9.87 (s, 1H, NH), 10.80 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-D
6
): δ (ppm) 0.78 (t, J 8.0 Hz, 3H, CH
3
2
3
4
1
2
2
6
): δ (ppm) 13.90, 19.65, 29.63, 116.77, 116.87, 116.95, 117.00, 121.68,
+
1
21.75, 12.66, 153.04, 153.06, 154.38, 156.71, 154.38, 156.71, 156.95, 173.33, 173.41. MS (EI): m/z (%) 266 [M ] (100), 223 (36), 166 (22), 152
+
–1
(
47), 125(22). HR-MS - calc. for C13
H
15FN
2
O Na [M+Na ]: 289.09644, obs. 289.09657. IR (ATR), ν (cm ) 824 (m), 985 (m), 1074 (m), 1145 (s),
3
1
185 (m-s), 1250 (m), 1341 (m), 1449 (s), 1506 (s), 1664 (vs), 1757 (m), 2930 (m), 2962 (m), 3070 (m), 3159 (m), 3350 (m). EA: calculated: C
8.64, H, 5.68, F 7.13, N 10.52; found: C 58.51, H 5.69, N 10.64.
5
5
-Fluoro-3-(n-pent-1-ylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6k)
(M = 280.30), chromatography of oily product on silica gel done twice (first: gradient hexane/AcOEt = 2:1 => 1:0, then:
0.37, mp 226-230 °C (dec.,
, broad signals): δ (ppm) 0.79 (t, J 6.7 Hz, 3H, CH ), 1.10-1.25 (m, 4H, 2 x CH
-N), 3.33 (dt, J 14.5 and 7.4 Hz, 1H, CH -N), 5.12 (s, 1H, 3-H), 6.81 (dd, J 8.7 Hz, J 4.7 Hz, 1H,
arom. H), 7.01-7.11 (m, overl., 2H, 2 x arom. H), 9.90 (s, vbr, 1H, NH), 10.79 (s, 1H, NH). 13C-NMR (63 MHz, DMSO-D
): δ (ppm) 15.06, 22.93,
8.07, 29.50, 62.68, 117.58, 117.75, 117.88, 117.94, 122.59, 122.71, 153.94, 153.97, 154.59, 157.86, 158.33, 157.86, 159.41, 160.02, 160.62,
C
14
H
17FN
2
O
3
AcOEt), very slow recrystallization from AcOEt/hexane at room temperature, finally -18 °C; yield 9 %; R
f
1
3
microcrystalline white powder), H-NMR (250 MHz, DMSO-D
.25-1.40 (m, 2H, CH ), 2.56-2.77 (m, 1H, CH
6
3
2
),
3
4
1
2
2
2
6
2
1
+
+
74.30. MS (EI): m/z (%) 280 [M ] (100), 166 (16), 139 (19). HR-MS - calc. for C14
H
17FN
2
O Na [M+Na ]: 303.112096, obs. 303.11271. IR (ATR), ν
3
–
1
(
cm ) 703 (s), 823 (m), 1144 (m-s), 1449 (s), 1506 (m-s), 1523 (m-s), 1684 (vs), 2940 (m), 2957 (m), 3051 (m), 3185 (m, br), 3356 (m, br).
5
-Fluoro-3-(benzylaminocarbonylamino)-benzo[b]furan-2-(3H)-one 6l)
(M = 300.29), recrystallized first from MeOH, then from EtOH, yield 21 %; R 0.27, mp 237-241 °C (dec., colorless needles), H-
1
C
16
H
13FN
2
O
3
f
2
2
3
4
NMR (250 MHz, DMSO-D
6
): δ (ppm) 3.79 (d, J 15.7 Hz, 1H, benzyl-H), 4.60 (d, J 15.7 Hz, 1H, benzyl-H), 4.99 (s, 1H, 3-CH), 6.75 (dd, J 8.9, J
3
4
4
.7 Hz;1H, arom. H), 6.88 (dd, J 9.0, J 3.1 Hz; 1H, arom. H), 6.96-7.07 (m, 1H, arom. H), 7.07-7.15 (m, 1H, arom. H), 7.20-77.32 (m, overl., 4H,
15
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Helvetica Chimica Acta
10.1002/hlca.201800243
HELVETICA
arom. H), 9.86 (s, 1H, NH), 10.96 (s, 1H, NH). 13C-NMR (63 MHz, DMSO-D
): δ (ppm) 44.30, 61.97, 116.74, 116.82, 116.95, 117.10, 121.08,
6
+
1
21.19, 227.62, 127.93, 128.80, 137.16, 152.98, 153.00, 153.60, 157.26, 157.33, 173.13.MS (EI): m/z (%) 300 [M ] (100), 209 (23), 192 (13),
–
1
1
66 (19), 106(38), 91 (100), 65 (23). HR-MS - calc. for C16
H
13FN
2
O Na: 323.08079, obs.: 323.08000. IR (ATR), ν (cm ) 1437 (s), 1449 (s), 1507
3
(s), 1676 (vs, sh), 1757 (m), 2350 (w), 2779 (w), 3029 (m), 3166 (m), 3354 (m).
5
-Fluoro-3-(acethylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6m)
C
11
H
9
FN
2
O
4
(M = 252.20), recrystallized from MeOH/THF, yield 23 %; R 0.40, mp 213-216 °C (above 185 °C decomp., amorphous white
f
1
3
3
powder), H-NMR (250 MHz, DMSO-D
6
): δ (ppm) 2.02 (s, 3H, CH ), 5.53 (d, J 7.2 Hz, 1H, 3-CH), 7.02-7.26 (m, 3H, arom. H), 9.24 (d, J 7.2 Hz,
3
1
1
H, NH), 10.05 (s, 1H, NH). 13C-NMR (63 MHz, DMSO-D
6
): δ (ppm) 23.94, 51.93, 111.60, 111.92, 112.00, 112.05, 115.79, 116.18, 128.53,
+
28.67, 149.87, 149.90, 153.15, 157.29, 161.10, 172.45, 173.71. MS (EI): m/z (%) 252 [M ] (64), 210 (100), 165 (16), 139 (100), 137 (29), 111
+
–1
(
18), 43 (40). HR-MS - calc. for C11
H
9
FN
2
O Na [M+Na ]: 275.04440, obs. 275.04438. IR (ATR), ν (cm ) 1066 (s-m), 1233 (m), 1480 (m), 1532
4
(m), 1675 (vs), 1817 (m, sh), 2358 (w), 3261 (w-m), 3285 (w-m).
5
-Fluoro-3-(benzoylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (6n)
1
C
16
H
11FN
2
O
4
(M = 314.27), recrystallized from EtOH, yield 63 %; R 0.71, mp 239-240 °C, dec. (amorphous white powder), H-NMR (250
f
3
MHz, DMSO-D
6
): δ (ppm) 5.62 (d, J 7.0 Hz, 1H, 3-CH), 7.11-7.27 (m, 3H, arom. H), 7.51 (t, J 7.4 Hz, 2H, phenyl H), 7.61 (d, J 7.3 Hz, 1H,
phenyl H), 7.97 (d, J 7.0 Hz, 2H, phenyl-H), 9.59 (d, J 7.4 Hz, 1H, NH), 10.91 (s, 1H, NH). 13C-NMR (63 MHz, DMSO-D
3
3
): δ (ppm) 52.11,
6
1
1
3
11.71, 111.93, 112.07, 112.12, 115.84, 116.22, 128.50, 128.67, 128.98, 132.78, 133.37, 149.97, 150.00, 153.55, 157.33, 161.13, 168.46,
+
+
73.75. MS (EI): m/z (%) 314 [M ] (100), 286 (15), 139 (23), 105 (96), 77 (26). HR-MS - calc. for C16
H
11FN
2
O Na [M+Na ]: 337.06005, obs.
4
–
1
37.05981. IR (ATR), ν (cm ) 782 (s), 826 (s), 890 (s), 1060 (vs), 1819 (s), 2363 (w), 3293 (m).
1
,3-Bis-(5-fluorobenzo[b]furan-2-(3H)-one-3yl) urea (6o)
(mixture of diastereomers, M = 360.27). This compound was obtained by reaction of each two equivalents of glyoxylic acid
C
17
H
10
F
2
N
2
O
5
monohydrate and 4-fluorophenol with one equivalent of urea. Reacting equimolar amounts of starting materials did not furnish 5-
fluoro-benzo[b]furan-2-(3H)-one-3-yl urea. The obtained product mixtures contained always predominantly 6o which precipitated.) The
crude product was filtered off from the reaction mixture, washed twice with small amounts of trifluoroactic acid and dried in vacuum.
The white powder was carefully digested in toluene, filtered off, digested in toluene again and the toluene removed using a rotary
evaporator (70 °C). Finally the product was washed twice with water and dried in vacuum at 50 °C. Yield: 44 %, off-white amorphous
powder, R
f
0, mp 160-180 °C (dec.). Due to a pronounced tendency to form oils only an analytical sample was further purified: A solution
in methanol was carefully added with water until opacity occurred (about 50 %) and allowed to stand at room temperature in an
unsealed vessel for several days. The precipitate was filtered off and dried: mp 183-187 °C (dec., amorphous white powder), R
f
0 (R
f
0.64
1
3
with solvent AcOEt/isopropanole/water = 7:7:5, v/v/v). H-NMR (400 MHz, DMSO-D
6
; comparatively broad signals): δ (ppm) 5.41 (d, J 8.1
3
Hz, 2H, 2 x 3-CH), 6.83 (d, J 4.2 Hz, 2H, 2 x arom. H), 6.90-7.15 (m, overl., 4H, 2 x 2 arom. H), 9.78 (s, broad, 2H, 2 x NH), 12.58 (s, vbr., 2H, 2 x
NH). 13C-NMR (100 MHz, DMSO-D
): δ (ppm) 52.12, 115.23, 115.31, 115.46, 115.55, 116.56, 126.98, 127.05, 151.61, 154.42, 156.75, 156.98,
6
+
+
–1
1
73.13. MS (EI): m/z (%) 360 [M ] (32), 139 (100). HR-MS - calc. for C17
H
10
F
2
N
2
O [M ]: 360.055777, obs. 360.055260. IR (ATR), ν (cm )815 (s),
5
964 (m-s), 1048 (m-s), 1157 (s), 1169 (s), 1229 (s), 1241 (s), 1437 (s), 1506 (vs), 1540 (s, sh), 1708 (m-s, sh), 1718 (m-s, sh), 3277 (m-w), 3383
(
m), 3505 (m-w).
5
-Fluoro-3-(1,3-dioxo-1,3-dihydro-2H-isoindolyl-2-yl-carbonylamino)-benzo[b]furan-2-(3H)-one (6p)
FN (M = 340.27), the oily crude product was suspended in AcOEt whereby slow crystallization occurred. The white precipitate
C
17
H
9
2
O
3
was filtered off and washed with diethyl ether. From a 5 mmol preparation after drying 630 mg of crude product were obtained (mp
90-193 °C, dec.).The product was suspended in 25 ml of boiling AcOEt, filtered and the remaining residue washed twice with diethyl
1
ether and dried: 45 mg (3 %), mp 235-238 °C (dec., amorphous white powder); R
f
0.47 and some traces of by-product, R
f
0.60
1
3
(
dichloromethane/MeOH = 9:1, v/v: R
f
0.90 and traces of by-product, R
f
0.80). H-NMR (400 MHz, DMSO-D
6
): δ (ppm) 5.72 (d, J 6.7 Hz, 1H,
-CH), 7.00-7.40 (m, overl., 3H, arom. H), 7.96 (d, J 11.3 Hz, 4H, 4 x arom. H), 9.38 (d, J 6.8 Hz, 1H, NH). 13C-NMR (100 MHz, DMSO-D
3
): δ
3
6
(ppm) 52.30, 92.80, 11.86, 112.12, 112.20, 112.28, 116.36, 116.60, 123.41, 124.49, 127.70, 127.79, 131.35, 136.04, 148.33, 150.10, 158.06,
+
+
1
66.73, 173.14.MS (EI): m/z (%) 340 [M ] (45), 174 (100). HR-MS - calc. for C17
H
9
FN
2
O Na [M+Na ]: 363.03931, obs.: 363.03988. IR (ATR), ν
5
–
1
(
cm ) 717 (s), 877 (s), 1070 (s), 1477 (s), 1507 (s), 1675 (m), 1685 (m), 1724 (s), 1744 (s, sh), 1800 (m), 1819 (m), 3313 (m-w), 3357 (m-w).
16
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Helvetica Chimica Acta
10.1002/hlca.201800243
HELVETICA
5
-Fluoro-3-[N-(aminocarbonyl-)N-methylamino)-benzo[b]furan-2-(3H)-one (7h)
FN (M = 224.19). This compound was prepared as described for 6h: Yield: 21-28 %, white micro crystalline powder, mp 280.5-
C
10
H
9
2
O
3
1
2
82.0 °C (from AcOEt, than from water or water/MeOH, 3:1, v/v; dec., above 220 °C sublimation to needles). H-NMR (400 MHz, DMSO-D
6
):
3
4
δ (ppm) 2.57 (s, 3 H, CH
3
), 5.07 (s, 1H, 3-CH), 6.82 (dd, J 9.6 Hz, J 4.6 Hz, 1H, arom. H), 6.99-7.11 (m, 2H, 2 x arom. H), 9.87 (s, 1H, NH), 10.82
(
s, 1H, NH’). 13C-NMR (100 MHz, DMSO-D
6
): δ (ppm) 27.40, 63.44, 116.75, 116.93, 116.98, 117.01, 117.67, 117.91, 121.42, 121.49, 152.98,
+
1
52.99, 154.42, 156.75, 157.18, 173.30. MS (EI): m/z (%) 224 [M ] (100), 180 (27), 152 (42), 138 (28). EA: Calculated: C 53.57, H 4.05, N 12.50;
–
1
found: C 53.44, H 4.09, N 12.35. IR (ATR), ν (cm ) 817 (s), 1135 (m), 1143 (m), 1180 (m), 1262 (s), 1278 (m), 1332 (m), 1369 (m), 1415 (m-s),
437 (s), 1512 (s, sh), 1685 (vs), 1717 (s), 1757 (s), 3236 (m-s, br).
1
5
-Fluoro-3-[2,5-dioxo-pyrrolidino-1-carbonyl-(N-cyclohexyl)-amino]-benzo[b]furan-2-(3H)-one (7q)
(M = 374.37). Mixture of products from which was isolated N-(cyclohexylcarbamoyl)succinamic acid as main component in
C
19
H
16FN
2
O
5
4
5 % yield:
Cyclohexylcarbamoylsuccinamic acid: C11
recrystallization from AcOEt/EtOH = 5:1, v/v (53 % recovery): mp 167-173 °C; [40]: mp 162-164 °C, [41]: mp 170-173 °C (needles, from from
H
18
N
2
O (M = 242.13), from EtOH/water = 2:1, v/v; colorless crystals (45 % yield); after second
4
1
3
water). H-NMR (250 MHz, DMSO-D
6
): δ (ppm) 1.02-1.42 (m, 5H, 5 x cycohexyl_H), 1.45-1.65 (m, 3H, 3 x cyclohexl-H),1.79 (d, J 10.6 Hz, 2H,
3
2
x cyclohexyl-H), 2.45-2.55 (m, overl., 4H, 2 x CH
2
CO), 3.48-3.55 (m, 1H, cyclohexyl-H), 8.25 (d, J 7.0 Hz, 1H, NH), 10.29 (s, 1 H, NH), 12.14
(
(
s, broad, 1H, OH). 13C-NMR (63 MHz, DMSO-D
6
): δ (ppm) 25.49, 26.41, 29.34, 31.92, 33.70, 48.91, 153.64, 174.88, 175.47. MS (neg. ESI): m/z
+
+
%) 241.3 [M-H ] (100). HR-MS - calc. for C17
H
22
N
2
O
3
Na [M+Na ]: 325.15281, obs. 325.15251. EA: calculated: C 67.53, H, 7.33, N 9.26; found:
–
1
C 67.49, H 7.30, N 9.14. IR (ATR), ν (cm ) 891 (m), 931 (m, sh), 1180 (m), 1215 (s), 1545 (s, sh), 1670 (s, sh), 1696 (s), 2856 (m), 2920 (m), 3115
w, br), 3303 (w, br).
(
5
-Fluoro-3-[phenylaminocarbonyl-(N-methyl)-amino]-benzo[b]furan-2-(3H)-one (7s)
1
C
16
H
13FN
2
O
3
(M = 300.29), recrystallized from EtOH/water (9:1, v/v), yield 85 %; R
f
0.55, mp 275-277 °C, dec. (colorless plates). H-NMR (250
3
3
MHz, DMSO-D
6
): δ (ppm) 2.50 (s, 3H, CH
3
), 5.26 (s, 1H, 3-CH), 6.86 (dd, J 8.9 Hz, J 4.7 Hz, 1H, arom. H), 7.12 (td, J 8.6 Hz, J 3.2 Hz, 1H, arom.
H), 7.27 (dd, J 8.9 Hz, J 3.1 Hz, 1H, arom. H), 7.33-7.42 (m, overl., 3H, 3 x arom. H), 7.50 (d, J 7.0 Hz, 1H, arom. H), 10.09 (s, 1H, NH). 13C-NMR
3
3
(
63 MHz, DMSO-D ): δ (ppm) 28.90, 63.59, 117.89, 117.92, 118.01, 118.28, 119.35, 119.73, 122.17, 122.29, 127.98, 129.18, 130.14, 134.03,
6
+
+
1
53.85, 153.88, 154.64, 156.61, 158.37, 176.06. MS (ESI): m/z (%) 299.2 [M-H ] (100). HR-MS - calc. for C16
H
13FN
2
O
3
Na [M ]: 323.080790, obs.
–
1
3
23.080870. IR (ATR), ν (cm ) 745 (s), 907 (s), 1188 (s), 1383 (s), 1438 (s), 1685 and 1689 (vs, overl. sh), 1756 (m-w), 3119 (m, br). EA,
calculated: C 64.00, H 4.36, N 9.33; found: C 64.28, H 4.41, N 9.40.
5
-Fluoro-3-[phenylaminocarbonyl-(N-cyclohexyl)-amino]-benzo[b]furan-2-(3H)-one (7t)
(M = 368.41), recrystallized from EtOH/water (4:1, v/v): White powder, yield 82 %, mp 190-191 °C (rods), NMR: Mixture of 7t
C
21
H
21FN
2
O
3
(80 %) and the isomeric 5-Fluoro-3-[(phenylaminocarbonyl-(N-cyclohexyl)-amino]-benzo[b]furan-2-(3H)-one (20 %, according to the 3-
CH signal at 5.82 ppm and NH-signal at 9.98 ppm). In order to remove the less polar minor isomer (R 0.70) the obtained product was
f
recrystallized further two times from AcOEt/heptane (1:1, v/v): Overall yield 32 %; R
f
0.66 (uniform), mp 195.0-197.5 °C (colorless
1
3
3
rhombuses and plates). H-NMR (400 MHz, DMSO-D
6
, NOESY, COSY): δ (ppm) 0.87 (dd, J 12.7 Hz, J 25.4 Hz, 1H, aliph. H), 0.99 (dd, J 12.1
3
Hz, J 22.7 Hz, 1H, aliph. H), 1.16 (dd, J 26.3 and 12.8 Hz, 2H, 2 x aliph. H), 1.40-1.83 (m, 6H, 6 x aliph. H), 3.63 (dd, J 7.3.8 Hz, J15.7 Hz, 1H,
3
3
3
aliph. H), 5.36 (s, 1H, 3-CH), 6.83 (dd, J 8.4 Hz, J 4.4 Hz, 1H, arom. H), 7.09 (td, J 8.6 Hz, J 2.9 Hz, 1H, arom. H), 7.33 (d, J 5.2 Hz, 1H, arom.
H), 7.40 (t, J 7.2 Hz, 3H, 3 x arom. H), 7.50 (t, J 7.6 Hz, 2H, 2 x arom. H), 10.05 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-D
3
3
): δ (ppm) 25.46,
6
25.73, 25.79, 30.16, 31.21, 53.25, 116.89, 116.98, 117.12, 123.52, 123.59, 127.16, 128.23, 129.18, 133.09, 153.05, 154.93, 156.66, 171.39. MS
+
+
–1
(
ESI): m/z (%) 367.3 [M-H ] (100). HR-MS - calc. for C21
H
21FN
2
O Na [M ]: 391.143390, obs. 391.142700. IR (ATR), ν (cm ) 745 (s), 753 (s), 1174
3
(s), 1429 (s, sh), 1501 (m), 1682 (vs, sh), 1766 (m), 2937 (m-w), 3197 (m-w, br).
5
-Fluoro-3-[methylaminocarbonyl-(N-methyl)-amino]-benzo[b]furan-2-(3H)-one (7u)
(M = 238.22). In order to obtain 1,3-bis-(5-fluorobenzo[b]furan-2-(3H)-one-3yl)-1,3-dimethyl urea, 1 equiv. of 1,3-
C
11
H
11FN
2
O
3
dimethylurea 1u and each two equiv. of glyoxylic acid monohydrate 2 and 4-fluorophenol were reacted as described in the general
1
procedure. After recrystallization from MeOH/EtOH (1:1, v/v): Glittering colorless crystals, yield 61 %; R
f
0.36, mp 237-239 °C (dec.). H-
17
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3
NMR (250 MHz, DMSO-D
6
): δ (ppm) 2.64 (s, 3H, CH
3
), 2.91 (s, 3H, CH ), 5.11 (s, 1H, 3-CH), 6.82 (dd, J 8.6 Hz, J 4.6 Hz, 1H, arom. H), 7.04-7.13
3
(
m, overl., 2H, arom. H), 9.87 (s, 1H, NH). 13C-NMR (63 MHz, DMSO-D
6
): δ (ppm) 25.97, 28.72, 63.15, 117.68, 117.83, 117.96, 118.04, 118.51,
+
1
4
1
18.94, 122.11, 122.23, 153.79, 153.82, 154.66, 157.89, 158.39, 173.00.MS (ESI): m/z (%) 238 [M ] (100), 152 (35). EA: Calculated: C 55.46, H
–
1
.65, N 11.76; found: C 55.71, H 4.67, N 11.68. IR (ATR), ν (cm ) 758 (s, sh), 825 (s), 872 (s), 1016 (m), 1196 (s), 1205 (s), 1283 (s), 1444 (m-s),
483 (s), 1689 (vs, sh), 1754 (m-w), 3150 (m, br).
5
-t-Butyl-3-(dimethylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (8a)
(M = 276.15), chromatography on silica gel (gradient hexane/AcOEt = 2:1 => 1:4, v/v) recrystallized from AcOEt/hexane,
C
15
H
20
N
2
O
3
finally cooled to -18 °C; yield 20 %; R
f
0.36 (AcOEt/hexane = 1:2, v/v: R 0.17), mp 211-215 °C (dec., colorless needles), sample again
f
1
recrystallized from MeOH/water: mp 216-220 °C (dec.). H-NMR (400 MHz, DMSO-D
6
): δ (ppm) 1.29 (s, 9H, t-Bu), 2.81 (s, 6H, 2 x CH
3
), 5.18
3
3
3
3
(
d, J 7.0 Hz, 1H, 3-CH), 7.05 (d, J 6.8 Hz, 1H, arom. H), 7.25 (s, 1 H, arom. H), 7.35 (ddd, J 7.0 Hz, J 3.8 Hz, J 1.25 Hz, 1H, arom. H), 7.55 (d, J
7
1
.0 Hz, 1H, arom. H). 13C-NMR (100 MHz, DMSO-D
6
): δ (ppm) 31.86, 34.78, 36.26, 52.48, 110.01, 120.72, 126.09, 127.65, 146.86, 151.76,
+
+
57.52, 175.18. MS (ESI): m/z (%) 299 [M+Na ] (100). HR-MS - calc. for C15
H
20FN
2
O
3
Na [M+Na ]: 299.13716, obs. 299.13710. EA: calculated:
–
1
C 65.20, H 7.30, N 10.14; found: C 64.47, H 7.24, N 9.96. IR (ATR), ν (cm )821 (s), 889 (m, sh), 896 (s), 1061 (s), 1247 (m-s), 1378 (m-s), 1462
m), 1486 (s), 1518 (s), 1620 (m, sh), 1642 (s), 1805 (s), 2960 (m-w, br), 3311 (m).
(
5
-t-Butyl-3-(1-azolidinylcarbonylamino)-benzo[b]furan-2-(3H)-one (8c)
C
17
H
22
N
2
O
3
(M = 302.27), chromatography on silica gel (gradient heptane/AcOEt = 2:1 => 1:4, v/v) recrystallized from
1
dichloromethane/hexane (1:2, v/v), yield 22 %; R
f
0.40, mp 209-212 °C (dec., colorless amorphous powder). H-NMR (400 MHz, CDCl
3
): δ
3
3
3
(
ppm) 1.31 (s, 9H, t-Bu), 1.93 (s, 4H, 2 x CH
2
), 3.39 (s, 4H, 2 x CH ), 5.33 (d, J 7.5 Hz, 1H, 3-CH), 5.41 (d, J 7.5 Hz, 1H, NH), 7.00 (d, J 8.4 Hz,
2
H, arom. H), 7.34 (d, J 8.4 Hz, 1H, arom. H), 7.37 (s, 1 H, arom. H). 13C-NMR (100 MHz, CDCl
3
): δ (ppm) 25.53, 31.57, 34.72, 45.76, 52.19,
1
1
3
+
10.27, 121.44, 125.45, 126.68, 147.66, 151.52, 155.46, 175.38. MS (ESI): m/z (%) 325.2 [M+Na ] (100). HR-MS - calc. for C17
H
22
N
2
O Na
3
+
–1
[
(
M+Na ]: 325.15281, obs. 325.15251. EA: calculated: C 67.53, H, 7.33, N 9.26; found: C 67.49, H 7.30, N 9.14. IR (ATR), ν (cm ) 823 (s), 1065
s), 1086 (m), 1133 (s), 1233 (m), 1404 (m), 1486 (s), 1515 (s-m), 1640 (s, sh), 1810 (s), 2901 (m-w), 2953 (m-w), 2969 (m-w), 3259 (m-w, br).
The corresponding lactone precursor 5c (Scheme 2) was isolated from polar chromatographic fractions:
2
-(1-Azolidinylcarbonylamino)-2-(2-hydroy-5-t-butylphenyl)-acetic acid (5c)
(M = 320.39), chromatography on silica gel (gradient heptane/AcOEt = 2:1 => 1:4, v/v), precipitated with dichloromethane
C
17
H
24
N
2
O
4
from the oily fraction, then recrystallized from tetrahydrofurane/AcOEt, yield 8 %; R
f
0.01 (dichloromethane/MeOH = 9:1, v/v: R 0.35), mp
f
1
3
1
93-195 °C (dec., colorless plates). H-NMR (400 MHz, DMSO-D
6
): δ (ppm) 1.23 (s, 9H, t-Bu), 1.80 (s, 4H, 2 x CH
2
), 3.24 (d, J 9.5 Hz, 4H, 2 x
3
3
3
3
4
CH
2
), 5.46 (d, J 8.4 Hz, 1H, 3-CH), 6.21 (d, J 8.4 Hz, 1H, arom. H), 6.74 (d, J 8.4 Hz, 1H, arom. H), 7.13 (dd, J 8.3 Hz, J 2.0 Hz, 1H, arom. H),
3
7
5
.55 (d, J 7.0 Hz, 1H, arom. H), 9.60 (s, br, 1H, OH), 12.44 (s, br, 1H, OH). 13C-NMR (100 MHz, DMSO-D
6
): δ (ppm) 25.48, 31.88, 34.19, 45.80,
+
3.49, 115.52, 124.51, 125.85, 126.42, 141.45, 153.19, 156.41, 173.72. MS (EI): m/z (%) 320 [M ] (100), 276 (88), 162 (57), 98 (45), 72 (45). MS
+
–1
(
(
(
ESI): m/z (%) 343.1 [M+Na ] (100). EA: calculated: C 63.73, H 7.55, N 8.74; found: C 63.25, H 7.49, N 8.56. IR (ATR), ν (cm ) 690 (s, sh), 829
m), 1129 (m), 1232 (m), 1243 (m), 1257 (m), 1412 (s), 1482 (m), 1504 (s), 1522 (m), 1526 (m), 1637 (vs), 1722 (s-m), 2863 (w), 2949 (w), 3182
m, br).
5
,7-Di-t-butyl-3-(dimethylaminocarbonylamino)-benzo[b]furan-2-(3H)-one (9a)
C
19
H
28
N
2
O
3
(M = 332.44), chromatography on silica gel (gradient hexane/AcOEt = 3:1 => 1:3, v/v), recrystallized from
1
dichloromethane/hexane (1:2, v/v), yield 24 %; R
f
0.50, mp 190-194 °C (dec., colorless needles). H-NMR (400 MHz, DMSO-D
6
): δ (ppm)
3
4
4
1
.28 (s, 9H, t-Bu), 1.36 (s, 9H, t-Bu), 2.80 (s, 6H, 2 x CH
3
), 5.18 (d, J 6.6 Hz, 1H, 3-CH), 7.08 (d, J 0.8 Hz, 1H, arom. H), 7.22 (d, J 1.7 Hz, arom.
H), 7.57 (d, J 7.1 Hz, 1H, NH). 13C-NMR (100 MHz, DMSO-D
): δ (ppm) 29.95, 31.91, 34.30, 34.88, 36.28, 51.94, 118.36, 122.79, 128.03,
32.28, 146.28, 149.57, 157.49, 175.15. MS (ESI): m/z (%) 299 [M+Na ] (100). IR (ATR), ν (cm ) 876 (s), 1070 (vs), 1527 (m-s), 1534 (m), 1636
s), 1811 (s), 2949 (m-w). EA: calculated: C 68.65, H 8.49, N 8.43; found: C 68.47, H 8.59, N 7.73.
3
6
+
–1
1
(
5
,7-Di-t-butyl-3-(1-azolidinylcarbonylamino)-benzo[b]furan-2-(3H)-one (9c)
(M = 358.48), two times chromatography on silica gel (first: gradient heptane/AcOEt = 3:1 => 1:4 v/v, then AcOEt),
C
21
H
30
N
2
O
3
precipitated from oily fraction with a small amount of dichloromethane, recrystallized from dichloromethane/hexane (1:2, v/v), yield
18
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3
)
1
2
2 % ; R
x CH
13C-NMR (100 MHz, DMSO-D
55.80, 175.17. MS (ESI): m/z (%) 381.3 [M+Na ] (100). HR-MS - calc. for C21
f
0.56, mp 200-204 °C (dec., colorless rods). H-NMR (400 MHz, DMSO-D
6
): δ (ppm) 1.28 (s, 9H, t-Bu), 1.36 (s, 9H, t-Bu), 1.82 (s, 4H, 2
3
4
3
2
), 3.23 (d, J 2.9 Hz, 4H, 2 x CH
2
), 5.21 (d, J 6.9 Hz, 1H, 3-CH), 7.09 (s, 1H, arom. H), 7.23 (d, J 2.9 Hz, 1H, arom. H), 7.38 (d, J 7.3 Hz, NH).
6
): δ (ppm) 25.46, 29.95, 31.92, 34.31, 34.90, 45.86, 51.69, 118.48, 122.83, 128.03, 132.32, 146.29, 149.57,
+
+
1
H
30FN
2
O
3
Na [M+Na ]: 381.121541, obs. 381.21529. EA:
–
1
calculated: C 70.36, H 8.44, N, 7.81; found: C 69.99, H 8.38, N 7.69. IR (ATR), ν (cm ) 876 (m), 882 (m-w, sh), 1075 (vs), 1110 (m), 1395 (m),
1523 (m-s), 1633 (s), 1805 (m-s), 2875 (m-w), 2959 (m), 3295 (m-w, br).
Physico-Chemical Investigations
CL-estimation
In order to select good candidates for further investigations, the capability to undergo CL reactions was assessed in a first step. To this
aim a small amount (of about 1 mg) of compounds 6 - 9 was dissolved in the dark in 1 ml of THF and 50 μl of 3 % hydrogen peroxide
followed by 100 μl of a 10 % solution of DBU in THF were added.
UV-Vis measurements
-4
Benzofuranone solutions (10 M) were prepared by diluting the stock solutions with acetonitrile or buffered solution (pH = 7.8) consisting
-3
of equal amounts of KH
2
PO
4
(1 M), KH
2
PO (10 M), EDTA (1 M), and destilled water. Absorption spectra were recorded from 250 to 500 nm
4
using an UV-Vis double-beam spectrophotometer with a double monochromator (Lambda650, Perkin Elmer). Micro- and ultra-micro
quartz cuvettes (Hellma Optics, Germany) with a path length of 1 cm and 0.3 cm were used for the measurements in acetonitrile or buffered
solution, respectively. All samples were measured at room temperature against their respective blank solutions. The molar extinction
coefficients were calculated at the maximum absorption wavelengths using Lambert-Beer’s law.
Fluorescence and CL measurements
Fluorescence spectra and the CL-kinetics were obtained with a fluorescence spectrofluorimeter (Quantamaster 30, PTI/Photomed,
Germany). Fluorescence spectra were recorded using an excitation wavelength of 360 nm and an emission wavelength range of 380 to
5
1
50 nm. Excitation and emission slits were set to 5 nm. All measurements were performed in macro quartz cuvettes with a path length of
cm (Hellma Optics) under magnetic stirring at (25.0  0.1) °C using a temperature-controlled cuvette holder. Temperature was monitored
by a controller (TC 425, Quantum Northwest, USA). At least three measurements were made for each substance, which have been purified
for the measurements, and the controls. Spectra were blank corrected, i.e. subtracting the contribution of the solvent.
Kinetic measurements in acetonitrile with DBU as CL-trigger
A reaction mixture containing acetonitrile (1.98 ml) and benzofuranone stock solution (0.02 ml, 10-3 M; final concentration 10-5 M) was
prepared and equilibrated to 25 °C under magnetic stirring. For time-resolved measurements, CL was recorded at 430 nm with the
emission slits adjusted to 5 nm. After at least 2 min baseline recording, the CL reaction was induced by injecting DBU stock solution (0.03
ml, 67x10-3 M) with a Hamilton syringe. The solution was added to the sample by using a port in the mechanical spectrometer housing
and a flexible tube connecting the syringe with a cuvette cap manufactured by our workshop. The kinetics of CL was recorded for 30 min.
Control experiments were recorded under the same conditions in the absence of DBU.
Kinetic measurements in phosphate buffered acetonitrile solution with HRP/H
2
2
O as CL-trigger
CL in presence of HRP and H
2
O
2
was recorded similar to the measurements in presence of DBU using a modified protocol of Matuszczak
(0.5 ml, 10-3 M) while
[
13]. Here, CL was triggered by injecting subsequently the benzofuranone stock solution (0.5 ml, 10-3 M) and H
2
O
2
19
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stirring the temperature-controlled reaction mixture consisting of phosphate buffer (0.5 ml, 1 M), EDTA (0.5 ml, 1 M), and HRP working
-3
solution (0.5 ml, 0.01 mg/ml in 10 M phosphate buffer). For control measurements, HRP and H
2
2
O were omitted.
Data analysis
For each measured CL intensity-time curve the value of maximum light intensity (Imax), the rise time (trise, i.e. the time to reach maximum
light intensity), the half time (t1/2, i.e. the time to half maximum value), and the total area under the curve (corresponding to the total
amount of light generated during the reaction) were determined using FelixGX software (version 4.0.1, PTI/Photomed) and MS Excel.
Values represent averaged data from at least three independent experiments. Standard errors of the mean (SEM) are shown.
Quantum yield determination
The total areas of CL intensity measured in counts were converted to absolute quantum yields using the photon emission standard luminol
following a modified protocol. [5,38] In brief, by relating the reference value for the luminol quantum yield to the total counts obtained in
our system for a known luminol concentration a correction factor was obtained, which then allowed for converting measured counts into
CL quantum yields.
The luminol stock solution (10-3 M) was prepared by dissolving luminol in carbonate buffer (K
CO , 0.1 M, pH = 11.5). The solution was
2
3
-1
-1
stored in the dark at 4 °C and the extinction coefficient was determined to be 7600 M cm at 348 nm. Hydrogen peroxide solution (0.3 %)
was prepared daily by diluting the concentrated hydrogen peroxide solution with water. A HRP stock solution was made by dissolving 1
mg HRP in 1 ml carbonate buffer (0.1 M). The stock solution was stored at 4°C. A macro quartz cuvette (Hellma Optics) containing 2.8 ml
-4
luminol (10 M) was placed in the temperature-controlled cuvette holder of the spectrofluorimeter (Quantamaster 30, PTI/Photomed). All
measurements were performed at (25.0  0.1) °C under stirring. The time-based data acquisition was started and intensity was recorded
at a wavelength of 428 nm, i.e. the wavelength of maximum luminol emission. The CL reaction was initiated by subsequent injection of
hydrogen peroxide (0.3 %, 100 ꢀl) and HRP (1 mg/ml, 100 ꢀl) to the luminol solution in the dark with syringes after recording the baseline.
The CL light intensity increased within seconds and then declined within a few minutes. When CL intensity approximately reached baseline
level, hydrogen peroxide solution (0.3 %, 100 ꢀl) was added again to ensure that all luminol molecules reacted. The CL decay was
monitored until the reaction was complete. The calibration factor was calculated by multiplying the luminol CL quantum yield with the
number of mols of luminol divided by the integrated CL intensity (measured in counts, obtained in seven experiments). A reference
luminol quantum yield of (1.23  0.20) % was used. [38]
The quantum yields of the benzofuranones were calculated using the total area of CL (i.e. the integral of CL intensity in counts) multiplied
with the calibration factor and the correction factor for the sensitivity of the photomultiplier at the respective wavelengths given by the
manufacturer divided by the number of mols of the respective reagents.
Supporting Information
Additional details of the experimental work are provided in the Supporting Information comprising the following issues: General
Remarks and Materials, Synthesis of Urea Derivatives, Isolated Intermediates, Physico-Chemical Investigations, NMR Spectra, and
Literature.
20
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Author Contribution Statement
Conception, synthesis, analysis and interpretation of analytical data: RK; layout, execution, analysis, and interpretation of spectral and CL
measurements: BH; conceptual and analysis support: DW and CB. RK wrote the manuscript with contributions and review from all
authors.
References
[1] L. J. Kricka, ‘Clinical applications of chemiluminescence’, Anal. Chim. Acta 2003, 500, 279-286.
[2] Z. Zhang, J. Lai, K. Wu, X. Huang, S. Guo, L. Zhang, J. Liu, `Peroxidase-catalyzed chemiluminescence system and its application in immunoassay, Talanta
2
018, 180, 260-270.
3] M. Vacher, I. F. Galvan, B.-W. Ding, S. Schramm, R. Berraud-Pache, P. Naumov, N. Ferre, Y.-J. Liu, I. Navizet, D. Roca-Sanjua, W. J. Baader, R. Lindh, `Chemi-
and bioluminescence of cyclic peroxides`, Chem. Rev. 2018, 118, 6927−6974.
[
[4] L. Yue, Y.-J. Liu, `Mechanism of AMPPD chemiluminescence in a different voice`, J. Chem. Theory Comput. 2013, 9, 2300−2312.
[5] C. V. Stevani, S. M. Silva, W. J. Baader, `Studies on the mechanism of the excitation step in peroxyoxalate chemiluminescence`, Eur. J. Org. Chem. 2000,
4
037-4046.
6] T. H. Fereja, A. Hymete, T. Gunasekaran, ‘A recent review on chemiluminescence reaction, principle and application on pharmaceutical analysis’, ISRN
Spectroscopy 2013, 2013, 1-12.
7] G. Chen, M. Jin, P. Du, C. Zhang, X. Cui, Y. Zhang, J. Wang, F. Jin, Y. She, H. Shao, S. Wang, L. Zheng, `A review of enhancers for chemiluminescence
enzyme immunoassay`, Food Agric. Immunol., 2017, 28(2), 315-327.
8] K. Aslan, C. D. Geddes, Metal-enhanced chemiluminescence: advanced chemiluminescence concepts for the 21st century`, Chem. Soc. Rev. 2009, 38,
556–2564.
9] M. Zhang, Y. Jia, J. Cao, G. Li, H. Ren, H. Li, H. Yao, `Carbon dots-enhanced luminol chemiluminescence and its application to 2-methoxyestradiol
determination`, GREEN CHEM. LETT. REV. 2018, 11 (4), 379–386.
10] M. Ma, F. Diao, X. Zheng, Z. Guo, ‘Selective light-triggered chemiluminescence between fluorescent dyes and luminol, and its analytical application’, Z.
Anal. Bioanal. Chem. 2012, 404, 585–592.
[
[
[
2
[
[
[
[
[
[
[
11] C. Dodeigne, L. Thunus, R. Lejeune, ‘Chemiluminescence as diagnostic tool. A review’, Talanta 2000, 51(3), 415-439.
12] Y. Su, H. Chen, Z. Wang, Y. Lu, ‘Recent advances in chemiluminescence’, Appl. Spectr. Rev. 2007, 42, 139-176.
13] L. Hu, G. Xu, ‘Applications and trends in electrochemiluminescence’, Chem. Soc. Rev. 2010, 39, 3275-3304.
14] R. F. Vasil'ev, Y. B. Tsaplev, ‘Light-created chemiluminescence’, Russ. Chem. Rev. 2006, 75, 989-1002.
15] R. A. Bowie, M. G. Sanders, P. J. Worsfold, ‘Analytical applications of liquid phase chemiluminescence reactions - a review’, J. Biolumin. Chemilumin. 1996,
1
1, 61-90.
16] G. J. Lofthouse, H. Suschitzky, B. J. Wakefield, R. A. Whittaker, B. Tuck, ‘Synthesis and chemiluminescent reactions of some 3-
alkoxycarbamoylbenzo[b]furan-2(3H)-ones’, J. Chem. Soc., Perkin Trans. 1 1979, 1634-1639.
[
[
17] B. Matuszczak, ‘Linear and cyclic N-acetyl-α-aryl-glycines: synthesis and chemiluminescence studies’, Monatshefte für Chemie 1996, 127, 1291-1303.
18] B. Matuszczak, ‘Zur Chemilumineszenz von N-Acyl-α-arylglycinen: Anwendungsmöglichkeiten für die klinisch-chemische Analytik‘, Pharmazie 1996, 51,
[
8
62-865.
19] B. Matuszczak, ‘Linear and cyclic N-Acyl-α-arylglycines III. Synthesis and chemiluminescence studies of naphthol and phenanthrol amidoalkylation
products’, Monatshefte für Chemie 1997, 128, 945 -951.
20] B. Matuszczak, ‘Linear and cyclic N-acyl-α-arylglycines. IV. Novel 3-Substituted 3-Acylaminobenzo[b]furan-2(3H)-ones: Synthesis and
chemiluminescence studies’, J. Prakt. Chem. 1998, 340, 20-25.
21] S. Schramm, I. Navizet, P. Naumov, N. K. Nath, R. Berraud-Pache, P. Oesau, D. Weiss, R. Beckert, ‘The light emitter of the 2-coumaranone
chemiluminescence: theoretical and experimental elucidation of a possible model for bioluminescent systems’, Europ. J. Org. Chem. 2016, 4, 678-681.
22] S. Schramm, L. F. M. L. Ciscato, P. Oesau, R. Krieg, J. F. Richter, I. Navizet, D. Roca-Sanjuán, D. Weiß, R. Beckert, ‘Investigations on the synthesis and
chemiluminescence of novel 2-coumaranones - II’, ARKIVOC 2015, 5, 44-59.
23] S. Schramm, D. Weiss, I. Navizet, D. Roca-Sanjuán, H. Brandl, R. Beckert, H. Görls, ‘Investigations on the synthesis and chemiluminescence of novel 2-
coumaranones’, ARKIVOC 2013, 3, 174-188.
24] L. F. M. L. Ciscato, F. H. Bartoloni, A. S. Colavite, D. Weiss, R. Beckert, S. Schramm, ‘Evidence supporting a 1,2-dioxetanone as an intermediate in the
benzofuran-2(3H)-one chemiluminescence’, Photochem. Photobiol. Sci. 2014, 13, 32-37.
[
[
[
[
[
[
21
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Helvetica Chimica Acta
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HELVETICA
[25] S. Schramm, I. Navizet, D. P. Karothu, P. Oesau, V. Bensmann, D. Weiss, R. Beckert, P. Naumov. `Mechanistic investigations of the 2-coumaranone
chemiluminescence´, Phys. Chem. Chem. Phys. 2017, 19, 22852-22859.
[
[
[
26] R. C. Valentine, R. S. Wolfe, ‘Glyoxlyurea’, Biochem. Biophys. Res. Commun. 1961, 5, 305-308.
27] D. Ben-Ishai, J. Altman, Z. Bernstein, ‘The reactions of ureas with glyoxylic acid and methyl glyoxylate’, Tetrahedron 1977, 33 (10), 1191–1196.
28] W. Traube, F. Schottländer, C. Goslich, R. Peter, F. A. Meyer, H. Schlüter, W. Steinbach, K. Bredow, ‘Über ortho-diamino-pyrimidine und ihre Überführung
in Purine‘, Liebigs Ann. Chem. 1923, 432 (1), 266–296.
[
[
[
29] R. Behrend, ‘Zur Geschichte der Harnsäuresynthesen‘, Liebigs Ann. Chem. 1925, 441 (1), 215–216.
30] J. G. Frick, Jr., R. J. Harper, Jr., ‘Reaction of dimethylurea and glyoxal’, Ind. Eng. Chem. Prod. Res. Dev. 1982, 21, 599 -600.
31] A. Einhorn, E. Bischkopff, B. Szelinski, G. Schupp, E. Spröngerts, C. Ladisch, T. Mauermayer, ‘Ueber die N-Methylolverbindungen der Säureamide [Erste
Abhandlung]‘, Liebigs Ann. Chem. 1905, 343, 207-305.
[32] D. Matthies, ‘Herstellung und Umsetzung von N-Acyl-α-chloroglycinen‘, Synthesis 1978, 1978(1), 53-54.
[33] W. Schroth, R. Krieg, H. Kluge, M. Gäbler, ‘Synthese aromatischer Acylisocyanate durch Thermolyse von 1,1-Diacyl-3,3-dialkylharstoffen‘. Z. Chem. 1985,
2
5 (11), 398-399.
[
[
[
34] H. B. Dunford, J. S. Stillman, `On the function and mechanism of action of peroxidases´, Coord. Chem. Rev. 1976, 19, 187-251.
35] M. A. Motsenbocker, K. Kondo, `Improvements to enhanced horseradish peroxidase detection sensitivity´, J. Biolumin. Chemilumin. 1994, 9, 15-20.
36] S. Gnaim, O. Greena, D. Shabat, `The emergence of aqueous chemiluminescence: new promising class of phenoxy 1,2-dioxetane luminophore`, Chem.
Commun., 2018, 54, 2073-2085.
37] L. P. Silva, C. M. Magalhaes, `Mechanistic insights into the efficient intramolecular chemiexcitation of dioxetanones from TD-DFT and multireference
calculations.´ Int. J. Quantum Chem. 2018, e25881.
38] Y. Ando, K. Niwa, N. Yamada, T. Irie, T. Enomoto, H. Kubota, Y. Ohmiya, H. Akiyama; `Development of a quantitative bio⁄chemiluminescence
spectrometer determining quantum yields: re-examination of the aqueous luminol chemiluminescence standard`, Photochem. Photobiol., 2007, 83, 1205–
210.
[
[
1
[39] P. Nesvadba, L. Bugnon, P. Dubs, S. Evans, ‘A versatile new synthesis of 3-aryl-3H-benzofuran-2-ones’, Synlett 1999, S1, 863-864.
[40] M. Okano, Y. Ito, T. Shono, R. Oda, ‘Studies of isocyanides. I. The formation of urethanes and ureas from isocyanides’, Bulletin of the Chemical Society of
Japan 1963, 36 (10), 1314 - 1316.
41] H. Yamada, Y. Wada, S. Tanimoto, M. Okano, ‘The reaction of isocyanides with halogens or N-halosuccinimides and alcohols’, Bulletin of the Chemical
Society of Japan1963, 36 (10), 1314 - 1316.
[
22
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Helvetica Chimica Acta
10.1002/hlca.201800243
HELVETICA
Graphical Abstract
First synthesis of highly chemiluminescent benzo[b]furan-2-(3H)-ones
bearing a urea substructure
Reimar Krieg, Birgit Hoffmann, Dieter Weiß, and Christoph Biskup
23
This article is protected by copyright. All rights reserved.