New isatin derivatives with antioxidant activity

Article abstract of DOI:10.1016/j.ejmech.2009.12.035

The reaction between isatin and 2,5-dimethoxyaniline is described. The main product was identified as 3,3-bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one. The antioxidant activity of the compounds isolated was evaluated with two methods. Three published antitumor E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones entered the same tests to search whether they are endowed with antioxidant activity too. 3,3-Bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one and the three antitumor agents showed a good chemical antioxidant activity.

Full text of DOI:10.1016/j.ejmech.2009.12.035

European Journal of Medicinal Chemistry 45 (2010) 1374–1378
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New isatin derivatives with antioxidant activity
,
a
a
a
a
Aldo Andreani a , Silvia Burnelli , Massimiliano Granaiola , Alberto Leoni , Alessandra Locatelli ,
*
Rita Morigi a, Mirella Rambaldi a, Lucilla Varoli a, Mauro Andrea Cremonini b, Giuseppe Placucci b,
Rinaldo Cervellati c, Emanuela Greco c
a
`
Universita di Bologna, Dipartimento di Scienze Farmaceutiche, Via Belmeloro 6, 40126 Bologna, Italy
Universita di Bologna – Sede distaccata di Cesena, Piazza Goidanich 60, Cesena, Italy
Universita di Bologna Dipartimento di Chimica ‘‘Giacomo Ciamician’’ Via Selmi 2, Bologna, Italy
b
c
`
`
a r t i c l e i n f o
a b s t r a c t
Article history:
The reaction between isatin and 2,5-dimethoxyaniline is described. The main product was identified as
3,3-bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one. The antioxidant activity of the
compounds isolated was evaluated with two methods. Three published antitumor E-3-(2-chloro-3-
indolylmethylene)1,3-dihydroindol-2-ones entered the same tests to search whether they are endowed
with antioxidant activity too. 3,3-Bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one and the
three antitumor agents showed a good chemical antioxidant activity.
Received 26 June 2009
Received in revised form
13 November 2009
Accepted 16 December 2009
Available online 28 December 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Isatin
2,5-Dimethoxyaniline
Antioxidant activity
Briggs–Rauscher reaction
1. Introduction
methine by a nitrogen. We thought that the reaction between isatin
4 (Scheme 2) and an aniline bearing selected substituents, could be
Most of our research is devoted tothe synthesis of newantitumor
agents and some of them have been prepared by means of the
Knoevenagel reaction between an aldehyde and an oxindole. A
recent example of this research is a paper describing antitumor
activity and effect on cell cycle of some substituted E-3-(2-chloro-3-
indolylmethylene)1,3-dihydroindol-2-ones 3 (Scheme 1) prepared
by means of the above mentioned reaction with the aldehyde 1 and
the oxindole 2 as the starting compounds [1].
In other papers we considered different aldehydes [2,3] and we
also planned the synthesis of compounds bearing bridges different
from the usual methine group for the connection of the two
moieties. The mechanism of action of these compounds has not
been completely understood but we believe they act by means of
multiple mechanisms [3].
the right route since the reaction between isatin and p-anisidine 5
is well documented in the literature under different experimental
conditions and leads always to the expected imine 6 [4–9]. This
reaction gave always the related imine even when employed with
different methoxy anilines such as 2-methoxy [10], 3-methoxy [11]
and 2,4-dimethoxyaniline [12] but when we attempted the reaction
of isatin with 2,5-dimethoxyaniline 7 to obtain the imine 8, we
were able to isolate only one compound whose spectroscopic data
were not in agreement with the structure of 8 and was later
identified as 3,3-bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihy-
droindol-2-one 9. Under the same experimental conditions, when
isatin was replaced by N-acetylisatin 10, the reaction proceeded
with nucleophilic attack at the C-2 carbonyl leading to heterocyclic
ring cleavage and formation of 2-(2-acetamidophenyl)-N-(2,5-
dimethoxyphenyl)-2-oxoacetamide 11 in agreement with previous
reports [13].
2. Chemistry
For the structure determination of compound 9 we planned to
use X-ray crystallography since any crystal was apparently shiny
but unfortunately it was not a single crystal but an agglomerate of
microcrystals useless for this kind of analysis. After several crys-
tallization attempts with different solvents we decided to leave this
approach.
To obtain a deeper insight into structure–activity relationships
of this class of compounds, we designed the replacement of the
* Corresponding author. Tel.: þ39 51 2099714; fax: þ39 51 2099734.
E-mail address: aldo.andreani@unibo.it (A. Andreani).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.035

A. Andreani et al. / European Journal of Medicinal Chemistry 45 (2010) 1374–1378
1375
R
N
O
NH2
O
N
O
Cl
O
O
+
O
R1
R2
O
N
H
R1
R2
N
H
Cl
O
+
O
O
N
R
N
H
N
H
4
6
5
2
1
3
a: R=CH3 R1=OH R2=CH3
b: R=CH3 R1=OH R2=H
O
NH2
Cl
c: R=
R1=OH R2=CH3
N
O
O
4
+
O
O
N
Scheme 1. Substituted E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones.
H
7
8
The mass spectrum of compound 9 (m/z ¼ 435) indicates that
one molecule of isatine reacted with two molecules of 2,5-dime-
thoxyaniline 7 by loss of two protons and one oxygen.
H2N
O
O
NH2
The NMR data in d6-DMSO (298 K) and CDCl3 (298 and 263 K)
show that one aromatic proton is missing from each of the two 2,5-
dimethoxyaniline moieties and an aliphatic quaternary carbon at
59.8 ppm replaces the carbonyl at C3 of the parent isatin, thus
forming a diaryloxindol similar to those reported in ref. 14.
A rough molecular mechanics calculation (Chemsketch 12, ACD,
Inc., Toronto, CA) shows that the two aryls are tilted with respect to
each other, thus making the molecule chiral and the atoms of each
2,5-dimethoxyaniline moiety diastereotopic. The two aryls undergo
a dynamic phenomenon (most probably a rotation about the C10 – C3
O
O
O
N
H
9
H
N
O
O
O
00
+
7
O
and C1 – C3 bonds) that broadens the proton and carbon signals of
O
H
N
the two 2,5-dimethoxyaniline moieties. The dynamic phenomenon
stops (on the NMR time scale) at 263 K.
O
N
O
O
The proposed structure of 9 (where each 2,5-dimethoxyaniline
moiety is bonded to the C3 of the aryloxindol via the former C4 of
2,5-dimethoxyaniline) is con0firmed at 263 K by strong HMBC
10
11
00
connections between both H6 , H6 and C3 and by the presence of
Scheme 2. Synthesis of compounds 9 and 11.
ROESY cross peaks only between each of the 2,5-dimethoxyaniline
aromatic protons and a different methoxy group. This rules out the
possibility of the 2,5-dimethoxyaniline moieties being bonded to C3
of the aryloxindol via the former C3 of 2,5-dimethoxyaniline (one
methoxy would give ROESY cross peaks with two aromatic protons,
while the other would give none) or via the former C6 (each
aromatic proton would give ROESY cross peaks with the nearby
proton and methoxy group): see Experimental section and
Supplementary data.
From a literature survey we found that, notwithstanding the
numerous aforementioned citations of the reaction between isatin
and methoxy anilines, no mention was reported about the reaction
between isatin and 2,5-dimethoxyaniline. Nevertheless it has been
reported that isatin may give Friedel–Crafts adducts [13–16]. We
believe that the different behavior of 2,5-dimethoxyaniline 7
(compared to other methoxy anilines) is due to the combined
electronic effect of the amino group and the methoxy groups which
activate the 4-position.
Cancer Institute (NCI, Bethesda, MD). Both these groups of
compounds were evaluated for their possible antioxidant activity.
In recent years many papers reported the potential beneficial
effects of different free-radical scavenger antioxidant polyphenols
as preventive agents against human neoplastic diseases at several
sites including stomach, duodenum, colon, liver, lung, breast, ovary
or skin [18]. A review of several papers reached the conclusion that
the proofs of the claimed beneficial effects on humans are incon-
clusive [19]. However, a very recent review on the impact of anti-
oxidant supplementation on chemotherapy suggests that the
concurrent use of antioxidants and chemotherapeutic drugs could
diminish the dose-limiting toxicity of these latter [20]. The chem-
ical antioxidant activity of compounds under test (3a–c, 9 and 11)
was assessed with two methods: the Briggs–Rauscher (BR) oscil-
lating reaction method that works in acidic conditions [21] and the
Trolox Equivalent Antioxidant Activity (TEAC) assay working at
pH ¼ 7.4 [22].
Therefore the reaction proceeds with nucleophilic attack at the
C-3 carbonyl of isatin leading to compound 9 according to Scheme
S1. A similar mechanism could explain the reaction products of
isatin with alifatic secondary diamines [17].
4. Results and discussion
Collected results of antioxidant activity with the BR method at
acidic pH (ca. 2), and with the TEAC assay at pH ¼ 7.4 are reported in
Table 1.
3. Antioxidant activity
As we wrote in the introduction, compounds 3a–c showed
antitumor activity [1] whereas the compounds resulting from the
above described reactions did not show any activity when sub-
jected to the standard antitumor tests performed by the National
In view of a possible development of these new compounds as
chemopreventive drugs in food integrators, it is quite important to
test antioxidant activity at acidic pH value near to that of gastric
fluids. In fact there are several evidences that stomach acts as

1376
A. Andreani et al. / European Journal of Medicinal Chemistry 45 (2010) 1374–1378
Table 1
Positive controls are the substances used as standard: resorcinol
for the BR method and Trolox for the TEAC assay. Compound 11
showed negligible activity either at pH ca. 2 and 7.4. Surprisingly,
compound 9 presents a good relative antioxidant activity with both
methods even if it contains four OMe groups that are found to have
very poor free radicals scavenger power at least in flavonoid
structures [30,31]. No doubt about this activity since the inhibitory
phase in the BR method is similar to that produced by OH groups
and in the TEAC assay the only species with which OMe groups can
Relative antioxidant activity of the examined compounds.
Compound
RACa
resorcinol)
(
m
M equivalents
TEACb (mM equivalents
Trolox)
3a
3b
3c
9
2.83 ꢂ 0.05
2.74 ꢂ 0.06
Negligible
1.28 ꢂ 0.04
Negligible
0.91 ꢂ 0.04
0.82 ꢂ 0.07
0.97 ꢂ 0.05
1.40 ꢂ 0.09
Negligible
11
a
RAC (Relative Antioxidant Capacity, standard resorcinol) values are the average
þ
react is the radical cation ABTSꢁ (see Supplementary data). A
of at least three measurements at different concentrations ꢂ SE.
b
mechanistic interpretation of this will be given in the next section.
TEAC (Trolox Equivalent Antioxidant Capacity) is given as the ratio of the slopes
of the straight lines Abs. vs. conc. of the sample and the standard respectively ꢂSE.
(See Supplementary data).
5. Mechanistic interpretation of inhibition in the Briggs–
Rauscher method
a bioreactor in which many drugs can interact [23,24]; moreover
recent in vivo studies demonstrated that some polyphenols are
promptly absorbed in the stomach [25,26]. TEAC measurements
were conducted at pH 7.4, the physiological pH of the blood.
Inspection of data in Table 1 revealed that the relative antioxi-
dant activity in acidic conditions of compounds 3a, 3b is quite high,
similar to that found for (ꢀ)catechin (2.2) and (ꢀ)epicatechin (2.6)
from green tea [27] that are among the strongest antioxidants. On
the contrary, compound 3c showed negligible activity under these
conditions, probably because it interferes with some of the
components of the BR mixture before the phenolic OH reacts with
the generated HOOꢁ radicals [28] (see also Supplementary data).
The antioxidant capacity values at pH ¼ 7.4 of compounds 3a
In 2002, Furrow et al. [32] reported a 13-step new mechanism
(named FCA model) for the BR reaction that takes into account the
important role played by HOOꢁ radicals in its oscillatory behavior.
To simulate the perturbations by a free-radical scavenger on the
oscillations, the following steps 1 and 2 were added to FCA model,
where ArOH indicates a generic compounds containing an OH
phenolic group, e.g. compound 3a.
IN ArOH D HOOꢁ / ArOꢁ D H2O2
DEG ArOH / Products
ð1Þ
ð2Þ
and 3b are quite good, similar to that of
lower than those of other common antioxidants, as quercetin (3.1),
cyanidin (2.48) or -carotene (2.57) [22]. Both the RAC and TEAC
a-tocopherol (0.97) but
The step IN represents the typical way of subtraction of a radical
by an antioxidant: an H atom transfer from a phenolic –OH group to
the radical [33].
b
values of compound 3a are a slight but significantly (t-Student test,
p < 0.05) higher than those of compound 3b. This can be inter-
preted in terms of the BDE (Bond Dissociation Enthalpy) theory by
Wright et al. [29], and with the proposed empirical additivity rules
The formed aroxyl radical ArOꢁ is quite stable and can react with
another radical or with oxygen to give diamagnetic stable
compounds. In the simulations ArOꢁ was considered an end
product. The 1st order step DEG represents the possible parallel
degradation of the inhibitor to unspecified products. The degra-
dation may be due to the oxidation (by acidic iodate) or iodination
(by I2 or HOI) of ArOH. The kinetics of these reactions was recently
studied in detail on some diphenols [28]: the results showed that
for simulation purposes they can be summarized by step DEG. The
kinetic constants of the FCA step were kept fixed to those reported
in ref. 28, while kIN and kDEG were allowed to vary for the best fit to
to calculate the BDE of phenolic OH groups and
DBDE with respect
to phenol (
D
BDE ¼ BDEcomp. ꢀ BDEPhOH) [29]. From the data
reported in ref. 29, it can be seen that the effect of a CH3 group in
ortho- position with respect to a phenolic group in a benzene ring at
a parity of other substituents, decreases the bond enthalpy of the
OH group of 8.4 kJ molꢀ1 indicating that this group acts as a better
radical scavenger than that of the same compound with an H atom
in the ortho- position.
b
a
640
600
560
0
200
400
600
time (s)
Fig. 1. (a) Experimental behavior of V(Iꢀ)/mV vs. time/s for an inhibited BR mixture (initial conditions: [MA] ¼ 0.050 M, [Mn2þ] ¼ 0.0067 M, [IO3ꢀ] ¼ 0.0667 M, [HClO4] ¼ 0.0266 M,
[H2O2] ¼ 1.2 M, 1.0 mL of 3a solution (conc. ¼ 1.01
m
M in mixture) added after the third oscillation). (b) Simulated behavior of [Iꢀ] vs. time for a mixture of the same initial
composition. The satisfactory agreement between the experimental (626 s) and simulated inhibition time (565 s) can be noted.

A. Andreani et al. / European Journal of Medicinal Chemistry 45 (2010) 1374–1378
1377
Table 2
IN and particularly DEG steps represent overall processes for which
individual steps and rate constants have not be determined.
The same agreement was obtained with all the explored
concentrations of 9, Table 3, finding the following unique values for
Experimental and calculated inhibition times (s) for compound 3a.
Conc. (
mM)
tinhib (exptl.)
tinhib (calcd.)
5.06
3.54
2.02
1.01
2917
2073
1106
629
2820
2268
1440
565
the rate constants: kIN ¼ 1.7 ꢃ 108
M
ꢀ1 sꢀ1, kDEG ¼ 1.8 ꢃ 10ꢀ3 sꢀ1
.
These mechanistic calculations are a proof of the possibility of
the reaction (10).
Even though it is not permissible to compare rate constants
from different-order rate equations, kINs are several orders of
magnitude higher than kDEGs, so we can conclude that in any case
the scavenging action by the present compounds with phenolic OH
or OMe groups against HOOꢁ radicals is the source of the observed
perturbations on the oscillations of the Briggs–Rauscher reaction.
experimental behaviors. Simulations were performed by using the
COPASI numerical integration program [34]. Experimental and
simulated behaviors of V(Iꢀ) and [Iꢀ] vs. time for a typical BR
mixture perturbed by compound 3a are reported in Fig. 1(a) and (b)
respectively. The satisfactory agreement between the experimental
and calculated inhibition times can be noted, although IN and
particularly DEG steps represent overall processes for which indi-
vidual steps and rate constants have not be determined.
6. Conclusion
Compounds 3a–c and 9 showed a good chemical antioxidant
activity according to the design of these molecules that include
a phenolic OH or OCH3 group(s) in their structures. The anti-
oxidative mechanism of compound 9 was not directly elucidated.
However perturbations of the Briggs–Rauscher oscillating system
The same agreement was obtained with all the explored
concentrations of 3a, Table 2, finding the following unique values
for the rate constants: kIN ¼ 1.6 ꢃ 108 Mꢀ1 sꢀ1, kDEG ¼ 6.0 ꢃ 10ꢀ4 sꢀ1
.
The obtained values of kIN and kDEG for the perturbed BR reac-
tion by 3a are in line with those obtained for ten substituted
þ
by this compound together with the decolorization of the ABTSꢁ
radical cation, strongly suggest a mechanism similar to the action of
polyphenols. From the bond enthalpies, the O–C(H3) bond
diphenols [28], ranging from 105 to 108 Mꢀ1
s
ꢀ1, and from 0 to
-tocopherol
and peroxyl radicals (ROOꢁ) a rate constant of about 106 Mꢀ1 sꢀ1
10ꢀ5 sꢀ1. It is to be noted that for the reaction between
a
(351 kJ molꢀ1) seemed to us the best candidate to release a CH3
ꢁ
,
radical by bond homolytic breaking. This hypothesis was confirmed
from mechanistic calculations, thus supporting the plausibility of
the proposed mechanism.
To confirm whether this action is also explicated in cultured cells,
further investigation is needed to test the antioxidant power on these
models.Inparticular, sincecompounds3a–bwereselectedforfurther
evaluation of their antitumor activity by the Developmental Thera-
peutics Program (DTP) at the NCI, it will be interesting to consider
these results when all the antitumor tests will be completed.
was experimentally found [33].
To simulate the inhibitory effects by compound 9, the step IN
was modified as follows:
IN ArðOCH3Þ3OCH3 D HOOꢁ / ArðOCH3Þ3Oꢁ D CH3OOH
ð10Þ
that hypothesizes a CH3 group transfer from one OCH3 group to the
hydroperoxyl radical with formation of the methyl hydroperoxide
CH3OOH. This compound was first synthesized in stable form by
Rieche and Hitz [35]. In acidic solution (pH ¼ 2) and in the presence
of metal ions and oxygen, the corresponding aldehyde is finally
formed.
7. Experimental section
The DEG step was left unchanged with the same meaning as
illustrated above.
7.1. Materials and methods. (for the antioxidant activity see
Supplementary data)
Typical experimental and simulated behaviors of V(Iꢀ) and [Iꢀ]
vs. time for a typical BR mixture perturbed by 9 are reported in
Fig. 2(a) and (b) respectively.
The melting points are uncorrected. Elemental analyses were
performed with a Fisons Carlo Erba Instrument EA1108 and
compounds used for tests were at least 95% pure. Bakerflex plates
(silica gel IB2-F) were used for TLC: the eluent was chloroform/
The very good agreement between the experimental and
calculated inhibition times can be noted, although, as stated above,
a
b
680
640
600
0
200
400
600
time (s)
Fig. 2. (a) Experimental behavior of V(Iꢀ)/mV vs. time/s for an inhibited BR mixture (initial conditions: [MA] ¼ 0.050 M, [Mn2þ] ¼ 0.0067 M, [IO3ꢀ] ¼ 0.0667 M, [HClO4] ¼ 0.0266 M,
[H2O2] ¼ 1.2 M, 1.0 mL of 9 solution (conc. ¼ 1.48
m
M in mixture) added after the third oscillation). (b) Simulated behavior of [Iꢀ] vs. time for a mixture of the same initial
composition. The very good agreement between the experimental (553 s) and experimental inhibition time (552 s) can be noted.

1378
A. Andreani et al. / European Journal of Medicinal Chemistry 45 (2010) 1374–1378
Table 3
interpretation of the antioxidant effects. We are grateful to the
University of Bologna for financial support.
Experimental and calculated inhibition times (s) for compound 9.
Conc. (
m
M)
tinhib (exptl.)
tinhib (calcd.)
3.71
3.34
2.97
2.25
1.48
1066
967
865
779
553
1051
993
928
776
552
Appendix. Supplementary data
Scheme S1 (Mechanism of reaction for the formation of
compound 9). NMR spectra of compound 9. Experimental section
and references related to the antioxidant activity. This material
can be found in the online version at doi:10.1016/j.ejmech.2009.
12.035.
methanol in various proportions. Kieselgel 60 was used for column
chromatography; the eluent was a mixture of chloroform/methanol
95/5. The IR spectra were recorded in nujol on a Nicolet Avatar 320
E.S.P.; nmax is expressed in cmꢀ1. The NMR spectra were acquired
with a Varian Mercury-plus spectrometer using library sequences;
the chemical shift (referenced to solvent signal) is expressed in
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Mat95XP apparatus.
7.1.1. Synthesis of 3,3-bis(4-amino-2,5-dimethoxyphenyl)-1,3-
dihydroindol-2-one 9
Isatin (3 mmol) was dissolved in methanol (20 mL) and treated
with 2,5-dimethoxyaniline (3 mmol). The reaction mixture was
refluxed for 18 h and concentrated under reduced pressure: the
resulting solid was purified with column chromatography which
gave 15% of unreacted isatin and 70% of compound 9. Crystallized
from methanol, m.p. 235–238 ꢄC dec..
IR: 3400–3260, 1700, 1210, 1040, 740. 1H NMR (400 MHz, CDCl3,
0
263 K): 3.31 (3H, s, OCH3 bonded to C2 ), 3.58 (3H, s, OCH3 bonded to
C200), 3.57 (3H, s, OCH3 bonded to C5 ), 3.70 (3H, s, OCH3 bonded to
0
C5000 þ 4H, broad s, NH2), 6.31 (1H, s, H3 ), 6.34 (1H, s, H300), 6.40 (1H, s,
0
H6 ), 6.86 (1H, s, H600), 6.82 (1H, d, H7, J ¼ 7.8), 6.88 (1H, td, H5, J ¼ 6.9,
J ¼ 1.3), 7.09 (1H, td, H6, J ¼ 7.6, J ¼ 1.2), 7.30 (1H, d, H4, J ¼ 7.6), 8.25
(1H, s, NH).13C NMR (400 MHz, 0CDCl3, 263 K): 55.9 (OCH3 bonded to
C200), 56.0 (OCH3 bonded to C5 ), 56.4 (OCH3 bonded to C500), 56.8
(OCH3 bonded to C2 ), 59.8 0(C3), 100.7 (C300H), 101.8 (C3 H), 109.0
0
0
(C7H), 112.7 (C600H), 113.9 (C6 H), 115.4 (C100), 116.5 (C1 ), 122.2 (C5H),
0
125.7 (C4H), 127.3 (C6H), 136.4 (C3a), 136.50 (C4 þ C400), 140.3 (C5 ),
140.7 (C500),141.2 (C7a),152.0 (C200),152.2 (C2 ),181.6 (C2). MS (70 eV):
m/z (%): 435 (100) [Mþ], 428 (7), 484 (8), 376 (53), 346 (18), 284 (6),
269 (6), 255 (6), 218 (10). Anal. for C24H25N3O5 (435.47) calcd (%) C
66.19, H 5.79, N 9.65; found (%) C 66.24, H 5.70, N 9.60.
0
0
´
´
´
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7.1.2. Synthesis of 2-(2-acetamidophenyl)-N-(2,5-
dimethoxyphenyl)-2-oxoacetamide 11
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Under the experimental conditions described above, by replac-
ing isatin with N-acetyl-isatin, compound 11 was obtained with
a yield of 40%. Crystallized from methanol, m.p. 154–155 ꢄC.
IR: 3360–3240, 1680, 1660, 1210, 870. 1H NMR (300 MHz,
[D6]DMSO, 25 ꢄC): 2.02 (3H, s, CH3), 3.72 (3H, s, CH3), 3.86 (3H, s,
CH3), 6.73 (1H, dd, ar, J ¼ 9, J ¼ 3), 7.06 (1H, d, ar, J ¼ 9), 7.27 (1H, m,
ar), 7.65 (3H, m, ar), 7.86 (1H, d, ar, J ¼ 3), 9.69 (1H, s, NH),10.59 (1H,
s, NH). 13C NMR (300 MHz, [D6]DMSO, 25 ꢄC): 23.79 (CH3), 55.52
(CH3), 56.46 (CH3), 106.71 (CH), 108.91 (CH), 112.00 (CH), 121.46
(CH), 123.72 (CH), 124.69 (C), 126.86 (C), 130.95 (CH), 133.51 (CH),
137.38 (C), 143.24 (C), 153.18 (C), 160.56 (C), 169.04 (C), 188.85 (C).
MS (70 eV): m/z (%): 342 (16) [Mþ], 189 (8), 179 (5), 162 (100), 153
(37), 146 (48), 138 (65), 128 (27), 118 (18), 98 (20), 65 (11); Anal. for
C18H18N2O5 (342.35) calcd (%) C 63.15, H 5.30, N 8.18; found (%) C
63.00, H 5.43, N 8.24.
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We wish to thank Prof. Dr. Stanley D. Furrow, Penn State Berks,
Reading, PA, USA for the helpful discussion about the mechanistic