Complete assignment of the 1H and 13C NMR spectra of antimicrobial 4-arylamino- 3-nitrocoumarin derivatives

Article abstract of DOI:10.1002/mrc.2681

Herein, we describe the synthesis and complete assignment of the ¹H and ¹³C NMR chemical shifts of a series of antimicrobial 4-arylamino-3-nitrocoumarin derivatives based on a combination of ¹H and ¹³C NMR,

Full text of DOI:10.1002/mrc.2681

MRC Letters
Received: 16 June 2010
Revised: 6 August 2010
Accepted: 10 August 2010
Published online in Wiley Online Library: 5 September 2010
(wileyonlinelibrary.com) DOI 10.1002/mrc.2681
Complete assignment of the 1H and 13C NMR
spectra of antimicrobial 4-arylamino-
3-nitrocoumarin derivatives
a
b∗
c
a
´
´
´
´
´
Vidoslav Dekic, Niko Radulovic, Rastko Vukicevic, Biljana Dekic,
d
b
´
Danielle Skropeta and Radosav Palic
Herein, we describe the synthesis and complete assignment of the 1H and 13C NMR chemical shifts of a series of antimicrobial
4-arylamino-3-nitrocoumarin derivatives based on a combination of 1H and 13C NMR, 1H-1H-COSY, NOESY, HSQC and HMBC
experiments. Conformational effects upon the chemical shifts of the coumarin moiety arising from the anisotropy of the
aryl side group are briefly discussed. This study provides the first complete and fully assigned NMR data for this important
group of antimicrobial compounds and bridges the gap existing in the literature with regard to NMR structural data for
c
4-arylamino-3-nitrocoumarins. Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: 1H NMR; 13C NMR; 2D NMR; 4-arylamino-3-nitrocoumarins; synthesis; structure elucidation; molecular conformation
Introduction
3-nitro-coumarin derivatives [4-(naphthalen-1-ylamino)-3-nitro-
chromen-2-one and 3-nitro-4-phenylamino-chromen-2-one].[13]
Thestructuralelucidationofthesecoumarinderivatives,whichis
importantintheareaoforganicandmedicinalchemistry, isheavily
Coumarins are an important class of naturally occurring
benzopyranones with a widespread occurrence in plants. They
have been used extensively in traditional medicine and are known
to have broad pharmacological functions including antibac-
terial, antifungal, antitumor, anticoagulant, anti-inflammatory,
anti-HIV, vasodilator, sedative, hypnotic, analgesic and hypother-
mic activities.[1–3] We have previously synthesized a number of
coumarin derivatives and tested their in vitro antimicrobial and
antioxidant activities.[4–9] Several of these compounds, in partic-
ular 4-arylamino-3-nitrocoumarin derivatives, showed promising
activity in reducing the microbial growth in both fungal and
bacterial cells.[8,9] Significant antimicrobial activity of a series of
4-substituted-3-nitrocoumarins was also recently confirmed by
a QSAR study.[10] Previous research has shown that 4-amino-
3-nitrocoumarins, tested for pharmacological activity in mice
and rabbits, had neurotropic activity, generally inhibiting spon-
taneous locomotor activity, decreasing hyperactivity induced
by phenamine, and prolonging thiopental sleep.[11] The same
study[11] also pointed out to the most active of the derivatives
containing a secondary amino group, 3-nitro-4-phenylamino-
chromen-2-one, that inhibited phenamine hyperactivity two- to
fivefold and prolonged thiopental sleep twofold. Compounds pos-
sessing a 4-amino-3-nitrocoumarin moiety were also interesting
due to the previously noted steric inhibition of resonance of
the C-3 NO2 group by the C-4 amino group in 4-morpholinyl-3-
nitro-chromen-2-one, evident from their IR and UV spectra.[12] The
analysisofcrystalstructuresandgas-phaseconformations,inferred
from single crystal X-ray and molecular modeling experiments,
showed that the changes in π-delocalization of the farmacoac-
tive formal 3-amino-2-nitro-acrylic acid derivatives might explain
the observed significant difference of the antimicrobial and
antioxidant activities and spectral properties of two 4-arylamino-
1
reliant on H and 13C NMR spectroscopy. However, many of the
reported studies of these derivatives either do not provide NMR
spectral data at all, or provide only limited, and often unassigned,
NMR data.[14–17] This situation is further complicated by the fact
that full NMR assignments are often not performed due to a partial
orcompleteoverlapoftheprotonsignalsofthecoumarinicmoiety
and also of the aryl side group. The same situation is found in the
literature regarding the assignment of the 13C NMR spectral data
for many coumarin derivatives.[18–23]
To address the paucity of fully assigned NMR spectral data
for these types of compounds, a series of seven 4-arylamino-
3-nitrocoumarins of varying complexity were synthesized and
their 1H and 13C NMR spectral data fully assigned based on a
combination of 1D and 2D NMR experiments including COSY,
NOESY, HSQC, and HMBC.
∗
´
Correspondence to: Niko Radulovic, Department of Chemistry, Faculty of
ˇ
ˇ
ˇ
Science and Mathematics, University of Nis, Visegradska 33, 18000 Nis, Serbia.
E-mail: vangelis0703@yahoo.com
a
b
c
Department of Chemistry, Faculty of Science and Mathematics, University of
ˇ
Pristina, Lole Ribara 29, 38220 Kosovska Mitrovica, Serbia
Department of Chemistry, Faculty of Science and Mathematics, University of
ˇ
ˇ
ˇ
Nis, Visegradska 33, 18000 Nis, Serbia
Department of Chemistry, Faculty of Science, University of Kragujevac,
´
R. Domanovica 12, 34000 Kragujevac, Serbia
d
School of Chemistry, University of Wollongong, Wollongong, NSW
2522, Australia
c
Magn. Reson. Chem. 2010, 48, 896–902
Copyright ꢀ 2010 John Wiley & Sons, Ltd.

1H and 13C NMR spectra of antimicrobial 4-arylamino-3-nitrocoumarin derivatives
From the HMBC spectrum (Fig. 2) it was possible to determine
which one of the two groups of aromatic protons mentioned
above was bound to which ring. A broad signal at δ 13.78 ppm
assigned to the carboxyl proton showed a single interaction with
a carbon at 125.9 ppm, attributed to the ipso aryl carbon at C-2ꢁ.
This carbon (C-2ꢁ) in turn showed cross peaks with the protons at
7.16 and 6.86 ppm. The latter was assigned to H-6ꢁ and appeared
as a doublet of doublets as expected due to the significantly
different coupling constants between H-6ꢁ and H-5ꢁ (J = 8.0 Hz)
and H-6ꢁ and H-4ꢁ (J = 1.0 Hz). The proton signal at 7.16 ppm,
which was assigned to H-4ꢁ, appeared as a doublet of doublet of
doublets as expected due to vicinal coupling with both H-3ꢁ and
H-5ꢁ (J = 8.0 and 9.0 Hz, respectively) and long-range coupling
with H-6ꢁ (J = 1.0 Hz).
The aromatic methine resonances appearing at 8.11 and
7.27 ppm were attributed to H-3ꢁ (dd, J = 8.0, 1.5 Hz) and H-5ꢁ
(ddd, J = 9.0, 8.0, 1.5 Hz), respectively, based on similar reasoning
to that just described. The carbon methine signals (Table 1) of the
C ring (i.e. C-3ꢁ, C-4ꢁ, C-5ꢁ, and C-6ꢁ) were readily connected to
the aforementioned 1H NMR signals according to correlations in
theHSQCspectrum, andfurthercorroboratedbyHMBCdata. Thus,
the group of protons with chemical shifts at 6.86, 7.16, 7.27, and
8.11 ppm was convincingly allocated to ring C.
The remaining two carbons of the anthranilic side group, C-1ꢁ
and the carboxyl carbon atom, were assigned as follows (Table 1):
carbon C-1ꢁ (139.2 ppm) interacted through three bonds with the
protons H-3ꢁ and H-5ꢁ as seen in the HMBC spectrum. Additionally,
the interaction of H-3ꢁ with a carbon appearing at 172.5 ppm in
HMBC, along with its distinctive chemical shift, made it possible to
assign the carboxyl carbon.
Scheme 1. Synthesis of 4-arylamino-3-nitrocoumarin derivatives 3a–g.
Results and Discussion
The reaction of 4-chloro-3-nitrocoumarin (1) with a range of
differently substituted arylamines (2a–g) in a 1 : 1 molar ratio
in ethyl acetate in the presence of triethylamine gave seven
4-arylamino-3-nitrocoumarin derivatives (3a–g), in moderate to
high yields (65–87%) (Scheme 1). The coumarin derivatives varied
inthenatureandpositionoftheirarylaminosubstituentsincluding
ortho-, meta-, and para-derivatives, along with a mixture of weak
and strong electron-donating groups (e.g. methyl, amino) and
electron-withdrawing groups (e.g. nitro, carboxylic acid). Five of
the derivatives prepared were novel (3b, 3c, 3d, 3f and 3g), while
two of the derivatives (3a and 3e) were reported previously.[11,12]
However, only limited NMR data are available for the two known
compounds (3a and 3e),[11,12] and thus their 13C NMR spectral
HMBC was also informative in assigning the peaks of the second
proton group. A signal at 7.88 ppm, which appeared as a doublets
of doublets, had significant cross peaks with quaternary carbons
at 146.0 and 152.5 ppm, as well as with a methine carbon at
133.9 ppm, attached to a proton of 7.60 ppm (HSQC). Additionally,
the doublet of doublets at 7.34 ppm showed correlations with
carbons at 115.1 and 152.5 ppm (quaternary) and with a carbon
at 124.1 ppm, having a HSQC interaction with the resonance at
1
7.22 ppm in the H-NMR spectrum. Thus, the proton occupying
position 5 is demonstrated to appear at 7.88 ppm, and interacts
through three bonds, as seen in the HMBC spectrum (Fig. 2), with
the quaternary carbons C-4 (146.0) and C-8a (152.5), while H-8
(7.34) showed interactions with C-4a (115.1, three bonds apart)
and a two-bond interaction with C-8a (152.5). This spectrum also
contained evidence that a carbon at 133.9 ppm is C-7 by the
presence of a cross peak with H-5, which then allowed assignment
of H-7 to the peak at 7.60 ppm (HSQC). Analogously, H-8 correlated
with a carbon at 124.1 ppm (C-6), and thus the shift of the proton
H-6 was deduced to be 7.22 ppm from HSQC. The assignment of
carbons C-4a and C-8a was supported through additional HMBC
correlations between H-6 and C-4a and H-7 and C-8a. Finally, the
last two carbon signals at 155.4 and 118.9 ppm were attributed to
the lactone carbonyl carbon (C-2) and nitro-bearing carbon (C-3),
respectively, based on their chemical shifts, since no H interactions
wereobservedinanyofthe2Dspectraandbycomparisonwiththe
analogous signals in the biphenyl derivative 3b (discussed below).
Further confirmation of the NMR assignments of 3a was
obtained from the NOESY spectrum. The proton H-5 was shown
to interact with both the carboxyl hydrogen and H-6ꢁ, indicating
the orientation of the aryl substituent bound to the coumarin
framework. The abovementioned cross peaks suggest that the C
ring is oriented away from the nitro group and that the A and C
1
data, as well as the H NMR spectral data for compound 3e, are
reported here for the first time.
The anthranilic acid derivative 3a was obtained as a yellow
crystalline solid. High-resolution electron impact mass spectrom-
etry (HR-EIMS) of 3a indicated a molecular formula of C16H10N2O6
([M]+ at m/z 326.0560, ꢀ = +2.1 mmu). The IR spectrum was
found to be identical with that previously described[11] showing
characteristic vibrations at 1330 and 1553 cm−1 (NO2), 1703 cm−1
1
(C O) and 3420 cm−1 (O–H, N–H). The H NMR spectrum (Ta-
ble 1) of compound 3a exhibited eight aromatic methine signals,
which appeared as four doublets of doublets and four doublets of
triplets. The cross peaks observed in the 1H–1H COSY and NOESY
(Fig. 1) spectra for 3a differentiated two groups of signals, that
is, two separate proton spin systems, belonging to rings A and C
(Fig. 1). The first group comprised the protons that appear in the
1H NMR spectrum (Table 1) as two sets of doublets of doublets
at 7.88 and 7.34 ppm and two sets of doublets of triplets at 7.22
and 7.60 ppm, while the second group consists of two sets of
doublets of doublets at 6.86 and 8.11 ppm and two sets of doublet
of doublet of doublets at 7.16 and 7.27 ppm, respectively.
c
Magn. Reson. Chem. 2010, 48, 896–902
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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V. Dekic´ et al.
Table 1. NMR dataa of compounds 3a and 3b in CDCl3
Compound
3a
3b
Position
δH, m (J, Hz)
δC
NOESY
HMBCb
δH, m (J, Hz)
δC
NOESY
HMBCb
2
–
–
155.4
118.9
146.0
115.1
125.7
–
–
–
154.2
117.5
152.1
113.2
126.9
–
–
–
–
6
–
3
–
–
–
–
–
4
–
–
–
–
–
–
4a
5
–
–
–
7.88, dd
(8.5, 1.0)
7.22, td
(8.5, 1.0)
7.60, td
(8.5, 1.0)
7.34, dd
(8.5, 1.0)
–
6, 6ꢁ, COOH
4, 7, 8a
7.55, dd
(8.5, 1.5)
7.00, td
(8.5, 1.0)
7.56, ddd
(8.5, 8.0, 1.5)
7.32, dd
(8.0, 1.0)
–
4, 7, 8a
6
7
8
124.1
133.9
117.7
5, 7
6, 8
7
4a, 8
5, 8a
123.6
134.8
118.1
5, 7
6, 8
7
4a, 8
5, 8a
4a, 6, 8a
4a, 6, 8a
8a
NH
1ꢁ
2ꢁ
3ꢁ
152.5
–
–
–
–
–
152.8
–
–
–
3, 4a
v
11.20, brs
–
11.18, brs
–
2ꢁ-OCH3
139.2
125.9
131.6
–
–
143.0
152.8
110.2
–
–
–
4ꢁ
–
–
–
–
8.11, dd
(8.0, 1.5)
7.16, ddd
(9.0, 8.0, 1.0)
7.27, ddd
(9.0, 8.0, 1.5)
6.86, dd
(8.0, 1.0)
13.78, brs
–
1ꢁ, 5ꢁ,
COOH
2ꢁ, 6ꢁ
7.13, d
(1.5)
2ꢁ-OCH3, 6ꢁꢁ
1ꢁ, 5ꢁ, 1ꢁꢁ
4ꢁ
5ꢁ
6ꢁ
124.5
130.2
118.8
3ꢁ, 5ꢁ
4ꢁ, 6ꢁ
5ꢁ, 5
–
125.4
119.2
125.3
–
6ꢁ, 2ꢁꢁ, 6ꢁꢁ
5ꢁ
–
1ꢁ, 3ꢁ
2ꢁ, 4ꢁ
7.15, dd
(8.0, 1.5)
7.19, d
(8.0)
1ꢁ, 3ꢁ, 1ꢁꢁ
2ꢁ, 4ꢁ
COOH
2ꢁ-OCH3
1ꢁꢁ
172.5
5
–
–
–
2ꢁ
–
–
–
–
3ꢁ, NH
–
–
2ꢁ
–
–
–
3.84, s
–
55.8
130.0
109.1
–
–
–
2ꢁꢁ
–
–
7.01, d
(2.0)
3ꢁꢁ- OCH3, 5ꢁ
4ꢁ, 4ꢁꢁ, 6ꢁꢁ
3ꢁꢁ
4ꢁꢁ
5ꢁꢁ
–
–
–
–
–
–
–
–
–
–
–
–
–
147.5
136.7
114.9
–
–
–
–
–
6.80, d
(8.0)
6ꢁꢁ
1ꢁꢁ, 3ꢁꢁ
6ꢁꢁ
–
–
–
–
7.07, dd
(8.0, 2.0)
3.95, mc
3.95, mc
120.0
3ꢁ, 5ꢁ, 5ꢁꢁ
4ꢁ, 2ꢁꢁ, 4ꢁꢁ
3ꢁꢁ-OCH3
4ꢁꢁ-NH2
–
–
–
–
–
–
–
–
55.7
–
2ꢁꢁ
–
3ꢁꢁ
–
a Measured at 500 (1H) and 125 MHz (13C).
b HMBC interactions of the hydrogen from the column Position with carbons from the column HMBC.
c Overlapping signals.
rings do not lie in the same plane, but rather are at a specific angle
to one another. Simultaneous interaction of H-5 with H-6ꢁ and the
carboxyl hydrogen is unlikely to occur in a single conformation of
the molecule, but may result from single-bond rotation around the
C1ꢁ –N bond giving rise to different conformations that are close
in energy (this was confirmed by energy minimization calculations
using MM2 force field from the ChemDraw 11.0 software package).
The biphenyl derivative 3b was obtained as a brown crystalline
solid. The molecular formula (C23H19N3O6) of 3b was deduced
from the HR-EIMS of the M+ ion at m/z 433.1285 (ꢀ = +1.1 mmu).
N–H and Ar–H absorptions in the range of 3039–3376 cm−1 and
a strong band at 1702 cm−1 corresponding to the C O group
were observed in the IR spectrum of the synthesized compound.
Evidence for the presence of the NO2 group was provided by the
IR absorptions that appeared at 1344 and 1537 cm−1
.
The 1H NMR spectrum of compound 3b contained ten aromatic
methine resonances, four of which (7.00, 7.32, 7.55, and 7.56 ppm)
were readily grouped together based on their mutual interactions
in the NOESY spectrum (Fig. 3). These four aromatic protons were
attributed to ring A, due to their splitting patterns (two dds and
two ddds) and 1H–1H COSY interactions. The chemical shift of
the carbon atoms to which these protons were bonded to was
subsequently determined from the HSQC spectrum. In the HMBC
spectrum(Fig. 4),theprotonresonanceat7.55 ppmwascorrelated
with a protonated carbon at 134.8 ppm, which in turn showed an
HSQC correlation to the proton signal at 7.56 ppm. Further HMBC
c
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Magn. Reson. Chem. 2010, 48, 896–902

1H and 13C NMR spectra of antimicrobial 4-arylamino-3-nitrocoumarin derivatives
Figure 1. The NOESY correlations of compound 3a.
Figure 4. The HMBC correlations of compound 3b.
coupling with H-7 of 8.5 and 1.5 Hz, respectively. The remaining
doublet of doublets from this group at 7.32 ppm was thereby
assigned to H-8, and confirmed by HMBC, which showed a three-
bond correlation with the C-6 (123.6 ppm) and C-4a (113.2 ppm)
signals, and through two bonds with C-8a at 152.8 ppm.
In addition to the aromatic protons, integration of the 1H
NMR spectrum of 3b shows an additional nine protons: one at
11.18 ppm, five at 3.95 ppm, and three at 3.84 ppm. The signal at
11.18 ppm was readily assigned to the proton of the secondary
amino group connecting the coumarin and dianisidine parts of
the molecule based on its chemical shift. The two protons of the
primary amino group and the three protons of one of the methoxy
groups were found to overlap as a multiplet at 3.95 ppm, while
the other methoxy protons appeared as a singlet at 3.84 ppm. A
NOESYinteractionbetweenthesignalsat11.18 ppmand3.84 ppm
allowed the assignment of the latter signal to the methoxy group
on the C ring and demonstrated that these two groups are close
in space to one another. A further NOESY cross peak between the
methoxy protons with a signal at 7.13 ppm was used to assign the
H-3ꢁ proton, and accordingly the C-3ꢁ carbon (110.2 ppm, HSQC).
The remaining protons of the C ring were determined from HMBC
data (Fig. 4). The three-bond coupling of the H-3ꢁ proton to the
carbon atom at 119.2 ppm, which was in turn correlated to the
proton at 7.15 ppm (by HSQC), allowed the assignment of these
two resonances as C-5ꢁ and H-5ꢁ, respectively. The proton H-6ꢁ was
resolved by its interaction with H-5ꢁ in the NOESY spectrum, and
C-6ꢁ assigned from HSQC data. The remaining carbons on the C
ring were identified on the basis of HMBC interactions with the
assigned protons.
Figure 2. The HMBC correlations of compound 3a.
Figure 3. The NOESY correlations of compound 3b.
The assignment of the D ring carbons and protons was
performed starting from the three-bond interaction of H-3ꢁ and
H-5ꢁ with a carbon at 130.0 ppm in the HMBC spectrum. Since this
carbon atom does not fit into the C ring, it was obvious that its
position should be C-1ꢁꢁ. This was also confirmed by a three-bond
correlation with a proton at 6.80 ppm identified as H-5ꢁꢁ. Further
interactions observed for H-5ꢁꢁ aided the assignment of C-3ꢁꢁ, H-6ꢁꢁ,
and C-6ꢁꢁ from the corresponding spectra. The carbon resonance
for C-3ꢁꢁ at 147.5 ppm is also correlated with the D ring methoxy
protons (3.95 ppm) through three bonds. The remaining aromatic
proton (H-2ꢁꢁ) was assigned to the signal at 7.01 ppm, which was
confirmed by its interaction with C-6ꢁꢁ (HMBC). Both H-2ꢁꢁ and H-6ꢁꢁ
showed a three-bond correlation with a C signal at 136.7 ppm,
assigned to the carbon atom bonded to the NH2 group (C-4ꢁꢁ).
correlations for the peak at 7.55 ppm were observed with two
quaternary carbons at 152.1 and 152.8 ppm. The proton signal
at 7.32 ppm showed HMBC cross peaks with a carbon signal at
123.6 ppm (to which a proton with a signal at 7.00 ppm was
bonded), and to two quaternary carbon signals at 113.2 and
152.8 ppm.
On the basis of the data described above, it can be concluded
that the proton resonance at 7.55 ppm (dd) occupies position 5 on
theAringofcompound3b.H-5showedthroughspaceinteractions
with C-7 (134.8 ppm, thus also making possible the assignment of
H-7, 7.56 ppm), C-4 (152.1 ppm), and C-8a (152.8 ppm). H-5 also
showed vicinal coupling with H-6 (which must then appear at
7.00 ppm, in accordance with the NOESY data) and long-range
c
Magn. Reson. Chem. 2010, 48, 896–902
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

V. Dekic´ et al.
Table 2. 1H NMR chemical shifts (δ, ppm), multiplicity and coupling constants (J, Hz) of coumarin derivatives 3c–g (500 MHz, CDCl3)
Compound
Position
H-5
3c
3d
3e
3f
3g
7.38, dd
(8.5, 1.5)
6.96, td
(8.5, 1.5)
7.54, td
(8.5, 1.5)
7.30, dd
(8.5, 1.5)
11.54, brs
7.03, d
(8.5)
7.41, dd
(8.5, 1.5)
7.06, ddd
(8.5, 7.5, 1.5)
7.64, ddd
(8.5, 7.5, 1.5)
7.28, dd
(8.5, 1.5)
10.71, brs
8.11, dd
7.36, dd
(8.5, 1.5)
6.99, td
(8.5, 1.0)
7.56, ddd
(8.5, 8.0, 1.5)
7.29, dd
(8.0, 1.0)
11.28, brs
7.15, d
7.70, dd
(8.5, 1.5)
7.08, td
(8.5, 1.5)
7.61, td
(8.5, 1.5)
7.32, dd
(8.5, 1.5)
11.42, brs
–
7.31, dd
(8.5, 1.5)
7.04, td
(8.5, 1.0)
7.60, td
(8.5, 1.5)
7.35, dd
(8.5, 1.0)
11.04, brs
6.99, d
(8.5)
H-6
H-7
H-8
NH
H-2ꢁ
(2.5, 2.0)
–
(8.0)
H-3ꢁ
H-4ꢁ
H-5ꢁ
H-6ꢁ
6.72, d
(8.5)
7.25, d
7.14, dd
(8.0, 1.0)
7.23, ddd
(8.0, 7.0, 1.0)
7.34, ddd
(8.5, 7.0, 1.0)
7.32, dd
(8.5, 1.0)
2.37, s
7.77, d
(8.5)
(8.0)
–
8.22, ddd
–
–
(8.0, 2.0, 1.0)
6.72, d
(8.5)
7.03, d
(8.5)
–
7.63, t
7.25, d
(8.5)
7.15, d
(8.5)
–
7.77, d
(8.5)
6.99, d
(8.5)
–
(8.0)
7.53, ddd
(8.0, 2.5, 1.0)
C-2ꢁ- CH3
C-4ꢁ-CH3
NH2
–
–
–
–
2.42, s
–
–
–
3.91, s
–
The HMBC spectrum of the biphenyl derivative 3b possessed an
important piece of information regarding the connection of the
coumarin and dianisidine moieties. The proton resonance for the
secondary amino group (N–H) showed a three-bond correlation
with both C-4a from the juncture of rings A and B and with the
carbon at 117.5 ppm assigned to C-3. This, coupled with NOESY
data for the NH signal, confirms the existence of the amino bridge.
The only remaining carbon to be assigned was C-2, the lactone
carbon, which was thereby attributed to the signal appearing
at 154.2 ppm in the 13C spectrum. The mutual spatial relation
between the C and D rings can be ascertained from proton NOESY
interactions. For example, interactions between both H-3ꢁ and
H-6ꢁꢁ and H-5ꢁ and H-6ꢁꢁ in the NOESY spectrum suggest that the
C and D rings are not in the same plane but rather are at an
angle to one another, and/or able to adopt different low-energy
conformationsthroughrotationaboutthesinglebondconnecting
C-4ꢁ and C-1ꢁꢁ.
The 1H and 13C NMR spectral data for the five other coumarin
derivatives (3c–g) were fully assigned using similar reasoning to
that described above, with the results presented in Tables 2 and 3.
In compounds having ortho-substituents on the aryl side groups
(3a, 3b, and 3f), the chemical shift of H-5 and H-6 is increased
in value compared to the analogous peaks in the compounds
without ortho-substituted aryl groups. This may be a consequence
of a different spatial arrangement resulting in differing anisotropic
influence of the aryl groups on these protons.
Table 3. 13C NMR chemical shifts (δ, ppm) of coumarin derivatives
3c–g (125 MHz, CDCl3)
Compound
Position
3c
3d
3e
3f
3g
C-2
154.7
117.4
152.2
112.3
128.4
123.5
134.8
118.2
153.2
128.8
126.3
116.0
147.0
116.0
126.3
–
153.4
115.3
151.3
114.5
127.3
124.0
135.4
118.5
151.8
143.4
119.2
148.8
122.0
131.1
129.7
–
154.1
117.3
151.6
112.3
128.0
123.6
134.9
118.0
153.1
135.6
124.6
130.7
138.5
130.7
124.6
–
154.5
116.6
150.7
112.8
126.7
124.0
134.6
117.7
152.4
136.4
136.4
125.7
127.1
131.4
128.5
18.0
153.8
110.1
151.3
111.8
128.0
123.8
135.3
118.4
153.6
138.5
126.4
139.4
92.8
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
C-1ꢁ
C-2ꢁ
C-3ꢁ
C-4ꢁ
C-5ꢁ
139.4
126.4
–
C-6ꢁ
C-2ꢁ-CH3
C-4ꢁ-CH3
–
–
21.1
–
–
Finnigan-MAT 8230 BE mass spectrometer. The IR measurements
(attenuated total reflectance, ATR) were carried out with a Thermo
Nicolet 6700 FTIR instrument. For TLC, silica gel plates (Kiesel 60
F254, Merck) were used. Visualization was affected by spraying
the plates with 1 : 1 aqueous sulfuric acid and then heating.
All the reagents and solvents were obtained from commercial
sources (Aldrich, USA; Merck, Germany; Fluka, Germany) and
Experimental
General remarks
Melting points were determined on a Kofler hot-plate apparatus
and are uncorrected. HRMS(EI) spectra were recorded on a
c
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2010, 48, 896–902

1H and 13C NMR spectra of antimicrobial 4-arylamino-3-nitrocoumarin derivatives
used as supplied, except the solvents, which were purified by
distillation.
2-[(3-nitro-2-oxo-2H-chromen-4-yl)amino]benzoic acid (3a,
C16H10N2O6)
Yellow crystals, yield 79%, mp 226–228 ◦C (Lit.[11] 227–228 ◦C).
HRMS(EI): M+ (C16H10N2O6) 326.0560, requires 326.0539 (ꢀ =
+2.1 mmu). IR (neat): 3420 (O–H, N–H), 1703 (C O), 1606 (C C),
NMR spectra
1553 and 1330 (NO2), 1289, 1032, 823, 782 cm−1. For 1H and 13
C
All NMR spectra were recorded at 25 ◦C in CDCl3 with TMS as
an internal standard. Chemical shifts are reported in ppm (δ) and
referenced to TMS (δH = 0 ppm) in 1H NMR spectra or to residual
CDCl3 (δH = 7.25 ppm, δC = 77 ppm) in heteronuclear 2D spectra.
Scalar couplings are reported in Hertz. Typically, 20–30 mg of
sample was dissolved in 1 ml of CDCl3, and 0.7 ml of the solution
transferred into a 5-mm Norell 507 NMR tube.
The1Hand13CNMRspectraofcompounds3a–gwererecorded
on a Bruker DMX 500 spectrometer operating at 500.26 and
125.8 MHz, respectively, equipped with a 5-mm dual 13C/1H probe
head. The 1H spectra were recorded with 16 scans, 1 s relaxation
delay, 4 s acquisition time, 0.125 Hz digital FID resolution, 51 280
FID size, with 6410 Hz spectral width, and an overall data point
resolution of 0.0003 ppm. The 13C spectra were recorded with
Waltz 161H broadband decoupling, 1024 scans, 0.5 s relaxation
delay, 1 s acquisition time, 0.5 Hz digital FID resolution, 65 536 FID
size, 31 850 Hz spectral width, and an overall data point resolution
of 0.005 ppm.
2D spectra were recorded on a Bruker DMX 500 spectrometer
(500.26 MHz for 1H, 125.8 MHz for 13C) equipped with an inverse
detection triple resonance 5-mm probe (TXI). Standard pulse
sequences were used for 2D spectra. COSY and NOESY spectra
were recorded at spectral widths of 5 kHz in both F2 and F1
domains; 1 K × 512 data points were acquired with 32 scans per
increment and the relaxation delays of 2.0 s. The mixing time
in NOESY experiments was 1 s. Data processing was performed
on a 1K × 1K data matrix. Inverse-detected 2D heteronuclear
correlated spectra were measured over 512 complex points in
F2 and 256 increments in F1, collecting 128 (HSQC) or 256
(HMBC) scans per increment with a relaxation delay of 1.0 s.
The spectral widths were 5 and 27 kHz in F2 and F1 dimensions,
respectively. The HSQC experiments were optimized for C–H
couplings of 145 Hz; the HMBC experiments were optimized
for long-range C–H couplings of 10 Hz. Fourier transforms were
performed on a 512 × 512 data matrix. π/2 Shifted sine-squared
window functions were used along F1 and F2 axes for all 2D
spectra.
NMR spectra, see Table 1. For NOESY, HMBC and HSQC spectra,
see Supporting information.
4-[(4ꢁ-amino-3,3ꢁ-dimethoxybiphenyl-4-yl)amino]-3-nitro-2H-
chromen-2-one (3b, C23H19N3O6)
Brown crystals, yield 72%, mp 215–218 ◦C. HRMS(EI): M+
(C23H19N3O6) 433.1285, requires 433.1274 (ꢀ = +1.1 mmu). IR
(neat): 3376–3039 (N–H and Ar–H), 2941 (C–H), 1702 (C O),
1608 (C C), 1537 and 1344 (NO2), 1224, 1032, 751, 690 cm−1. For
1H and 13C NMR spectra, see Table 1. For NOESY, HMBC and HSQC
spectra, see Supporting information.
4-[(4-aminophenyl)amino]-3-nitro-2H-chromen-2-one(3c,
C15H11N3O4)
Brown crystals, yield 84%, mp 228–230 ◦C. HRMS(EI): M+
(C15H11N3O4) 297.0743, requires 297.0750 (ꢀ = −0.7 mmu). IR
(neat): 3371–3068 (N–H and Ar–H), 1710 (C O), 1605 (C C),
1549 and 1332 (NO2), 1240, 1056, 945, 782, 756 cm−1. For 1H and
13C NMR spectra, see Tables 2 and 3.
3-nitro-4-[(3-nitrophenyl)amino]-2H-chromen-2-one(3d,
C
15H9N3O6)
Yellow crystals, yield 72%, mp 253–255 ◦C. HRMS(EI): M+
(C15H9N3O6) 327.0474, requires 327.0491 (ꢀ = −1.7 mmu). IR
(neat): 3348–3080 (N–H and Ar–H), 1689 (C O), 1606 (C C),
1548 and 1354 (NO2), 1266, 1064, 821, 761 cm−1. For 1H and 13
NMR spectra, see Tables 2 and 3.
C
4-[(4-methylphenyl)amino]-3-nitro-2H-chromen-2-one(3e,
C
16H12N2O4)
Yellow crystals, yield 70%, mp 192–194 ◦C (Lit.[12] 193–195 ◦C).
HRMS(EI): M+ (C16H12N2O4) 296.0815, requires 296.0797 (ꢀ =
+1.8 mmu). IR (neat), 3276–3072 (N–H and Ar–H), 2919 (CH3),
1693 (C O), 1610 (C C), 1548 and 1322 (NO2), 1208, 1054, 813,
757 cm−1. For 1H and 13C NMR spectra, see Tables 2 and 3.
Synthesis of 4-chloro-3-nitrocoumarin (1)
4-[(2-methylphenyl)amino]-3-nitro-2H-chromen-2-one(3f,
4-Hydroxycoumarin (Merck, Germany) was nitrated using 72%
HNO3 in glacial AcOH according to a published procedure,[12] to
afford 4-hydroxy-3-nitrocoumarin. The starting compound 1 was
prepared from 4-hydroxy-3-nitrocoumarin following the method
of Chechi et al.,[24] modified by Kaljaj et al.[25] Melting point, IR and
1H NMR spectral data were identical to those described.[24]
C
16H12N2O4)
Yellow crystals, yield 81%, mp 180–182 ◦C. HRMS(EI): M+
(C16H12N2O4) 296.0781, requires 296.0797 (ꢀ = −1.6 mmu). IR
(neat): 3120–3036 (N–H and Ar–H), 2947 (CH3), 1705 (C O), 1612
(C C), 1556 and 1323 (NO2), 1218, 1054, 798, 749 cm−1. For H
1
and 13C NMR spectra, see Tables 2 and 3.
General synthesis of 4-arylamino-3-nitrocoumarins (3a–g)
4-[(4-iodophenyl)amino]-3-nitro-2H-chromen-2-one (3g,
C
15H9IN2O4)
The solution of 4-chloro-3-nitrocoumarin (1 g, 4.4 mmol) and the
appropriate arylamine (2a–g) (4.4 mmol) in ethyl acetate (10 cm3)
in the presence of triethylamine (1 cm3, 7.2 mmol) was refluxed for
1–3 h. After cooling, the precipitated solid was filtered off, washed
with ethyl acetate and then water. The purity of the synthesized
compounds was assessed by TLC.
Yellow crystals, yield 76%, mp 250–253 ◦C. HRMS(EI): M+
(C15H9IN2O4) 407.9616, requires 407.9607 (ꢀ = +0.9 mmu). IR
(neat): 3286 (N–H), 1686 (C O), 1604 (C C), 1548 and 1334
(NO2), 1207, 1057, 843, 784 cm−1. For 1H and 13C NMR spectra, see
Tables 2 and 3.
c
Magn. Reson. Chem. 2010, 48, 896–902
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

V. Dekic´ et al.
ˇ
[10] Z. Debeljak, A. Skrbo, I. Jasprica, A. Mornar, V. Plecˇko, M. Banjanac,
Acknowledgements
ˇ
M. Medic´-Saric´, J. Chem. Inf. Model. 2007, 47, 918.
TheauthorsaregratefultoProf.SlobodanMilosavljevic´ andDrNina
Todorovic´, from the Faculty of Chemistry, University of Belgrade,
fortheNMRmeasurements. TheauthorsacknowledgetheMinistry
of Science and Technological Development of Serbia for financial
support (project 172061).
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2010, 48, 896–902