Structural elucidation of a series of 6-methyl-3-carboxamidocoumarins

Full text of DOI:10.1002/mrc.4541

Structural elucidation of a series of 6-methyl-3-carboxamidocoumarins
André Fonseca,a Alexandra Gaspar,a Maria João Matos,a Ligia R. Gomes,b,c John N. Low,d
Eugenio Uriarte,e Fernanda Borgesa
a) CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto,
4169-007, Porto, Portugal.
b) FP-ENAS-Faculdade de Ciências de Saúde, Escola Superior de Saúde da UFP, Universidade
Fernando Pessoa, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal
c) REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of
Porto, 4169-007, Porto, Portugal.
d) Department of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen, AB24 3UE,
Scotland
e) Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela,
15782 Santiago de Compostela, Spain.
Introduction
The original concept of privileged structure introduced by Evans in 1988 has evolved from
the original meaning.[1] Nowadays, privileged structures can be defined as molecular
frameworks that are able to provide potent and selective ligands for more than one type of
biological targets through judicious structural modifications.[2] Benzopyrone based systems,
namely chromones and coumarins, are actually recognized as privileged structures[3] and used
as templates for the design of new chemical libraries for drug discovery programs.[4]
Simple coumarins are considered an unparalleled template to perform structural
modifications allowing the synthesis of an array of compounds with a remarkable
pharmacological profile exhibiting diverse activities such as anticancer, antiviral, anti-
inflammatory and antimicrobial.[5] Due to the synthetic accessibility and substitution
variability these heterocyclic compounds play an important role not only in the Organic
Chemistry but also in the Medicinal Chemistry field.
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The application of coumarins in the development of therapeutic solutions for aging related
diseases is still an emerging field even though the data acquired so far point out their
importance in the development of novel drug candidates for targets ascribed to Alzheimer’s
and Parkinson’s diseases. In the past years our group has screened a large library of
benzopyrane based derivatives towards monoaminoxidase isoforms (MAO-A and MAO-B).
MAO-B is an important target in Parkinson’s disease as it plays a key role in the metabolism
of dopamine, a neurotransmitter critical for the maintenance of cognitive function.[6] Within
this framework some coumarin-based MAO-B inhibitors have been discovered.[5] Such
knowledge led to an ongoing effort to improve their potency and selectivity, and as a result a
small library of 6-methyl-3-carboxamidocoumarin derivatives (Table 1) was designed and
synthesized. Along this framework, a complete structural elucidation by one- (1D) and two-
dimensional (2D) NMR techniques of the heterocyclic compounds is found to be relevant.
Therefore, in this work some coumarins of our library containing different electron donor
groups (EDG) in diverse positions of the exocyclic aromatic ring (Scheme 1, Table 1) were
selected for complete structural analysis. Finally, to complete the study, single crystal X-ray
crystallographic
analysis
of
compound
3
(N-(4’-hydroxyphenyl)-6-methyl-3-
carboxamidocoumarin) was carried out.
Experimental
General procedures
1H and 13C NMR were recorded on a Bruker Avance III 400 NMR spectrometer operating at
1
400.14 MHz and 100.62 MHz, respectively. For the H NMR experiments, the relaxation
13
delay was 90° pulse, spectral width of 8012 Hz and 65K data points. In the case of the C
NMR experiments the corresponding parameters were 30° pulse, 24038 Hz and 65K
respectively and 2.0s relaxation delay. For the Distortionless Enhancement by Polarization
Transfer (DEPT) sequence the width of the 90◦ pulse for 13C was 7.7 μs, and the 90º pulse for
1H was 9.8 μs; the delay 2JC, H was set to 2.0 ms. For correlation spectroscopy (COSY) and
heteronuclear single quantum coherence (HSQC) the data points were set to 2ꢀ Kꢀ ×ꢀ 256
(t2ꢀ ×ꢀ t1) with a relaxation delay D1 of 1.5s. The heteronuclear multiple bond connectivity
(HMBC) was acquired with data points set to 4ꢀ Kꢀ ×ꢀ 256 (t2ꢀ ×ꢀ t1) and relaxation delay
D1 of 1s. Furthermore, the long-range coupling time for HMBC was set to 71ms. The data
were processed using quadratic sine-bell weighting functions in both dimensions. The spectra
were recorded at room temperature in 5 mm outer-diameter tubes. Samples were prepared in
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deuterated dimethyl sulfoxide (DMSO-d6) for compounds 1-3 and deuterated chloroform
(CDCl3) for compounds 4-6. Tetramethylsilane (TMS) was used as internal standard, and
chemical shifts (δ) were expressed in parts per million (ppm) and coupling constants (J) in
Hertz. The 1H and 13C chemical shifts of DMSO-d6 were 2.5 and 40.0 ppm and of CDCl3 7.3
and 77.0 ppm.
Electronic impact mass spectroscopy (EI/MS) was carried out on a VG AutoSpec (Fison,
Ipswich, United Kingdom) instrument and data was reported as m/z (percentage of relative
intensity of the most important fragments).
X-Ray diffraction
A crystal of compound 3 (recrystallized from methanol) was used for X-ray crystallographic
analysis. Information about crystal data, experimental data collection conditions and
refinement as well as the structural geometric parameters are available as an Electronic
Supplementary Publication from the Cambridge Crystallographic Data Centre,[7] (CCDC-
1479922) at https://www.ccdc.cam.ac.uk/structures in CIF format. The compound
crystallized in space group P-1 with two molecules in the asymmetric unit as a hemisolvate.
Materials
Starting materials and reagents were obtained from commercial suppliers and were used
without further purification (Sigma–Aldrich, Portugal). Melting points (mp) are uncorrected
and were determined with a Reichert Kofler thermopan or with a Büchi 510 apparatus. Flash
chromatography was performed on silica gel (Merck 60, 230–400 mesh) and thin-layer
chromatography (TLC) was performed on pre-coated silica gel plates (Merck 60 F254). The
spots were visualized under UV detection (254 and 366 nm). Following the workup, the
organic solutions were dried over Na2SO4. Solvents were evaporated in a Buchi Rotavapor.
Organic solutions were dried over anhydrous Na2SO4.
Synthesis of coumarin derivatives (1-6)
The starting material 6-methylcoumarin-3-carboxylic acid (A) was synthesized in a 89%
yield according to a previously described method.[8] The structural data (NMR and MS) was
in accordance to the literature. [9]
General synthetic method.. Compound A (1 mmol) was dissolved in dichloromethane (10
mL) and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.1 mmol) and 4-
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dimethylaminopyridine (1.1 mmol) were added. The mixture was stirred under a positive
argon pressure at 0 °C for five minutes.[10] After this period, the aromatic amine with the
appropriated substitution pattern was added. The reaction mixture was stirred for four hours
at room temperature. Upon completion, the crude products were filtered and purified by flash
chromatography (hexane/ethyl acetate 9:1) or recrystallization (ethanol) to give the desired
products (1-6) in yields ranging from 56% to 82% (see Table 1).[11]
Results and Discussion
A three step synthetic strategy, briefly described in Scheme 1, was used to efficiently obtain
the coumarin derivatives (1-6) efficiently with an overall yield between 56% and 82%.
Scheme 1 - Reagents and conditions: a) EDC, DMAP, DCM, aromatic amine with the
appropriate substituent, 0 ºC to r.t., 4h.
All synthesized coumarins (1-6) are listed in Table 1. To complement compound´s
characterization the melting point and mass spectrometry of all the derivatives were also
acquired and the data included in Table 1. The unambiguous assignment of all protons and
carbons of coumarin derivatives was achieved by means of 1D and 2D resonance techniques.
The NMR data are depicted in Tables 2 and 3.
Firstly, the NMR data of compound 1 was acquired. From 1H and 13C spectra thirteen protons
1
and seventeen carbons were observed. In the H NMR spectrum (Table 2) the signal at 8.98
ppm was set up as H-4, a characteristic singlet of coumarin nucleus. The protons of the
methyl substituent were readily assigned from the spectrum and are found to resonate at 2.41
ppm. Additionally, the protons at 7.83, 7.61 and 7.46 ppm were found to correlate with each
other. Taking into account COSY data and proton multiplicity pattern it was concluded that
these protons take part of the coumarin core: a meta coupling of 1.5 Hz at 7.83 ppm
consistent with H-5, an ortho and meta coupling of 8.5 and 1.5 Hz at 7.61 ppm, and an ortho
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coupling of 8.5 Hz at 7.46 ppm, that are consistent with H-7 and H-8, respectively, were
observed. From HSQC data it was possible to assign the carbons bonded to the protons of the
coumarin nucleus. Accordingly, the peaks located at 148.7, 130.4, 135.9, and 116.5 ppm were
assigned as C-4, C-5, C-7, and C-8, respectively. As COSY indicates the correlation with
coupled protons it was concluded that the four protons located at 6.83, 6.92, 6.96 and 8.39
ppm correlate to each other. Based on their multiplicity pattern and HMBC data they were
unequivocally assigned to the protons H-5’, H-3’, H-4’ and H-6´, respectively. The exocyclic
aromatic ring protons have been found to be bonded to carbons resonating at 119.6, 115.1,
124.8 and 120.4 respectively. By a linear combination of DEPT-135 and proton decoupled
13C spectra eight quaternary carbons at 118.8, 119.1, 127.0, 135.1, 147.1, 152.7, 159.4 and
161.6 ppm were observed. From 2D spectra analysis it was concluded that the signals at 7.61
ppm (H-7) and at 7.46 ppm (H-8) have a long range correlation with the carbons at 152.7
ppm and 118.8 ppm, respectively. Based on their dissimilar chemical environment the signals
were assigned to carbons C-8a and C-4a, respectively. The signals at 161.6 and at 159.4 ppm
were assigned to C-2 and C-9, respectively, as they exhibit long range interactions with H-4
and NH/H-4, respectively (Figure 1A). Moreover, a long range interaction was observed
between the signal of the proton H-4 and the signal at 119.1 ppm that was assigned as C-3.
However, the unequivocal assignment of the quaternary carbons C-1’ and C-2’ was not
possible to be performed from analysis of the HMBC spectrum. For that reason, C-1’ was
assigned at 127.0 ppm and C-2’ at 147.1 ppm using the increment system to estimate carbon
chemical shifts of substituted benzene.[12] Conversely, the long range couplings that occur
between the proton peak at 10.22 ppm and three carbon peaks (C1’, C2’ and C3’) observed in
HMBC (Figure 1B) allowed the unequivocal assign the proton of the hydroxyl function. The
chemical shifts of OH and NH protons have been strongly affected by the presence of the two
intramolecular hydrogen bonds.
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A
NH
OH
C3’
C6’
C1’
C2’
A
C2’
C9
B
NH
C9
H4
C9
C2
B
Figure 1 –HMBC spectrum of compound 1. Sections of the long-range couplings of proton
peaks (A) at 10.22 ppm (-OH) and 11.12 ppm (NH) and (B) at 8.98 (H4) and at 11.12 ppm
(NH) with structural and carbonyl carbons peaks, respectively, are shown.
As all the compounds herein reported (compounds 1-6, Table 1) own the same coumarin
nucleus substitution pattern (3-carboxamide-6-methylcoumarin) as compound 1, the
unambiguous assignment of the sub substructure was performed directly from their 1D and
2D NMR spectra (Tables 2 and 3). However, as they have different substitution patterns on
the exocyclic aromatic substituent a thorough spectral analysis was done for compound 2,
which has a hydroxyl group in the meta position, and compound 3, which has an hydroxyl
group in para position. A correlation analysis similar to that performed compound 1 was
tracked.
For compound 2, H-2’, H-4’, H-5’ and H-6’ protons were assigned taking into account the
1
multiplicity pattern of each signal in the H NMR spectrum (Table 2), COSY and HMBC
correlation data (Figure 2). The peak at 7.31 ppm was assigned as H-2’ since it shows a
multiplicity pattern of doublet of doublets with a constant of 2.1 Hz, owed to the coupling of
two meta protons. H-4’ was attributed to the signal at 6.56 ppm while the peaks at 7.16 and
7.03 ppm were identified as H-5’ and H-6’, respectively, based on their multiplicity patterns.
The corresponding carbons were ascribed based on the HSQC data. Thus, the carbon directly
attached to proton H was assigned as 107.3 ppm and C-4’ resonate at 111.9 ppm. The
quaternary carbons of the exocyclic aromatic ring, namely C-1’ and C-3’, were assigned
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taking into account DEPT-135 and HMBC data. The peak at 139.4 ppm was labelled as C-
1’as it has a long range interaction with the proton of the hydroxyl group. The signal at 158.3
ppm was attributed to C-3’.
1
Regarding compound 3, the H NMR spectrum showed the presence of equivalent protons
with the signals at 7.51 and 6.77 ppm. Bearing in mind that the proton of the amide group
shows a long range interaction with two carbons at 122.0 ppm while the proton of the
hydroxyl group possess a long range interaction with other two carbons at 115.3 ppm, C-2’
and C-6’ were then assigned at 122.0 ppm. As a result, C-3’ and C-5’ were found to resonate
at 115.3 ppm. From HSQC data the peak at 7.51 ppm was attributed to both H-2’and H-6’,
whereas H-3’ and H-5’ were assigned with the signal at 6.77 ppm. In both cases a difference
of NH and OH proton shifts were observed, when compared with compound 1, due to the
absence of one of the intramolecular hydrogen bonds.
Figure 2 – Correlations observed in COSY (blue dotted lines) and HMBC (red solid lines)
spectra of compound 2.
The NMR signal assignments of compounds 4, 5 and 6 were performed by a correlation with
the data acquired for compounds 1, 2 and 3 (Tables 2 and 3). However, due to solubility
constrains the spectra were recorded using CDCl3 as solvent instead of DMSO-d6. So, a slight
1
alteration on chemical shifts values was observed. The main changes were found in the H
NMR multiplicity signals of the coumarin nucleus. In the 1H NMR spectra of compounds 4, 5
and 6 the H-5 and H-7 appeared as multiplets. In the case of compound 5 it was found a
similar multiplet that was assigned as H-5, H-7 and H-2’, as it integrates for 3 protons. From
the HSQC spectra (compounds 4 and 6) we can observe that H-7 is more downshielded than
H-5. In the case of compound 5 proton H-2' is upshielded in relation to H-5 and H-7.
X-ray diffraction
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Compound 3 provided suitable crystals for X-ray analysis. The thermal ellipsoid plot for
compound 3 together with the adopted numbering scheme is shown in Figure 3. There are
two molecules in the asymmetric unit connected by hydrogen bonding via a methanol solvent
molecule. The configuration of the molecule is given by the C-N rotamer of the amide which
defines the position of the aromatic rings with respect to one other: both molecule displays an
anti configuration, with respect to carbonyl of the coumarin (Table 1), thus allowing for the
establishment of an N–H···O intramolecular H bond (geometric parameters are given in Table
4). In addition, there is a weak hydrogen bond linking the exocyclic aromatic ring, namely
between H-2’ and the carbonyl of the amide group. Both intramolecular bonds form pseudo
S6 rings interactions in each molecule. The two molecules are linked by one molecule of
methanol solvent via hydrogen bonding where the methanol acts as a bridge since its oxygen
atom acts as acceptor of the 4’-hydroxyl group of one molecule of 3 and as a donor to the
amide carbonyl oxygen atom in the other molecule. An inspection of the bond lengths shows
that there is a slightly asymmetry of the electronic distribution around the pyrone ring: the
C23–C24 and C13–C14 bond (Figure 3), correspondent to the pyrone double bond of each
molecule, distances have values of 1.350(3) and 1.352(2) Å, respectively. The shorter
distance values, which are lower than the expected for a Car–Car bond, indicate the existence
of a higher electronic density area in pyrone ring. The values for distances of C13–C131 and
C23-C231 bonds connecting the coumarin to the amide spacer are typical of a Csp3–Csp3 bond
conferring freedom for the rotation of the phenylamide substituent around it. The dihedral
angles between the mean planes of the coumarin, OCN spacer and exocyclic aromatic ring
(Table 5), indicate that the molecules are essentially planar.
Figure 3 – Structure and the adopted numbering scheme of compound 3.
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Conclusions
In the present work 6-methyl-3-carboxamidocoumarin derivatives (compounds 1-6), bearing
electron-donating substituents in different positions of the exocyclic aromatic ring, were
synthesized in good yields and in mild reaction conditions and characterized by homo- and
hetero-nuclear NMR techniques. Their unequivocal identification constitutes a valuable
database for the accurate identification of the coumarins of our library. In addition, these
results can be used as reference for structural elucidation of newer naturally occurring and
synthetic coumarins.
Acknowledgments
This project was supported by Foundation for Science and Technology (FCT) and
FEDER/COMPETE2020 (UID/QUI/00081/2015 and POCI-01-0145-FEDER-006980).
The authors thank Fundação para a Ciência e Tecnologia (FCT) for their grants. A. Fonseca
(SFRH/BD/80831/2011), A. Gaspar (SFRH/BPD/93331/2013) and M. J. Matos
(SFRH/BPD/95345/2013) were supported by grants from FCT, POPH and QREN.
Crystallographic data were collected at the Engineering and Physical Sciences Research
Council (EPSRC) X-ray crystallography service UK. Thanks are due to the staff at the
National Crystallographic Service, University of Southampton for the data collection, help
and advice.[13]
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Table 1 – Structure, names, melting points, yields and mass spectroscopy analysis of compounds 1-6.
Compound
R
R1
H
R2
H
Name
MS/EI (rel. int.)
Melting point / ºC
274-275
Yield
56%
OH
N-(2’-hydroxyphenyl)- 6-methyl-3-carboxamidocoumarin
295 (M, 46), 187
(100), 115 (12)
1
H
H
OH
H
H
OH
H
N-(3’-hydroxyphenyl)- 6-methyl-3-carboxamidocoumarin
N-(4’-hydroxyphenyl)- 6-methyl-3-carboxamidocoumarin
N-(2’-methoxyphenyl)- 6-methyl-3-carboxamidocoumarin
N-(3’-methoxyphenyl)- 6-methyl-3-carboxamidocoumarin
N-(4’-methoxyphenyl)- 6-methyl-3-carboxamidocoumarin
296 (M+, 40), 295
(M, 99), 187 (100),
115 (64), 103 (51)
296 (M+, 26), 295
(M, 92), 187 (100),
115 (25), 103 (18)
310 (M+, 29), 309
(M, 78), 187 (100),
115 (34), 77 (20).
310 (M+, 16), 309
(M, 66), 187 (100),
115 (33), 77 (20).
310 (M+, 70), 309
(M, 96), 187 (100),
115 (62), 77 (44)
217-218
230-231
204-205
174-175
199-200
61%
61%
82%
74%
73%
2
3
4
5
6
OCH3
H
H
OCH3
H
H
H
OCH3
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Table 2 – 1H NMR chemical shifts, multiplicity and coupling constants of coumarins 1-6.
Compounds
Position
1a)
2 a)
3 a)
4b)
5 b)
6 b)
4
5
8.98 (s)
8.82 (s)
8.82 (s)
8.94 (s)
*
8.96 (s)
**
8.95 (s)
*
7.83 (d, 1.5)
7.78 (d, 1.5)
7.78 (d, 1.6)
7.46-7.50 (m) 7.46-7.50 (m) 7.46-7.50 (m)
7
7.61 (dd, 1.5, 7.60 (dd, 1.5, 7.58 (dd, 1.6,
*
**
*
8.5)
8.5)
7.45 (d, 8.5)
7.31 (dd, 2.1,
2.1)
8.5)
8
7.46 (d, 8.5)
-
7.44 (d, 8.5)
7.51 (d, 8.7)
7.32 (d, 8.1)
-
7.34 (d, 9.1)
**
7.33 (d, 9.1)
7.65 (d, 9.0)
2’
3’
4’
5’
6’
6.92 (dd, 1.7,
8.0)
-
6.77 (d, 8.7)
6.94 (dd, 1.6,
8.0)
-
6.91 (d, 9.0)
6.94-6.98(m)
6.56 (ddd,
1.0, 2.1, 8.1)
7.16 (dd, 8.1,
8.1)
-
7.11 (ddd,
1.6, 7.8, 8.0)
7.00 (ddd,
1.6, 7.8, 8.0)
8.54 (dd, 1.6,
8.0)
6.72 (ddd,
1.4, 2.5, 7.8)
7.27 (dd, 7.8,
8.1)
-
6.83 (ddd,
1.7, 6.8, 8.0)
8.39 (dd, 1.4,
8.0)
6.77 (d, 8.7)
7.51 d, 8.7)
6.91 (d, 9.0)
7.65 (d, 9.0)
7.01- 7.05
(m)
7.22 (ddd,
1.4, 1.6, 8.1)
2.46 (s)
CH3
OH
2.41 (s)
2.40 (s)
9.54 (s)
-
2.40 (s)
9.38 (s)
-
2.45 (s)
2.46 (s)
-
10.22 (s)
-
-
-
OCH3
NH
3.97 (s)
3.84 (s)
3.82 (s)
10.75 (s)
11.12 (s)
10.58 (s)
10.47 (s)
11.31 (s)
10.87 (s)
a) Solvent was DMSO-d6.
b) Solvent was CDCl3
* H-7 is more downshielded than H-5 (from HSQC data).
** The multiplet integrates for 3 protons. H-2' is upshielded in relation to H-5 and H-7 (from HSQC data).
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Table 3 – 13C NMR chemical shifts of compounds 1-6.
Compounds
Position
1 a)
2 a)
3 a)
4 b)
5 b)
6 b)
2
161.6
119.1
148.7
118.8
130.4
135.1
135.9
116.5
152.7
159.4
127.0
147.1
115.1
124.7
161.2
120.3
147.7
118.7
130.2
135.2
135.7
116.5
152.5
160.2
139.4
107.3
158.3
111.9
161.2
120.2
147.6
118.7
130.1
135.1
135.6
116.4
152.5
159.6
130.0
122.0
115.3
154.6
159.3
119.0
148.5
118.5
129.4
135.2
135.3
116.4
152.8
161.7
127.8
149.2
110.3
124.5
159.5
118.6
148.5
118.4
129.5
135.4
135.6
116.4
152.7
162.0
138.9
106.0
160.2
111.1
159.2
118.6
148.6
118.6
129.5
135.4
135.4
116.4
152.7
162.0
131.0
122.1
114.2
156.7
3
4
4a
5
6
7
8
8a
9
1’
2’
3’
4’
5’
6’
119.4
120.4
130.2
111.0
115.3
122.0
120.7
121.0
129.7
112.8
114.2
122.1
CH3
20.7
-
20.7
-
20.7
-
20.8
56.0
20.8
55.4
20.8
55.5
OCH3
a) Solvent was DMSO-d6.;
b) Solvent was CDCl3
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Table 4 - Hydrogen-bond geometry (distances in Å, angles in º) for compound 3.
D—H···A
D—H
0.83
H···A
1.89
1.80
D···A
D—H···A
176
O5—H5···O231
2.719 (2)
2.655 (2)
2.655 (2)
2.972 (2)
2.675 (2)
2.936 (2)
O334—H334···O5
N132—H132···O12
C332—H332···O131
N232—H232···O22
C432—H432···O231
0.86
173
0.92 (3) 1.83 (3)
0.95 2.38
0.94 (3) 1.87 (3)
0.95 2.34
148 (2)
120
143 (2)
120
Table 5 - Selected dihedral angles, ° for compound 3.
Compound
θ1°
θ2°
θ3°
3.28 (11)
2.06 (12)
3.0 (2)
2.2 (3)
4.4 (2)
1.1 (3)
1
2
θ1 is the dihedral angle between the mean planes of the coumarin and exocyclic benzene ring . θ2 is the dihedral
angles between the mean planes of the coumarin ring and the plane defined by the OCN amide atoms. θ3 is the
dihedral angle between the mean planes of the exocyclic benzene ring and the plane defined by the OCN amide
atoms.
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