New coumarin-based fluorescent melatonin ligands. Design, synthesis and pharmacological characterization

Article abstract of DOI:10.1016/j.ejmech.2015.09.003

The design and synthesis of a series of new fluorescent coumarin-containing melatonin analogues is presented. The combination of high-binding affinities for human melatonergic receptors (h-MT₁R and h-MT₂R) and fluorescent properties, derived from the inclusion of melatonin pharmacophoric elements in the coumarin scaffold, yielded suitable candidates for the development of MT₁R and MT₂R fluorescent probes for imaging in biological media.

Full text of DOI:10.1016/j.ejmech.2015.09.003

Accepted Manuscript
New coumarin-based fluorescent melatonin ligands. design, synthesis and
pharmacological characterization
Mario de la Fuente Revenga, Clara Herrera-Arozamena, Nerea Fernández-Sáez,
Gema Barco, Itxaso García-Orue, David Sugden, Silvia Rivara, María Isabel
Rodríguez-Franco
PII:
S0223-5234(15)30247-6
10.1016/j.ejmech.2015.09.003
EJMECH 8100
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 May 2015
Revised Date: 1 September 2015
Accepted Date: 2 September 2015
Please cite this article as: M. de la Fuente Revenga, C. Herrera-Arozamena, N. Fernández-Sáez, G.
Barco, I. García-Orue, D. Sugden, S. Rivara, M.I. Rodríguez-Franco, New coumarin-based fluorescent
melatonin ligands. design, synthesis and pharmacological characterization, European Journal of
Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.09.003.
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ACCEPTED MANUSCRIPT

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New Coumarin-Based Fluorescent Melatonin Ligands.
Design, Synthesis and Pharmacological Characterization
1
1
1
Mario de la Fuente Revenga, Clara Herrera-Arozamena, Nerea Fernández-Sáez,
1
1
2
Gema Barco, Itxaso García-Orue, David Sugden,
3
,1
Silvia Rivara, and María Isabel Rodríguez-Franco*
1
Instituto de Química Médica, Consejo Superior de Investigaciones Científicas (IQM-
CSIC), C/ Juan de la Cierva 3, 28006-Madrid, Spain
2
Division of Women’s Health, King’s College London, London SE1 1UL, U.K.
3
Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze
2
7/A, 43124 Parma, Italy
Corresponding author:
María Isabel Rodríguez-Franco, PhD
Tel.: +34 915622900; fax: +34 915644853
E-mail: isabelrguez@iqm.csic.es
1

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Abstract: The design and synthesis of a series of new fluorescent coumarin-containing
melatonin analogues is presented. The combination of high-binding affinities for human
melatonergic receptors (h-MT R and h-MT R) and fluorescent properties, derived from
1
2
the inclusion of melatonin pharmacophoric elements in the coumarin scaffold, yielded
suitable candidates for the development of MT R and MT R fluorescent probes for
1
2
imaging in biological media.
Keywords: Melatonin; GPCRs fluorescent probes; coumarins; bioisosterism.
Abbreviations
Boc, tert-butyloxycarbonyl protecting group; CDI, 1,1'-carbonyldiimidazole; GPCRs,
G-protein-coupled receptors; HPLC-MS, liquid chromatography–mass spectrometry;
MSA, methanesulphonic acid; MTRs, melatonergic receptors; h-MT R: human
1
melatonin receptor subtype-1; h-MT R: human melatonin receptor subtype-2; TEA,
2
triethylamine; TFA, trifluoroacetic acid.
2

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Fluorescence-based techniques for studying pharmacological and biochemical processes
have undergone a huge development in the last decade. Given the demanding technical
set-up, hazards and expense of using radioligands for these studies, fluorescent-based
methodologies appear more desirable and easier to implement [1].
Melatonin is produced in the pineal gland during the dark-phase of the day-night cycle.
Most of its functions are mediated by two G protein–coupled receptors (GPCRs), named
MT and MT . Besides the regulation of circadian rhythms, melatonergic receptors
1
2
(MTRs) are involved in numerous physiological processes whose underlying molecular
basis has not been fully elucidated [2]. Several fluorescence-spectroscopy techniques
have been developed for studying different aspects of the pharmacology of GPCRs [3].
Melatonin itself bears an indole moiety that confers a certain intrinsic fluorescence to
the molecule, although the photochemical properties of this heterocycle are not
appropriate for the development of fluorescent-based methodologies. Different
melatonin ligands bearing fluorescent cores are reported in the literature, out of which
only 7-azamelatonin and boron-dipyrromethene derivatives were reportedly designed
and developed specifically for receptor labelling purposes [4,5].
Coumarins are a class of fluorophores from the benzopyrone family. As a representative
example, umbelliferone has an emission wavelength above 450 nm that would prevent
interference with the naturally-present tryptophan or other indole-containing
biomolecules. Moreover, their generous Stokes shift and high quantum yield make
coumarins suitable structures for the development of fluorescence-based imaging
techniques for the visualization of metabolic processes and interactions with receptors
[
6,7]. To continue the development of novel fluorescent probes capable of identifying
the generally low-expressed melatonin receptors, together with our interest in the
development of melatonin-based potential drugs [8,9], we wished to integrate a suitable
3

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fluorophore within the structure of melatonin receptor ligands. Herein, we report the
design, synthesis, and characterization of a series of novel fluorescent melatonin
analogues in which the indole nucleus of melatonin has been bioisosterically replaced
by a fluorescent 2H-chromen-2-one ring, with varying anchorage points of the
substituents attached to it (Figure 1).
Figure 1. Structure of coumarin-based fluorescent melatonin ligands (1-8) designed by
adding pharmacophoric elements of melatonin to the coumarin core.
Coumarins bearing the acetylaminoalkyl chain linked to position 4 (1-4) were obtained
in moderate to good yields by a Pechmann condensation between a β-ketoester (10 or
1
1) and the corresponding phenol in methanesulphonic acid (MSA) at room temperature
(Scheme 1). The relative position of the alkyloxy group in the phenol greatly
determined the course of the reaction; reactivity was clearly favored in m-alkyloxy
substituted phenols, whereas p-alkyloxy substitution greatly retarded it. In fact, the
reaction that afforded compound 3 required up to 30 days for achieving completion (see
Supplementary Material for further details).
4

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For obtaining coumarin 5, bearing the alkyl acetamido chain in position 3 and the
methoxy group in 6, an adapted Perkin condensation was employed. Preliminary
attempts to promote base-catalysed intramolecular condensation of intermediate ester 12
did not led to the formation of the desired coumarin. Conversely, 1-acetylpyrrolidin-2-
one and 2-hydroxy-5-methoxybenzaldehyde were identified by HPLC-MS, suggesting
that the ester group underwent a nucleophilic attack from the amide nitrogen. In order to
avoid such cyclization, 12 was protected with a tert-butyloxycarbonyl group (Boc) prior
to its treatment with trifluoroacetic acid (TFA). Nevertheless, in the reaction mixture a
significant amount of 1-acetylpyrrolidin-2-one was also identified, revealing that
undesired cyclization was only partially prevented, isolating coumarin 5 in very low
yield (9%).
HO
R2
R1
O
O
NHAc
)n
(
4
O
O
KO
OEt
R6
R7
AcHN
(
)n
OEt MSA, 0ºC then rt
CDI, THF,
MgCl2,
rt, overnight
O
O
1
1
0, n = 2 (34%)
1, n = 3 (32%)
1
2
3
4
, n = 2, R = H, R = OMe (81%)
, n = 2, R -R = -OCH O- (80%)
, n = 2, R = OMe, R = H (67%)
, n = 3, R = H, R = OMe (69%)
6 7
6
7
2
6
7
6
7
MeO
CHO
OH
MeO
CHO
O
NHAc
1
) Boc2O, DMAP,
MeCN, rt, 24h
MeO
6
3
AcHN
CO H
2
(
)
n
EDCI·HCl, DMAP,
TEA, CH2Cl2, rt, 5h
2) Et Si, TFA:DCM
NHAc (1:2), 0ºC, 1.5h
3
O
( )3
O
O
12 (76%)
5 (9%)
OMe
5
1) CDI, DMF, MW (120 ºC, 5 min)
( )n
3
NHAc
2
) TEA, MW (200 ºC) OMe
O
O
CHO
OH
6
7
, n = 0 (68%)
, n = 1 (65%)
NHAc
Cl
+
AcHN
HO
HO
O
O
K CO , DMF,
O
7
O
O
N
7
O
O
2
3
H
2
00ºC (mw), 15 min
8
(22%)
9
(11%)
Scheme 1. Synthesis of new coumarin-based melatonin ligands 1-8.
5

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Coumarins 6 and 7, bearing the alkyl acetamido chain in position 3 and the methoxy
group in 5, were obtained in moderate yield (65-68%) from 2-hydroxy-6-
methoxybenzaldehyde and N-acetylglycine or N-acetyl-β-alanine, using 1,1'-
carbonyldiimidazole (CDI) and triethylamine (TEA) in a microwave reactor at 200 ºC.
Finally, to link the acetamide chain to position 7, 7-hydroxy-2H-chromen-2-one
(
umbelliferone) was reacted with 2-chloroethylacetamide in basic medium, using a
microwave reactor at 200 °C for 5 h. In addition to the expected product (8, 22% yield),
-((2-hydroxyethyl)amino)-2H-chromen-2-one (9, 11% yield) was also isolated, as the
7
result of a Smiles rearrangement, favored by the harsh conditions of basicity, pressure,
and temperature that were needed to promote the primary attack of the phenolate over
the chlorinated carbon.
The fluorescent properties of the coumarin-bearing compounds 1-8 are summarized in
Table 1. Compounds 1, 2, 4, and 8 showed fluorescence intensities in the same
wavelength and concentration range as umbelliferone. Derivatives 3, 6, and 7 required
higher concentration for measurable fluorescence detection; conversely, their Stokes
shifts were greater than the other compounds in the series that, in general, showed much
narrower shifts than umbelliferone. The major difference between the excitation and
emission wavelengths was found in coumarin 7, greater than umbelliferone itself. In the
case of derivative 5, no fluorescent emission could be recorded in the fluorimeter after
excitation at its maximum UV band (368 nm).
6

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Table 1. Photophysical properties of compounds at concentration 0.1 µM in phosphate
buffer 0.1 M pH 7.4
Fluorescence Intensity
Compd.
λexc (nm) λem (nm) Stokes shift (nm)
a
(A.U.)
Umbelliferone
328
324
346
340
322
323
305
324
453
389
424
435
388
463
467
391
125
65
367
336
325
320
323
845
724
187
1
2
78
b
3
95
4
66
b
6
140
162
67
b
7
8
a
b
Fluorescence intensity expressed in arbitrary units. Measured at 5 µM.
Compounds’ affinity for human MT R and MT R was screened at 100 nM
1
2
concentration (Table 2) [10]. Coumarin 3 showed the greatest degree of specific
displacement of radioligand at both receptors (69% at h-MT and 83% at h-MT ). Its
1
2
affinity constants were also determined, giving K s in the nanomolar range: h-MT (K ,
i
1
i
1
3 ± 0.5 nM) and h-MT (K , 3.4 ± 0.1 nM). These results confirmed that coumarin 3
2 i
strongly binds to both receptors, although with lower potency than melatonin: h-MT1
K , 0.091 nM) and h-MT (K , 0.15 nM) [10].
(
i
2
i
7

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1
25
Table 2. Percentage of radioligand 2-[ I]iodomelatonin displacement (%)
a,b
from recombinant human MTRs, elicited by compounds at 100 nM.
Compd.
h-MT1
h-MT2
1
2
3
4
5
6
7
8
31.8 ± 4.1
10.0 ± 1.4
4.5
2.3
69.0 ± 0.2 83.3 ± 1.2
43.0 ± 0.8
3.4
3.0
1.0
0
18.8 ± 1.8
25.1 ± 5.2
3.2
0.2
0.4
a
Results are expressed as percentage inhibition ± SEM (n=3) of specific melatonin
b
binding (for values below 10% SEM are not given). Melatonin at 100 nM gives 100 %
of radioligand displacement at both MTRs.
The relative positions of the acetamido chain and alkyloxy group on the coumarin core
were determinant for receptor recognition, as it could be explained by superposition
studies using the bioactive conformation of melatonin. The most potent melatonergic
ligand of the series, 3, is able to properly reproduce the putative active conformation of
the natural ligand (Figure 2, left), defined by pharmacophore analysis and molecular
superposition with conformationally-constrained compounds [11]. The lower binding
affinity of coumarin 3 compared to melatonin is likely due to the presence of the
carbonyl group in position 2, as it is known that both a 6,6-bicyclic nucleus [12] and a
benzofuran oxygen [13] are well tolerated at melatonin receptors.
8

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Figure 2. Melatonin (yellow carbons) in its putative active conformation, superposed to
compound 3 (left, gray carbons), compound 4 (middle, green carbons) and compound 7
(right, orange carbons).
The binding drop of 7-methoxycoumarin 1 highlights the strong dependence of MTRs
affinity from the presence and position of the methoxy group, in the same way that
moving the methoxy group of melatonin from position 6 to position 5 had brought more
than 1.000-fold reduction in binding affinity [14]. The compensation offered by chain
elongation in compound 4 can be explained by the possibility to get a good
superposition of its pharmacophoric elements on those of melatonin, with a partially
folded conformation of the side chain (Figure 2, middle).
Compounds 6 and 7, having shorter amide chains in position 3 and a methoxy group in
position 5, showed very limited MT binding affinity, probably related to the poor
1
fitting to the melatonergic pharmacophore (Figure 2, right).
Compound 2 with a methylene-dioxy bridge connecting positions 6 and 7 has very low
potency, likely due to an unsuitable orientation of the oxygen lone pairs, which
probably hampers a proper interaction with the receptor.
9

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Additionally, the most active compounds 3 and 4 were functionally characterized in a
well-established cellular model of melatonin action, cultured Xenopus laevis
melanophores as previously described (Figure 3) [15]. Compound 4 lacked any
-5
significant effect on pigment aggregation, with no agonist action up to 10
M
concentration. On the other hand, 3 was a potent agonist showing close to a full agonist
response (Emax = 89 ± 2 % compared to melatonin) with an EC = 117.8 ± 20.6 nM. At
50
-4
a higher dose (10 M) derivative 3 acted as a melatonin receptor antagonist as did 4
(
data not shown). The drop in potency of 3 compared to melatonin (EC = 0.074 ±
50
0
.006 nM; Emax = 100%) is more marked in this activity model than in radioligand
binding experiments on recombinant h-MTRs. Taking into account that Xenopus laevis
melanophores express native frog melatonin receptors which are likely to be a different
subtype (Mel1c), receptor-subtype and interspecies-differences could be responsible for
the observed differences in potency.
Figure 3. Concentration-response curves for melatonin, 3, and 4 on Xenopus laevis
melanophores. Each point on the Y axis is the mean pigment aggregation response
(relative change in absorbance before [Ai] and 60 min. after [Af] drug treatment). SEM
values are omitted as they were all less than the area covered by the symbol.
1
0

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In summary, we have designed a series of fluorescent melatonin analogues in which the
indole nucleus of melatonin has been replaced with O-substituted coumarin scaffolds.
The relative position of the pharmacophoric elements on the coumarin ring greatly
determined the affinity for h-MT and h-MT and the fluorescence properties. Best
1
2
binding results were obtained when the acetamido chain was attached to the coumarin
position 4. Among compounds 1-4, the methoxy substituent, either in position 6 or 7 of
the ring, affected in an inverse manner either property. N-(2-(6-Methoxy-2-oxo-2H-
chromen-4-yl)ethyl)acetamide (3) showed the best human MT R / MT R binding, in the
1
2
nanomolar range, and demonstrated its agonistic properties in Xenopus laevis
melanophores. Thus, coumarin derivative 3 behaved as a nonselective nanomolar
melatonergic fluorescent probe that could be used for imaging in cells and tissues
expressing melatonin receptors.
1
1

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Acknowledgements
This work was supported by the Spanish Ministry of Economy and Competitiveness
MINECO, Grant SAF2012-31035), Fundación de Investigación Médica Mutua
(
Madrileña Automovilística (Grant AP103952012) and Consejo Superior de
Investigaciones Científicas (CSIC, Grant PIE-201280E074). M.F.R. was the recipient of
a JAE-Predoctoral Contract (Grant JAE-Pre-2009-106) and I.G.-O. of a fellowship for
Research Starting (Grant JAE-Intro-2012-0047), both from the Program “Junta para la
Ampliación de Estudios” co-financed by the CSIC and the European Social Fund.
Supplementary data
Experimental details for the synthesis of coumarin-based compounds and intermediates.
Determination of spectroscopic and biological properties.
1
2

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Table 1. Photophysical properties of compounds at concentration 0.1 µM in phosphate
buffer 0.1 M pH 7.4
Fluorescence Intensity
Compd.
λexc (nm) λem (nm) Stokes shift (nm)
a
(A.U.)
Umbelliferone
328
324
346
340
322
323
305
324
453
389
424
435
388
463
467
391
125
65
367
336
325
320
323
845
724
187
1
2
78
b
3
95
4
66
b
6
140
162
67
b
7
8
a
b
Fluorescence intensity expressed in arbitrary units. Measured at 5 µM.

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1
25
Table 2. Percentage of radioligand 2-[ I]iodomelatonin displacement (%)
a,b
from recombinant human MTRs, elicited by compounds at 100 nM.
Compd.
h-MT1
h-MT2
1
2
3
4
5
6
7
8
31.8 ± 4.1
10.0 ± 1.4
4.5
2.3
69.0 ± 0.2 83.3 ± 1.2
43.0 ± 0.8
3.4
3.0
1.0
0
18.8 ± 1.8
25.1 ± 5.2
3.2
0.2
0.4
a
Results are expressed as percentage inhibition ± SEM (n=3) of specific melatonin
b
binding (for values below 10% SEM are not given). Melatonin at 100 nM gives 100 %
of radioligand displacement at both MTRs.

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Highlights for
New Coumarin-Based Fluorescent Melatonin Ligands. Design, Synthesis and
Pharmacological Characterization
•
•
•
New coumarin-based fluorescent ligands at human melatonergic receptors (h-
MTRs)
N-(2-(6-Methoxy-2-oxo-2H-chromen-4-yl)ethyl)acetamide: nanomolar affinity
at h-MTRs
Coumarin – melatonin superposition studies: a rationale for h-MTRs affinities

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Supplementary data for
New Coumarin-Based Fluorescent Melatonin Ligands.
Design, Synthesis and Pharmacological Characterization
1
1
1
Mario de la Fuente Revenga, Clara Herrera-Arozamena, Nerea Fernández-Sáez,
1
1
2
Gema Barco, Itxaso García-Orue, David Sugden,
3
,1
Silvia Rivara, and María Isabel Rodríguez-Franco*
1
Instituto de Química Médica, Consejo Superior de Investigaciones Científicas (IQM-
CSIC), C/ Juan de la Cierva 3, 28006-Madrid, Spain
2
Division of Women’s Health, King’s College London, London SE1 1UL, U.K.
3
Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze
2
7/A, 43124 Parma, Italy
S1

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EXPERIMENTAL SECTION
Chemistry. General Methods. Reagents and solvents were purchased from common
commercial suppliers, mostly Sigma-Aldrich, and were used without further
purification. Analytical thin-layer chromatography (TLC) was carried out using Merck
silica gel 60 F254 plates, and the compounds were visualized under UV-light ( = 254
or 365 nm) and/or stained with phosphomolybdic acid 10% wt. in ethanol. Automatized
chromatographic separation was carried out in an IsoleraOne (Biotage) using different
silica Si50 cartridges from Agilent Technologies. High-performance liquid
chromatography was performed on a Waters analytical HPLC-MS (Alliance Watters
2
690) equipped with a SunFire C18 4.6 x 50 mm column, a UV photodiode array
detector ( = 214–274 nm) and quadrupole mass spectrometer (Micromass ZQ). HPLC
analyses were used to confirm the purity of all compounds (>95%) and were performed
on Waters 6000 equipment, at a flow rate of 1.0 mL/min, with a UV photodiode array
detector ( = 214–274 nm), and using a Delta Pak C 5 µm, 300 Å column. The elution
18
was performed in a gradient mixture of MeCN/water.
Melting points (uncorrected) were determined in a MP70 apparatus (Mettler Toledo).
1
13
H NMR and C NMR spectra were recorded in CDCl or DMSO-d solutions using the
3
6
following NMR spectrometers: Varian INOVA-300, Varian INOVA-400, Varian
Mercury-400 or Varian Unity-500. Chemical shifts are reported in δ scale (ppm) relative
to internal Me Si. J values are given in hertz, and spin multiplicities are expressed as s
4
(singlet), d (doublet), t (triplet), q (quartet), p (pentet) or m (multiplet). High Resolution
Mass Spectra (HRMS) were obtained by electron spray ionization (ESI) in positive
mode using a Hewlett-Packard MSD 1100 spectrometer. Reactions under microwave
irradiation (MW) were performed in a Biotage Initiator 2.5 reactor.
S2

ACCEPTED MANUSCRIPT
Ethyl 5-acetamido-3-oxopentanoate (10). N-acetyl-β-alanine (1 g, 7.62 mmol) and
carbonyldiimidazole (CDI, 1.5 g, 9.3 mmol) were dissolved and stirred for 1 h in
tetrahydrofurane (THF, 26 mL). Potassium monoethylmalonate (2 g, 11.43 mmol) and
magnesium chloride (MgCl , 0.72 g, 7.62 mmol) were then added. The reaction was
2
stirred overnight at rt. Water was then added and the mixture extracted with EtOAc (5 x
2
0 mL), the organics were dried over MgSO and solvent removed. The crude was
4
chromatographed (DCM : MeOH gradient) to afford 10 (530 mg, 34%) as a pure oil.
+
1
HPLC-MS (m/z) [MH ] 202.14. H NMR (300 MHz, CDCl ) δ 6.02 (s, 1H), 4.21 (q, J =
3
7
3
.1 Hz, 2H), 3.51 (d, J = 5.9 Hz, 2H), 3.46 (s, 2H), 2.81 (d, J = 5.9 Hz, 2H), 1.95 (s,
H), 1.29 (t, J = 7.1 Hz, 3H).
Ethyl 6-acetamido-3-oxohexanoate (11). Following the method described for 7, from
-acetamidobutanoic acid (2.5 g, 17.2 mmol), 11 (1.18 g, 32% yield) was obtained as a
4
+
1
yellow oil. HPLC-MS (m/z) [MH ] 216.26. H NMR (300 MHz, CDCl ) δ 5.88 (s, 1H),
3
4
1
.19 (q, J = 7.1 Hz, 2H), 3.44 (s, 2H), 3.25 (q, J = 6.6 Hz, 3H), 2.61 (d, J = 6.7 Hz, 2H),
.96 (s, 3H), 1.81 (p, J = 6.7 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H).
Pechmann condensantion. General procedure. Intermediate 10 (1 eq.) was mixed
with the corresponding phenol (1.5 eq.) and methanesulfonic acid (MSA, 25 eq.) was
added at 0ºC, and then stirred at rt. Reaction evolution was followed by TLC. Reaction
times were highly dependent on phenol substituents. When the reaction reached
completion, sat. aq. NaHCO was added over the mixture and the aqueous phase
3
extracted several times with EtOAc. After drying the organics over MgSO , and
4
removal of the solvent, the resulting crude was purified to afford the corresponding 2-
oxo-2H-chromen acetamide.
S3

ACCEPTED MANUSCRIPT
N-(2-(7-Methoxy-2-oxo-2H-chromen-4-yl)ethyl)acetamide (1). Obtained from 10 and
resorcinol in 84% yield as a white solid of mp 164-166 ºC. Reaction time: 1h. Crude
1
chromatographed (DCM:MeOH gradient). HPLC purity 99% (230 to 400 nm). H NMR
(
500 MHz, CDCl ) δ 7.68 (d, J = 8.9 Hz, 1H), 6.88 (dd, J = 8.9, 2.5 Hz, 1H), 6.79 (d, J
3
=
2.5 Hz, 1H), 6.11 (s, 1H), 5.91 (s, 1H), 3.86 (s, 3H), 3.56 (d, J = 7.0 Hz, 2H), 2.97 (d,
13
J = 7.0 Hz, 2H), 1.99 (s, 3H). C NMR (126 MHz, CDCl ) δ 170.53, 162.80, 162.50,
3
1
61.17, 155.58, 153.56, 125.64, 112.60, 111.63, 101.07, 55.78, 38.66, 32.00, 23.25.
+
+
HRMS (ESI ): m/z calcd for C H NO (M) 261.1005, found 261.1001.
1
4
15
4
N-(2-(6-Oxo-6H-[1,3]dioxolo[4,5-g]chromen-8-yl)ethyl)acetamide (2). Obtained
from 10 and resorcinol in 80% yield as a white solid of mp 196-198 ºC. Reaction time:
3
4
0 min. Crude chromatographed in DCM:MeOH gradient. HPLC purity 100% (230 to
1
00 nm). H NMR (400 MHz, DMSO-d ) δ 8.03 (t, J = 5.6 Hz, 1H), 7.42 (s, 1H), 7.09
6
13
(
s, 1H), 6.17 (s, 2H), 3.31 (q, J = 6.8 Hz, 2H), 2.84 (t, J = 6.8 Hz, 2H), 1.78 (s, 3H). C
NMR (101 MHz, DMSO-d ) δ 169.51, 160.18, 154.26, 150.74, 150.36, 144.74, 112.70,
6
+
1
11.40, 102.62, 102.60, 98.10, 37.55, 31.80, 22.59. HRMS (ESI ): m/z calcd for
+
C H NO (M) 275.0794, found 275.0793.
14
13
5
N-(2-(6-Methoxy-2-oxo-2H-chromen-4-yl)ethyl)acetamide (3). Obtained from 10 and
-methoxyphenol. Reaction time: 30 days. The compound was crystallised from
4
EtOH:Et O and isolated as a white powder of mp 206-207 ºC. Yield: 67%. HPLC purity
2
1
1
00% (230 to 400 nm). H NMR (500 MHz, DMSO-d ) δ 8.09 (t, J = 5.8 Hz, 1H), 7.42
6
(d, J = 2.9 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.22 (dd, J = 9.0, 2.9 Hz, 1H), 6.36 (s,
13
1
H), 3.86 (s, 3H), 3.36 (q, J = 6.8 Hz, 2H), 2.93 (d, J = 6.8 Hz, 2H), 1.79 (s, 3H). C
NMR (126 MHz, DMSO-d ) δ 169.54, 159.88, 155.69, 153.64, 147.50, 119.39, 119.30,
6
+
1
17.75, 114.80, 107.74, 55.83, 37.52, 31.36, 22.54. HRMS (ESI ): m/z calcd for
+
C H NO (M+H) 261.1001, found 261.0999.
14
15
4
S4

ACCEPTED MANUSCRIPT
In an attempt to improve the synthesis of 3, a wide variety of methodologies reported
for the synthesis of coumarins via Pechmann condensation were tested, namely TiCl4
[
(
1], 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate)
®
Selectfluor ) [2], BiCl [3], TsOH [4], ZrCl [5], Bi(NO ) ·H O [6], H SO [7], and
3
4
3 3
2
2
4
MSA [8]. Among them, only MSA catalysis was able to afford the desired compound,
despite the extremely low rate of the reaction turnover.
N-(3-(7-Methoxy-2-oxo-2H-chromen-4-yl)propyl)acetamide (4). Obtained from 11
and resorcinol in 69% yield as an off-white solid of mp 99.5 – 101.5 ºC. Reaction time:
3
4
1
3
0 min. Crude chromatographed (DCM : MeOH gradient). HPLC purity 100% (230 to
1
00 nm). H NMR (400 MHz, DMSO-d ) δ 7.93 (t, J = 5.5 Hz, 1H), 7.72 (d, J = 8.8 Hz,
6
H), 7.00 (d, J = 2.6 Hz, 1H), 6.97 (dd, J = 8.8, 2.6 Hz, 1H), 6.20 (s, 1H), 3.86 (s, 3H),
13
.12 (q, J = 6.7 Hz, 2H), 2.83 – 2.65 (m, 2H), 1.81 (s, 3H), 1.77 – 1.68 (m, 2H). C
NMR (101 MHz, DMSO-d ) δ 169.80, 162.95, 160.91, 157.12, 155.74, 126.79, 112.88,
6
+
1
10.96, 101.63, 56.60, 38.67, 29.11, 28.58, 23.32. HRMS (ESI ): m/z calcd for
+
C H NO (M+H) 275.1158, found 275.1164.
15
17
4
2
-Formyl-4-methoxyphenyl 4-acetamidobutanoate (12). 1-Acetamidobutyric acid
(
960 mg, 6.62 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
EDCI·HCl, 1.65 g, 8.61 mmol), dimethylaminopyridine (DMAP, 162 mg, 1.32 mmol)
(
and triethylamine (TEA, 1.48 ml, 10.60 mmol) were dissolved in dichloromethane
DCM, 60 mL) and then 2-hydroxy-5-methoxybenzaldehyde (1 g, 6.62 mmol) was
(
added. The reaction was kept stirring at room temperature (rt) for 5h, then HCl 0.1M
was added till pH stayed acidic. The aqueous phase was extracted with ethylacetate
(
EtOAc, 3 x 40 mL). The organics were pooled together, dried over MgSO , solvent
4
evaporated and the resulting crude was chromatographed (DCM: MeOH) to afford
intermediate 12 (1.40 g, 76%) as a white solid. HPLC-MS (m/z) 100% (230 to 400 nm)
S5

ACCEPTED MANUSCRIPT
1
[
MH+] 280.15. H NMR (300 MHz, CDCl ) δ 10.03 (s, 1H), 7.34 (d, J = 3.1 Hz, 1H),
3
7
.17 (dd, J = 8.9, 3.1 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H), 5.85 (s, 1H), 3.87 (s, 3H), 3.40
(q, J = 6.2 Hz, 2H), 2.72 (t, J = 7.3 Hz, 2H), 2.18 – 1.87 (m, 5H).
N-(2-(6-Methoxy-2-oxo-2H-chromen-3-yl)ethyl)acetamide (5). Intermediate 12 (500
mg, 1.79 mmol), di-tert-butyldicarbonate (Boc O, 699 mg, 1.79 mmol) and DMAP (547
2
mg, 2.69 mmol) were dissolved in anhydrous acetonitrile (MeCN, 5.5 mL). The mixture
was stirred at rt for 24h. The solvent was removed to afford a crude that was
chromatographed (hexane : EtOAc) to give 230 mg of a mixture of Boc-protected-5
(HPLC-MS (m/z) [MH+] 362.35, Amax 286 nm) and 1-acetylpyrrolidin-2-one (HPLC-
MS (m/z), [MH+] 127.79, no abs. 230 to 400 nm) in a proportion of ca. 1:2 as
1
determined by H-NMR. This mixture was dissolved in triethylsilane (253 µL, 1.6
mmol), treated at 0 ºC with trifluoroacetic acid (TFA) and DCM (1.8 mL, 1:2) and then,
allowed to reach rt. After 90 min the reaction was finished and DCM partially removed.
The mixture was then added over a cooled sat. solution of ammonia in MeOH (6 mL).
Solvent and excess of ammonia were removed under vacuum and the solid mixture of
salts and organics chromatographed (DCM : MeOH) to afford 5 (40 mg, 9% two steps)
1
as a yellow shiny solid of mp 205 – 208 ºC. HPLC purity 95% (230 to 400 nm). H
NMR (500 MHz, DMSO-d ) δ 9.66 (s, 1H), 7.71 (t, J = 2.9 Hz, 1H), 6.95 (d, J = 2.4 Hz,
6
1
H), 6.87 – 6.83 (m, 2H), 3.72 (d, J = 7.2 Hz, 2H), 3.70 (s, 3H), 3.02 (td, J = 7.2, 2.9
1
3
Hz, 2H), 2.46 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 171.30, 169.04, 152.32,
6
1
51.42, 130.43, 128.43, 122.33, 117.69, 116.93, 113.52, 55.81, 42.56, 25.21, 22.89.
+
+
HRMS (ESI ): m/z calcd for C H NO (M) 261.1001, found 261.0995.
1
4
15
4
General procedure for coumarins 6 and 7. Corresponding acid (2 eq) and CDI (2.1
eq) were dissolved in the minimum amount of anhydrous DMF and microwaved at 120
ºC for 5 minutes. 2-Hydroxy-6-methoxybenzaldehyde (1 eq) and TEA (1 eq) were
S6