Semisynthesis, ex vivo evaluation, and SAR studies of coumarin derivatives as potential antiasthmatic drugs

Article abstract of DOI:10.1016/j.ejmech.2014.03.029

Asthma is a chronic inflammatory disorder that causes contraction in the smooth muscle of the airway and blocking of airflow. Reversal the contractile process is a strategy for the search of new drugs that could be used for the treatment of asthma. This work reports the semisynthesis, ex vivo relaxing evaluation and SAR studies of a series of 18 coumarins. The results pointed that the ether derivatives 1-3, 7-9 and 13-15 showed the best activity (E _max = 100%), where compound 2 (42 μM) was the most potent, being 4-times more active than theophylline (positive control). The ether homologation (methyl, ethyl and propyl) in position 7 or positions 6 and 7 of coumarins lead to relaxing effect, meanwhile formation of esters generated less active compounds than ethers. The SAR analysis showed that it is necessary the presence of two small ether groups and the methyl group at position 4 (site 3) encourage biological activity through soft hydrophobic changes in the molecule, without drastically affecting the cLogP.

Full text of DOI:10.1016/j.ejmech.2014.03.029

European Journal of Medicinal Chemistry 77 (2014) 400e408
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Semisynthesis, ex vivo evaluation, and SAR studies of coumarin
derivatives as potential antiasthmatic drugs
,
, ,
*
Amanda Sánchez-Recillas a 1, Gabriel Navarrete-Vázquez b 2, Sergio Hidalgo-Figueroa b,
María Yolanda Rios c, Maximiliano Ibarra-Barajas d, Samuel Estrada-Soto a
, ,
2
*
a Laboratorio de Farmacognosia y Química de Productos Naturales, Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida
Universidad 1001, Col. Chamilpa, 62209 Cuernavaca, Morelos, Mexico
b Laboratorio de Química Farmacéutica, Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa,
62209 Cuernavaca, Morelos, Mexico
c Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Col. Chamilpa, 62209 Cuernavaca,
Morelos, Mexico
d Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, 54090 Tlalnepantla,
Estado de México, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Asthma is a chronic inflammatory disorder that causes contraction in the smooth muscle of the airway
and blocking of airflow. Reversal the contractile process is a strategy for the search of new drugs
that could be used for the treatment of asthma. This work reports the semisynthesis, ex vivo relaxing
evaluation and SAR studies of a series of 18 coumarins. The results pointed that the ether derivatives 1e3,
Received 5 November 2013
Received in revised form
8 March 2014
Accepted 11 March 2014
Available online 12 March 2014
7e9 and 13e15 showed the best activity (Emax ¼ 100%), where compound 2 (42
mM) was the most potent,
being 4-times more active than theophylline (positive control). The ether homologation (methyl, ethyl
and propyl) in position 7 or positions 6 and 7 of coumarins lead to relaxing effect, meanwhile formation
of esters generated less active compounds than ethers. The SAR analysis showed that it is necessary the
presence of two small ether groups and the methyl group at position 4 (site 3) encourage biological
activity through soft hydrophobic changes in the molecule, without drastically affecting the cLogP.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Asthma
Coumarins
Natural products
Pharmacophoric group
SAR
Tracheal rings
1. Introduction
Abbreviations: (CH3CO)2O, acetic anhydride; mL, microliters; mM, micromolar;
C4H7BrO2, ethyl bromoacetate; CaCl2, calcium chloride; cAMP, cyclic adenosine
monophosphate; CDCl3, Deuterated chloroform; cGMP, cyclic guanosine mono-
phosphate; CH3CN, acetonitrile; CO2, carbon dioxide; CRC, concentrationeresponse
curve; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EC50, media effective
concentration; EDTA, ethylenediaminetetraacetic acid; EIeMS [Mþ], electron
impactemass spectrometry; Emax, maximum effect; Et3N, triethylamine; g, gram; H,
hour; H2SO4, sulfuric acid; Hz, hertz; J, coupling constants; K2CO3, potassium car-
bonate; KCl, potassium chloride; KH2PO4, potassium phosphate monobasic; KI,
potassium iodide; KOH, potassium hydroxide; Me2SO4, dimethyl sulfate; MgSO4,
magnesium sulfate; cLogP, partition coefficient calculated; mL, milliliters; mm,
millimeters; Mp, melting point; NaCl, sodium chloride; NaHCO3, Sodium bicar-
bonate; NMR, nuclear magnetic resonance; O2, oxygen; Oct/wat, octanolewater
mixture; ppm, parts per million; SM1, starting material 1; SM2, starting material 2;
SM3, starting material 3; SAR, structureeactivity relationship; SN2, bimolecular
nucleophilic substitution; TLC, thin layer chromatography; TMS, tetramethylsilane;
UV, ultra violet.
Asthma is a chronic inflammatory disorder of the airways. The
chronic inflammation of airway is characterized by the hyperre-
activity causing limited airflow (by bronchoconstriction, mucus
plug and increased inflammation) when airways are exposed to
allergens such as those from house dust mites, animals with fur,
cockroaches, pollens and molds, occupational irritants, tobacco
smoke, respiratory infections, exercise, chemical irritants and drugs
[1]. These affect 300 million people worldwide, especially to child
population [2]. In the first place, drug therapy is centered on
counteracting bronchoconstriction and/or inflammation processes
using b2-agonist and inhaled glucocorticoids, respectively. Besides,
there are some other therapeutic alternatives as leukotriene re-
ceptor antagonist, phosphodiesterases inhibitors, anticholinergics
and anti-IgE, which are indicated for the successful treatment of
asthma. However, they are not completely effective in chronic
treatment [1]. Plants, especially those with ethnopharmacological
uses, have been the primary source for early drug discovery.
* Corresponding authors. Facultad de Farmacia, Avenida Universidad 1001, Col.
Chamilpa, 62209 Cuernavaca, Morelos, Mexico.
E-mail addresses: gabriel_navarrete@uaem.mx (G. Navarrete-Vázquez), enoch@
uaem.mx (S. Estrada-Soto).
1
Taken from the. Ph. D. thesis of Amanda Sánchez-Recillas.
These authors contribute equally in current work.
2
http://dx.doi.org/10.1016/j.ejmech.2014.03.029
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

A. Sánchez-Recillas et al. / European Journal of Medicinal Chemistry 77 (2014) 400e408
401
Furthermore, many bioactive compounds have been isolated from
these or have served as a prototype to develop new molecules with
therapeutic interest [3], e.g. coumarins that are phenolic com-
pounds with a common structure of 2-H-1-benzopyran-2-one with
multiple biological effects such as anti-inflammatory, anticoagu-
lant, antibacterial, antifungal, antiviral, anticancer, antihyperten-
of median effective concentration (EC50), maximum effect (Emax) as
well as calculated LogP for each compound (cLogP).
Esculetin (6,7edihydroxycoumarin, SM1) is a di-hydroxylated
coumarin, which has significant pharmacological effects such as
anticoagulant, anti-inflammatory, vasoconstriction inhibitor, hep-
atoprotective, anti-obesity, hypolipidemic, and neuroprotective
agent [7e12]. Compounds designed from SM1 have great potential
to show important pharmacological effects [4]. The most active
compounds derived from SM1 were di-ethers: (1) methoxy-, (2)
ethoxy- and (3) propoxy-, which achieved 100% of relaxation and
the CRC moved toward left with respect to SM1 (Emax ¼ 48%) and
sive,
antitubercular,
anticonvulsant,
antiadipogenic,
antihyperglycemic, antioxidant, and neuroprotective properties
[4e6]. Therefore, the search for novel molecules based on coumarin
scaffold with therapeutic activity represents pharmacological al-
ternatives to treat prevalent diseases such as asthma.
theophylline (Emax ¼ 100%, EC50 ¼ 144
mM) used as positive control
(Fig. 1); this fact suggests that compounds were more potent than
2. Results and discussion
SM1 and theophylline. Compounds
1
and
3
are equipotent
(EC50 ¼ 80
m
M), and the compound 2 (EC50 ¼ 42
mM) achieved the
2.1. Chemistry
most potent tracheal relaxation of the entire series, which was 4-
times more potent than theophylline. Compounds 4, 5 and 6 (CCR
not shown) didn’t showed significant relaxant effect on the
contraction induced by carbachol.
In this work was carried out the semisynthesis of 18 coumarins
(1e18), on the basis of 6,7-dihydroxycoumarin (compounds 1e6),
7-hydroxycoumarin (compounds 7e12) and 4-methyl-7-
hydroxycoumarin (compounds 13e18) through alkylation and
esterification of hydroxyl groups in position 6, or 6 and 7 of
coumarin structure as previously described (1e3) (Table 1). Com-
pounds were obtained in discrete to moderate yields, and were
purified by recrystallization. The chemical structures of the syn-
thesized compounds were determined based on their spectral data
(1H, 13C NMR and mass spectra MSeEI [Mþ]).
Umbelliferone (7-hydroxycoumarin, SM2) is a mono-hydroxyl-
ated coumarin that also showed significant pharmacological effects
such as antidiabetic, hypolipidemic, anti-inflammatory, anti-
allergic, anti-oxidant, and bronchodilator [13e16]. This compound
was previously isolated from plant species that are used in tradi-
tional medicine as anti-asthmatic [17]. Therefore, compounds
designed from umbelliferone have great potential to show impor-
tant pharmacological effects, including as antiasthmatic [4]. The
mono derivatives of SM2 such as 7, 8 and 9 reached 100% of
relaxation, but were less potent than the di-ethers, however their
CCR were moved to the left compared than SM2 (Fig. 2). Compound
2.2. Rat tracheal ring assay
9 was the most potent (EC50 ¼ 133
mM) in this series, it is consid-
All the compounds were evaluated [0.1e500
mM] on the
ered to be equipotent with theophylline. The ester derivatives 10,11
and 12 didn’t show significant relaxing effect (Emax ¼ <30%) on the
contraction induced by carbachol.
contraction induced by carbachol (1 M, muscarinic cholinergic
m
agonist) in the trachea rat rings; and theophylline (phosphodies-
terases inhibitor) was used as positive control. Table 2 shows values
Table 1
Physicochemical data of the semisynthetic coumarin derivatives.
Compound number
R1
R2
R3
Molecular weight
(g/mol)
Melting
point (ꢀC)
Reaction
time (h)
Unoptimized
yield (%)
M.W. EI MS:
(m/z) [M]þ
SM1
1
2
3
4
5
6
SM2
7
8
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eMe
eMe
eMe
eMe
eMe
eMe
eMe
eOH
eOMe
eOEt
eOPr
eOAc
eOCOPh
eOCH2COOEt
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eOH
eOMe
eOEt
eOPr
178
206
234
262
262
386
350
162
176
190
204
204
266
248
176
190
204
218
218
280
262
271.0e273.0
128.0e130.0
105.5e106.5
79.5e81.5
127.6e130.4
185.1e186.6
107.0e108.8
230.0
e
e
178
206
234
262
178
386
350
162
176
190
204
204
266
248
176
190
204
218
219
280
262
80
48
48
48
48
6
38
54
85
78
85
91
e
eOAc
eOCOPh
eOCH2COOEt
eOH
eOMe
eOEt
eOPr
eOAc
eOCOPh
eOCH2COOEt
eOH
eOMe
eOEt
eOPr
eOAc
eOCOPh
eOCH2COOEt
e
117.8e119.8
86.7e88.7
62.6e63.9
24
60
36
0.5
12
6
95
46
27
90
78
96
e
9
10
11
12
SM3
13
14
15
16
17
18
133.5e135.0
158.0e159.3
112.2e114.2
190.0e192.0
158.6e161.7
108.2e110.5
74.4e75.2
152.5e153.5
161.5e163.5
100.1e103.1
e
48
80
240
0.5
12
8
77
37
96
92
86
71
eH
SM: Starting Materials, [M]þ ¼ molecular ion found.

402
A. Sánchez-Recillas et al. / European Journal of Medicinal Chemistry 77 (2014) 400e408
Table 2 (continued)
Table 2
Ex vivo pharmacological parameters of the relaxant effect of coumarin derivates
used in the SAR study.
Compound number Structure
Emax (%) EC50
(
mM) miLogP
Compound number Structure
Emax (%) EC50
(
m
M) miLogP
1.021
17
34
70
NS
4.234
SM1
47.9
NS
80
1
100
1.636
18
200
2.661
Bold represents the most active compounds.
Emax: maximum effect; EC50: median effective concentration; RM: Raw materials;
NS. Not shown; given that Emax < 40%.
2
3
100
42
2.388
3.394
100
89
4-Methylumbelliferone (SM3) is a coumarin that has demon-
strated pharmacological effects as analgesic, anti-arthritis, anti-
inflammatory, antipyretic, antibacterial, anti-viral, anti-cancer,
among others [18]. However, it has not been demonstrated the
possible relaxing effect of the tracheal smooth muscle. SM3 showed
significant relaxing effect (Emax ¼ 90%), and the ether derivatives
13, 14 and 15 showed 100% of relaxation. Compound 14
4
5
6
77
12
33
231
NS
0.627
5.259
2.113
(EC50 ¼ 80
mM) was the most potent in this series. The CCR of 14 is
shifted to the left compared with theophylline (Fig. 3). Once again,
the ester derivatives 16, 17 and 18 of SM3 also did not showed
significant relaxant effect.
These findings suggest that increasing in the lipophilic chain in
position 6 and/or 7 of the coumarin skeleton increases the relaxing
effect in rat tracheal rings, while reducing the lipophilic chain the
effect reduces too. As described above, one of the strategies to treat
asthma is to reduce the bronchoconstriction. The main target could
be to generate relaxation of the smooth muscle cells of the airway
by the increasing of cyclic nucleotide second messengers (cAMP
and cGMP), decrease of intracellular calcium (calcium channel
blockers), the inhibition of excitatory response (anti-cholinergic,
antihistamines, anti-leukotriens) or induce relaxing direct response
NS
SM2
7
67
100
100
100
16
449
244
245
133
NS
1.511
2.046
2.422
2.925
1.542
8
(b-agonists or phosphodiesterases inhibition) [19]. The coumarin
ether derivatives may act by inhibition of cyclic nucleotide phos-
phodiesterases that generate cAMP and cGMP increasing, as
established by Hoult and Payá [20] and Billington et al. [21]. Also,
can increase the intracellular concentration of cAMP by activation
of b2-adrenergic receptors or prostaglandins-E2 production [19],
with subsequent decrease of the influx of calcium through the
9
10
11
26
NS
3.858
12
31
90
NS
2.285
1.887
2.423
SM3
13
260
230
100
14
100
80
2.799
15
16
100
62
267
360
3.302
1.918
Fig. 1. Concentrationeresponse curves of ether compounds 1e3 and SM1 on trachea
rings pre-contracted with carbachol [1
mM]. All results are expressed as the
mean ꢁ S.E.M of six experiments (*p < 0.05).

A. Sánchez-Recillas et al. / European Journal of Medicinal Chemistry 77 (2014) 400e408
403
Fig. 4. Pharmacophoric pattern based on experimental results.
and grouped in the ALOGPS 2.1 software available online [30,31]. In
addition, the experimental EC50 and Emax values from relaxant ef-
fect were considered. SM1 and SM2 bear hydroxyl groups (OH) in
position 6 and 7 or 7, respectively, and it seems that these polar
substituents did not contributes with the relaxing effect in the
trachea, while the SM3, with a hydroxyl group in position C-7 and a
small lipophilic group (eCH3) in position C-4, increased the relax-
atory effect. Homologated ether derivatives (with a maximum of
three carbons) in the position C-6 and C-7 of the coumarins,
drastically increase relaxant effect generating the most potent
compounds as 1, 2 and 3. The ether formation only in position C-7
with and without a methyl group in C-4 also increases the relaxant
effect; however, all of them are less potent than 1, 2 and 3, which
indicates that is necessary to add liphophilic groups such as di-
ether derivatives to improve the relaxant effect. Ester derivatives
(acetyl and benzoyl) reduced relaxant effect. SAR analysis revealed
the minimal stereo-electronic requirements for good relaxation
activity: 1) Positions C-6 and C-7 substituted with homologous
aliphatic ether chains (1e3 carbon atoms, preferable two) reflect
possible steric and lipophilic molecular recognition from the cav-
ities in the active site. Strong improvements of ligand affinity for
many therapeutic targets are frequently accompanied by modest
lipophilicity increases [32]. 2) Sometimes, a methyl group with its
lipophilic surface well included into a protein hydrophobic pocket
is expected to provide 1.5 kcal/mol binding energy and so give up to
a 10-fold improvement in binding energy [33]. In this case, position
C-4 of the coumarin replaced with a methyl group (compound 14),
improved the activity 3-fold versus compound 8, but it was not as
active as the most potent compound (2).
Fig. 2. Concentrationeresponse curves of ether compounds 7e9 and SM2 on trachea
rings pre-contracted with carbachol [1 M]. All results are expressed as the
mean ꢁ S.E.M of six experiments (*p < 0.05).
m
blockade of calcium channels of membrane [22], as well as blocking
of calcium channels from intracellular, generating the decrease in
intracellular calcium concentrations [23,24]. Meurs et al. [25] sug-
gest that the importance of muscarinic receptors not only involves
the blockage of the airway, but also have an important regulatory
role in the function of b2-adrenergic receptors agonists and
inflammation; thus, the coumarin derivatives can act through a
muscarinic antagonism [26]. It is worth mentioning that previous
studies have shown that the compound 1 has significant vaso-
relaxant [27] and anti-allergic [28] effects. Finally, the underlying
mechanism of relaxant action and the possible anti-inflammatory
activity of the most active compounds 1, 2, 3, 9 and 14 are cur-
rent studying in order to develop new potential drugs for the
treatment of asthma [24,29].
2.3. Structureeactivity relationship (SAR) of coumarin derivatives
For SAR analysis it was used descriptors of LogP values (Table 2)
calculated with different programs and after that were averaged
Based on the most active molecules treated in this study
(compounds 2 and 14), we examine the effect of different sub-
stituents in the molecular scaffold of coumarin derivatives. The
construction of pharmacophoric model (Fig. 4) and a proposed new
molecule designed (Fig. 5) was based on experimental results and is
Fig. 3. Concentrationeresponse curves of ether compounds 13e15 and SM3 on trachea
rings pre-contracted with carbachol [1
mM]. All results are expressed as the
Fig. 5. New coumarin derivative molecule designed based on the data generated in
mean ꢁ S.E.M of six experiments (*p < 0.05).
this work.

404
A. Sánchez-Recillas et al. / European Journal of Medicinal Chemistry 77 (2014) 400e408
describing the main chemical features shared by the most active
coumarin derivatives. A chemical inspection of molecular structure
suggest an incorporation of methyl group at position 4, as well as
ethoxy substitutions at positions 6 and 7. The three dimensional
display was built with the program DSV3.5. [34].
4.1.2.1. 6,7-Dimethoxy-2H-chromen-2-one (1). Pale yellow powder,
60% yield, mp: 128.0e130.0 ꢀC, recrystallized from 100% ethanol. 1H
NMR (CDCl3) d: 3.93 (3H, s, CH3), 3.95 (3H, s, CH3), 6.28 (1H, d,
J ¼ 9.6 Hz), 6.83 (1H, s), 6.87 (1H, s) 7.64 (1H, d, J ¼ 9.6 Hz); 13C NMR
(50 MHz, CDCl3): dC 56.2 CH3, 100.1 (CH), 108.1 (CH), 111.5 (C), 113.5
(CH), 143.4 (CH), 146.4 (C), 150.1 (C), 152.9 (C), 161.4 (C).
3. Conclusions
4.1.2.2. 7-Methoxy-2H-chromen-2-one (7). White crystals, 95%
yield, mp: 117.8e119.8 ꢀC, recrystallized from 100% methanol. 1H
The skeleton of 2H-1-benzopyran-2-one (coumarin) represents
a remarkable scaffold with interesting airway relaxant properties to
develop new therapeutic alternatives that are useful in the treat-
ment of asthma. Compounds 2, 9 and 14, from each series,
respectively, showed the best activity, being compound 2 the most
potent of all of three series. The SAR analysis shown that the
presence of two small ether groups (6 and/or 7 position) and the
methyl group at position 4, increases biological activity through
soft changes in the hydrophobic characteristic of the molecule,
since it doesn’t drastically affects the LogP. Last should be between
2.0 and 2.7 and/or less than 3, because after that value the hydro-
phobic molecule would lose effectiveness and potency.
NMR (CDCl3)
d
: 3.88 (3H, s), 6.25 (1H, d, J ¼ 9.6 Hz), 6.86 (1H, d,
J ¼ 1.6 Hz), 7.63 (2H, d, J ¼ 9.6 Hz), 7.37 (1H, dd, J ¼ 9.6 Hz, 1.6 Hz);
13C NMR (200 MHz, CDCl3): dC 55.9 (CH3), 101.0 (CH), 112.7 (CH),
113.2 (CH), 113.2 (C), 128.9 (CH), 143.5 (CH), 160.0 (C), 161.2 (C),
163.0 (C).
4.1.2.3. 7-Methoxy-4-methyl-2H-chromen-2-one (13). White pow-
der, 77% yield, mp: 158.6e161.7 ꢀC, recrystallized from 100%
ethanol. 1H NMR (CDCl3)
d: 2.40 (3H, s), 3.88 (3H, s), 6.14 (1H, s),
6.82 (1H, d, J ¼ 2.6 Hz), 6.86 (1H, dd, J ¼ 8.8, 2.6 Hz), 7.50 (1H, d,
J ¼ 8.8 Hz); 13C NMR (200 MHz, CDCl3): dC 18.9 (CH3), 55.9 (CH3),
101.1 (CH), 112.0 (CH), 112.5 (CH), 113.8 (C), 125.7 (CH), 152.7 (C),
155.4 (C), 161.4 (C), 162.8 (C).
4. Experimental
4.1.3. Preparation of 2, 8 and 14 (Scheme 1b)
4.1. Chemistry
0.4 g of SM1 (2.2 mmol), SM2 (2.4 mmol) and SM3 (2.2 mmol),
respectively, were dissolved with 2 mL of DMF (compound 2) or
acetone (compound 8 and 14). After that, solid K2CO3 (1.9 or 3.8
equivalents) were add, and stirred during thirty minutes. 2.2
equivalents of ethyl iodide and potassium iodide (as catalyst) were
added to the mixture and heating for 2, 2.5 and 5 days at 40 ꢀC,
respectively. Finally, when reaction was completed (monitored by
TLC analysis), the solid was filtered off and dried to afford the
corresponding compounds 2 (6,7-diethoxy-2H-chromen-2-one), 8
(7-ethoxy-2H-chromen-2-one) and 14 (7-ethoxy-4-methyl-2H-
chromen-2-one).
All chemicals were ACS grade and all starting materials and
reagents were obtained commercially from SigmaeAldrich and
were used as received. Melting points were determined on an EZ-
Melt MPA120 automated melting point apparatus from Stanford
Research Systems and are uncorrected. TLC monitored reactions on
1.5 ꢂ 3.0 cm pre-coated silica gel 60 F254 plates (E. Merck KGaA,
Darmstadt, Germany) and visualized with 254-nm UV light. The
reactions that lasted for more than 24 h were kept in refrigerator for
overnight.
NMR studies were carried out with a Varian Inova 400 instru-
ment. Chemical shifts (dH, dC) and coupling constants values (J) are
given in ppm and Hz, respectively. The following abbreviations for
signal multiplicities are represented by s (singlet), d (doublet), t
(triplet), q (quartet) and m (multiplet). Standard reference was
used: TMS (dH ¼ 0, dC ¼ 0) in CDCl3. Mass spectra were recorded on
a Jeol JMS-700 equipment (JEOL USA Inc., Peabody, MA, USA).
4.1.3.1. 6,7-Diethoxy-2H-chromen-2-one (2). Yellow powder, 54%
yield, mp: 105.5e106.5 ꢀC, recrystallized from 100% ethanol. 1H
NMR (CDCl3)
d
: 1.48 (3H, t, J ¼ 7.2 Hz), 1.51 (3H, t, J ¼ 7.2 Hz), 4.10
(2H, q, J ¼ 7.2 Hz), 4.15 (2H, q, J ¼ 7.2 Hz), 6.26 (1H, d, J ¼ 9.6 Hz),
6.82 (1H, s), 6.87 (1H, s), 7.60 (1H, d, J ¼ 9.6 Hz); 13C NMR (400 MHz,
CDCl3): dC 14.6 (CH2), 14.9 (CH2), 65.0 (CH2), 65.4 (CH2), 101.2 (CH),
110.3 (CH),111.5 (C),113.4 (CH),143.6 (CH),145.9 (C),150.2 (C),152.9
(C), 161.7 (C).
4.1.1. General method of semisynthesis of compounds 1e3, 7e9 and
13e15
Compounds were prepared using bimolecular nucleophilic sub-
stitution (SN2) reaction, starting from 6,7-dihydroxycoumarin (SM1),
7-hydroxycoumarin (SM2) or 7-hydroxy-4-methylcoumarin (SM3)
as starting materials (SM), polar aprotic solvents [acetone, acetoni-
trile (CH3CN), dimethylformamide (DMF)] and potassium carbonate
(K2CO3) or potassium hydroxide (KOH) as a base. All reactions were
carried out with moderate heating (<40 ꢀC). The crude products
were washed with cold water, filtered, dried and purified according
to the procedures reported [35].
4.1.3.2. 7-Ethoxy-2H-chromen-2-one (8). White powder, 46% yield,
mp: 86.7e88.7 ꢀC, recrystallized from 100% ethanol. 1H NMR
(CDCl3)
d
: 1.46 (3H, t, J ¼ 7.0 Hz), 4.10 (2H, q, J ¼ 7.0 Hz), 6.24 (1H, d,
J ¼ 9.4 Hz), 6.81 (1H, s), 7.34 (1H, d, J ¼ 8.8 Hz), 7.63 (1H, d,
J ¼ 9.4 Hz), 7.82 (1H, dd, J ¼ 8.8, 2.4 Hz); 13C NMR (200 MHz, CDCl3):
dC 14.8 (CH3), 64.4 (CH2), 101.5 (CH), 112.5 (C), 113.1 (CH), 113.1 (CH),
128.8 (CH), 143.6 (CH), 156.0 (C), 161.4 (C), 162.3 (C).
4.1.3.3. 7-Ethoxy-4-methyl-2H-chromen-2-one (14). White powder,
37% yield, mp: 108.2e110.5 ꢀC, recrystallized from 100% ethanol. 1H
4.1.2. Preparation of 1, 7 and 13 (Scheme 1a)
NMR (CDCl3)
d
: 1.42 (3H, t, J ¼ 7.2 Hz), 2.36 (3H, d, J ¼ 1.2 Hz), 4.06
0.4 g of SM1 (2.2 mmol), SM2 (2.4 mmol) and SM3 (2.2 mmol),
respectively, were dissolved with CH3CN (2.0 mL). After that, solid
KOH (1.5 or 3 equivalents) was added, and stirred during thirty
minutes. 2.1 equivalents of dimethyl sulfate (Me2SO4) were added to
the mixture and heating for 48 h at 40 ꢀC. Finally, when reaction was
completed (monitored by TLC analysis), the solid was filtered off and
dried to afford the corresponding compounds 1 (6,7-dimethoxy-2H-
chromen-2-one), 7 (7-methoxy-2H-chromen-2-one) and 13 (7-
methoxy-4-methyl-2H-chromen-2-one), respectively.
(2H, q, J ¼ 7.2 Hz), 6.09 (1H, s), 6.76 (1H, d, J ¼ 2.8 Hz), 6.82 (1H, dd,
J ¼ 8.8, 2.8 Hz), 7.45 (1H, d, J ¼ 8.8 Hz); 13C NMR (400 MHz, CDCl3):
dC 14.5 (CH3), 18.7 (CH3), 64.1 (CH2), 101.3 (CH), 111.8 (CH), 112.6
(CH), 113.5 (C), 125.5 (CH), 152.7 (C), 155.2 (C), 161.5 (C), 162.0 (C).
4.1.4. Preparation of 3, 9 and 15 (Scheme 1c)
0.4 g of SM1 (2.2 mmol), SM2 (2.4 mmol) and SM3 (2.2 mmol),
respectively, were dissolved with 2 mL of acetone (for compound 9
and 15) and DMF (for compound 3). After that, solid K2CO3 (4 molar

A. Sánchez-Recillas et al. / European Journal of Medicinal Chemistry 77 (2014) 400e408
405
Scheme 1. Synthesis of homologous coumarin derivates: methyl (a), ethyl (b), propyl (c), acetyl (d), benzoyl and (e) Ethoxy-2-oxoethyl (f). Reagents and conditions: (i) CH3CN, KOH,
Me2SO4/reflux, (ii) DMF or acetone, K2CO3, ethyl iodide, KI/reflux, (iii) DMF or acetone, K2CO3, n-propyl bromide, Et3N or KI/reflux, (iv) acetic anhydride, H2SO4, Et3N, (v) benzoyl
chloride, Et3N (vi), acetone, K2CO3, ethyl bromoacetate.
equivalent) was added and stirred during thirty minutes. Then 3
equivalents of triethylamine (Et3N) and 3 equivalents of n-propyl
bromide were added. All of three reactions were heating for 2, 2.5
and 5 days at 40 ꢀC, respectively. After reactions were completed
(analyzed by TLC), the solid was filtered off and dried to afford the
corresponding compounds 3 (6,7-dipropoxy-2H-chromen-2-one),
9 (7-propoxy-2H-chromen-2-one) and 15 (4-methyl-7-propoxy-
2H-chromen-2-one).
4.1.4.1. 6,7-Dipropoxy-2H-chromen-2-one (3). Beige powder, 85%
yield, mp: 79.2e81.8 ꢀC, recrystallized from 100% methanol. 1H
NMR (CDCl3)
d: 1.06 (3H, t, J ¼ 7.6), 1.08 (3H, t, J ¼ 7.6), 1.85 (2H, q,

406
A. Sánchez-Recillas et al. / European Journal of Medicinal Chemistry 77 (2014) 400e408
J ¼ 7.2 Hz), 1.90 (2H, q, J ¼ 7.2 Hz), 3.98 (2H, t, J ¼ 6.4 Hz), 4.01 (2H, t,
J ¼ 6.4 Hz), 6.25 (1H, d, J ¼ 9.6 Hz), 6.81 (1H, s), 6.87 (1H, s), 7.60
(1H, d, J ¼ 9.6 Hz); 13C NMR (400 MHz, CDCl3): dC 10.6 (CH3), 10.6
(CH3), 22.4 (CH2), 22.5 (CH2), 70.9 (CH2), 71.6 (CH2),101.1 (CH), 110.8
(CH), 111.5 (C), 113.3 (CH), 143.6 (CH), 146.3(C), 150.3 (C), 153.4 (C),
161.7 (C).
(1H, d, J ¼ 8.4 Hz); 13C NMR (200 MHz, CDCl3): dC 18.9 (CH3), 21.4
(CH3), 110.6 (CH), 114.7 (CH), 118.0 (C), 118.2 (CH), 125.5 (CH), 152.0
(C), 153.2 (C), 154.3 (C), 160.5 (C), 168.8 (C).
4.1.7. Preparation of 5, 11 and 17 (Scheme 1e)
0.4 g of SM1 (2.2 mmol), SM2 (2.4 mmol) and SM3 (2.2 mmol),
respectively, were stirred in 2 mL of CH2Cl2. After that, 2.5 molar
equivalents of Et3N, and 5 molar equivalents of benzoyl chloride
were added to the mixture. Reaction was agitated for 2 days and
12 h for compounds 5, 11 and 17, respectively. After reactions were
completed (analyzed by TLC), the solid was filtered off and dried to
afford the corresponding compounds 5 (2-oxo-2H-chromen-7-yl
benzoate), 11 (2-oxo-2H-chromene-7-yl benzoate) 17. (4-methyl-
2-oxo-2H-chromene-7-yl benzoate) 17.
4.1.4.2. 7-Propoxy-2H-chromen-2-one (9). White powder, 27%
yield, mp: 62.6e63.9 ꢀC, recrystallized from 1:1 methanol: ethanol
mixture. 1H NMR (CDCl3)
d
: 1.05 (3H, t, J ¼ 7.6 Hz), 1.83 (2H, q,
J ¼ 7.2 Hz), 3.97 (2H, t, J ¼ 6.4 Hz), 6.23 (1H, d, J ¼ 9.6 Hz), 6.80 (1H,
d, J ¼ 2.4 Hz), 6.84 (1H, d, 2.4 Hz), 7.36 (1H, d, J ¼ 8.4 Hz), 7.63 (1H, d,
J ¼ 9.6 Hz); 13C NMR (100 MHz, CDCl3): dC 10.6 (CH3), 22.5 (CH2),
70.3 (CH2), 101.5 (CH), 112.5 (CH), 113.1 (C), 113.1 (CH), 128.9 (CH),
143.7 (CH), 156.9 (C), 161.5 (C), 162.6 (C).
4.1.7.1. 2-Oxo-2H-chromene-6,7-diyl
Translucent powder, 82% yield, mp: 185.1e186.6 ꢀC, recrystallized
from 100% acetonitrile. 1H NMR (CDCl3)
7.26 (1H, s), 7.39 (4H, dd, J ¼ 8.0, 8.0 Hz), 7.44 (1H, s), 7.56 (2H, m),
7.72 (1H, d, J ¼ 9.6 Hz), 8.05 (4H, d, J ¼ 7.6 Hz); 13C NMR (200 MHz,
CDCl3): dC 113.2 (CH), 117.7 (C), 117.7 (CH), 121.6 (CH), 128.2 (C),
129.4 (CH), 131.1 (CH), 134.8 (CH), 134.9 (CH), 139.4 (C), 143.2 (CH),
146.7 (C), 152.7 (C), 160.8 (C), 164.9 (C).
dibenzoate
(5).
4.1.4.3. 4-Methyl-7-propoxy-2H-chromen-2-one (15). White crys-
tals, 96% yield, mp: 74.4e75.2 ꢀC, recrystallized from 100% meth-
d
: 6.48 (1H, d, J ¼ 9.6 Hz),
anol. 1H NMR (CDCl3)
d
: 1.03 (3H, t, J ¼ 7.6 Hz), 1.81 (2H, m), 1.83
(2H, q, J ¼ 7.2 Hz), 2.36 (3H, d, J ¼ 1.2 Hz), 3.94 (2H, t, J ¼ 6.4 Hz),
6.23 (1H, s), 6.82 (1H, dd, J ¼ 2.4, 2.4 Hz), 6.75 (1H, d, J ¼ 2.4 Hz),
7.44 (1H, d, J ¼ 8.8 Hz); 13C NMR (400 MHz, CDCl3): dC 10.41 (CH3),
18.7 (CH3), 22.3 (CH2), 70.0 (CH2), 101.3 (CH), 111.8 (CH), 112.6 (CH),
113.4 (C), 125.4 (CH), 152.5 (C), 155.3 (C), 161.3 (C), 162.2 (C).
4.1.7.2. 2-Oxo-2H-chromen-7-yl benzoate (11). White powder, 78%
yield, mp: 158.0e159.3 ꢀC, recrystallized from 100% methanol. 1H
4.1.5. General methods of synthesis of compounds 4e5, 10e11 and
16e17
NMR (CDCl3)
d: 6.42 (1H, d, J ¼ 9.6 Hz), 7.19 (1H, dd, J ¼ 8.4, 2.2 Hz),
Esterification of SM1 (2.2 mmol), SM2 (2.4 mmol) and SM3
(2.2 mmol) were carried out with acetic anhydride [(CH3CO)2O] and
sulfuric acid as catalyst, or benzoyl chloride and Et3N. All reactions
were carried out in agitation at room temperature. The crude re-
actions were washed with cold water, filtered, dried and purified
according to the procedures reported [35].
7.53 (1H, d, J ¼ 2.2 Hz), 7.48e7.60 (3H, m), 7.66 (1H, d, J ¼ 8.4 Hz),
7.72 (1H, d, J ¼ 9.6 Hz), 8.21 (2H, d, J ¼ 7.6 Hz); 13C NMR (200 MHz,
CDCl3): dC 110.8 (CH), 116.3 (CH), 116.9 (C), 118.7 (CH), 128.8 (CH),
128.9 (CH), 130.4 (CH), 134.2 (C), 134.2 (CH), 143.0 (CH), 153.6 (C),
154.9 (C), 160.4 (C), 164.6 (C).
4.1.7.3. 4-Methyl-2-oxo-2H-chromen-7-yl benzoate (17). White
powder, 86% yield, mp: 161.5e163.5 ꢀC, recrystallized from 100%
4.1.6. Preparation of 4, 10 and 16 (Scheme 1d)
0.4 g of SM1 (2.2 mmol), SM2 (2.4 mmol) and SM3 (2.2 mmol),
respectively, were stirred with 4 molar equivalents of acetic an-
hydride and two drops of H2SO4 (as catalyst) under ice bath. After
that, 2.5 molar equivalents of Et3N were added to the mixture and
agitated for 2 days, 10 min and 30 min, respectively, at room tem-
perature. After reactions were completed (analyzed by TLC), the
solid was filtered off and dried to afford the corresponding com-
pounds 4 (2-oxo-2H-chromene-6,7-diyl diacetate), 10 (2-oxo-2H-
chromen-7-yl acetate) and 16 (4-methyl-2-oxo-2H-chromen-7-yl
acetate), respectively.
ethanol. 1H NMR (CDCl3)
d
: 2.46 (3H, d, J ¼ 1.2 Hz), 6.30 (1H, d,
J ¼ 1.2 Hz), 7.21 (1H, dd, J ¼ 8.4,1.8 Hz), 7.25 (1H, d, J ¼ 1.8 Hz), 7.48e
7.50 (3H, m), 7.67 (1H, d, J ¼ 8.4 Hz), 8.22 (2H, dd, J ¼ 8.4,1.4 Hz); 13
C
NMR (200 MHz, CDCl3): dC 19.0 (CH3), 110.8 (CH), 114.7 (CH), 119.0
(C), 118.4 (CH), 125.6 (CH), 128.9 (CH), 130.4 (CH), 134.2 (CH), 152.0
(C), 153.5 (C), 154.4 (C), 160.6 (C), 164.6 (C), 190.0 (C).
4.1.8. General methods of synthesis of compounds 6, 12 and 18
Reactions were carried out by etherification of SM1 (2.2 mmol),
SM2 (2.4 mmol) and SM3 (2.2 mmol) with ethyl bromoacetate
(C4H7BrO2), K2CO3, and acetone as solvent. The mixture was stirred
at room temperature. The crude products were washed with cold
water, filtered, dried and purified according to the procedures re-
ported [35].
4.1.6.1. 2-Oxo-2H-chromene-6,7-diyl diacetate (4). Gray powder,
78% yield, mp: 127.6e130.4 ꢀC, recrystallized from 100% ethanol. 1H
NMR (CDCl3)
d
: 2.32 (3H, s), 2.34 (3H, s), 6.43 (1H, d, J ¼ 9.6 Hz), 7.26
(1H, s), 7.35 (1H, s), 7.64 (1H, d, J ¼ 9.6 Hz); 13C NMR (200 MHz,
CDCl3): dC 20.8 (CH3), 112.5 (CH), 117.0 (C), 117.1 (CH), 121.8 (CH),
138.9 (C), 142.5 (CH), 144.9 (C), 151.8 (C), 160.0 (C), 167.7 (C).
4.1.9. Preparation of 6, 12 and 18 (Scheme 1f)
0.4 g of SM1 (2.2 mmol), SM2 (2.4 mmol) and SM3 (2.2 mmol),
respectively, were dissolved in acetone (2.0 mL) and 1.75 or 3.5
molar equivalents of K2CO3 was add, and stirred during thirty min.
After that, it was added drop wise 1.75 molar equivalents of ethyl
bromoacetate. The mixture was reacted at room temperature dur-
ing 6 h for compounds 6 and 12, and 8 h for 18. After reactions were
completed (analyzed by TLC), the solid was filtered off and dried to
afford the corresponding compounds 6 ethyl {[6-(2-ethoxy-2-
oxoethoxy)-2-oxo-2H-chromen-7-yl]oxy}acetate, 12 [ethyl 2-(2-
oxo-2H-chromen-7-yloxy)acetate] and 18 [ethyl 2-(4-methyl-2-
oxo-2H-chromen-7-yloxy)acetate].
4.1.6.2. 2-Oxo-2H-chromen-7-yl acetate (10). White powder, 90%
yield, mp: 133.5e135.0 ꢀC, recrystallized from 100% ethanol. 1H
NMR (CDCl3)
d
: 2.34 (3H, d, J ¼ 2.4 Hz), 6.40 (1H, d, J ¼ 10.2 Hz), 7.05
(1H, dd, J ¼ 8.4, 2.2 Hz), 7.12 (1H, d, J ¼ 2.2 Hz), 7.49 (1H, d,
J ¼ 8.4 Hz), 7.70 (1H, d, J ¼ 10.2 Hz); 13C NMR (50 MHz, CDCl3): dC
20.4 (CH3), 110.6 (CH), 116.2 (CH), 116.8 (C), 118.5 (CH), 128.7 (CH),
143.0 (CH), 153.3 (C), 154.8 (C), 160.4 (C), 168.8 (C).
4.1.6.3. 4-Methyl-2-oxo-2H-chromen-7-yl
powder, 92% yield, mp: 152.5e153.5 ꢀC, recrystallized from ethanol.
1H NMR (CDCl3)
: 2.34 (3H, s), 2.43 (3H, d, J ¼ 1.2 Hz), 6.27 (1H, d,
J ¼ 1.2 Hz), 7.06 (1H, d, J ¼ 1.8 Hz), 7.11 (1H, dd, J ¼ 8.4, 1.8 Hz), 7.61
acetate
(16). White
d
4.1.9.1. Ethyl {[6-(2-ethoxy-2-oxoethoxy)-2-oxo-2H-chromen-7-yl]
oxy}acetate (6). White powder, 91% yield, mp: 107.0e108.8 ꢀC,

A. Sánchez-Recillas et al. / European Journal of Medicinal Chemistry 77 (2014) 400e408
407
recrystallized from 100% ethanol. 1H NMR (CDCl3)
d
: 1.30 (3H, t,
contraction before and after addition of the test materials. Muscular
tone was calculated from the tracings, using Acknowledge software
(BiopacÒ).
J ¼ 7.0 Hz), 1.32 (3H, t, J ¼ 7.0 Hz), 4.27 (2H, q, J ¼ 7.0, 3.4 Hz), 4.28
(2H, q, J ¼ 7.0, 3.4 Hz), 4.75 (4H, d, J ¼ 7.6 Hz), 6.30 (1H, d, J ¼ 9.6 Hz),
6.76 (1H, s), 7.01 (1H, s), 7.59 (1H, d, J ¼ 9.6 Hz); 13C NMR (200 MHz,
CDCl3): dC 14.4 (CH3), 61.6 (CH2), 61.9 (CH2), 66.2 (CH2), 67.6 (CH2),
102.4 (CH), 112.7 (C), 114.2 (CH), 114.6 (CH), 143.1 (CH), 144.9 (C),
150.5 (C), 151.9 (C), 161.0 (C), 167.8 (C), 168.8 (C).
4.3. Theoretical evaluation of lipophilicity
For a better understanding of the overall properties of the
described compounds, the lipophilicity values were estimated
trough theoretical determination with miLogP (expressed as the
oct/wat) using the Molinspiration calculation program [37,38], also
were calculated with different programs, and after that were
averaged and grouped in the ALOGPS 2.1 software available online
[30,31].
4.1.9.2. Ethyl
Yellow powder, 96% yield, mp: 112.2e114.4 ꢀC, recrystallized from
100% ethanol. 1H NMR (CDCl3)
2-(2-oxo-2H-chromen-7-yloxy)acetate
(12).
d: 1.32 (3H, t, J ¼ 7.0 Hz), 4.29 (2H, q,
J ¼ 7.0 Hz), 4.68 (2H, s), 6.28 (1H, d, J ¼ 9.6 Hz), 6.78 (1H, d,
J ¼ 2.2 Hz), 6.90 (1H, dd, J ¼ 8.4, 2.2 Hz), 7.40 (1H, d, J ¼ 8.4 Hz), 7.64
(1H, d, J ¼ 9.6 Hz); 13C NMR (200 MHz, Acetone-d6): dC 14.4 (CH3),
61.9 (CH2), 65.6 (CH2), 101.9 (CH), 113.0 (CH), 113.5 (C), 113.9 (CH),
129.1 (CH), 143.3 (CH), 155.8 (C), 161.0 (C), 168.0 (C).
4.4. Data analysis
Data are expressed as mean of six experiments ꢁ standard error
of mean (SEM). Concentration responses curves (CRC) were plotted
and experimental data of the CRC were adjusted by the nonlinear,
curve-fitting program Microcal TM origin 8.0 (Microcal Software
Inc., USA). Significance was evaluated using the Student’s t test.
Values of *p < 0.05 imply significance of the pharmacological ef-
fects in the experiments.
4.1.9.3. Ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetate (18).
White crystals, 71% yield, mp: 100.1e103.2 ꢀC, recrystallized from
100% ethanol. 1H NMR (CDCl3)
d: 1.29 (3H, t, J ¼ 7.2 Hz), 2.37 (3H, d,
J ¼ 1.2 Hz), 4.25 (2H, q, J ¼ 7.2 Hz), 4.65 (2H, s), 6.13 (1H, q, 1.2 Hz),
6.75 (1H, d, J ¼ 2.8 Hz), 6.88 (1H, dd, J ¼ 8.8, 2.8 Hz), 7.49 (1H, d,
J ¼ 8.8 Hz); 13C NMR (400 MHz, CDCl3): dC 14.1 (CH3), 18.6 (CH3),
61.7 (CH2), 65.3 (CH2), 101.7 (CH), 112.5 (CH), 112.5 (C), 114.4 (CH),
125.7 (CH), 152.3 (C), 155.0 (C), 160.6 (C), 161.0 (C), 167.9 (C).
Acknowledgments
4.2. Pharmacological evaluation
This study was supported by Consejo Nacional de Ciencia y
Tecnología (CONACYT) Proyecto de Ciencia Básica 2011-01
(167044) and Fellowship grant (298553) for the Ph.D studies of A.
Sánchez-Recillas.
4.2.1. Chemicals and drugs
Carbamylcholine (carbachol), teophylline, dimethyl sulfoxide
(DMSO) and ethyl ether were purchased from SigmaeAldrich Co.
(St. Louis, MO, USA).
Appendix A. Supplementary data
4.2.2. Animals
Healthy male Wistar rats (250e300 g) were used and main-
tained under standard laboratory conditions with free access to
food and water. All animal procedures were conducted in accor-
dance with our Federal Regulations for Animal Experimentation
and Care (SAGARPA, NOM-062-ZOO-1999, México), and approved
by the Institutional Animal Care and Use Committee based on US
National Institute of Health publication (No. 85-23, revised 1985).
All experiments were carried out using six animals per group.
Animals used were euthanized by exposure to ethylic ether and
cervical dislocation.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.029.
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