Natural organic acid as green catalyst for xanthenones synthesis: Methodology, mechanism and calcium channel blocking activity

Article abstract of DOI:10.21577/0103-5053.20170082

Xanthenones were synthesized via one-pot tricomponent reaction, under solvent-free conditions, using aldehydes, phenolic and cyclic 1,3-dicarbonyl compounds. Natural organic acids (NOAs), compounds present in many living metabolisms, were used as potentia

Full text of DOI:10.21577/0103-5053.20170082

http://dx.doi.org/10.21577/0103-5053.20170082
J. Braz. Chem. Soc., Vol. 28, No. 12, 2313-2325, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
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103 - 5053 $6.00+0.00
Article
Natural Organic Acid as Green Catalyst for Xanthenones Synthesis: Methodology,
Mechanism and Calcium Channel Blocking Activity
a
a
a
b
Bruna S. Terra, Aura M. B. Osorio, Aline de Oliveira, Rebeca P. M. Santos,
b
b
c
Andressa P. Mouro, Natália F. de Araújo, Cameron C. da Silva,
c
b
b
a
Felipe T. Martins, Luciene B. Vieira, Daniella Bonaventura, Heitor A. de Abreu,
a
,a
Antonio F. C. Alcântara and Ângelo de Fátima*
a
b
Departamento de Química and Departamento de Farmacologia,
Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos, 6627,
1270-901 Belo Horizonte-MG, Brazil
3
c
Instituto de Química, Universidade Federal de Goiás, Campus Samambaia,
CP 131, 74001-970 Goiânia-GO, Brazil
Xanthenones were synthesized via one-pot tricomponent reaction, under solvent-free
conditions, using aldehydes, phenolic and cyclic 1,3-dicarbonyl compounds. Natural organic
acids (NOAs), compounds present in many living metabolisms, were used as potential green
catalysts. NOA are considered to be more eco-friendly and user-friendly alternative to traditional
methodologies. Optimization studies showed that oxalic acid was the best NOA catalyst for
such reaction furnishing the xanthenones with up to 93% of yield. Theoretical calculations
were performed to evaluate this reaction mechanism and regioselectivity. The results showed
that the regiospecificity of this three-component reaction is kinetically and thermodynamically
controlled by the addition of b-naphthol C2, instead of C10, to the aldehyde. Our results also
disclosed two xanthenones as novel calcium channels blockers. Eco-friendly reaction conditions,
easy workup procedure, short reaction times and good yields are some of the advantages of our
methodology.
Keywords: xanthenones, natural organic acid, theoretical calculations, calcium channels
blockers
Introduction
Xanthones (Figure 1) are a class of oxygenated
heterocycles that contain an aromatic ring fused to
1
4
-carbonyl-pyran ring as the basic skeleton. They
present several biological and pharmacologic properties:
antivascular, antiarrhythmic, antihypertensive, antioxidant,
anti-inflammatory, anticancer and calcium channel
Figure 1. Xanthone and xanthenone base structure (R = aromatic or
alkyl group).
2
4
blocking. On the other hand, xanthenones (Figure 1),
ceric ammonium nitrate, polyethyleneglycol bound sulfonic
5
6
8
xanthone derivatives, are poorly explored regarding
their biological properties, and described only for its
acid, Hy zeolite, triflate proline, tetrabutyl ammonium
9
10
fluoride, tri-methyl tetradecyl ammonium bromide,
sulfamic acid, p-toluenesulfonic acid, triphenylphosphine-
m-sulfonate/carbon tetrabromide, phosphorus(V) oxide,
indium(III) chloride, scandium(II) triflate, boron
trifluoride diethyl etherate,
liquids, 2,4,6-trichloro-1,3,5-triazine, silica-supported
perchloric acid, succinimide-N-sulfonic acid, ytterbium
perfluorooctanoate, thiamine hydrochloride and trityl
3,4
5
6
11
12
anti-proliferative, antiviral and anti-microbial properties.
13
14
The literature describes the synthesis of xanthenones
via a multicomponent reaction using a phenolic compound,
1
4
15
7
16,17
an aldehyde, and a cyclic 1,3-dicarbonyl compound. Many
functionalized ionic
3
18
19
LewisorBrönstedacidsmaybeusedascatalysts:calixarenes,
20
21
2
2
23
*e-mail: adefatima@qui.ufmg.br

2
314
Natural Organic Acid as Green Catalyst for Xanthenones Synthesis
J. Braz. Chem. Soc.
2
4
chloride. However, most of these methods do not
comply with environmental-friendly protocols, presenting
one or more of the drawbacks: less availability or hard
We developed a green methodology to synthesize
xanthenones from the one-pot condensation of benzaldehyde
derivatives, b-naphthol, and dimedone, employing
NOAs as organic acid catalysts. The mechanism and
regioselectivity were investigated using theoretical
calculations HF/6-31G(d) and LC-ωPBE/6-311++G(d,p).
Moreover, due to the high biological potential of
xanthenones, we also investigated this class of molecules
as calcium channel blockers.
4,5,18,21,22
8,11,16,17,19,23
catalyst preparation,
cumbersome product isolation procedures. They also use
long reaction time,
16
14,15,22
expensive and/or detrimental metal precursors,
toxic
1
1,14
catalysts,
reaction or purify the product.
and toxic organic solvents to perform the
13,16,17,20
A readily available
and non-hazardous catalyst that promotes the formation
of xanthenones in a short reaction time, under solvent-free
condition, and simple work-up is highly desirable.
Results and Discussion
A more eco-friendly and user-friendly alternative
employs natural organic acids (NOAs), which are
biodegradable compounds found in the metabolism of
Optimization of the reaction conditions
2
5,26
many organisms.
For example, acetic acid (the second
Initially, an investigation was performed to find the
best catalyst and reaction condition to synthesize the
xanthenone 4a (Table 1).As a reaction model, benzaldehyde
(1.0 equivalent), b-naphthol (1.2 equivalent) and dimedone
(1.5 equivalent) were mixed and catalytic amounts of oxalic
(pK = 1.2), malonic (pK = 2.8), succinic (pK = 4.2),
simplest monocarboxylic acid after only to formic acid)
catalyzes multicomponent reactions in the synthesis of
2
7
28
3
-alkoxyisoindolin-1-imines, piperidines, bicyclic
2
9
pyro, quinazolinones and (dihydroquinazolinylphenyl)
3
0
oxazolones. NOAs bearing two carboxylic groups and
used as catalysts include the oxalic, malonic and succinic
acids. Oxalic acid was reported as an efficient catalyst for
a1
a1
a1
and acetic acids (pK = 4.7) (5, 10 and 20 mmol%) were
a
added to reaction mixture. The evaluated organic acids
follow a homologous series in which the number of carbons
between the carboxyl groups varies; the oxalic having no
carbon among them; the malonic acid has one carbon;
the succinic acid has two carbons and the acetic acid is
31
32
33
the synthesis of coumarins, quinoxalines, quinolines,
3
4,35
36
bis(indolyl)methanes,
tetrahydroquinolines and
flavone. Although less explored in multicomponent
reactions, oxalic acid has also shown to be an efficient
catalyst in such reactions for obtaining quinazolinones,
piperidines and N-aryl-3-aminodihydropyrrol-2-one-
3
7
38
the corresponding monocarboxylic acid. The pK value
a1
3
9
of dicarboxylic acids increases with the increase in the
number of carbons between the carboxylates; a fact that
can be explained by the inductive effect of the neighboring
carboxyl and the stability of the corresponding conjugated
base. The oxalic acid has the most stable conjugate base,
because the negative charge can be stabilized by resonance
and can establish an intramolecular hydrogen bond.
Besides that, the other carbonyl in α, increases its acidity
by inductive effect.
40
4
-carboxylates compounds. Malonic acid was reported
as a catalyst in polymerization reactions
4
1,42
and for
43
obtaining 1,3-dichloropropanols from glycerol; however,
its use in multicomponent reactions has not been
described yet. Succinic acid was reported as a catalyst in a
multicomponent reaction for the preparation of amidoalkyl
naphthols and α-amino phosphonates. Most of the NOAs
come from economically renewable resources turning them
as targets for developing green multicomponent approach
for substances of pharmaceutical, agricultural, cosmetic
44
45
We chose to develop a solvent-free under microwave
radiation (MW) methodology because it makes the
synthesis of xanthenones simpler, cleaner and more
efficient. It also provides an increment on safety, easy
work-up, cost reduction, and prevents solvent waste,
25,45-48
and food purposes.
Despite the several described uses
of acetic, oxalic, malonic and succinic acids in organic
synthesis, the literature has not registered their use to
furnish xanthenones.
49-51
hazards and toxicity.
A probable mechanistic rationalization to produce
xanthenones involves an ortho-quinone methide
intermediate, which is generated by the nucleophilic attack
of the phenolic compound on the aldehyde, followed by
The catalyst quantity influences the xanthenone yields.
Oxalic acid demonstrated itself to be the most efficient as
a catalyst, providing similar or better yields than the other
acids in all conditions.
The best condition was using 20 mmol% of oxalic acid
and a reaction time of 5 min (82% yield; Table 1). This acid
also showed that higher concentrations or longer reaction
times lower the product yield. The behavior is opposite to
the other acids, which improved the reaction yield with the
3,11,14,15,20
dehydration.
Although this nucleophilic addition is
reasonable, there is no discussion about the regioselectivity
of the intermediate. Also, to the best of our knowledge,
there are no theoretical and/or experimental studies about
the mechanism proposition in the literature.

Vol. 28, No. 12, 2017
Terra et al.
2315
a
Table 1. Effect of the type and quantity of natural organic acids (NOAs) for obtaining xanthenone 4a under microwave radiation (MW) and solvent-free
conditions
b
entry
Natural organic acid (NOA)
oxalic acid
NOA quantity / mmol%
time / min
Yield / %
58
1
2
3
4
5
6
7
8
9
5
5
10
5
oxalic acid
5
51
oxalic acid
10
10
20
20
25
5
62
oxalic acid
10
5
52
oxalic acid
82
oxalic acid
10
5
65
oxalic acid
66
malonic acid
malonic acid
malonic acid
malonic acid
malonic acid
malonic acid
succinic acid
succinic acid
succinic acid
succinic acid
succinic acid
succinic acid
acetic acid
5
10
5
10
5
26
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
1
2
3
5
5
6
7
8
9
0
1
2
3
5
5
6
7
10
10
20
20
5
2
10
5
51
14
10
5
55
9
5
10
5
35
10
10
20
20
5
16
10
5
21
17
10
5
54
traces
58
acetic acid
5
10
5
acetic acid
10
10
20
20
-
traces
51
acetic acid
10
5
acetic acid
51
acetic acid
10
5
67
no catalyst
16
2
no catalyst
-
10
20
a
b
Reagents and conditions: benzaldehyde/b-naphthol/dimedone (molar ratio = 1.0:1.2:1.5); yields for purified xanthenone 4a.
increase on the reaction time. The reaction carried out in
absence of catalyst furnished the desirable product 4a in low
yields (16 and 20% after 5 and 10 min of MW, respectively).
Oxalic acid is the most acid catalyst herein tested. Since
the formation of xanthenones involves various protonated
intermediates, the stronger Brönsted-Lowry acid should be
the best organic catalyst.
protocols using microwave radiation lasted less than
1
5,18,22
5-15 min;
however, these methodologies have
1
8,22
disadvantages such as the need to prepare the catalysts,
the use of detrimental metals as catalysts,
use of toxic solvents to purify the desirable products.
1
5,22
and the
1
5
Our protocol takes only a few minutes to complete the
reaction, uses a cheap, eco-friendly and user-friendly
catalyst and all the xanthenones were purified by
recrystallization.
Most protocols describe long reaction times (30 minutes
8
,11,16,17,19,23
to hours) for the xanthenone synthesis.
The

2
316
Natural Organic Acid as Green Catalyst for Xanthenones Synthesis
J. Braz. Chem. Soc.
The scope of the substrates
between the dimedone and the aldehyde. In order to prevent
this side-reaction, the stoichiometry of b-naphthol and
dimedone was inverted, for a ratio of aldehyde/b-naphthol/
dimedone of 1.0:1.5:1.2, and we achieved better yields
(Table 2, entries 7 and 10-12).
In all the cases, the pure product was readily isolated
by recrystallization from methanol. The xanthenones
products were characterized by melting point, infrared (IR),
nuclear magnetic resonance (NMR) spectroscopy and an
electrospray ionization high-resolution mass spectrometry
(ESI-HRMS). Additional information was obtained from
X-ray diffraction spectroscopy.
We further investigated the scope of this methodology
since the optimal conditions have been determined for the
synthesis of xanthenone 4a, using oxalic acid as the catalyst.
Fifteen different xanthenones were synthesized with yields
ranging 51-93% (Table 2). Three kinds of aldehydes were
tested: containing electron-donating groups, electron-
withdrawing groups and heteroaromatic rings. The reaction
yields were not considerably affected by the presence of
electron-withdrawing groups (best result using 4-nitro-
benzaldehyde; 80% yield) or electron-donating groups
(
best result using 2-hydroxy-benzaldehyde; 93% yield) in
the aldehydes. The heteroaromatic aldehyde 2-pyridinyl
provided the best yield at 130 °C (85%), demonstrating a
greater stability at this temperature.
X-Ray
The crystal structure of 4e was determined and analyzed
in a diffractometer Enraf-Nonius Kappa-CCD. The ORTEP
representation of 4e is shown in Figure 2 and data related
to the collection and refinement of its crystal structure are
in the Table S6 of the Supplementary Information (SI).
Compound 4e crystallized in centrosymmetric space
groups, as expected from their achiral synthesis. The four
Low yields were observed for 4-hydroxybenzaldehyde
(
35%), 4-cyano-benzaldehyde (45%), 3-methoxy-
4
4
-hydroxy-benzaldehyde (46%) and 3,4-dimethoxy-
-hydroxy-benzaldehyde (55%), using the ratio aldehyde/
b-naphthol/dimedone of 1.0:1.2:1.5. This is explained
by the formation of subproducts from a side-reaction
Table 2. Synthesis of xanthenones catalyzed by oxalic acid under solvent-free condition
entry
Aldehyde
C H
Yield / %
a,c
1
2
3
4
5
6
7
8
9
4a (82)
6
5
a,d
4-O NC H
4
4b (80)
2
6
a,d
4-FC H
4c (72)
6
4
a,d
4-ClC H
4d (81)
6
4
a,c
2-OHC H
4e (93)
6
4
a,c
3-OHC H
4f (60)
6
4
b,d
4-OHC H
4g (68)
6
4
a,d
4-SMeC H
4h (86)
6
4
a,d
4-OMeC H
4i (70)
6
4
b,d
1
1
1
1
1
1
0
1
2
3
4
5
4-CNC H
4j (67)
6
4
b,d
3-OMe, 4-OHC H
4k (81)
6
3
b,d
3,5-OMe, 4-OHC H
4l (73)
6
2
b,d,e
2-thienyl
4m (58)
4n (85)
a,c
2-pyridinyl
b,c,e
5-nitro-2-furyl
4o (51)
a
b
Molar ratio of the reagents used: aldehyde/b-naphthol/dimedone (1.0:1.2:1.5) or aldehyde/b-naphthol/dimedone (1.0:1.5:1.2).Yield: for the pure xanthenone
c
d
e
after 5 min or 10 min of reaction. The temperature reaction used was 100 °C instead of 130 °C.

Vol. 28, No. 12, 2017
Terra et al.
2317
of the C15-C16-C17-C12-C13 fitted atoms is 0.0194 Å
in 4e). Ring B is distorted in with the C1 carbon deviating by
0.1857(14) Å in 4e from the mean plane calculated through
the C2-C11-O1-C12-C17 atoms (r.m.s.d. of the fitted atoms
is 0.0245Å in 4e). In the structure, the C1 and C14 are on the
same side of the substituted phenyl ring that is perpendicular
to the xanthenone mean plane. The mean plane calculated
through ring E forms ca. 90° angle 81.06(4)° in 4e with the
least-squares plane encompassing the non-hydrogen atoms of
the four fused ringsA-D, except for C1 and C14. In addition,
1
there is a S (8) motif in compound 4e due to the formation
of an intramolecular hydrogen bonded cycle.
1
Figure 2. ORTEP image of 4e. Non-hydrogen atoms are shown with
their 30% probability ellipsoids. Rings and non-hydrogen atoms are
arbitrarily labeled.
Theoretical investigation of the reaction mechanism
fused rings formed a nearly planar xanthenone core except
for ring A, which adopted a half-chair conformation with
the C14 carbon removed from the least-squares plane,
calculated through the other five coplanar atoms, by
Aprobablemechanismtoobtainxanthenonesisviainitial
nucleophilic attack of b-naphthol (2) C2 on the carbonyl
3,11,14,15,20,24
carbon of the benzaldehyde (1) (Scheme 1).
An
0.6451(15) Å in 4e (root-mean-square deviation (r.m.s.d.)
alternative mechanism would involve a nucleophilic attack
Scheme 1. Probable mechanism for the synthesis of xanthenone (4) using b-naphthol (2) via C2 nucleophilic on the carbonyl carbon of the protonated
benzaldehyde (5).

2
318
Natural Organic Acid as Green Catalyst for Xanthenones Synthesis
J. Braz. Chem. Soc.
of b-naphthol C10 (Scheme 2); however, this pathway leads
to the xanthenone 20 that is not observed for this reaction. In
order to verify this regioselectivity, theoretical calculations
were performed.
The energy variation of each reaction step was calculated
using HF/6-31G(d) level of theory. The electronic-nuclear
energies, Gibbs free energies, and entropy values for
all compounds in Schemes 1 and 2 are available in the
Supplementary Information (Tables S1-S4).
optimized structures and their energies are available in the
Supplementary Information (Table S5).
The theoretical study at 130 °C showed that the
activation energy (process 5 + 2 → TS, Table 3) to TS1
-
1
formation (DG = 6.84 kcal mol ) is lower than TS2
-1
formation (DG = 15.45 kcal mol ). This demonstrates that
the formation of 6 is thermodynamically and kinetically
favorable, while the formation of 13 is not favorable
(Figure 3).
Comparing both schemes and the energy step-by-step,
we concluded that step 2 is crucial to explain the
regioselectivity. In C2 pathway, this step is more favored
The rate constants k of the reaction were estimated
according to Eyring’s equation (equation 1):
-
1
by energy (DG = -1.72 kcal mol ); however, in C10
(1)
pathway, this step is thermodynamically disfavored
-1
(
DG = 10.51 kcal mol , Tables S3 and S4).
In equation 1, T is temperature, k is the Boltzmann’s
B
In order to have a better regioselectivity understanding,
constant, h is the Planck’s constant and R is the universal
7
1
-1
a mechanistic study was also performed employing an
improved level of theory (LC-ωPBE/6-311++G(d,p)).
Scheme 3 shows the step 2 of both C2 and C10 pathways
with their respective transition states (TS1 and TS2). All
gas constant. The values were 6 × 10 and 3 × 10 s at
130 °C for steps 2(a) and 2(b), respectively. This means
that the formation of 6 is one billion times faster than the
formation of 13. This study justifies the reason product 4
Scheme 2. Probable mechanism for the synthesis of xanthenone (20) using b-naphthol (2) via C10 nucleophilic on the carbonyl carbon of the protonated
benzaldehyde (5).

Vol. 28, No. 12, 2017
Terra et al.
2319
Scheme 3. Addition of b-naphthol (2) to protonated benzaldehyde (5) through nucleophilic attack of (a) C2 and (b) C10.
Table 3. Variations of electronic energy (DEelect), entropy (DS) and Gibbs
charge density from its highest occupied molecular orbital
HOMO) passing through C2 and leading to 6. The formation
free energy (DGtotal) to the studied steps at 130 °C
(
DEelect
kcal mol )
/
DS /
(cal mol K )
DGtotal
(kcal mol )
/
of 13 might occur by the nucleophilic attack of b-naphthol
through its energetic nearest occupied orbital to the HOMO
Process
-
1
-1
-1
-1
(
a
Step 2(a)
(i.e. HOMO – 1 orbital), which presents charge density
5
+ 2 → TS1
-11.97
-11.54
-23.51
4.65
1.35
6.00
6.84
-10.03
-3.19
passing through C10. This attack is less favorable than the
first one because the HOMO – 1 is less available than HOMO,
TS1 → 6
5
+ 2 → 6
-1
and 19 kcal mol highest in energy. Furthermore, the LUMO
a
Step 2(b)
of 5 is energetically closer to the HOMO than to HOMO – 1
of b-naphthol. This corroborates with the thermodynamic
and kinetics observations. The frontier orbitals of all studied
species at LC-ωPBE/6-311++G(d,p) level of theory are
available in the Supplementary Information.
5
+ 2 → TS2
-2.73
-8.61
2.05
0.40
2.45
15.45
-8.52
6.93
TS2 → 13
5
+ 2 → 13
-11.34
a
The step 2 is completely sketched in Scheme 3.
Figure 3. Energy profiles along the pp aa tt hh ww aa yy ff oo rr ss t tee pp 22 ( (aa ))( (dd aa t taa i inn l ri ge dh )t
ga nr ed y s) t ae np d2 s( bt e) p( d2 a( bt a) i( nd ab t lau ei n) ad ta 1r k3 0g r°e Cy .) at 130 °C.
Figu rr ee 4 4. .F rFo rno t ni et ri eo rr b oi tr ab l is t aL l Us ML UO Mo f Op ro tf o np ar ot et do nb ae tn ez da l db e hn yz da el d( e5 h) ,y Hd eO M( 5 O) ,
aH n Od MH O Ma nO d– H1 Oo Mf b O- n a– p h1 t ho of l b( 2- n) .a Ap ht ot hm ole (g 2e ) n. dAs :t oC m(b ll ea gc ek n) ,d Os :( Cd a r( kb l ga rc ek y) ),
aO n d( r eH d )( l ai gn hd t Hg r (e gy r )e. y).
is obtained at the synthetic procedure while the product
20 is not observed.
The protonated benzaldehyde (5) suffers a nucleophilic
Calcium influx assay
attack receiving the charge density in its lowest unoccupied
molecular orbital (LUMO) of the carbonyl carbon atom
52
Previously presented data byWang et al., suggested the
(Figure 4). The b-naphthol acts as a nucleophile donating
activity of several xanthones on blocking calcium channels

2
320
Natural Organic Acid as Green Catalyst for Xanthenones Synthesis
J. Braz. Chem. Soc.
2+
2+
57
(
Ca ) and also beta adrenergic receptors. As xanthones are
where L-type Ca channels are mostly expressed. Thus,
chemically related to xanthenones, we decided to verify
the potential of the synthesized xanthenones as calcium
channel blockers, and for that we performed an in vitro
calcium influx assay using cortical brain synaptosomes. All
xanthenones were evaluated to determine their potential
we performed an assay to evaluate vascular contraction
induced by KCl. KCl induces vascular contraction due
to vascular smooth muscle cell membrane depolarization
by changing the potassium equilibrium potential and
clamping the membrane potential at some value above
2+
58
to inhibit cytosolic Ca entry when synaptosomes were
depolarized by potassium chloride (KCl) 33 mM. The
the resting level. Membrane depolarization increases
2
+
2+
[Ca ] (cytosolic calcium) through L-type Ca channels,
C
2+
2+
2+
changes in cytosolic Ca may reflect the Ca influx and
were monitored continuously in fura-2-loaded synaptosomes
activating Ca /calmodulin-dependent protein kinase II
(CaMKII), which induces myosin light chain kinase
59
(
as described in Experimental section). Figure 5 shows the
phosphorylation and, consequently, vascular contraction.
2+
results of the most promising xanthenones in inhibiting Ca
An important component of blood pression (BP) regulation
is dependent on the contractile state of vascular smooth
muscle cells (VSMCs), which is controlled to a large extent
by the activities of KCNQ (subtype of potassium channel
influx induced by high potassium. Depolarization by KCl
led to an increase in intracellular calcium concentration
2+
[
Ca ], which was efficiently inhibited (ranging from 60
i
2
+
60
to 80% inhibition) by xanthenones (4c, 4d, 4e, 4g, 4h, 4j,
from K 7 family) and also L-type Ca channels. Indeed,
v
2
+
all at 200 mM) and also partially inhibited by a selective
the clinical importance of L-type Ca channels blockers is
sustained by its involvement in BP regulation.
2
+
L-type Ca channels blocker, nifedipine (5 μM). As
53
described in literature, the synaptosomes are commonly
used to study synaptic transmission since they hold the
molecular machinery necessary for the uptake, storage, and
release of neurotransmitters. In addition to that, they have
Our results show that after all xanthenones from Figure 5
were tested, the pre-treatment with xanthenones 4c or 4e was
able to reduce the maximum contraction effect (E ) induced
max
by KCl in endothelium-denuded aortic rings (Figure 6
and Table 4) compared to the untreated control group.
These results were in accordance to vascular effect induced
53,54
become common tools for drug testing.
According to
our data, nifedipine blockage did not completely abolish
2+
2+
the increase in Ca influx observed in synaptosomes. This
data corroborates literature evidences which show that
by nifedipine which is a selective L-type Ca channels
blocker with most initial studies being performed in the
2
+
61
in synaptosomes just around 10% of Ca channels are
early 1970s. Mikkelsen et al. (1979), as well as Pedersen
55,56
62
L-type and mostly are N-, P/Q-type or R-type channels.
Furthermore, xanthenones displayed at Figure 5 were able to
et al. (1978), verified that nifedipine induced almost
completely relaxation of arteries and veins pre-contracted
2+
63
reduce Ca influx to lower levels than nifedipine, suggesting
by potassium. In the same way, Mikkelsen et al. (1978)
2+
that other Ca channels might be blocked by those drugs.
To clarify which channels are specifically blocked by those
xanthenones detailed electrophysiological experiments
would be required to test this hypothesis, which were beyond
the scope of the present study.
showed that nifedipine depressed the potassium contracture
of human vessels. In vessels, vasoconstriction induced by
2+
KCl was inhibited by calciseptine (L-type Ca blocker) or
2+
mibefradil (not selective T-type Ca blocker) indicating
Figure 5. Effect of several xanthenones on the KCl induced cytosolic
2
+
Ca entry. The results are expressed as the mean ± S.E.M. of three
independent experiments. *Indicates a significant difference compared
2+
to KCl induced Ca entry (p < 0.01). #Indicates a significant difference
compared to nifedipine (p < 0.01).
Figure 6. Effect of xanthenones on KCl-induced contractile response in
endothelium-denuded aortic rings. The concentration-response curves for
KCl were determined in the absence of control or after incubation with
2+
As several xanthenones were able to block Ca influx
in synaptosomes, we decided to test their effects on a model
-1
4c or 4e (100 μmol L ).

Vol. 28, No. 12, 2017
Terra et al.
2321
that in this vascular preparation, these calcium channels
are necessary for vasoconstriction. Based in all these
and are referenced to signals from tetramethylsilane (TMS) in
6
4
1
13
the H NMR spectra and deuterated solvent in the C NMR
1
informations, our results suggest that xanthenones 4c and
spectra. H NMR data are presented in the following order:
2
+
4
e could be partially blocking L-type Ca channels, that
chemical shift in ppm (multiplicity, integration, coupling
constant (J) in hertz (Hz)). Melting points (uncorrected)
were obtained on a Mettler FP 80 HT apparatus. Infrared
spectra were recorded on a PerkinElmer Spectrum One
spectrophotometer. The high-resolution mass spectra were
obtained using a mass spectrometer with an electrospray
ionization source (ESI-MS) model Shimadzu LC-ITTOF.
Well-shaped single crystals of the xanthenone
derivatives were chosen, mounted on a k-goniostat and
exposed to graphite-monochromated X-ray beam (Mo Kα,
l = 0.71073 Å) using an Enraf-Nonius Kappa-CCD
diffractometer equipped with a CCD camera of 95 mm lens.
X-Ray diffraction experiments were carried out at room
temperature. The data collection strategy was calculated by
setting j scans and ω scans with k offsets. No absorption
correction was applied to the raw dataset due to the
negligible absorption coefficient of the compounds using
a Mo Kα beam. The crystallographic software employed
61,62
are one of the main subtypes present in vascular tissue.
More experiments to determine if those xanthenones may
2
+
block another specific subtype of Ca channel or may
2+
act in receptor-operated Ca channels are needed to be
studied to elucidate this point. We believed this data may
be interesting in the identification of hypotensive and
vasorelaxing activities of synthetic xanthenones.
Table 4. Effect of xanthenone 4c and 4e on the Emax for KCl
-
1
Xanthenone (100 μmol L )
Control
Emax / mN
4.1 ± 0.2 (8)
a
4
4
c
e
3.2 ± 0.4 (4)
a
2.9 ± 0.1 (5)
a
Compared to respective control group (p < 0.05, ANOVA followed by
Newman-Keuls). The number within parentheses indicates the number
of animals. Values are mean ± S.E.M.
65
was as follows: COLLECT (X-ray diffraction experiment
6
6
Conclusions
monitoring), HKL Denzo-Scalepack software package
6
7
(
indexing, integration and scaling of raw data), SIR2004
68
The present work described an alternative green protocol
for the preparation of xanthenones using natural organic
acids as catalysts, under solvent-free and microwave
irradiation conditions. The oxalic acid was the best catalyst
and furnished the xanthenones in moderate to excellent
yields (51-93%). This protocol presented itself as being
more eco-friendly and user-friendly alternative to traditional
methodologies. The unprecedented theoretical study showed
that the regioselectivity observed for such reaction is a
consequence of the kinetic and thermodynamic control of
the addition of b-naphthol C2, instead of the C10 atom, to
the aldehyde. Additionally, our results have disclosed the
xanthenones 4c and 4e as novel calcium channels blockers
since these compounds were able to reduce the maximum
contraction effect induced by KCl in endothelium-denuded
(structure solving), SHELXL-97 (structure refinement),
6
9
70
MERCURY and ORTEP-3 (structure analysis and
graphical representations). Direct methods of phase retrieval
were used to solve the crystal structures.All the non-hydrogen
atoms in the asymmetric unit were located from the electronic
density Fourier map. The early model was refined by
the full-matrix least squares method based on F2. In the
refinements, free anisotropic and fixed isotropic thermal
displacement parameters were adopted for non-hydrogen
and hydrogen atoms, respectively. The isotropic thermal
displacement parameters of the hydrogens were either 20%
(C-H hydrogens, except those in methyl moieties) or 50%
(hydrogens in methyl and hydroxyl moieties) greater than
the equivalent isotropic parameter of the bonded atom.
For the positions of the hydrogens, bond distances were
stereochemically constrained according to the riding model.
2+
aortic rings and also partially blocking L-type Ca channels.
Experimental
General procedure for the xanthenone synthesis
General techniques
For the xanthenone synthesis, aldehyde (1.0 mmol),
b-naphthol (1.2 mmol), dimedone (1.5 mmol) and organic
acid (5, 10 or 20 mol%) were added to a 50 mL round-
bottom flask under solvent-free conditions. The reaction
All starting materials were obtained from commercially
available sources with high-grade purity and used without
1
13
®
further purification. H and C NMR spectra at 200 and
00 MHz were obtained on a BrukerAVANCE DPX 200 and
proceeded under MW in a DISCOVER CEM reactor,
4
in a open flask mode, using the following conditions:
temperature 130 °C, maximum power 250 W, hold time
3 min, and run time 5 or 10 min at high stirring with an open
a BrukerAVANCE DRX 400 spectrometer, respectively. The
chemical shifts (d) are expressed in parts per million (ppm)

2
322
Natural Organic Acid as Green Catalyst for Xanthenones Synthesis
J. Braz. Chem. Soc.
8
1
tube.After completion of the reaction, methanol was added
to the mixture, and the solid was filtered and recrystallized
from methanol to afford the xanthenones in high purity.
a Biotek spectrofluorimeter according to Grynkiewicz et al.
Calcium chloride was added to the synaptosomal suspension
at the beginning of each fluorimetric assay (1.0 mM final
concentration). Synaptosomes were stirred throughout the
experiment in a plate maintained at 35 °C. Xanthenones
were added to the synaptosomal suspension 10 minutes prior
to membrane depolarization with KCl (33 mM). Baseline
(240 s) measurements were obtained prior to the addition
of drugs. At the end of each experiment, sodium dodecyl
sulfate (SDS) 10% (0.1% final) was added to obtain maximal
fluorescence (Rmax) by 3.0 M Tris + 400.0 mM EGTA
Theoretical methodology
Theoretical calculations were performed using the
71
Gaussian 09 program package. Gas phase optimization
and evaluation of the harmonic frequencies at the
HF/6-31G(d,p) level of theory were performed. Geometries
were characterized as true minima in the potential energy
surface (PES) when all vibrational modes were real.
It was employed the LC-ωPBE/6-311++G(d,p) level
81
(pH 8.6) to obtain minimal fluorescence (Rmin).
7
2-76
71
of theory
as implemented at Gaussian 09 program.
Animals, vessel preparation and isometric tension
measurements
As criteria to geometry optimization convergence it was
-
7
-6
-1
considered 10 Hartree to the energy and 10 Hartree Bohr
to forces. The search for the transition states (TS) was done
Male Balb/c mice were housed under standard
laboratory conditions with free access to food and water.
Male Balb/c mice 8 to 12 weeks old (28-32 g) were killed
by decapitation. The thoracic aorta was quickly removed,
cleaned of adherent connective tissues and cut into rings
(2-3 mm length). In all the rings, the endothelium was
mechanically removed by gently rolling the lumen of the
vessel on a thin wire. The aortic rings were placed between
two stainless-steel stirrups and connected to an isometric
force transducer (Letica Scientific Instruments; Barcelona,
Spain) to measure the tension in the vessels. The rings were
placed in an organ chamber containing Krebs solution with
7
7
using QST3 method. Harmonic vibrational frequency
calculations were conducted analytically for vibrational
analysis. Intermediates of the mechanisms were species
with all real harmonic frequencies in the PES, while the
TSs present one imaginary frequency. Intrinsic reaction
7
8
coordinate (IRC) calculations were carried out from
each TS.
Purification of synaptosomes
Adult Swiss mice of both sexes (25-30 g) were decapitated
and had their cortices removed and homogenized in
-1
the following composition (mmol L ): sodium chloride,
135.0; KCl, 5.0; potassium dihydrogen phosphate, 1.17;
calcium chloride, 2.5; MgSO , 1.4; sodium bicarbonate,
1
(
1
:10 (m/v) 0.32 M sucrose solution containing dithiothreitol
0.25 mM) and ethylenediamine tetraacetic acid (EDTA;
mM). Then, the homogenates were submitted to low-speed
4
20.0; glucose, 11.0. The solution was maintained at pH 7.4
and gassed with 95% oxygen and 5% carbon dioxide at
37 °C. The rings were initially stretched to a basal tension
of 0.5 g, and then, they were allowed to equilibrate for
60 min. Endothelial denudation was assessed qualitatively
by the degree of relaxation induced by acetylcholine EC
centrifugation (1000 × g, 10 min), and the synaptosomes
were purified from the supernatant by discontinuous
7
9
Percoll-density gradient centrifugation, as previously
80
described. The isolated nerve terminals were resuspended
in Krebs-Ringer-HEPES solution (KRH; 124 mM sodium
chloride, 4 mM KCl, 1.2 mM magnesium sulfate, 10 mM
glucose, 25 mM HEPES, pH 7.4) with no added calcium
5
0
-1
(1 μmol L ) in the presence of the contractile tone induced
-1
by phenylephrine (0.1 μmol L ). For these studies with the
endothelium-denuded vessels, the rings were discharged
when there was more than 10% relaxation. The Ethics
Committee in Animal Experimentation of the Federal
University of Minas Gerais (CETEA/UFMG) approved
all the in vivo experimental procedures under protocol
number 164/2009.
-1
chloride to a concentration of approximately 10 mg mL
followed by division into 200 μL aliquots that were kept
on ice until loaded with Fura-2 AM (stock solution 1 mM
in dimethyl sulfoxide) for 30 min. The Ethics Committee
in Animal Experimentation of the Federal University of
Minas Gerais (CETEA/UFMG) approved all the in vivo
experimental procedures under protocol number 139/2013.
Vascular contraction induced by potassium chloride (KCl) in
2+
2+
Measurements of cytosolic Ca concentration [Ca ]
denuded mice aorta in the presence or absence of 4c and 4e
i
Fura-2 AM measurements of the cytosolic calcium
The aortic rings without the endothelium were
incubated with and without 4c or 4e (100 μmol L ) for
2+
-1
concentration [Ca ] in synaptosomes were performed on
initial

Vol. 28, No. 12, 2017
Terra et al.
2323
3
0 minutes. After this time, the cumulative concentration-
6. Rama, V.; Kanagara, K.; Pitchumani, K.; Tetrahedron Lett.
2012, 53, 1018.
response curves for KCl (10-120 mM) were determined.
7
. Khanna, R.;Dalal,A.;Kumar, R.;Kamboj, R. C.;ChemistrySelect
2016, 4, 840.
Statistics
8
. Li, J.; Lu, L.; Su, W.; Tetrahedron Lett. 2010, 51, 2434.
The maximum effect (Emax) was considered to be the
maximal amplitude response reached in the concentration-
effect curves for the relaxant agent. The data are expressed
as the mean ± S.E.M. (standard error of mean). In each set
of experiments, the number of animals used (n) indicates
the number of tissues studied. The differences between
the mean values were assessed using one-way analysis
of variance (ANOVA) followed by a Newman Keuls
post-hoc test and values of p < 0.05 were considered to
be significant.
9. Gao, S.; Tsai, C. H.; Yao, C. F.; Synlett 2009, 35, 3523.
10. Shinde, P. V.; Kategaonkar, A. H.; Shingate, B. B.; Shingare,
M. S.; Beilstein J. Org. Chem. 2011, 7, 53.
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12. Khurana, M.; Magoo, D.; Tetrahedron Lett. 2009, 50, 4777.
13. Huo, C.-D.; Bao, X.-Z.; Hu, D.-C.; Jia, X.-D.; Sun, C.-G.;Wang,
C.; Chin. Chem. Lett. 2014, 25, 699.
14. Nandi, G. C.; Samai, S.; Kumar, R.; Singh, M. S.; Tetrahedron
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1
5. Rao, M. S.; Chhikara, B. S.; Tiwari, R.; Shirazi, A. N.; Parang,
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Supplementary Information
1
6. Sethukumar, A.; Chandy, M. M.; Prakasam, B. A.; Pallepogu,
R.; Struct. Chem. 2011, 22, 671.
1
13
The experimental data, copies of H NMR and
C
spectra of all xanthenones synthesized, crystal data
and refinement parameters of 4e and all information
of theoretical studies are available free of charge at
http://jbcs.org.br as a PDF file.
17. Sethukumar, A.; Vithya, V.; Kumar, C. U.; Prakasam, B. A.;
J. Mol. Struct. 2012, 1008, 8.
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Acknowledgments
2
0. Wu, L.; Wu, Y.; Yan, F.; Fang, L.; Monatsh. Chem. 2010, 141,
871.
Financial support was obtained from the Coordenação
deAperfeiçoamento de Pessoal de Nível Superior (CAPES),
Fundação deAmparo à Pesquisa do Estado de Minas Gerais
21. Shirini, F.; Khaligh, N. G.; Dyes Pigm. 2012, 95, 789.
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Prasad, S. S.; Reddy, C. S.; Catal. Sci. Technol. 2012, 2, 1382.
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J.; Tetrahedron Lett. 2013, 54, 6732.
(FAPEMIG) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq). The authors are also
grateful to Prof Rosemeire Brondi Alves (Department of
Chemistry at Universidade Federal de Minas Gerais, Brazil)
and Prof Fernando César de Macedo Júnior (Department
of Chemisty at Universidade Estadual de Londrina, Brazil)
for the suggestions and critical reading of this manuscript.
24. Khazaei, A.; Zolfigol, M. A.; Moosavi-Zare, A. R.; Zare, A.;
Khojasteh, M.; Asgari, Z.; Khakyzadeh, V.; Khalafi-Nezhad,
A.; Catal. Commun. 2012, 20, 54.
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Submitted: January 30, 2017
Published online: May 9, 2017