Synthesis of new chiral xanthone derivatives acting as nerve conduction blockers in the rat sciatic nerve

Article abstract of DOI:10.1016/j.ejmech.2012.06.049

The synthesis and structure elucidation of three new chiral xanthone (9H-xanthon-9-one) derivatives (2-4) are fully reported. The coupling reactions of the synthesized building block 6-methoxy-9-oxo-9H-xanthene-2-carboxylic acid (1) with two enantiomerically pure amino alcohols ((S)-(+)-valinol and (S)-(+)-leucinol) and one amine ((S)-(-)-α-4-dimethylbenzylamine), were carried out using the coupling reagent O-(benzotriazol-1-yl-)-N,N,N′, N′-tetramethylluronium tetrafluoroborate (TBTU). The coupling reactions were performed with yields higher than 97% and enantiomeric excess higher than 99%. The structures of the compounds were established by IR, MS, and NMR ( ¹H, ¹³C, HSQC, and HMBC) techniques. Taking into account that these new chiral xanthone derivatives have molecular moieties structurally very similar to local anaesthetics, the ability to block compound action potentials (CAP) at the isolated rat sciatic nerve was also investigated. Nerve conduction blockade might result from a selective interference with Na ⁺ ionic currents or from a non-selective modification of membrane stabilizing properties. Thus, the mechanism, by which the three chiral xanthone derivatives cause conduction blockade in the rat sciatic nerve and their ability to prevent hypotonic haemolysis, given that erythrocytes are non-excitable cells devoid of voltage-gated Na⁺ channels, are also described. Data suggest that nerve conduction blockade caused by newly-synthesized xanthone derivatives might result predominantly from an action on Na⁺ ionic currents. This effect can be dissociated from their ability to stabilize cell membranes, which became apparent only upon increasing the concentration of compounds 2-4 to the higher micromolar range.

Full text of DOI:10.1016/j.ejmech.2012.06.049

European Journal of Medicinal Chemistry 55 (2012) 1e11
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new chiral xanthone derivatives acting as nerve conduction blockers
in the rat sciatic nerve
,
b
,
,
,
Carla Fernandes a 1, Laura Oliveira c 1, Maria Elizabeth Tiritan b d, Luís Leitao c, Angelo Pozzi c,
José Bernardo Noronha-Matos c, Paulo Correia-de-Sá c , Madalena M. Pinto
,
a,b,
**
*
a Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Portugal
b Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313
Porto, Portugal
c Laboratório de Farmacologia e Neurobiologia, UMIB, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
d Centro de Investigação em Ciências da Saúde, Instituto Superior de Ciências da Saúde-Norte, (CICS-ISCS-N), CESPU e Gandra, PRD, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure elucidation of three new chiral xanthone (9H-xanthon-9-one) derivatives
(2e4) are fully reported. The coupling reactions of the synthesized building block 6-methoxy-9-oxo-9H-
xanthene-2-carboxylic acid (1) with two enantiomerically pure amino alcohols ((S)-(þ)-valinol and (S)-
Received 6 January 2012
Received in revised form
18 June 2012
Accepted 25 June 2012
Available online 2 July 2012
(þ)-leucinol) and one amine ((S)-(ꢀ)-
a-4-dimethylbenzylamine), were carried out using the coupling
reagent O-(benzotriazol-1-yl-)-N,N,N0,N0-tetramethylluronium tetrafluoroborate (TBTU). The coupling
reactions were performed with yields higher than 97% and enantiomeric excess higher than 99%. The
structures of the compounds were established by IR, MS, and NMR (1H, 13C, HSQC, and HMBC) tech-
niques. Taking into account that these new chiral xanthone derivatives have molecular moieties
structurally very similar to local anaesthetics, the ability to block compound action potentials (CAP) at
the isolated rat sciatic nerve was also investigated. Nerve conduction blockade might result from
a selective interference with Naþ ionic currents or from a non-selective modification of membrane
stabilizing properties. Thus, the mechanism, by which the three chiral xanthone derivatives cause
conduction blockade in the rat sciatic nerve and their ability to prevent hypotonic haemolysis, given
that erythrocytes are non-excitable cells devoid of voltage-gated Naþ channels, are also described. Data
suggest that nerve conduction blockade caused by newly-synthesized xanthone derivatives might
result predominantly from an action on Naþ ionic currents. This effect can be dissociated from their
ability to stabilize cell membranes, which became apparent only upon increasing the concentration of
compounds 2e4 to the higher micromolar range.
Keywords:
Chiral xanthone derivatives
TBTU
Sciatic nerve
Hypotonic haemolysis
Local anaesthetic-like properties
Action potential blockade
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
(esomeprazole magnesium), are single enantiomeric drugs [2,3].
The differences normally exhibited by the enantiomers in phar-
Chiral compounds represent almost two-third of all drug sales
worldwide and are of great importance in Medicinal Chemistry
and in drug discovery [1]. According to a recently survey, the
three top-selling drugs for 2008 and 2009, namely LipitorÔ (ator-
vastatin calcium), PlavixÔ (clopidogrel bisulfate) and NexiumÔ
macodynamics, pharmacokinetics and toxicity, makes the thera-
peutics with single enantiomers, comparing with racemates, of
unquestionable advantages [4,5]. Therefore, regulatory authorities
recommend that chiral drugs should be marketed as pure enan-
tiomers [6,7]. Consequently, the development of efficient meth-
odologies for synthesis of chiral compounds in high yields and with
high enantiomeric purity is becoming one of the most important
tasks in the field of Chemistry.
Xanthone (9H-xanthon-9-one) derivatives have emerged as
a class of compounds with increasing interest, possessing a broad
spectrum of biological and pharmacological activities [8] and many
have proved to be important building blocks for the synthesis of
new interesting compounds [9e13]. Despite the large structural
multiplicity of bioactive xanthonic compounds only a handful of
* Corresponding author. Tel.: þ351 22 2062242; fax: þ351 22 2062232.
** Corresponding author. Laboratório de Química Orgânica
e Farmacêutica,
Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto,
Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. Tel.: þ351 222078692;
fax: þ351 226093390.
E-mail addresses: farmacol@icbas.up.pt (P. Correia-de-Sá), madalena@ff.up.pt
(M.M. Pinto).
1
These authors equally contributed to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.049

2
C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
synthetic chiral derivatives have been reported to date [14e23].
Some chiral xanthone derivatives (CXDs) exhibit heterogeneous
activities at the central nervous system (CNS). These compounds
can either stimulate neuronal activity, acting as analeptics, or
decrease nerve firing like antiepileptic drugs [17]. CXDs have also
revealed antiarrhythmic [16,18], antitumoral [14,19], anticonvul-
sant [15,18,20,21], local anaesthetic [22], antifungal and antibac-
terial [23] effects.
however the methodologies were improved in order to decrease
the reaction time and to improve the final yield. The compound 1
was then used as building block to synthesize CXDs 2e4, by
coupling reactions with (S)-(þ)-valinol (13), (S)-(þ)-leucinol (14)
and (S)-(ꢀ)-
a-dimethylbenzylamine (15), respectively. The reac-
tions were performed with the coupling reagent O-(benzotriazol-1-
yl-)-N,N,N0,N0-tetramethylluronium tetrafluoroborate (TBTU) based
on previously reports (Scheme 2) [27,28].
By inhibiting Ca2þ influx through receptor-operated and/or
voltage-sensitive channels, some of these compounds have been
shown to cause antihypertensive and vasorelaxant effects [24]. The
reported mechanisms of action are frequently associated with
enantioselectivity.
During the last few years, a huge number of coupling reagents
has appeared in the literature, which has been widely used for the
synthesis of peptides [29e31]. TBTU is one of the reagents that have
become popular due to its higher efficiency and lower tendency
towards racemization [27]. Thus, we used TBTU for the synthesis of
the CXDs by coupling a suitable functionalized xanthonic building
block (1) to different chiral ligands, in enantiomeric pure form.
The CXDs 2e4 (Scheme 2) are structurally similar to many local
anaesthetics, namely dibucaine, mepivacaine, lidocaine or bupiva-
caine, with which they might share common molecular moieties,
and, consequently, the same pharmacophore. Local anaesthetics are
known to block the peripheral nerve conduction by inhibiting
voltage-gated Naþ channels [32]. The mechanisms of nerve
conduction blockade caused by local anaesthetics can be differen-
tiated according to their structure and the way they cross the cell
membrane. The hydrophilic compounds diffuse to the axoplasm
through opened Naþ channel pores, whereas lipid soluble drugs
In this paper, we report the synthesis and biological activity
as nerve conduction blockers of three new CXDs, namely com-
pounds (S)-N-(1-hydroxy-3-methylbutan-2-yl)-6-methoxy-9-oxo-
9H-xanthene-2-carboxamide (2), (S)-N-(1-hydroxy-4-methylpentan-
2-yl)-6-methoxy-9-oxo-9H-xanthene-2-carboxamide (3) and (S)-
6-methoxy-9-oxo-N-(1-(p-tolyl)ethyl)-9H-xanthene-2-carboxamide
(4), as well as the synthesis of the building block, 6-methoxy-9-oxo-
9H-xanthene-2-carboxylic acid (1).
The total synthesis of 6-methoxy-9-oxo-9H-xanthene-2-
carboxylic acid (1) has already been accomplished by a multi-step
pathway [25,26]. Herein, the synthetic approach to obtain the
compound 1 (Scheme 1) was based on the previous procedure,
Scheme 1. Reagents and conditions: a) Methanol, H2SO4, reflux, 17 h; b) CuI, Cs2CO3, N,N-Dimethyl glycine, Dioxane, N2, 90 ꢁC, 14 h; c) Methanol/Tetrahydrofuran (1:1 v/v), 5 M
NaOH, room temp., 18 h; d) P2O5, CH3SO3H, room temp., 22 h; e) Methanol, H2SO4, reflux, 19 h; f) Methanol/Dichloromethane (1:1 v/v), 5 M NaOH, room temp., 22 h; g) Methanol/
Dichloromethane (1:1 v/v), 5 M NaOH, room temp., 22 h.

C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
3
Scheme 2. Reagents and conditions: a) TBTU, triethylamine, dry tetrahydrofuran, room temp., 30 mine2 h (the numbering used concerns the NMR assignments).
can easily pass the nerve membrane and gain access to the Naþ
channels even if they are closed (hydrophobic pathway).
isomer 11. After separation, compounds 11 and 12 were hydrolyzed
in alkaline medium yielding the xanthone derivative 1 and its
isomer 10, respectively (Scheme 1).
Due to the structure similarity between the three CXDs (2e4)
and local anaesthetic drugs, these compounds can act putatively
as modulators of Naþ influx, either from selective interference with
Naþ ionic currents or from non-selective modifications of the
membrane stabilizing properties. This prompted us to investigate
the effect of these CXDs on the amplitude of compound action
potentials (CAP) at the isolated rat sciatic nerve, as this electro-
physiological parameter is positively correlated with Naþ transport.
We also evaluated the ability of compounds 2e4 to prevent hypo-
tonic haemolysis, given that erythrocytes are non-excitable cells
devoid of voltage-gated Naþ channels.
2.1.2. Synthesis of chiral xanthone derivatives 2e4
Chiral xanthone derivatives 2e4 were obtained by the coupling
reaction of the building block 1 with (S)-(þ)-valinol (13), (S)-
(þ)-leucinol (14) and (S)-(ꢀ)-a-dimethylbenzylamine (15),
respectively, using TBTU and a catalytic amount of triethylamine in
dry tetrahydrofuran (Scheme 2). Despite a high amount of coupling
reagents described in the literature [29e31], TBTU is widely used
due to its higher efficiency and lower tendency towards racemi-
zation [27]. Thus, we used TBTU for the synthesis of the CXDs by
coupling the xanthone building block 1 to different chiral ligands,
in enantiomeric pure form. These reactions were carried out at
room temperature and showed high yields (above 97%), short
reaction times (30 mine2 h) and no racemization were observed
(enantiomeric excess higher than 99%). The purification
procedures were easy, involving extraction followed by
crystallization.
2. Results and discussion
2.1. Chemical synthesis
2.1.1. Synthesis of building block 1
Xanthone derivative 1 was synthesized, via Ullmann reaction,
with the formation of diaryl ether intermediate from the aryl
bromide 6 and the phenol 7 (Scheme 1). The aryl bromide 6 was
previous prepared from the corresponding carboxylic acid 5 by
Fisher esterification. The Ullmann condensation between 6 and 7
under the catalytic action of N,N-dimethyl glycine, CuI and Cs2CO3 in
dioxane [33] afforded the diaryl ether 8. This coupling reaction was
carried out at 90 ꢁC for 14 h to give the compound 8 in good yield
(54%), instead of 115 ꢁC, 24 h and 32%, respectively, used in the
traditionally copper-catalyzed Ullmann reaction with pyridine and
K2CO3 as described before [26]. The N,N-dimethyl glycine and the
stronger base Cs2CO3 was preferred to promote Ullmann coupling
reaction due to the lower reaction temperature, short reaction time
and higher yield was achieved. The diaryl ether 8 was dissolved in
a mixture of tetrahydrofuran/methanol and 5M NaOH solution to
hydrolyze the methyl esters, providing the compound 9. An intra-
molecular acylation of diaryl ether 9, using phosphorus pentoxide
and methane sulfonic acid at room temperature afford two
compounds, the xanthone 1 and its isomer 10 in different yields. The
separation of the two isomers was easily achieved through a Fisher
esterification followed by filtration due to the great solubility
difference in methanol. The compound 12 (described here for the
first time) presented a higher solubility in methanol than the ester
2.2. Structure elucidation
The structure of all synthesized compounds was established by
IR, MS, and NMR techniques. Although the synthesis of compound 1
and its intermediates have already been described before [25,26]
(except compounds 11 and 12) their structural data were not
reported.
The IR spectra of compounds 1e4 showed the presence of the
absorption bands corresponding to the C]O (1652e1687 cmꢀ1
)
and to aromatic C]C (1575e1433 cmꢀ1) associated with the
xanthone scaffold and a band at 1264e1272 cmꢀ1 corresponding to
the AreOCH3 group. Comparing IR data for the xanthonic building
block (1) and for the CXDs (2e4), the main changes are: the pres-
ence of two strong bands corresponding to the NeH
(3371e3244 cmꢀ1) and C]O of amide (1611e1617 cmꢀ1) groups,
as well as the absence of the broad band at 2901 cmꢀ1 corre-
sponding to the OeH carboxyl group.
The 1H NMR and 13C NMR data of CXDs (2e4) are reported in
Tables 1 and 2, respectively. The NMR data of compound 1 are also
included. Compounds 1e4 showed similar NMR profiles with
respect to the six aromatic protons of the xanthone scaffold and to

4
C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
Table 1
1H NMR chemical shifts of building block 1 and of chiral xanthone derivatives 2e4.
1
2
3
4
H-1
H-3
H-4
H-5
H-7
H-8
OCH3
NH
8.68 (d, J ¼ 3.2)
8.73 (d, J ¼ 2.2)
8.31 (dd, J ¼ 8.7, 2.2)
7.71 (d, J ¼ 8.7)
7.21 (d, J ¼ 2.3)
7.09 (dd, J ¼ 8.7, 2.3)
8.14 (d, J ¼ 8.7)
3.95 (s)
8.38 (d, J ¼ 8.7)
4.63 (t, J ¼ 5.6)
3.85 (m)
8.72 (d, J ¼ 2.2)
8.30 (dd, J ¼ 8.7, 2.2)
7.70 (d, J ¼ 8.7)
7.21 (d, J ¼ 2.3)
7.10 (dd, J ¼ 8.9, 2.3)
8.14 (d, J ¼ 8.9)
3.95 (s)
8.40 (d, J ¼ 8.7)
4.75 (t, J ¼ 5.7)
4.11 (m)
1.64 (m)
1.45 (m)
0.90, 0.91 (2 d, J ¼ 6.0)
3.43 (dd, J ¼ 13.3, 5.3)
e
8.56 (d, J ¼ 2.2)
8.32 (dd, J ¼ 8.7, 2.2)
7.53 (d, J ¼ 8.7)
6.92 (d, J ¼ 2.3)
6.99 (dd, J ¼ 8.9, 2.3)
8.25 (d, J ¼ 8.9)
3.93 (s)
8.29 (dd, J ¼ 13.3, 3.2)
7.69 (d, J ¼ 13.3)
7.19 (d, J ¼ 3.3)
7.07 (dd, J ¼ 13.3, 3.3)
8.09 (d, J ¼ 13.3)
3.93 (s)
e
e
e
e
e
e
e
e
e
e
6.58 (d, J ¼ 7.3)
OH
e
H-10
5.33 (m)
H-20
1.97 (m)
1.63 (d, J ¼ 6.9)
H-30
0.90, 0.93 (2 d, J ¼ 7.0)
e
H-40
e
e
CH2OH
AreCH3
H-200, H-600
H-300, H-500
3.54 (dd, J ¼ 10.4, 5.8)
e
e
2.34 (s)
e
e
7.31 (d, J ¼ 8.0)
e
e
7.18 (d, J ¼ 8.0)
the three protons of the methoxyl group, with similar chemical
shifts and coupling constants.
For compounds 2e4 were provided spectral data from HSQC and
HMBC experiments to clarify the structural elucidation of these
compounds.
The MS spectra of all compounds showed the respective
molecular ion peaks at the expected m/z values (see Experimental
Section).
Thus, the spectral data confirm the success of the coupling of the
xanthonic building block (1) with the three different chiral units in
order to obtain the compounds 2e4.
The 1H NMR spectra of all CXDs (2e4) had in common the
presence of the signals corresponding to the proton of the amide
group at 6.58e8.40 ppm. The 1H NMR spectra of compounds 2 and
3 contained signals due to a hydroxyl group on the side-chain
primary alcohol, at 4.63 and 4.75 ppm respectively, while the
spectra of CXD 4 showed signals concerning the presence of
a phenyl ring (side-chain). It is also important to point out the
following chemical shifts, with respect to the side chain of the
CXDs: 1.45e5.33 ppm for the proton of one (4) or two (2 and 3)
methine groups, 1.64e3.54 ppm for the protons of one (2) or two
(3) methylene groups, and 0.90e2.34 ppm for the protons of two
(2e4) methyl groups.
2.3. Biological activity studies
2.3.1. Chiral xanthone derivatives decrease sciatic nerve conduction
The synthesized CXDs (2e4) (Scheme 2) show chemical struc-
tures related to those found in local anaesthetics, which include an
aromatic end (xanthone scaffold) and an amide linkage [34]. The
absence of a tertiary amine is the most significant difference
regarding the pharmacophoric descriptors for local anaesthetics.
The main targets of local anaesthetic drugs are voltage-gated Naþ
channels. Therefore, compounds with anaesthetic-like properties
should block the initiation and propagation of nerve action
potential. This prompted us to investigate the effect of the newly-
synthesized CXDs on the amplitude of compound action poten-
tials (CAP) in the isolated rat sciatic nerve.
The 13C NMR spectra revealed signals for one (1) or two carbonyl
groups (2e4), one methoxyl group (1e4), two (1e3) or three
aromatic rings (4), one (4) or two (2 and 3) methine groups, one (2)
or two (3) methylene groups and two (2e4) methyl groups.
Table 2
13C NMR chemical shifts of building block 1 and of chiral xanthone derivatives 2e4.
1
2
3
4
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-4a
C-10a
C-8a
C-9a
124.9
127.6
135.7
116.7
100.7
164.9
113.7
126.6
175.3
156.3
157.6
115.0
120.2
e
125.4
130.9
134.1
118.0
100.8
165.1
114.0
127.7
174.9
157.0
157.6
114.9
120.6
167.7
e
125.3
130.7
134.0
118.1
100.8
164.7
114.0
127.7
174.9
157.1
157.5
114.9
120.7
165.2
e
124.0
130.1
134.3
118.5
100.3
164.8
113.8
128.3
175.9
157.8
158.0
115.5
121.0
165.5
e
CXDs (2e4) concentration-dependently decrease the amplitude
of CAP in the isolated rat sciatic nerve (Fig. 1). Compounds 2e4 and
the xanthone itself were about equipotent. Nerve conduction
blockade caused by CXDs was observed in the low micromolar
range (0.1e3 mM), but their effects were hardly washable even
when superfusion with the physiological salt solution was pro-
longed for several hours (data not shown). Blockade of nerve
conduction by CXDs mimicked the effect of a prototypical local
anaesthetic drug, dibucaine, when this compound was used in the
same concentration range (0.1e3
mM) (Fig. 1A). Conversely, no
CONH2
COOH
AreOCH3
C-10
significant effect on CAP amplitude was observed when the rat
sciatic nerve was challenged with sucrose to increase the osmotic
strength of the physiological salt solution to the levels comparable
to the application of the CXDs (Fig. 1A). These results suggest that
nerve conduction blockade caused by the newly-synthesized
xanthone derivatives might result from an action on Naþ ionic
currents, acting in a similar manner to local anaesthetic drug. The
local anaesthetic-like property was shared by all xanthonic
compounds tested, yet a higher potency was expected for the CXDs
(2e4) compared to the xanthone itself. The absence of the tertiary
amine may contribute to nerve conduction blocking potency of
167.6
56.2
e
57.0
56.3
28.7
18.8, 19.7
e
56.3
49.8
31.9
21.9, 24.5
23.5
63.9
e
55.9
49.3
21.0
e
C-20
e
C-30
e
C-40
e
e
CH2OH
AreCH3
C-100
e
61.3
e
e
e
29.7
e
e
e
139.8
126.2
137.2
129.4
C-200, C-600
C-400
e
e
e
e
e
e
C-300, C-500
e
e
e

C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
5
Fig. 1. Effect of chiral xanthone derivatives on rat sciatic nerve conduction. (AeD) Chiral xanthone derivatives (2e4) concentration-dependently decrease the amplitude of
compound action potentials (CAP) in the isolated rat sciatic nerve. Control experiments with sucrose (ꢂ), to access the effect of osmolarity, and with the prototypical local
anaesthetic, dibucaine (,), were also performed. Drugs were applied in a cumulative manner (from 0.1 to 3 mM) and contacted the sciatic nerve for at least 15 min. The ordinates
are percentage of compound action potential amplitude (100%) observed in control conditions (in the absence of test drugs). The vertical bars represent ꢃS.E.M. of an n number of
experiments (shown in parenthesis) and are shown when they exceed the symbols in size.
CXDs, while increasing their hydrophobic characteristics that make
these compounds long-acting agents as they are hardly washable
from nerve membranes within several hours. In spite of this, when
used in the low micromolar concentration range compounds 2e4
did not cause axonal damage as predicted from the absence of
significant changes (P > 0.05) in the activity of lactate dehydroge-
nase (LDH) leaked into the incubation medium over time. Local
anaesthetics must penetrate the epineurium, perineurium and
endoneurium in order to reach their intended site of action.
Consequently, local anaesthetics require much higher concentra-
tions to be effective when used clinically than when tested in iso-
lated nerves [34]. The hydrophobic characteristics of these new
molecules (2e4) may, thus, become an interesting feature for
anaesthetic drug improvement.
2.3.2. Chiral xanthone derivatives had no protective effect on
hypotonic haemolysis
Local anaesthetics also display non-specific membrane stabi-
lizing properties besides their action on voltage-gated Naþ chan-
nels. This prompted us to investigate if the CXDs 2e4 could prevent
hypotonic haemolysis on rat erythrocytes, given that these are non-
excitable cells lacking voltage-gated Naþ channels.
The membrane protective effects of sucrose and dibucaine
(0.1e100
mM) on hypotonic haemolysis were initially evaluated
(Fig. 2A). Sucrose had a protective effect on hypotonic haemolysis,
but its effect was not concentration-dependent. Just this fact
suggests that osmolarity is not responsible for the membrane
stabilizing effect of sucrose in the micromolar concentration range.
Membrane stabilizing properties of sugars have been described by

6
C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
Fig. 2. Anti-haemolytic effects of chiral xanthone derivatives (0.01e100
mM) compared with those produced by the prototypical local anaesthetic drug, dibucaine, and by changes in
fluid osmolarity caused by sucrose. Chiral xanthone derivatives (2e4), as well as dibucaine (,) and sucrose (ꢂ), were applied in concentrations ranging from 0.01 to 100
m
M. The
ordinates are relative haemolysis (RH) caused on rat erythrocytes incubated for 10 min with an haemolytic solution containing a predetermined concentration of NaCl in 10 mM
phosphate buffer, at pH 7.0; Value one represents no haemolytic protection and corresponds to 50% erythrocyte haemolysis obtained with NaCl ranging from 63 to 67 mM in the
absence of test drugs. The vertical bars represent ꢃS.E.M. of an n number of experiments (shown in parenthesis) and are shown when they exceed the symbols in size. *p < 0.05
(one-way ANOVA followed by Dunnett’s modified t test) when compared with the situation with no haemolytic protection.
Crowe [35], being sucrose one of the most potent compounds of
this class. Strauss and Hauser [36] suggested a direct interaction of
sucrose with the phosphate group of membrane phospholipids. The
xanthone exceeded the sucrose protective effect. Compound 2
applied in the low micromolar concentration range (0.1e1 M),
increased significantly (P 0.05) haemolytic protection as
m
<
local anaesthetic dibucaine (0.1e100
molysis in a concentration-dependent manner (Fig. 2A). At the
highest concentration tested (100 M), dibucaine reduced relative
m
M) reduced hypotonic hae-
compared to xanthone (Fig. 2B). Nevertheless, neither of these
compounds exceeded the membrane protective effect of sucrose,
m
except when they were used in the 100
results were obtained with compounds 3 and 4 (0.3e1
and D). However, in contrast with compounds 2 and 3, compound 4
was devoid of protective effect when it was applied in the 100 mM
mM concentration. Similar
haemolysis (RH) to 0.421 ꢃ 0.034. Similar results were reported
m
M) (Fig. 2C
with this compound in a previous study [37].
The xanthone had little or no protective effect on hypotonic
haemolysis in the low micromolar concentration range
concentration; in these circumstances, RH was 0.967 ꢃ 0.037 (n ¼ 6,
(0.1e30
only upon increase of the concentration to 100
decreased to 0.686 ꢃ 0.021, n ¼ 4), a situation in which the
m
M) (Fig. 2A). A significant protective effect was observed
Fig. 2C).
m
M (RH was
Thus, in contrast with the more potent action of dibucaine, the
anti-haemolytic effect of CXDs (2e4) was only observed when

C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
7
these compounds were tested in higher micromolar concentrations
(30e100 M). No significant membrane protective effects were
verified in the low micromolar concentration range (0.1e3 M); in
laboratory as described elsewhere [38,39] and consisted of amylose
tris-3,5-dimethylphenylcarbamate coated onto APS-Nucleosil
(500 Å, 7 mm, 20% w/w), and packed into a stainless-steel
m
m
these circumstances, CXDs cause blockade of sciatic nerve
conduction (see above), but their effects on hypotonic haemolysis
were of smaller magnitude than that produced by sucrose.
15 ꢂ 0.46 cm ID size column. Working solutions of enantiomeric
mixtures of CXDs were prepared mixing equal aliquots of each
enantiomer at the concentration of 20
mg/mL. Optical pure (S)-
(þ)-valinol, (S)-(þ)-leucinol and (S)-(ꢀ)-
a-dimethylbenzylamine
3. Conclusions
were commercial reagents, from Fluka and SigmaeAldrich. Other
reagents and solvents were commercially available materials at pro
analysis or HPLC grade, from SigmaeAldrich, Merck and Fluka, and
used without further purification.
The synthetic approach used to synthesize the xanthonic
building block (1) was improved when compared to the described
procedure in the literature. TBTU demonstrated to be very efficient,
allowing the coupling of the xanthone derivative 1 with two chiral
amino alcohols and one chiral amine, at room temperature. The
three new CXDs were obtained, 2e4, with excellent yields, short
reaction times and no racemization. Long-range C, H correlations
led to an unambiguous establishment of the structures of the new
compounds.
Data from biological activity studies suggest that CXDs (2e4)
cause no significant protection against hypotonic haemolysis
when applied in concentrations high enough to block the sciatic
nerve conduction in the rat. It, thus, appears that nerve conduction
blockade caused by the newly-synthesized CXDs result predomi-
nantly from an action on Naþ ionic currents. This effect can be
dissociated from their ability to stabilize cell membranes, which
only became apparent upon increasing the concentration of CXDs
(2e4) to the high micromolar range.
4.2. Synthesis of the building block 1
The synthetic route used to synthesize the building block 1 is
outlined in Scheme 1, which involved a multi-step pathway.
4.2.1. Esterification of 4-bromoisophthalic acid (5). dimethyl 4-
bromoisophthalate (6)
To
a solution of 4-bromoisophthalic acid (5) (16.40 g,
66.93 mmol) in methanol (600 mL) was added 12 mL of concen-
trated H2SO4. Then, the reaction mixture was refluxed for 17 h.
After evaporation of the methanol, water (100 mL) was added and
the crude product was extracted with diethyl ether (3 ꢂ 100 mL).
The organic layer was washed with water (100 mL), saturated
NaHCO3 solution (3 ꢂ 150 mL) and water (2 ꢂ 100 mL), successively.
After drying with anhydrous sodium sulfate and filtered, the
solvent was evaporated under reduced pressure. During overnight
at room temperature the dimethyl 4-bromoisophthalate (6)
4. Experimental
appeared as a white solid. Yield: 92% m.p.: 56e58 ꢁC; IR nmax (cmꢀ1
(KBr): 1754, 1309, 1253, 929, 565; 1H NMR (CDCl3, 300.13 MHz)
)
:
4.1. General methods
d
8.43 (1H, d, J ¼ 2.2 Hz, H-2), 7.95 (1H, dd, J ¼ 8.3 and 2.2 Hz, H-6),
Melting points were obtained in a Köfler microscope and are
uncorrected. IR spectra were measured on an ATIMattson Genesis
series FTIR (software: WinFirst v.2.10) spectrophotometer in KBr
microplates (cmꢀ1). 1H and 13C NMR spectra were taken in CDCl3 or
DMSO-d6 at room temperature, on Bruker Avance 300 instrument
(300.13 MHz for 1H and 75.47 or 125.77 MHz for 13C). Chemical
7.75 (1H, d, J ¼ 8.3 Hz, H-5), 3.95 (3H, s, C(100)OOCH3), 3.93 (3H, s,
C(1`)OOCH3); 13C NMR (CDCl3, 75.47 MHz) : 165.7 (C-100), 165.5 (C-
d
1`), 134.7 (C-4), 133.0 (C-6), 132.3 (C-2), 132.2 (C-5), 129.3 (C-3),
127.0 (C-1), 52.7 (C(100)OOCH3), 52.5 (C(1`)OOCH3); MS (EI) m/z (%):
273 [M]þ. (100), 256 (9), 240 (13), 221 (6), 209 (8), 203 (11).
shifts are expressed in
d
(ppm) values relative to tetramethylsilane
4.2.2. Ullmann diaryl ether coupling. dimethyl 4-(30-methoxyphenoxy)
(TMS) as an internal reference. Coupling constants are reported in
hertz (Hz). 13C NMR assignments were made by 2D HSQC and
HMBC experiments (long-range C, H coupling constants were
optimized to 7 and 1 Hz). MS spectra were recorded as EI (electronic
impact) mode on a VG Autospec Q spectrometer (m/z) and HRMS
mass spectra were measured on a Bruker Daltonics micrOTOF Mass
Spectrometer, recorded as ESI (electrospray) mode in Centro de
Apoio Científico e Tecnolóxico á Investigation (C.A.C.T.I.), University
of Vigo, Spain.
TLC was performed using Merck silica gel 60 (GF254) plates, with
appropriate mobile phases. Compounds were visually detected by
absorbance at 254 and/or 365 nm. Column chromatography was
carried out using Merck silica gel 60 (0.040e0.063 mm). Optical
rotation measurements were carried out on a Polartronic Universal
polarimeter. Liquid chromatography was performed using a HPLC
system consisted of two Shimadzu LC 10-ADvp pumps, a FCV-10AL
solvent selector valve, an automatic injector SIL10-Advp, a SPD-
10AV UV/VIS detector with a SCL-10Avp interface or a HPLC
system consisted of two Shimadzu LC 10-AD pumps, an automatic
injector SIL10-AF, a SPD-10A UV/VIS detector with a CBM-10A
interface or a System 880-PU Intelligent HPLC Pump (Jasco)
isophthalate (8)
A
mixture of dimethyl 4-bromoisophthalate (6) (10.10 g,
36.98 mmol), 3-methoxyphenol (7) (6.88 g, 55.45 mmol), CuI
(0.70 g, 3.70 mmol), Cs2CO3 (24.09 g, 73.94 mmol), N,N-dimethyl
glycine (1.14 g, 11.09 mmol) and dioxane (74 mL) was heated in
a sealed flask at 90 ꢁC under nitrogen atmosphere for 14 h. After
cooling water (100 mL) was added and the crude product was
extracted with ethyl acetate (100 mL). The organic layer was
separated, and the aqueous layer was extracted with ethyl acetate
(2 ꢂ 100 mL). The combined organic layers were washed with
brine, dried with anhydrous sodium sulfate, filtered, and evapo-
rated under reduced pressure. The oily dark product was purified
by column chromatography (silica gel, petroleum ether/diethyl
ether in gradient) to provide dimethyl 4-(30-methoxyphenoxy)
isophthalate (8). Yield: 54% m.p. 88e90 ꢁC; IR nmax (cmꢀ1) (KBr):
1724, 1719, 1613, 1488, 1274, 1229, 1153, 948, 763; 1H NMR (CDCl3,
300.13 MHz)
d
: 8.58 (1H, d, J ¼ 2.2 Hz, H-2), 8.07 (1H, dd, J ¼ 8.7 and
2.2 Hz, H-6), 7.27 (1H, dd, J ¼ 8.4 and 8.3 Hz, H-50), 6.95 (1H, d,
J ¼ 8.7 Hz, H-5), 6.74 (1H, dd, J ¼ 8.3 and 3.1 Hz, H-60), 6.62 (1H, dd,
J ¼ 3.6 and 3.1 Hz, H-20), 6.61 (1H, dd, J ¼ 8.4 and 3.6 Hz, H-40), 3.93
(3H, s, C(10)OOCH3), 3.89 (3H, s, C(100)OOCH3), 3.79 (3H, s,
equipped with a Rheodyne 7125 injector fitted with a 20
mL loop,
AreOCH3); 13C NMR (CDCl3, 75.47 MHz) : 165.7 (C-10), 165.3 (C-
d
a 875-UV Intelligent UV/VIS Detector, and a Chromatography
Station for Windows, version 1.7 DLL. Chirobiotic T column
(15 ꢂ 0.46 cm ID size column) was commercially available from
SigmaeAldrich. Polysaccharide column was prepared in the
100), 161.1 (C-3), 160.4 (C-1), 156.9 (C-4), 134.6 (C-2), 133.6 (C-6),
130.4 (C-5), 124.6 (C-30), 122.0 (C-10), 118.7 (C-50), 111.6 (C-60), 110.3
(C-40), 105.6 (C-20), 55.4 (AreOCH3), 52.3 (C(10)OOCH3), 52.2 (C(10)
OOCH3); MS (EI) m/z (%): 316 [M]þ. (100), 285 [M-OCH3]þ. (55), 253

8
C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
(54), 242 (10), 225 (16), 213 (12),198 (25),138 (15),127 (10), 92 (15),
83 (14), 64 (11).
(C-9a), 118.1 (C-4), 115.6 (C-8a), 113.7 (C-7), 100.4 (C-5), 55.9 (AreOCH3),
52.3 (COOCH3); MS (EI) m/z (%): 284 [M]þ. (89), 253 [M-OCH3]þ. (100),
225 (32), 197 (14), 182 (30), 169 (17), 154 (16), 142 (14), 126 (28), 111 (12),
75 (12), 63 (14).
4.2.3. Hydrolysis of dimethyl ester. 4-(30-Methoxyphenoxy)isophthalic
acid (9)
Dimethyl 4-(30-methoxyphenoxy)isophthalate (8) (7.28 g,
23.01 mmol) was dissolved in methanol/tetrahydrofuran (1:1 v/v)
and stirred at room temperature with 5M NaOH solution (25 mL)
for 18 h. After evaporation of the organic solvents, water was added
(150 mL) and the crude product was washed with dichloromethane
(2 ꢂ 200 mL). The organic layer was extracted with water
(2 ꢂ 150 mL). The aqueous layer was acidified with 5M HCl solution
resulting in the formation of a precipitate that was collected by
filtration under reduced pressure and washed with water, to
provide 4-(30-methoxyphenoxy)isophthalic acid (9) as a white
solid. Yield: 91%.
4.2.4.2. Methyl 8-methoxy-9-oxo-9H-xanthene-2-carboxylate (12). m.p.:
206e207 ꢁC; IR nmax (cmꢀ1) (KBr): 1726, 1668, 1611, 1480, 1431, 1264,
1079, 760; 1H NMR (CDCl3, 300.13 MHz)
d: 8.98 (1H, d, J ¼ 2.1 Hz, H-1),
8.33 (1H, dd, J ¼ 8.7 and 2.1 Hz, H-3), 7.65 (1H, dd, J ¼ 8.4 and 8.4 Hz, H-6),
7.47 (1H, d, J ¼ 8.7 Hz, H-4), 7.09 (1H, dd, J ¼ 8.4 and 0.8 Hz, H-7), 6.86
(1H, dd, J ¼ 8.4 and 0.8 Hz, H-5), 4.05 (3H, s, COOCH3), 3.98 (3H, s,
AreOCH3); 13C NMR (CDCl3, 125.77 MHz)
d: 175.8 (C-9), 166.0 (COOCH3),
160.8 (C-8), 157.9 (C-10a), 157.8 (C-4a), 135.3 (C-3), 134.9 (C-6), 129.4 (C-
2), 126.0 (C-1), 122.6 (C-9a), 117.7 (C-4), 112.5 (C-8a), 110.1 (C-7), 106.1 (C-
5), 56.5 (AreOCH3), 52.4 (COOCH3); MS (EI) m/z (%): 284 [M]þ. (100), 255
(56), 253 (30), 238 (56), 223 (47), 195 (27), 139 (33), 126 (20), 112 (18), 70
(15), 63 (12).
m.p.: 232e234 ꢁC; IR nmax (cmꢀ1) (KBr): 2907, 1694, 1680, 1601,
1486,1271,1265, 911, 758; 1H NMR (CDCl3, 300.13 MHz)
d: 8.53 (1H, d,
J ¼ 3.3 Hz, H-2), 8.09 (1H, dd, J ¼ 13.0 and 3.3 Hz, H-6), 7.29 (1H, dd,
J ¼ 12.3 and 11.9 Hz, H-50), 6.95 (1H, d, J ¼ 13.0 Hz, H-5), 6.76 (1H, dd,
J ¼ 11.9 and 3.4 Hz, H-60), 6.62 (1H, dd, J ¼ 3.4 and 3.3 Hz, H-20), 6.59
(1H, dd, J ¼ 12.3 and 3.3 Hz, H-40), 3.77 (3H, s, AreOCH3); 13C NMR
4.2.4.3. 6-Methoxy-9-oxo-9H-xanthene-2-carboxylic acid (1). m.p.:
>300 ꢁC; IR nmax (cmꢀ1) (KBr): 3411, 1687, 1610, 1575, 1500, 1433,
1271, 766; 1H NMR (DMSO-d6, 300.13 MHz)
d: 8.68 (1H, d,
J ¼ 3.2 Hz, H-1), 8.29 (1H, dd, J ¼ 13.3 and 3.2 Hz, H-3), 8.09 (1H, d,
J ¼ 13.3 Hz, H-8), 7.69 (1H, d, J ¼ 13.3 Hz, H-4), 7.19 (1H, d, J ¼ 3.3 Hz,
H-5), 7.07 (1H, dd, J ¼ 13.3 and 3.3 Hz, H-7), 3.93 (3H, s, AreOCH3);
(CDCl3, 75.47 MHz) d
: 179.3 (C-100), 179.3 (C-10), 163.5 (C-3), 162.5 (C-
1), 159.2 (C-4), 145.3 (C-2), 136.6 (C-6), 135.5 (C-5), 132.3 (C-30), 127.3
(C-10), 120.6 (C-50), 113.4 (C-60), 112.1 (C-40), 107.5 (C-20), 56.7
(AreOCH3);MS(EI)m/z(%):288[M]þ. (100), 271(10), 257[M-OCH3]þ.
(12), 227 (11), 199 (23), 165 (61), 124 (63), 92 (23), 77 (20), 64 (19).
13C NMR (DMSO-d6, 75.47 MHz)
d: 175.3 (C-9), 167.6 (COOH), 164.9
(C-6), 157.6 (C-10a), 156.3 (C-4a), 135.7 (C-3), 127.6 (C-2), 126.6
(C-8), 124.9 (C-1), 120.2 (C-9a), 116.7 (C-4), 115.0 (C-8a), 113.7 (C-7),
100.7 (C-5), 56.2 (AreOCH3); MS (EI) m/z (%): 270 [M]þ. (100), 253
[M ꢀ OH]þ. (26), 226 (27), 199 (15), 182 (15), 169 (8), 154 (7), 139 (7),
126 (17), 115 (10), 63 (16).
4.2.4. Intramolecular acilation. Xanthones formation
To a solution of 4-(30-methoxyphenoxy)isophthalic acid (9)
(4.15 g, 14.40 mmol) in methane sulfonic acid (60 mL) was added
phosphorus pentoxide (6.53 g, 45.99 mmol) and the reaction
mixture was stirred at room temperature for 22 h. The mixture was
poured over ice, resulting in the formation of a cream-coloured solid
that was collected by filtration under reduced pressure and dried at
room temperature. The crude product was dissolved in methanol
(800 mL) and H2SO4 (16 mL) was added. The mixture was refluxed
for approximately 19 h. The products were separated by filtration
and the solid was washed with cooled methanol, providing methyl
6-methoxy-9-oxo-9H-xanthene-2-carboxylate (11) with 85% yield.
It was also obtained, at lower yield (2%), the compound methyl 8-
methoxy-9-oxo-9H-xanthene-2-carboxylate (12), after the evapo-
ration of the methanol and purification by column chromatography
(silica gel, ethyl acetate/n-hexane in gradient). Methyl 6-methoxy-
9-oxo-9H-xanthene-2-carboxylate (11) (3.94 g, 13.86 mmol) was
dissolved in methanol/dichloromethane (670 mL, 1:1 v/v) and 5M
NaOH solution (54 mL) was added. The mixture was stirred at room
temperature for 22 h. After evaporation of the organic solvents,
water was added (100 mL) and the solution was acidified with 5M
HCl solution resulting in the formation of a white precipitate. The
suspension was filtered under reduced pressure and the white solid
was washed with water, to afford 6-methoxy-9-oxo-9H-xanthene-
2-carboxylic acid (1). Yield: 94%. The same procedure was followed
to hydrolyze the methyl 8-methoxy-9-oxo-9H-xanthene-2-
carboxylate (12) (37 mg, 0.14 mmol) to afford 8-methoxy-9-oxo-
9H-xanthene-2-carboxylic acid (10). Yield: 95%.
4.2.4.4. 8-Methoxy-9-oxo-9H-xanthene-2-carboxylic acid (10). m.p.:
268e270 ꢁC; IR nmax (cmꢀ1) (KBr): 3460,1687,1663,1603,1469,1420,
1266, 763; 1H NMR (DMSO-d6, 300.13 MHz)
d: 8.66 (1H, d, J ¼ 2.1 Hz,
H-1), 8.28 (1H, dd, J ¼ 8.7 and 2.1 Hz, H-3), 7.79 (1H, dd, J ¼ 8.4 and
8.4 Hz, H-6), 7.67 (1H, d, J ¼ 8.7 Hz, H-4), 7.20 (1H, dd, J ¼ 8.4 and
0.7 Hz, H-7), 7.06 (1H, dd, J ¼ 8.4 and 0.7 Hz, H-5), 3.94 (3H, s,
AreOCH3); 13C NMR (DMSO-d6, 125.77 MHz)
d: 174.3 (C-9), 166.2
(COOH), 160.2 (C-8), 157.2 (C-10a), 157.0 (C-4a), 136.1 (C-3), 134.9
(C-6), 127.9 (C-2), 125.0 (C-1), 122.1 (C-9a), 118.2 (C-4), 111.6 (C-8a),
109.7 (C-7), 107.0 (C-5), 56.3 (AreOCH3); HRMS (ESI) m/z: calcd for
(C15H10O5 þ H): 271.16994, found: 271.06010.
4.3. General procedure for the synthesis of chiral xanthone derivatives 2e4
The 6-methoxy-9-oxo-9H-xanthene-2-carboxylic acid (1)
(100 mg, 0.37 mmol) was dissolved in dry tetrahydrofuran (20 mL)
and triethylamine (103 mL, 0.74 mmol) was added. Following, TBTU
(120 mg, 0.37 mmol) and an appropriate chiral reagent (0.37 mmol)
were added. The mixture was stirred at room temperature for
30 min (4) or 2 h (2e3). After completion of the reaction, the
solvent was evaporated under reduced pressure and the crude
product was dissolved in dichloromethane (50 mL). This solution
was washed with 1M HCl solution (2 ꢂ 25 mL), saturated solution
of NaHCO3 (2 ꢂ 30 mL) and water (3 ꢂ 50 mL). The organic layer
was dried with anhydrous sodium sulphate, filtered and the solvent
was evaporated under reduced pressure. The product was recrys-
tallized from ethanol (2) or ethyl acetate/n-hexane (3e4), to afford
the chiral xanthone derivative.
4.2.4.1. Methyl 6-methoxy-9-oxo-9H-xanthene-2-carboxylate (11). m.p.:
176e178 ꢁC; IR nmax (cmꢀ1) (KBr): 1730,1663, 1581,1467, 1438,1270,1117,
764; 1H NMR (CDCl3, 300.13 MHz)
d
: 8.98 (1H, d, J ¼ 3.2 Hz, H-1), 8.32
(1H, dd, J ¼ 13.1 and 3.2 Hz, H-3), 8.24 (1H, d, J ¼ 13.2 Hz, H-8), 7.48 (1H,
d, J ¼ 13.1 Hz, H-4), 6.96 (1H, dd, J ¼ 13.2 and 3.4 Hz, H-7), 6.89 (1H, d,
J ¼ 3.4 Hz, H-5), 3.95 (3H, s, COOCH3), 3.93 (3H, s, AreOCH3); 13C NMR
4.3.1. (S)-N-(1-hydroxy-3-methylbutan-2-yl)-6-methoxy-9-oxo-9H-
xanthene-2-carboxamide (2)
Compound 2 was obtained as white solid. Yield: 99%; m.p.:
ꢁ
(CDCl3, 75.47 MHz)
d: 175.5 (C-9), 165.9 (COOCH3), 165.3 (C-6), 158.7 (C-
180e182 ꢁC (ethanol); ½a 2D5 C
ꢄ
ꢀ14.89ꢁ (10.08 mg/mL, dichloromethane);
10a), 157.8 (C-4a), 134.8 (C-3), 129.1 (C-2), 128.3 (C-8), 125.9 (C-1), 121.5
IR nmax (KBr): 3371, 3244,1656,1616,1539,1476,1444,1272, 834 cmꢀ1;1H

C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
9
NMR (DMSO-d6, 300.13 MHz)
d
: 8.73 (1H, d, J ¼ 2.2 Hz, H-1), 8.38 (1H, d,
4.4. Biological activity assay
J¼ 8.7 Hz, NH), 8.31 (1H, dd, J¼ 8.7 and 2.2 Hz, H-3), 8.14 (1H, d, J¼ 8.7 Hz,
H-8), 7.71 (1H, d, J ¼ 8.7 Hz, H-4), 7.21 (1H, d, J ¼ 2.3 Hz, H-5), 7.09 (1H, dd,
J ¼ 8.7 and 2.3 Hz, H-7), 4.63 (1H, t, J ¼ 5.6 Hz, OH), 3.95 (3H, s, AreOCH3),
3.85 (1H, m,H-10), 3.54 (2H, dd,J¼ 10.4 and 5.8 Hz, CH2OH),1.97 (1H, m,H-
20), 0.93 (3H, d, J ¼ 7.0 Hz, H-30a)*, 0.90 (3H, d, J¼ 7.0 Hz, H-30b)*; 13C NMR
4.4.1. Preparation and experimental conditions
In this study, sciatic nerve bundles, 3e4 cm in length, were
taken from adult Wistar rats (200e400 g), obtained from Charles
Rivers (Barcelona, Spain). Rats were kept at a constant temperature
(21 ꢁC) and a regular light (06.30e19.30 h)-dark (19.30e06.30 h)
cycle, with food and water ad libitum. Animal handling and
experiments followed the guidelines defined by the European
Communities Council Directive (86/609/EEC).
(DMSO-d6, 75.47 MHz) d: 174.9 (C-9), 167.7 (C]O, amide), 165.1 (C-6),
157.6 (C-10a), 157.0 (C-4a), 134.1 (C-3), 130.9 (C-2), 127.7 (C-8), 125.4 (C-1),
120.6 (C-9a), 118.0 (C-4), 114.9 (C-8a), 114.0 (C-7), 100.8 (C-5); 61.3
(CH2OH), 57.0 (AreOCH3), 56.3 (C-10), 28.7 (C-20), 19.7 (C-30a)*, 18.8
(C-30b)*; MS (EI) m/z (%): 357 [M þ H]þ.þ1 (15), 356 [M þ H]þ. (43), 340
[M ꢀ CH3]þ. (8), 270 (11), 254 (20), 253 (100), 239 (12), 227 (12), 226 (15);
HRMS (ESI) m/z:calcdfor(C20H21NO5 þ H): 356.14925, found: 356.14967;
Enantiomeric purity: e.e. > 99% (HPLC; Column: Chirobiotic T, Mobile
phase: n-Hexane:Ethanol (80:20 v:v), 0.5 mL/min, lmax 254 nm).
* Asterisks denote assignments that may be interchanged.
The rats were killed by decapitation and the sciatic nerves were
exposed from the spinal cord to the knee. A portion of the sciatic
nerve was rapidly isolated free from surrounding structures and
their ends were tied, in order to allow the setting of the nerve in the
recording chamber (Marsh Ganglion Bath). This process was per-
formed in the presence of gassed (95% O2 and 5% CO2) Tyrode’s
solution (pH 7.4) containing (mM): KCl 2.7, CaCl2 1.8, NaH2PO4 0.4,
MgCl2 1, NaCl 137, NaHCO3 11.9 and glucose 11.2, at 25 ꢁC. Before
electrophysiological recordings, desheathed sciatic nerves were
equilibrated with gassed Tyrode’s solution superfused with a flow
rate of 1 mL/min for at least 3 h at 25 ꢁC, in order to achieve a stable
baseline and reproducible electrically-evoked compound action
potentials (see e.g [40].). Measurement of lactate dehydrogenase
(LDH, EC 1.1.1.27) activity leaked into the incubation medium from
nerve fibre bundles was taken as a measure of neuronal cell damage
and followed the method described by Bergmeyer [41]. LDH activity
showed no significant changes over time of incubation, suggesting
that experimental manipulations did not affect sciatic nerve
viability.
4.3.2. (S)-N-(1-hydroxy-4-methylpentan-2-yl)-6-methoxy-9-oxo-9H-
xanthene-2-carboxamide (3)
Compound 3 was obtained as white ꢁsolid. Yield: 98%; m.p.:
156e158 ꢁC (ethyl acetate/n-hexane); ½a 2D5 C
ꢄ
- 25.00ꢁ (11.40 mg/mL,
dichloromethane); IR nmax (cmꢀ1) (KBr): 3360, 3285, 1652, 1617,
1546,1468,1443,1264, 830; 1H NMR (DMSO-d6, 300.13 MHz)
d: 8.72
(1H, d, J ¼ 2.2 Hz, H-1), 8.40 (1H, d, J ¼ 8.7 Hz, NH), 8.30 (1H, dd, J ¼ 8.7
and 2.2 Hz, H-3), 8.14 (1H, d, J ¼ 8.9 Hz, H-8), 7.70 (1H, d, J ¼ 8.7 Hz, H-
4), 7.21 (1H, d, J ¼ 2.3 Hz, H-5), 7.10 (1H, dd, J ¼ 8.9 and 2.3 Hz, H-7),
4.75 (1H, t, J ¼ 5.7 Hz, OH), 4.11 (1H, m, H-10), 3.95 (3H, s, AreOCH3),
3.43 (2H, dd, J ¼ 13.3 and 5.3 Hz, CH2OH),1.64 (2H, m, H-20),1.45 (1H,
m, H-30), 0.91 (3H, d, J ¼ 6.0 Hz, H-40a) *, 0.90 (3H, d, J ¼ 6.0 Hz, H-
40b)*; 13C NMR (DMSO-d6, 75.47 MHz)
d: 174.9 (C-9), 165.2 (C]O,
amide), 164.7 (C-6), 157.5 (C-10a), 157.1 (C-4a), 134.0 (C-3), 130.7 (C-
2), 127.7 (C-8), 125.3 (C-1), 120.7 (C-9a), 118.1 (C-4), 114.9 (C-8a),
114.0 (C-7), 100.8 (C-5), 63.9 (CH2OH), 56.3 (AreOCH3), 49.8 (C-10),
39.0 (C-20), 24.5 (C-30), 23.5 (C-40a)*, 21.9 (C-40b)*; MS (ESI) m/z (%):
371 [M þ H]þ.þ1 (28), 370 [M þ H]þ. (100), 328 (7), 306 (7), 253 (2),
197 (3), 171 (11); HRMS (ESI) m/z: calcd for (C21H23NO5 þ H):
370.16490, found: 370.16488; Enantiomeric purity: e.e. > 99%
(HPLC; Column: Amylose tris-3,5-dimethylphenylcarbamate coated
4.4.2. Electrophysiology experiments
Electrophysiology experiments were performed using an
extracellular partition recording technique, in order to monitor
changes in membrane polarization associated with electrical sciatic
nerve stimulation - compound action potentials (CAP). Sciatic nerve
trunks were mounted in a Marsh Ganglion Bath recording chamber.
The recording chamber was partitioned into three compartments:
a) a stimulating pool, containing a pair of silver electrodes con-
nected to a Grass SD9 stimulator (Quincy, MA, USA); b) a test-pool,
where drugs were applied; and c) a recording pool, containing
a pair of silver electrodes connected to a Hitachi VC-6025A digital
storage oscilloscope (Tokyo, Japan) coupled to an ink-recording
plotter.
onto APS-Nucleosil (500 Å, 7 mm, 20% w/w), Mobile phase: Etha-
nol:Acetonitrile (50:50 v:v), 0.5 mL/min, lmax 254 nm).
* Asterisks denote assignments that may be interchanged.
4.3.3. (S)-6-methoxy-9-oxo-N-(1-(p-tolyl)ethyl)-9H-xanthene-2-
carboxamide (4)
The rat sciatic nerve trunk was stimulated with a 0.2 Hz
frequency throughout the experiment. Supramaximal intensity
rectangular pulses of 0.03e0.05 ms duration were used to achieve
nerve firing synchronization, thus reducing the number of silent
units that might confound data interpretation. The stimulation
voltage was set 2 times the threshold stimulus required to achieve
maximal CAP amplitude, which did not exceed 2e3 V. The pulses
were delivered via a Grass SD9 stimulator (Quincy, MA, USA).
Solutions were changed by transferring the inlet tube of the
peristaltic pump (Gilson, Minipuls3, France) from one flask to
another. The flow rate was kept at 1 mL/min throughout the
experiments. Test drugs were applied in a cumulative manner and
were allowed to contact with the preparations for at least 15 min.
Nerve impulse blockade caused by test drugs was expressed as
a percentage decrement in amplitude of the CAP in control condi-
tions, i.e. without addition of any drug to the Tyrode’s solution.
Compound 4 was obtained as white solid. Yield: 97%; m.p.:
ꢁ
221e223 ꢁC (ethyl acetate/n-hexane); ½a 2D5 C
ꢄ
þ 125.70ꢁ (10.70 mg/
mL, dichloromethane); IR nmax (cmꢀ1) (KBr): 3333, 1660, 1611, 1530,
1476,1438,1266, 831, 827; 1H NMR (CDCl3, 300.13 MHz)
d: 8.56 (1H,
d, J ¼ 2.2 Hz, H-1), 8.32 (1H, dd, J ¼ 8.7 and 2.2 Hz, H-3), 8.25 (1H, d,
J ¼ 8.9 Hz, H-8), 7.53 (1H, d, J ¼ 8.7 Hz, H-4), 7.31 (2H, d, J ¼ 8.0 Hz,
H-200 and H-600), 7.18 (2H, d, J ¼ 8.0 Hz, H-300 and H-500), 6.99 (1H, dd,
J ¼ 8.9 and 2.3 Hz, H-7), 6.92 (1H, d, J ¼ 2.3 Hz, H-5), 6.58 (1H, d,
J ¼ 7.3 Hz, NH), 5.33 (1H, m, H-10), 3.93 (3H, s, AreOCH3), 2.34 (3H, s,
AreCH3), 1.63 (3H, d, J ¼ 6.9 Hz, H-20); 13C NMR (CDCl3, 75.47 MHz)
d: 175.9 (C-9), 165.5 (C]O, amide), 164.8 (C-6), 158.0 (C-10a), 157.8
(C-4a),139.8 (C-100),137.2 (C-400), 134.3 (C-3),130.1 (C-2),129.4 (C-300
and C-500), 128.3 (C-8), 126.2 (C-200 and C-600), 124.0 (C-1), 121.0 (C-
9a), 118.5 (C-4), 115.5 (C-8a), 113.8 (C-7), 100.3 (C-5), 55.9
(AreOCH3), 49.3 (C-10), 21.7 (AreCH3), 21.0 (C-20); MS (ESI) m/z (%):
389 [M þ H]þ.þ1 (27), 388 [M þ H]þ. (100), 354 (7), 338 (13), 270
(8), 219 (6), 197 (10); HRMS (ESI) m/z: calcd for (C24H21NO4 þ H):
388.15433, found: 388.15357; Enantiomeric purity: e.e. > 98%
(HPLC; Column: Amylose tris-3,5-dimethylphenylcarbamate coated
4.4.3. Hypotonic haemolysis on rat erythrocytes
Experiments were carried out on erythrocytes isolated from rat
blood samples. The technique used to produce haemolysis followed
the one described by Seeman and Weinstein [42], modified by
Timóteo and Ribeiro [43]. Blood samples were obtained by heart
onto APS-Nucleosil (500 Å, 7
nol:Acetonitrile (50:50 v:v), 0.5 mL/min, lmax 254 nm).
mm, 20% w/w), Mobile phase: Etha-

10
C. Fernandes et al. / European Journal of Medicinal Chemistry 55 (2012) 1e11
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M. Pinto, M.H. Vasconcelos, Insights into the in vitro antitumor mechanism of
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puncture from Wistar rats anaesthetized with urethane (1.2 g/kg).
Aliquots of 0.9 mL heparinised (100 units/mL) blood samples were
centrifuged for 15 min at 2000 rpm. The plasma, including the
leukocyte layer, was carefully removed, and then 154 mM NaCl in
10 mM phosphate buffer pH 7.0 was added, to make a total volume
of 12.5 mL. Erythrocytes were re-suspended by a gentle and
repeated inversion of the test tube. Aliquots of 0.2 mL of the stock
erythrocyte suspension were added to 4 mL of test drug solutions
(various concentrations) diluted in haemolytic solution containing
a predetermined concentration of NaCl in 10 mM phosphate buffer,
at pH 7.0; 50% erythrocyte haemolysis is usually obtained with NaCl
ranging from 63 to 67 mM. The mixture was kept at room
temperature for 10 min and then it was centrifuged at 3000 rpm for
15 min. The haemoglobin content of the supernatant was evaluated
at 540 nm using a spectrophotometer. All experiments were done
in duplicate.
4.4.4. Materials and solutions
Stock solutions of chiral xanthone derivatives (2e4), xanthone
(SigmaeAldrich) and dibucaine (SigmaeAldrich) were prepared in
dimethylsulfoxide (DMSO); solutions were kept protected from the
light to prevent photodecomposition. All stock solutions were
stored as frozen aliquots at ꢀ20 ꢁC. Dilutions of these stock solu-
tions were made daily (0.100, 0.300, 0.420, 0.560, 0.750, 1.00 and
3.00
mM) in Tyrode’s solution and appropriate solvent controls
were done. No statistically significant differences between control
experiments, made in the absence or in the presence of the solvents
at the maximal concentrations used (0.05% v/v), were observed.
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4.4.5. Statistical analysis
The data is expressed as mean ꢃ S.E.M., from an n number of
experiments. Statistical significance of experimental results was
analyzed by one-way analysis of variance (ANOVA) followed by
Dunnett’s modified t-test. P < 0.05 was considered to represent
significant differences.
Acknowledgements
This research was partially supported by Fundação para a Ciên-
cia e a Tecnologia (FCT, FEDER and POCI funding, 226/2003,
CEQUIMED-UP, I&D 4040/2007, PEst-OE/SAU/UI4040/2011 and
UMIB-215/94, PEst-OE/SAU/UI0215/2011) and by University of
Porto/Caixa Geral de Depósitos (Investigação Científica na Pré-
Graduação) projects. J.B.N.M. was in receipt of a PhD Fellowship
from FCT (SRFH/BD/68584/2010). We would also like to thank Mrs.
M. Helena Costa e Silva and Mrs. Belmira Silva for technical assis-
tance in biological activity assays and Mrs Milaydis Sosa Napolskij
for proof reading the manuscript.
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synthesis, Tetrahedron 60 (2004) 2447e2467.
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