Comparative inhibitory effects of niflumic acid and novel synthetic derivatives on the rat isolated stomach fundus

Article abstract of DOI:10.1211/0022357021778321

Novel derivatives of 2-[3-(trifluoromethyl)-analino]nicotinic acid (niflumic acid) were synthesized. The compounds were compared for their inhibitory effects on 5-hydroxytryptamine (5-HT)- and KCl-induced contraction of the rat fundus. The aim was to assess structure-activity relationships regarding the selectivity and potency of these compounds. Niflumic acid (1-100 μM) concentration-dependently inhibited 5-HT-induced tonic contractions with an IC50 value (concentration reducing the control contractile response by 50%, calculated from semi-log graphs) of 0.24 × 10^-4 M (n = 9). In contrast, it was significantly less potent at inhibiting KCl-induced responses (IC50 = 1.49 × 10^-4 M, n = 9). The methyl ester (NFAme) and amido (NFAm) analogues showed no selectivity between 5-HT- and KCl-induced contractions with IC50 values of 1.64 × 10^-4 M (n = 8) and 1.87 × 10^-4 M (n = 9) for 5-HT responses, and 2.61 × 10^-4 M (n = 8) and 2.55 × 10^-4 M (n = 7) for KCl-induced responses, respectively. Our results suggest that alteration of the carboxylic acid moiety of niflumic acid reduces the selectivity and potency of its inhibitory action on 5-HT-induced contractile responses of the rat fundus, possibly via a reduced interaction with calcium-activated chloride channels.

Full text of DOI:10.1211/0022357021778321

Journal of
JPP 2002, 54: 283–288
# 2002 The Authors
Received May 18, 2001
Accepted September 21, 2001
ISSN 0022-3573
Comparative inhibitory effects of ni umic acid and
novel synthetic derivatives on the rat isolated stomach
fundus
David N. Criddle, Ana Vanesca P. Meireles, Liana B. Macedo, Jose´ H.
Leal-Cardoso, Henrique C. Scarparo and Mohammed Jaffar
Abstract
Novel derivatives of 2-[3-(tri uoromethyl)-analino]nicotinic acid (ni umic acid) were syn-
thesized. The compounds were compared for their inhibitory effects on 5-hydroxytryptamine
(5-HT)- and KCl-induced contraction of the rat fundus. The aim was to assess structure–activity
relationships regarding the selectivity and potency of these compounds. Ni umic acid
lM) concentration-dependently inhibited 5-HT-induced tonic contractions with an IC50
(1–100
value (concentration reducing the control contractile response by 50%, calculated from semi-
4
-
.
log graphs) of 0 24¬10 M (n ¯ 9). In contrast, it was signiŽ cantly less potent at inhibiting KCl-
Laboratorio de Farmacologia
´
4
-
.
induced responses (IC50¯ 1 49¬10 M, n ¯ 9). The methyl ester (NFAme) and amido (NFAm)
dos Canais Ionicos,
Departamento de Ciencias
Fisiologicas, CCS, Universidade
´
Estadual do Ceara, Av.
´
Paranjana 1700, Fortaleza CE
60740-000, Brazil
ˆ
analogues showed no selectivity between 5-HT- and KCl-induced contractions with IC50 values
ˆ
4
4
4
-
-
-
.
.
.
of 1 64¬10 M (n¯ 8) and 1 87¬10 M (n¯ 9) for 5-HT responses, and 2 61¬10 M (n ¯ 8)
4
-
.
and 2 55¬10 M (n¯ 7) for KCl-induced responses, respectively. Our results suggest that
alteration of the carboxylic acid moiety of ni umic acid reduces the selectivity and potency of
its inhibitory action on 5-HT-induced contractile responses of the rat fundus, possibly via a
reduced interaction with calcium-activated chloride channels.
David N. Criddle, Ana Vanesca P.
Meireles, Liana B. Macedo, Jose
´
ˆ
H. Leal-Cardoso
Departamento de Farmacologia
e Fisiologia, Faculdade de
Medicina, Universidade Federal
Introduction
do Ceara, Cel. Nunes de Melo
´
1127, Porangabussu, Fortaleza,
CE, Brazil
The putative excitatory role of calcium-activated chloride (Cl ) channels in the
(Ca)
contractile action of various neurotransmitters on vascular and non-vascular
smooth muscle has received growing interest (Large & Wang 1996; Chipper®eld &
Harper 2000). This has largely been due to the development of relatively selective
inhibitors of these channels, such as the fenamates, typi®ed by ni¯ umic acid. This
compound has been shown to inhibit calcium-activated chloride currents (ICl(Ca)) in
isolated smooth muscle cells (Pacaud et al 1989; Akbarali & Giles 1993; Hogg et al
1994; Lamb et al 1994), although the exact mechanism of interaction of ni¯ umic
acid with the ion channel remains unclear. Nevertheless, ni¯ umic acid has been
Henrique C. Scarparo
Department of Pharmacy and
Pharmaceutical Sciences,
University of Manchester,
Manchester, M13 9PL, UK
Mohammed Jaffar
Correspondence: D. N. Criddle,
increasingly utilized as a pharmacological tool for investigation of Cl
channels
(Ca)
Departamento de Ciencias
ˆ
in functional studies in isolated tissues. We have previously demonstrated that
ni¯ umic acid inhibits noradrenaline (norepinephrine)-induced contraction of
isolated rat aorta and, on the basis of comparative data with nifedipine, suggested
Fisiologicas, CCS, Universidade
Estadual do Ceara, Av.
´
Paranjana 1700, Fortaleza CE
60740-000, Brazil. E-mail:
criddle!uece.br
´
that activation of Cl
channels may lead to a depolarization-induced opening of
(Ca)
voltage-dependent calcium channels (VDCCs) causing extracellular calcium entry
and consequent contraction (Criddle et al 1996). Subsequent studies have provided
supportive data for this hypothesis in a number of smooth muscle preparations
Funding: This study was
supported by the CNPq and
FUNCAP.
283

284
David N. Criddle et al
270 MHz spectrometer. Chemical shifts (d ppm) and
coupling constants (J Hz) were measured using tri-
methylsilane as the internal standard. Microanalytical
data were carried out at the Department of Chemistry,
University of Manchester.
Methyl 2-[3-(tri¯ uoromethyl)-analino]nicotinate
(NFAme)
To a solution of ni¯ umic acid (500 mg, 1.77 mmol)
in methanol (30 mL) was added K2CO3 (450 mg,
3.26 mmol), and dimethyl sulfate (0.5 mL, 670 mg,
5.3 mmol). The mixture was stirred at room temperature
for 2 h. The resulting yellow suspension was ®ltered.
The ®ltrate was extracted with ethyl acetate (2¬75 mL),
washed with water (2¬25 mL) and brine (50 mL), dried
over MgSO4, and condensed at the pump to give a
yellow oil which was chromatographed (eluting with
ethyl acetate) to give NFAme (380 mg, 73% ) as white
solid: m.p. 74±75 °C, 1H NMR (CDCl3): d 10.38 (1H, s,
NH ), 8.40 (1H, m, Ar-H ), 8.29 (1H, dd, J ¯ 6.0, 9.0 Hz,
Ar-H ), 8.10 (1H, t, J ¯ 6.0 Hz, Ar-H ), 7.89 (1H, m, Ar-
H ), 7.41 (1H, t, J ¯ 6.0 Hz, Ar-H ), 7.17 (1H, m, Ar-H ),
6.79 (1H, dd, J¯ 6.0, 9.0 Hz, Ar-H ), 3.95 (3H, s,
CO2CH3). Calculated for C14H11F3N2O2 : C, 56.76; N,
9.46; H, 3.74; found : C, 56.40; N, 9.31; H, 3.97% .
Figure
1
Synthesis of methyl 2-[3-(tri¯ uoromethyl)-analino]-
nicotinate (NFAme) and 2-[3-(tri¯ uoromethyl)-analino]nicotin-
amide (NFAm). Reagents and conditions: (i) (MeO)2SO2, K2CO3,
MeOH, rt, 2 h; (ii) SOCl2, THF, rt, 16 h; (iii) NH4OH (conc.),
EtOAc, rt, 2 h.
(Criddle et al 1997; Yuan 1997; Hyvelin et al 1998;
Lamb & Barna 1998). More recently, we have demon-
strated a potent and selective inhibition of 5-HT-
induced contraction of rat isolated stomach fundus by
ni¯ umic acid (Scarparo et al 2000), and this tissue may
provide a simple and convenient preparation for the
functional assay of analogues of ni¯ umic acid. Methyl
2-[3-(tri¯ uoromethyl)-analino]nicotinate (NFAme) and
2-[3-(tri¯ uoromethyl)-analino]nicotinamide (NFAm)
were synthesized to ascertain the importance of the
carboxylic acid moiety that is present in ni¯ umic acid
but not in these derivatives (for synthetic scheme, see
Figure 1). The inhibitory eŒects of these compounds on
5-HT- and KCl-induced contraction of the rat fundus
were assessed. The preliminary results of this study have
recently been communicated to the British Pharma-
cology Society (Birmingham BPS Meeting, December
2000; Criddle et al 2001).
Ni¯ umic acid chloride
To a solution of ni¯ umic acid (500 mg, 1.77 mmol) in
anhydrous tetrahydrofuran (10 mL) was added excess
thionyl chloride (1 mL). The mixture was stirred under
N2 for 16 h. The resulting yellow solution was condensed
at the pump and chromatographed (eluting with 1:1,
hexane±ethyl acetate) to give a yellow solid (420 mg,
1
79% ), m.p. 215±216°C, H NMR (DMSO-d6): d 10.67
(1H, s, NH ), 8.47 (1H, m, Ar-H ), 8.13 (2H, m, 2¬Ar-
H ), 7.87 (1H, m, Ar-H ), 7.58 (1H, t, J ¯ 6.0 Hz, Ar-H ),
7.36 (1H, m, Ar-H ), 6.99 (1H, m, Ar-H ).
2-[3-(Tri¯ uoromethyl)-analino]nicotinamide (NFAm)
To a solution of ni¯ umic acid chloride (400 mg,
1.33 mmol)in ethyl acetate(20 mL)wasadded ammonia
solution (conc., 20 mL). The mixture was stirred for 4 h
at room temperature and the product was extracted
with ethyl acetate (2¬150 mL), washed with water
(2¬50 mL) and brine (50 mL), dried over MgSO4, and
condensed at the pump. The crude product was re-
Materials and Methods
Chemical procedures
Melting points were determined using a Gallenkamp crystallized from ethyl acetate to aŒord NFAm (220 mg,
1
1
melting point apparatus and are uncorrected. The H 59% ) as white crystals: m.p. 284±285°C, H NMR
NMR data were recorded on a Jeol JNMR-EX270 (DMSO-d6): 10.73 (1H, s, NH ), 8.37 (2H, m, CONH2),

285
Inhibitory effects of niumic acid analogues
lm
m
) and KCl-(60 m ) induced contractions of the rat isolated fundus by
Table 1 Comparison of IC50 values for inhibition of 5-HT- (10
ni¯ umic acid, NFAme and NFAm.
Relative potency ratioa
m
Compound
5-HT-induced contraction
IC50
(
) KCl-induced contraction
4
4
-
-
.
.
.
.
.
.
.
6 2
.
1 6
Ni¯ umic acid
NFAme
0 24¬10 (0 11±0 37)
1 49¬10 (0 76±2 22)g
4
4
-
-
.
.
.
.
.
.
1 64¬10 (0 51±2 77)*
2 61¬10 (0 46±4 76)
4
4
-
-
.
.
.
.
.
.
.
1 4
NFAm
1 87¬10 (0 56±3 18)*
2 55¬10 (1 30±3 80)
Values are expressed as the mean of 7±9 observations, with 95% con®dence limits included in brackets. aKCl-induced contraction divided by
!
!
.
0 05, comparing 5-HT- and KCl-induced
P
.
P
5-HT-induced contraction. *
contraction.
0 05, compared with NFA-induced inhibition of contraction. g
8.31 (1H, m, Ar-H ), 8.21 (1H, dd, J ¯ 5.9, 9.0 Hz, Ar- ution was a standard Tyrode’s solution of the following
H ), 7.80 (1H, m, Ar-H ), 7.54 (1H, t, J ¯ 5.9 Hz, Ar-H ), composition (mm): NaCl 136, KCl 5, MgCl2 0.98, CaCl2
7.30 (1H, m, Ar-H ), 6.95 (1H, dd, J ¯ 5.9, 9.0 Hz, Ar- 2, NaH2PO4 0.36, NaHCO3 11.9, glucose 5.5. In sol-
H ). Calculated for C13H10F3N3O: C, 55.52; N, 14.94; H, utions in which the potassium concentration was raised
3.58; found: C, 55.81; N, 14.52; H, 3.30% .
(60 mm), the NaCl concentration was concomitantly
reduced to maintain osmolarity. The pH was maintained
at 7.4 throughout the experimental period.
Data are expressed graphically as the mean of n
observations³s.e.m. and curves were ®tted using
Microcal Origin. Inhibitory eŒects are expressed as %
of control responses in the absence of the drug. IC50
values (concentration producing 50% of control re-
sponse in absence of drug) were calculated by inter-
polation from semi-logarithmic plots and are expressed
as mean values with 95% con®dence limits. Statistical
analysis was performed using analysis of variance and
values were taken to be signi®cantly diŒerent when
Inhibition assays
Institutional approval for the protocols used for animal
experimentation was obtained. Male Wistar rats (250±
350 g) were killed by stunning and cervical dislocation.
The abdomen was opened and the stomach removed.
The fundus was dissected and washed in fresh Tyrode’s
solution. Longitudinal strips (2 cm long) were carefully
prepared and mounted vertically in an organ bath
(10 mL capacity) containing Tyrode’s solution bubbled
with air (37°C, pH 7.4). Tissues were mounted under an
initial resting tension of 2 g and left to equilibrate for a
period of 1 h, following which a further 2 g was applied
before starting the experimental protocol. Tension
changes were recorded using isometric force transducers
connected to a computerized data acquisition system
(WINDAQ). The eŒects of ni¯ umic acid (Sigma Chemi-
cal Company), and its analogues, NFAme and NFAm,
were assessed on contractions induced by KCl (60 mm)
!
P
0.05.
Results
Inhibitory effects of ni umic acid on fundus
contractions
and 5-HT (10 lm) with each agonist being evaluated in Application of 5-HT (10 lm) to the rat fundus induced
a separate experimental group. Following stable con- stable tonic contractions (1.67³0.16 g, n ¯ 26). In the
tractions to the contractile agent, ni¯ umic acid (or presence of ni¯ umic acid (1±100 lm) these tonic con-
analogue) was applied in increasing concentrations and tractions were completely inhibited in a concentration-
further responses obtained (contact time of 15 min). dependent manner, with an IC50 value of 20 lm (Table
Since dimethyl sulfoxide (DMSO) was used as a solvent 1 and Figure 2). This inhibitory eŒect of ni¯ umic acid
for stock solutions of ni¯ umic acid, the eŒects of this was fully reversible on washout of the drug from the
solvent at equivalent concentrations were assessed as tissue. In control-paired tissues exposed only to the
time-matched controls, on 5-HT-induced contraction of solvent DMSO at equivalent concentrations, the re-
the fundus.
sponse to 5-HT did not diminish over the experimental
Ni¯ umic acid and analogues were prepared as stock period (n ¯ 8). Application of KCl (60 mm) to the rat
solutions (10 mm) in DMSO and diluted on the day of fundus induced reproducible tonic contractions (2.61
the experiment in Tyrode’s solution. The bathing sol- ³0.34 g, n ¯ 24), which were inhibited less potently by

286
David N. Criddle et al
120
and depolarization are inhibited by ni¯ umic acid (Yuan
1997). In this respect the contractile pathway utilized by
5-HT is likely to be fundamentally diŒerent to that
involved in KCl-induced contraction, which appears to
be exclusively mediated by calcium in¯ ux through
VDCCs (Bolton 1972) and is completely blocked by
nifedipine in the rat fundus (Smaili et al 1991; Scarparo
et al 2000). Therefore the bulk of current evidence
suggests that the inhibitory actions of ni¯ umic acid on
5-HT-induced contraction in this tissue are mediated
100
80
60
40
20
0
via blockade of Cl
channels, leading to a closure of
(Ca)
VDCCs. Previous work in the rat mesenteric vascular
bed has shown that the inhibitory eŒects of ni¯ umic acid
on 5-HT-induced pressor responses are not mediated
via a direct antagonistic action at plasmalemmal
receptors, since no blockade was observed under
calcium-free conditions (Criddle et al 1997), further
strengthening the argument that the site of action of
ni¯ umic acid is distal to agonist-induced intracellular
calcium release.
10–6
10–5
Drug concn (
10–4
10–3
M
)
Figure 2 Inhibitory eŒects of ni¯ umic acid (D), NFAme (_) and
lm
NFAm (V) on contractions induced by 5-HT (10
) in the rat
isolated stomach fundus. Values are shown as means³s.e.m.
ni¯ umic acid (IC50 value of 120 lm; Figure 3 and Table
1) as previously demonstrated (Scarparo et al 2000).
These data show that analogues NFAme and NFAm
are less potent than the parent compound in inhibiting
5-HT-induced contraction in this tissue. This indicates
that the interaction of NFAme and NFAm with Cl
Inhibitory effects of ni umic acid analogues on
fundus contractions
(Ca)
channels may be weaker, as a result of the derivatisation
of the carboxylic acid function of the molecule. Previous
studies in smooth muscle have suggested that ni¯ umic
acid induces open-channel block by binding to a site
within the chloride channel (Hogg et al 1994). It may be
that an ionic interaction with a functional group of the
ion-channel protein has been impaired by these struc-
tural modi®cations, or alternatively may re¯ ect a more
general change due to alteration of the net charge of the
molecule. For example, ni¯ umic acid would be pre-
dominantly in its zwitterionic form, whereas analogues
NFAme and NFAm do not exist in this state.
Analogues NFAme and NFAm were signi®cantly less
potent inhibitors of 5-HT-induced contractions than
ni¯ umic acid (6±8 fold order of magnitude; Figures 2
and 3), producing inhibition of both 5-HT- and KCl-
induced responses with similar IC50 values (164±
261 lm; Table 1 and Figure 3).
Discussion
This study has shown that alteration of the carboxylic
As would be predicted by both of these hypotheses,
acid moiety of ni¯ umic acid changes the pharmaco- we found no diŒerence between the pharmacological
logical pro®le of this class of compounds, reducing both pro®les of the analogues in this study. Thus both in-
its inhibitory selectivity and potency on 5-HT-induced hibited 5-HT- and KCl-induced contractions with a
contractions of the rat fundus. 5-HT-induced contrac- similar potency, in contrast to ni¯ umic acid, which was
tion in this tissue, mediated via the 5-HT2B subtype of signi®cantly more potent at inhibiting 5-HT-induced
the receptor (Foguet et al 1992; Baxter et al 1994; Hoyer contractions than KCl-induced changesin the ratfundus
et al 1994; Kursar et al 1994), is likely to involve the (Scarparo et al 2000; this study). The inhibitory eŒects
activation of ICl(Ca) as an integral part of the phar- of ni¯ umic acidand synthetic analogueson KCl-induced
macological transduction mechanism. Foguet et al contraction may occur as a result of actions unrelated to
(1992) have shown that Cl
channels are activated by inhibition of ICl(Ca). For example, a recent study in
(Ca)
stimulation of rat fundus 5-HT2B receptors expressed in vascular smooth muscle has demonstrated relaxant
Xenopus oocytes, while 5-HT-induced activation of eŒects of ni¯ umic acid that appear to be independent of
ICl(Ca) has been reported in epithelial cells (Garner et al chloride-channel blockade (Kato et al 1999), while
1993). In addition, a more recent study in pulmonary ni¯ umic acid (& 50 lm) has been shown to directly
artery has shown that both 5-HT-induced contraction modulate a variety of ion channels in addition to chlor-

287
Inhibitory effects of niumic acid analogues
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60
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40
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0
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10–6
10–5
Drug concn (
10–4
10–3
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