Molecular determinants for the activating/blocking actions of the 2H-1,4-benzoxazine derivatives, a class of potassium channel modulators targeting the skeletal muscle KATP channels

Article abstract of DOI:10.1124/mol.108.046615

The 2H-1,4-benzoxazine derivatives are modulators of the skeletal muscle ATP-sensitive-K⁺ channels (K_ATP), activating it in the presence of ATP but inhibiting it in the absence of nucleotide. To investigate the molecular determinants

Full text of DOI:10.1124/mol.108.046615

0026-895X/08/7401-50–58$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 74:50–58, 2008
Vol. 74, No. 1
46615/3354164
Printed in U.S.A.
Molecular Determinants for the Activating/Blocking Actions of
the 2H-1,4-Benzoxazine Derivatives, a Class of Potassium
Channel Modulators Targeting the Skeletal Muscle KATP
Channels
Domenico Tricarico, Antonietta Mele, Giulia Maria Camerino, Antonio Laghezza,
Giuseppe Carbonara, Giuseppe Fracchiolla, Paolo Tortorella, Fulvio Loiodice, and
Diana Conte Camerino
Departments of Pharmacobiology (D.T., A.M., G.M.C., D.C.C.) and Medicinal Chemistry (A.L., G.C., G.F., P.T., F.L.), Faculty of
Pharmacy, University of Bari, Bari, Italy
Received February 21, 2008; accepted April 10, 2008
ABSTRACT
The 2H-1,4-benzoxazine derivatives are modulators of the skel- hexylmethyl Ͼ2-isopropyl ϭ 2-n-butyl Ն 2-phenyl Ն 2-ben-
etal muscle ATP-sensitive-Kϩ channels (KATP), activating it in zyl ϭ 2-isobutyl analogs. In the absence of ATP, the 2-phenyl
the presence of ATP but inhibiting it in the absence of nucleo- analog was the most potent inhibitor (IC50 ϭ 2.5 ϫ 10Ϫ11 M);
tide. To investigate the molecular determinants for the activat- the rank order of efficacy of the blockers was 2-phenyl Ն
ing/blocking actions of these compounds, novel molecules with 2-n-hexyl Ͼ 2-n-butyl Ͼ 2-cyclohexylmethyl, whereas the
different alkyl or aryl-alkyl substitutes at position 2 of the 1,4- 2-phenylethyl, 2-benzyl, and 2-isobutyl 1,4-benzoxazine ana-
benzoxazine ring were prepared. The effects of the lengthening logs were not effective; the 2-isopropyl analog activated the
of the alkyl chain and of branched substitutes, as well as of the KATP channel even in the absence of nucleotide. Therefore,
introduction of aliphatic/aromatic rings on the activity of the distinct molecular determinants for the activating or blocking
molecules, were investigated on the skeletal muscle KATP chan- actions for these compounds can be found. For example, the
nels of the rat, in excised-patch experiments, in the presence or replacement of the linear with the branched alkyl substitutes at
absence of internal ATP (10Ϫ4 M). In the presence of ATP, the the position 2 of the 1,4-benzoxazine nucleus determines the
2-n-hexyl analog was the most potent activator (DE50 ϭ 1.08 ϫ molecular switch from blockers to openers. These compounds
10Ϫ10 M), whereas the 2-phenylethyl was not effective. The were 100-fold more potent and effective as openers than other
rank order of efficacy of the openers was 2-n-hexyl Ն2-cyclo- KCO against the muscle KATP channels.
Potassium channel openers (KCO) are chemically diverse sized and tested against the ATP sensitive Kϩ-channel
compounds that belong to a number of structural classes,
including benzopyrans (cromakalim, bimakalim), benzo-
thiadiazines (diazoxide), cyanoguanidines (pinacidil), cyclo-
butenediones (WAY-151616), nicotinamides (nicorandil),
pyrimidines (minoxidil), tertiary carbinoles (ZD-6169), thio-
formamides (aprikalim), and dihydropyridine-like structures
(ZM-244085) (Mannhold, 2004; Jahangir and Terzic, 2005).
Several derivatives of these compounds have been synthe-
(KATP) subunits expressed in cell lines, which is the primary
target for KCO action.
These compounds show a broad spectrum of therapeutic
applications, including asthma, urinary incontinence, hyper-
tension, angina, hypoglycemia, neuromuscular disorders,
and some forms of epilepsy (Andersson, 1992; Longman and
Hamilton, 1992). KCO drugs exert their effects on pancreatic
␤ cells, neurons, and vascular/nonvascular smooth muscle
and cardiac muscle by opening KATP channels, thus shifting
the membrane potential toward the reversal potential for
potassium and reducing cellular electrical activity. In skele-
tal muscle, nicorandil is effective in restoring the depressed
This work was supported by Telethon Grant GGP04140.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.108.046615.
ABBREVIATIONS: KCO, potassium channel openers; WAY-151616, 3-(2,4-dichloro-6-methylbenzylamino)-4-(1,1-dimethyl-propylamino)cy-
clobut-3-ene-1,2-dione; ZD-6169, (s)-N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methyl-propionamide; ZM-244085, 9-(3-cyanophenyl-
)hexahydro-1,8-acridinedione; DE50, drug concentration needed to enhance the current by 50%; IC50, drug concentration needed to inhibit the
current by 50%; hypoPP, hypokalemic periodic paralyses; MOPS, 3-(N-morpholino)propane-sulfonic acid; DMSO, dimethyl sulfoxide.
50

Potassium Channel Modulators and Skeletal Muscle
51
contractility function in humans after neuromuscular block- activation of the fast-twitching muscle KATP channels at
er-induced paralysis (Saitoh, 2005). Pinacidil is capable of 10Ϫ7 M concentration (Tricarico et al., 2003). A downturn in
reducing the severity and frequency of paralysis in patients response is observed with this compound when a certain dose
affected by hypokalemic periodic paralyses (hypoPP); how- is exceeded, suggesting that its effectiveness is reduced at
ever, the patients suffer severe hypotension, which limits its higher concentrations. Furthermore, the same compound in
use in this muscle disorder (Links et al., 1992). Cromakalim the absence of internal ATP, caused a 41% inhibition of the
is able to repolarize the insulin-depolarized muscle fibers of KATP channels at 10Ϫ4 M concentration, which seems to be
patients with hypoPP “in vitro” as well as of Kϩ-depleted mediated by interaction with the Kir6.2 subunit (Tricarico et
rats, an animal model of hypoPP, by opening the skeletal al., 2003; Rolland et al., 2006). Dual hypotheses have been
muscle KATP channels (Tricarico et al., 1998, 1999). In some proposed to explain this peculiar behavior: first, the drug
myotonic patients, it is also able to suppress “in vitro” the action is dependent on the ability to bind two distinct sites of
abnormal hyperexcitability of the fibers; however, this com- skeletal muscle KATP channel complex modulating opposite
pound is not available for clinical use (Quasthoff et al., 1990). actions; second, the drug binds to a single site whose affinity
Blockers of the skeletal muscle KATP channels may instead and/or effect are modulated by ATP or generally by tissue
be useful in those conditions associated with insulin resis- metabolism (Tricarico et al., 2003).
tance and neuromuscular symptoms (Koster et al., 2005;
To investigate on the molecular determinants responsible for
Flechtner et al., 2006). Sulfonylureas used for the treatment the opening/blocking actions of these compounds, several new
of diabetes type II also block the skeletal muscle types. Un- molecules were prepared and tested on the muscular KATP
fortunately, the currently available openers and blockers of channels. The influence of the lengthening of the alkyl chain
the KATP channels were not developed against the skeletal and of the branched alkyl chain substitutes on the activity of
muscle channel types and are therefore not indicated for the molecules against the KATP channels was therefore inves-
neuromuscular disorders (Camerino et al., 2007).
tigated by preparing the 2-n-hexyl and 2-n-butyl-1,4-benzox-
Recent data show differences in the biophysical properties, azine derivatives, and the 2-isopropyl and 2-isobutyl-1,4-benz-
subunit expression, and drug responses between muscles oxazine derivatives, respectively. Whereas the influence of the
types and phenotypes, which corroborates the idea that mo- introduction of aliphatic/aromatic rings on the activity of the
lecular composition of the skeletal muscle KATP channels is molecules was investigated by preparing the 2-cyclohexyl-
complex (Tricarico et al., 2006). In native skeletal muscle methyl, 2-phenyl, 2-benzyl and 2-phenylethyl-1,4-benzoxazine
fibers, the KATP channel is indeed an hybrid assembly of derivatives. The drug experiments were performed “in vitro” in
Kir6.2/SUR2A and Kir6.2/SUR1 subunits organized as ho- excised-patches on KATP channels, cromakalim-glibenclamide-
momeric complexes with the possible contribution of SUR2B sensitive and tolbutamide-insensitive, of native fibers of the rat,
to the functional channels. However, the cromakalim-sensi- in the presence or absence of internal ATP (10Ϫ4 M).
tive KATP channels are the main complexes found in the
different muscle types and phenotypes, thereby representing
a valuable drug target in this tissue (Tricarico et al., 2006).
We have shown previously that the 2-n-propyl-1,4-benzox-
azine derivative 1 (Fig. 1), a novel modulator of the muscular
Materials and Methods
Muscle Preparations and Single Fiber Isolation. The flexor
digitorum brevis muscles were dissected from male Wistar rats un-
KATP channels, in the presence of internal ATP leads to 54%
der urethane anesthesia (1.2 g/kg). After dissection, the animals
were rapidly killed with an overdose of urethane according to the
Guide for the Care and Use of Laboratory Animals prepared by the
U.S. National Academy of Sciences. Single muscle fibers were pre-
pared by enzymatic dissociation (Tricarico et al., 1998).
Drugs and Solutions. The normal Ringer solution contained
145 ϫ 10Ϫ3 M NaCl, 5 ϫ 10Ϫ3 M KCl, 10Ϫ3 M MgCl2, 0.5 ϫ 10Ϫ3
M
CaCl2, 5 ϫ 10Ϫ3 M glucose, and 10Ϫ2 M MOPS, pH 7.2. The patch
pipette solution contained 150 ϫ 10Ϫ3 M KCl, 2 ϫ 10Ϫ3 M CaCl2, and
10Ϫ2 M MOPS, pH 7.2. The bath solution contained 150 ϫ 10Ϫ3
M
KCl, 5 ϫ 10Ϫ3 M EGTA, and 10Ϫ2 M MOPS, pH 7.2. Stock solution
of ATPK2 (5 ϫ 10Ϫ3 M) was prepared by dissolving the chemical in
the bath solution.
Stock solutions of the 2H-1,4-benzoxazine derivatives (30 ϫ 10Ϫ3
M), cromakalim, tolbutamide and glibenclamide (20 ϫ 10Ϫ3 M)
(SIGMA, Mi) were prepared by dissolving the compounds in dimethyl
sulfoxide (DMSO). Microliter amounts of the stock solutions were
then added to the bath solution as needed to obtain concentrations of
2H-1,4-benzoxazine derivatives ranging between 10Ϫ12 and 10Ϫ4
M
(DMSO 0.33%). Because of the low solubility of these compounds in
the aqueous solvent, the drugs were tested at concentrations Ͻ10Ϫ4
M. DMSO applied at 0.33% concentration to the excised patches in
the presence of ATP (10Ϫ4 M) did not increase the KATP channel
activity (solvent control). DMSO did not affect KATP current even in
the absence of internal nucleotide. The drugs were tested in the
presence or in the absence of internal ATP (10Ϫ4 M) with no added
Mg2ϩ ions to reduce possible ATPase activity in the patches (Russ et
al., 2003; Tricarico et al., 2003).
Fig. 1. Molecular structures of 2H-1,4-benzoxazine derivatives. R indi-
cates the substitutes at position 2 of the 2H-1,4-benzoxazine nucleus.

52
Tricarico et al.
Synthesis of the New 2H-1,4-Benzoxazine Derivatives. The washout after the drug solution applications were excluded from the
key intermediate in the synthetic pathway of all benzoxazine deriv- analysis. Patches containing channels unresponsive to cromakalim
atives 2–9 (Fig. 1) was the appropriate 2-substituted-6-chloro-2H- but inhibited by glibenclamide and tolbutamide, possibly composed
1,4-benzoxazine-3-one, which was condensed with 3-aminopyridine by SUR1 subunit, as well as channels unresponsive to any of these
either in refluxing dry toluene in the presence of TiCl4 and anisole as drugs, were excluded from the mean.
previously reported for derivative 1 (compounds 3 and 9) (Tricarico et
Electronic Quantum Chemical Calculations and Conforma-
al., 2003), or in dry acetonitrile in the presence of triethyl amine and tional Analysis. The 2H-1,4-benzoxazine derivatives were first con-
structed by fragments, and the molecular geometry was optimized to
DFT B3LYP/6–31G* level theory; we got the ATP and ADP opti-
mized starting geometry by the Spartan “06 internal database. The
optimized structures were submitted for a systematic Merck Molec-
ular Force Field conformational analysis as described previously
(Tricarico et al., 2004). We searched the most populated low-energy
conformer families within the 3.0 kcal/mol range for each compound
and within 7 kcal/mol for nucleotides by a systematic conformational
analysis; from among them, some low energy conformers were se-
lected and used for the superimposition. The electrostatic potentials
of the selected conformers were also calculated. All calculations were
performed by using the SPARTAN ‘06 software package (Wavefunc-
tion Inc. Irvine, CA). Energies were corrected for aqueous solvation
by using the Cramer-Truhler SM54 solvation model. Graphical rep-
resentations and superimpositions were performed by DS Visualizer
v1.7 (Accelrys Inc., San Diego, CA) (Tricarico et al., 2004). Two
criteria were used for selection and representation of the conformers.
First, the threshold of energy levels chosen for selection was 3 kcal/
mol, which is generally considered sufficiently low to allow the in-
terconversion between conformers in physiological condition. Sec-
ond, within the low energy conformers we identified those showing a
conformation matching with that adopted by the ATP molecule into
the Kir6.2 task (Haider et al., 2007).
POCl3 (compounds 2 and 4–8). In addition, the preparation of ben-
zoxazinones followed two different synthetic pathways. The former
started from the commercially available ␣-bromo acids, which were
converted to the corresponding acyl chloride with SOCl2 and con-
densed with 2-amino-4-chloro-phenol in the presence of triethyl ben-
zyl ammonium chloride and NaHCO3 in CHCl3 to give a one-pot
cyclization reaction (benzoxazinone intermediate for compounds 2, 4,
and 9). The latter started from ␣-hydroxy ethyl esters whose conden-
sation with 2-nitro-4-chloro-phenol under Mitsunobu conditions fol-
lowed by reduction and cyclization with 6N HCl and Fe powder in
1,4-dioxane afforded the desired compounds in high yields (benzox-
azinone intermediate for compounds 3 and 5-8). All synthetic details
will be reported elsewhere. Microanalyses of all final molecules were
within Ϯ 0.4% of theoretical values. For pharmacological experi-
ments, these molecules were used as racemates and free bases.
The generic names of the 2H-1,4-benzoxazine derivatives (Fig. 1)
were: (R/S)-6-chloro-2-butyl-3- (pyridin-3-yl-amino)-2H-1,4-benzox-
azine (2), (R/S)-6-chloro-2-hexyl-3- (pyridin-3-yl-amino)-2H-1,4-benzox-
azine (3), (R/S)-6-chloro-2-isopropyl-3- (pyridin-3-yl-amino)-2H-1,4-ben-
zoxazine (4), (R/S)-6-chloro-2-isobutyl-3- (pyridin-3-yl-amino)-2H-1,4-
benzoxazine (5), (R/S)-6-chloro-2-cyclohexylmethyl-3- (pyridin-3-yl-
amino)-2H-1,4-benzoxazine (6), (R/S)-6-chloro-2-benzyl-3- (pyridin-3-yl-
amino)-2H-1,4-benzoxazine (7), (R/S)-6-chloro-2–2- (phenyl)ethyl-3-
(pyridin-3-yl-amino)-2H-1,4-benzoxazine (8), and (R/S)-6-chloro-2-phe-
nyl-3- (pyridin-3-yl-amino)-2H-1,4-benzoxazine (9).
Patch Clamp Experiments. Experiments were performed in
inside-out configurations using the standard patch-clamp technique.
Recordings of channel currents were performed during voltage steps
of 10 s, going from 0 mV of holding potential to Ϫ60 mV immediately
after excision, at 20°C, in the presence of 150 ϫ 10Ϫ3 M KCl on both
sides of the membrane in the absence (controls) or in the presence of
ATP in the bath solution. The macropatch currents with a mean
amplitude of Ϫ510.5 Ϯ 31 pA (number of macropatches ϭ 211) were
recorded at 1-kHz sampling rates (filter ϭ 0.2 kHz) using an Axo-
patch-1D amplifier equipped with a CV-4 headstage (Molecular De-
vices, Sunnyvale, CA). Pipettes having a resistance of 1 Ϯ 0.2 M⍀
(number of macropatches ϭ 211) measured in KCl on both sides of
the membrane patches were used to measure the currents sustained
by multiple KATP channels and their pharmacological properties.
The currents flowing through the macropatches excised from dif-
ferent fibers were digitally averaged and were calculated by sub-
tracting the baseline level of the currents from the open channel
level. The baseline level for the KATP current was measured in the
presence of ATP (5 ϫ 10Ϫ3 M). Macropatches containing voltage-
dependent channels or inward-rectifier Kϩ channels were excluded
from the analysis. Current amplitude was measured using the
Clampfit program (Molecular Devices). No correction for liquid junc-
Statistics. The concentration – response relationships of the KATP
currents constructed in the presence of internal ATP fits the product
of two equations describing the interaction of a ligand with two sites
mediating opposite effects, the stimulatory effect or the inhibitory
effect (Rovati and Nicosia, 1994; Tricarico et al., 2003). However, the
concentration-response relationships of the KATP currents versus
drug concentrations constructed in the absence of ATP are well fitted
by one inhibitory term.
The stimulatory component can be described by the term:
͑IdrugϩATP Ϫ 1͒ ϫ 100 ϭ Amax/͑1 ϩ ͑DE50/[Drug])n͒
whereas the inhibitory component can be described by the term:
͑Idrug Ϫ 1͒ ϫ 100 ϭ Imax/͑1 ϩ ͓͑Drug]/IC50)n͒
(1)
(2)
For eq. 1, IdrugϩATP is the KATP current measured in the presence of
the molecules under study, in the presence of internal ATP (10Ϫ4 M)
and normalized to that in the absence of drugs, Amax is the percent-
age maximal activation of the KATP currents produced by the mole-
cules under study, DE50 is the concentration of the drug needed to
enhance the current by 50%, [Drug] is the concentration of the drug
tested, and n is the slope factor of the concentration-response rela-
tionships. For eq. 2, Idrug is the KATP current measured in the
tion potential was made, estimated to be Ͻ1.9 mV in our experimen- presence of the molecules under study, in the absence of ATP (con-
tal conditions.
trols) and normalized to that in the absence of drugs; Imax is the
Concentration-response relationships were constructed as de- percentage maximal inhibition of the KATP currents produced by the
scribed previously (Tricarico et al., 2006). No more than two different molecules under study; IC50 is the concentration of the drug needed
concentrations of the drugs on the same excised macropatch were to reduce the current by 50%; and n is the slope factor of the curves
applied. Washout periods followed the first and second applications calculated in the absence of ATP. The algorithms of the fitting
of the drug solutions. A solution enriched with the Kir6.2/SUR2A-2B procedures used are based on a Marquardt least-squares fitting
agonist cromakalim (100 nM) followed the second washout period. routine. Data analysis and plot were performed by using SigmaPlot
The cromakalim-sensitive channels were then exposed to solutions software (Systat Software, Inc., San Jose, CA). The data are ex-
enriched with the Kir6.2/SUR1 selective blocker tolbutamide (1.5 pressed as mean Ϯ S.E. unless otherwise specified. The unpaired t
mM) and/or to glibenclamide (100 nM), an nonselective channel test was used to compare the best fit values pooled from the concen-
blocker. Drug solutions were applied to the patches by using the tration-response relationships analysis. The validity of the test is
ValveLink8 fast perfusion system (AutoMate Scientific, Berkeley, based on the assumption that the best fit values follows the Gaussian
CA). Patches showing rundown or that did not fully recover during distribution and that the sample sizes (number of data points) used

Potassium Channel Modulators and Skeletal Muscle
53
to calculate the degree of freedom are similar between groups. The previously observed with other 2-linear alkyl chain structur-
data are significantly different for p Ͻ 0.05 or less.
ally related analogs, significant inhibitory responses have
been observed also with the 2-n-butyl-1,4-benzoxazine and
2-n-hexyl-1,4-benzoxazine derivatives 2 and 3 at concentra-
tions Ͼ10Ϫ8 M (Fig. 2A) (Table 1) (Tricarico et al., 2003).
In the absence of internal ATP, the application of increas-
ing concentrations (10Ϫ9 M–10Ϫ4 M) of compounds 2 and 3 to
the excised patches significantly reduced dose dependently
the KATP currents (Fig. 2B). The calculation of the parame-
ters of the eq. 2 by fitting routine showed that the rank order
of efficacy and potency of the blockers expressed as Imax and
IC50 was 2– n-hexyl Ͼ2-n-butyl, whereas no differences 2were
obse2rved in the relative slopes of the concentration-response
curves (Tab.2). The 2-linear alkyl chain analogs were also
significantly more effective and potent as blockers than the
2-branched alkyl chain, 2-cyclohexylmethyl, 2-benzyl, and
2-phenylethyl analogs (Table 2).
Effects of 2-Branched Alkyl Chain Substitutes on the
Opening/Blocking Actions of 2H-1,4-Benzoxazine Deriv-
atives on the KATP Channels. In the presence of internal
ATP (10Ϫ4 M), the exposure of the macropatches to solutions of
the 2-isopropyl-1,4-benzoxazine 4 and of the 2-isobutyl-1,4-ben-
zoxazine derivative 5, in the range of concentrations from 10Ϫ9
M to 10Ϫ6 M, dose dependently enhanced the inward currents
in respect with the current levels recorded in the presence of
Results
Effects of the Lengthening of the 2-Alkyl Chain Sub-
stitutes on the Opening/Blocking Actions of 2H-1,4-
Benzoxazine Derivatives on the KATP Channels. The
effects of increasing concentrations of the 2-n-butyl-1,4-ben-
zoxazine and of the 2-n-hexyl-1,4-benzoxazine derivatives 2
and 3 on muscle KATP currents of excised macropatches re-
corded at Ϫ60 mV (Vm) were investigated. In the presence of
internal ATP (10Ϫ4 M), the exposure of the macropatches to
solutions of 2 and 3, in the range of concentrations from
10Ϫ11 M to 10Ϫ8 M, dose dependently enhanced the inward
currents with respect to the current levels recorded in the
presence of ATP alone (Fig. 2A). The 2-n-hexyl-1,4-benzox-
azine derivative 3 was more effective in activating the KATP
channels than its analog 2, producing a significant enhance-
ment of the KATP current as determined by t test. The calcu-
lation of the parameters of the eq. 1 by fitting routine showed
that the rank order of efficacy of the compounds as openers
expressed as Amax was 2-n-hexyl Ͼ2-n-butyl, whereas the
1
DE50 and slope values were not statistically different
(Tab.11). The 2-n-hexyl analog was significantly more effective
and potent than the 2-branched alkyl chain and 2-cyclic
aromatic analogs in activating the KATP channels (Tab. 1). As ATP alone (Fig. 3A). The 2-isopropyl-1,4-benzoxazine derivative
Fig. 2. Effects of the lengthening of the 2-alkyl chain substitutes on the opening/blocking actions of 2H-1,4-benzoxazine derivatives on the KATP
channels. The effects of the 2-n-hexyl and of the 2-n-butyl-1,4-benzoxazine derivatives were tested on KATP currents recorded in the excised
macropatches during voltage step going from 0 mV of holding potential to Ϫ60 mV (Vm), in the presence of KCl 150 mM on both sides of the membrane,
at 20°C. C and O in the traces indicated closed and open channel levels, respectively. The drug solutions were applied on the internal side of the patches
in the presence or absence of ATP. No more than two drug concentrations were applied on the same patches. Sample traces of KATP currents recorded
in control condition (Control), in the presence of internal ATP (Control ϩ ATP 10Ϫ4 M), in the presence of different concentrations of drug solutions
enriched with ATP (10Ϫ4 M) or in the absence of nucleotide. A, in the presence of internal ATP both compounds, at 10Ϫ9 M concentration, enhanced
the KATP current in respect with the current levels recorded in the presence of ATP alone. At 10Ϫ6 M concentration an inhibitory response was observed
with both compounds. Concentration-response data were fitted by the sum of a stimulatory and an inhibitory sites function (continuous lines). B, in
the absence of ATP, both compounds reduced the KATP current in respect with the current levels recorded in control condition. Concentration-response
relationships analysis showed that the two compounds were effective in inhibiting the KATP channels. Concentration-response data of both compounds
were fitted by a single inhibitory site function. Each experimental point represents the mean Ϯ S.E. of the percentage activation or inhibition of the
KATP currents versus the drug concentrations of a minimum of three and a maximum of six macropatches.

54
Tricarico et al.
4 was significantly more effective in activating the KATP chan- fects on the KATP current (Fig. 3B). Concentration-response
nels than its analog 5. The calculation of the parameters of the relationship experiments showed that the 2-isopropyl-1,4-
eq. 1 by fitting routine showed that the rank order of efficacy of benzoxazine derivative 4 was indeed capable to significantly
the compounds as openers expressed as Amax was 2-isopropyl enhance the KATP current even in the absence of internal
1
Ͼ2-isobutyl, whereas no differences were observed in DE50 and nucleotide, whereas the 2-isobutyl analog 5 in the same
1
slope of the concentration-response curves for these compounds range of concentrations did not affect the channel current
(Table 1). No inhibitory responses have been observed with (Fig. 3B; Tab. 2).
these compounds (Fig. 3A).
Effects of the Introduction of the Aliphatic or Aro-
In the absence of internal ATP, the application of increas- matic Rings at Position 2 of the 1,4-Benzoxazine Nu-
ing concentrations (10Ϫ12 M–5 ϫ 10Ϫ4 M) of the 2-branched cleus on the Opening/Blocking Actions of the 2H-1,4-
alkyl chain-1,4-benzoxazine derivatives showed different ef- Benzoxazine Derivatives on the KATP Channels. In the
TABLE 1
Fitting parameters of the concentration–response curves of 2H-1,4-benzoxazine derivatives versus the skeletal muscle KATP currents in the
presence of ATP
The parameters reported in the table were calculated by using the fitting routine based on the sum of the terms 1 and 2 as described under Materials and Methods.
Compounds are the 2H-1,4-benzoxazine derivatives with 2–n-hexyl, 2-n-butyl, 2-isopropyl, 2-isobutyl, 2-cyclohexylmethyl, 2-benzyl, 2-phenyl, or 2-phenylethyl groups at
position 2 of the benzoxazine nucleus. Amax is the maximal activation of the KATP currents produced by the molecules under study and it is calculated with respect to the
1
current levels measured in the presence of ATP (10Ϫ4M). DE501 is the concentration of the drug needed to enhance the current by 50% calculated with respect to the maximal
activation produced by the compounds in the presence of internal ATP. Imax is the maximal inhibition of the KATP currents produced by the molecules under study and it
1
is calculated with respect to the maximal current levels measured in the presence of drugs ϩ ATP. IC501 is the concentration of the drug needed to reduce the current by 50%,
calculated with respect to the maximal inhibition produced by the molecules. n is slope factor of the concentration-response relationships. In some cases, the reduced drug
responses and the low number of data points did not allow the evaluation of the plateau phase and calculation of the IC50 , DE501, and slopes of the concentration-response
1
relationships of the compounds.
Compounds
Amax
%
DE50
n
Imax
%
IC50
1
n
1
1
1
M
M
Linear
2-n-Hexyl
2-n-Butyl
Cyclic
2-Cyclohexylmethyl
2-Benzyl
2-Phenyl
2-Phenylethyl
Branched
2-Isopropyl
2-Isobutyl
ϩ105 Ϯ 2*†‡
ϩ75 Ϯ 2
1.08 Ϯ 0.99 ϫ 10Ϫ10†‡
3.24 Ϯ 0.9 ϫ 10Ϫ10
0.93 Ϯ 0.1†
0.9 Ϯ 0.2
Ϫ95 Ϯ 9†§
Ϫ97 Ϯ 6§
4.1 Ϯ 3 ϫ 10Ϫ8†§
8.0 Ϯ 1 ϫ 10Ϫ8§
Ϫ0.5 Ϯ 0.1
Ϫ0.6 Ϯ 0.1
ϩ88 Ϯ 5†‡
ϩ59 Ϯ 2
ϩ69 Ϯ 1
ϩ10 Ϯ 3
1.81 Ϯ 0.9 ϫ 10Ϫ10†‡
7.21 Ϯ 0.1 ϫ 10Ϫ10
3.92 Ϯ 5 ϫ 10Ϫ10
0.99 Ϯ 0.1‡
0.95 Ϯ 0.1
0.96 Ϯ 0.1
Ϫ30 Ϯ 2†‡
Ϫ26 Ϯ 5
Ϫ24 Ϯ 3
ϩ76 Ϯ 0.55¶
ϩ58 Ϯ 0.5
1.9 Ϯ 0.9 ϫ 10Ϫ9
3.8 Ϯ 0.7 ϫ 10Ϫ9
0.71 Ϯ 0.1
0.72 Ϯ 0.3
Ϫ21 Ϯ 5
Ϫ19 Ϯ 6
Symbols indicates data significantly different for P Ͻ 0.05 as determined by unpaired t test:
* Significantly different from 2-n-butyl analog data.
† Significantly different from 2-branched analog data.
‡ Significantly different from 2-cyclic aromatic analogs data.
§ Significantly different from 2-cyclic analog data.
¶ Significantly different from 2-isobutyl analog data.
TABLE 2
Fitting parameters of the concentration–response curves of 2H-1,4-benzoxazine derivatives versus the skeletal muscle KATP currents in the
absence of ATP
The parameters reported in the table were calculated by using the fitting routine based on the equation 2 as described under Materials and Methods. Compounds are the
2H-1,4-benzoxazine derivatives with 2–n-hexyl, 2-n-butyl, 2-isopropyl, 2-isobutyl, 2- cyclohexylmethyl, 2-benzyl, 2-phenyl, or 2-phenylethyl groups at position 2 of the
benzoxazine nucleus. Imax is the maximal inhibition of the KATP currents produced by the molecules under study and it is calculated with respect to the maximal current
levels measured in the pr2esence of drugs. IC50 is the concentration of the drug needed to reduce the current by 50%, calculated with respect to the maximal inhibition
2
produced by the molecules; n is slope factor of the concentration-response relationships. Amax is the maximal activation of the KATP currents produced by the molecules under
2
study. DE502 is the concentration of the drug needed to enhance the current by 50% calculated with respect to the maximal activation produced by the compounds. In some
cases, the reduced drug responses and the low number of data points did not allow the evaluation of the plateau phase and calculation of the IC50 and slopes of the
1
concentration-response relationships of the compounds.
Compounds
Imax
Amax
IC50
DE50
2
n
2
2
2
%
M
Linear
2-n-Hexyl*
2-n-Butyl
Cyclic
2-Cyclohexylmethyl
2-Benzyl
2-Phenyl
2-Phenylethyl
Branched
2-Isopropyl
2-Isobutyl
†‡
Ϫ85 Ϯ 8*†
Ϫ62 Ϯ 6*†
1.0 Ϯ 0.1 ϫ 10Ϫ8
9.0 Ϯ 3 ϫ 10Ϫ8†
*
Ϫ0.49 Ϯ 0.02*
Ϫ0.32 Ϯ 0.03
Ϫ35 Ϯ 2
Ϫ5 Ϯ 0.1
Ϫ98 Ϯ 5§
Ϫ15 Ϯ 3
2.5 Ϯ 2 ϫ 10Ϫ11¶
Ϫ0.18 Ϯ 0.09
0.71 Ϯ 0.1
ϩ52 Ϯ 5
6 Ϯ 1 ϫ 10Ϫ11
0
Symbols indicate data significantly different for P Ͻ 0.05 as determined by unpaired T test:
* Significantly different from 2-n-butyl data.
† Significantly different from 2-cyclohexylmethyl, 2-benzyl, and 2-phenylethyl analogs data.
‡ Significantly different from 2-cyclic aromatic analogs data.
§ significantly different from 2-cyclic analog data.
¶ Significantly different from 2-linear alkyl chain analog.

Potassium Channel Modulators and Skeletal Muscle
55
presence of internal ATP 10Ϫ4 M, the exposure of the mac- Slight inhibitory responses have been observed with these
compounds at concentrations Ͼ10Ϫ7 M (Fig. 4A; Table 1).
ropatches to drug 6-9 solutions, in the range of concentra-
tions from 10Ϫ11 M to 10Ϫ7 M, enhanced dose dependently
In the absence of internal ATP, in the range of concentra-
tion tested (10Ϫ12–10Ϫ4 M), the 2-phenyl-1,4-benzoxazine de-
the inward currents with respect to the current levels re-
rivative 9 was significantly more potent and effective as an
KATP channel blocker than its analogs that did not produce
more than 35% inhibition of the channel currents (Fig. 4B;
Table 2). This compound was also more potent and effec-
tive than the 2-linear and 2-branched alkyl chain analogs
(Table 2).
corded in the presence of ATP alone, however, showing dif-
ferent efficacy (Fig. 4A). The 2-cyclohexylmethyl-1,4-benzox-
azine derivative 6 was significantly more effective and potent
than its structurally related analogs in enhancing the KATP
current, whereas the 2-phenylethyl-1,4-benzoxazine deriva-
tive 8 was the least effective compound (Fig. 4A). The calcu-
lation of the parameters of eq. 1 showed that the rank order
of efficacy and potency of the openers expressed as Amax and
1
Discussion
DE50 was 2-cyclohexylmethyl Ͼ 2-phenyl Ն 2-benzyl. No
1
differences were observed in the slopes of the concentration-
response curves for these compounds (Table 1). The 2-cyclo-
hexylmethyl analog was also significantly more effective and
In the present work, several new molecules belonging to
the class of 2H-1,4-benzoxazine derivatives were synthesized
and tested against the native skeletal muscle KATP channels
potent than the 2-branched alkyl chain analogs (Table 1). with the goal of investigating the molecular determinants for
Fig. 3. Effects of the 2-branched alkyl chain substitutes on the opening/blocking actions of 2H-1,4-benzoxazine derivatives on the muscle KATP
channels. The effects of the 2-isobutyl and of the 2-isopropyl 1,4-benzoxazine derivatives were tested on KATP currents recorded in the excised
macropatches during voltage step going from 0 mV of holding potential to Ϫ60 mV (Vm), in the presence of 150 mM KCl on both sides of the membrane
at 20°C. C and O in the traces indicated closed and open channel levels, respectively. The drug solutions were applied on the internal side of the patches
in the presence or absence of ATP. No more than two drug concentrations were applied on the same patches. Sample traces of KATP currents recorded
in control condition (Control), in the presence of internal ATP (Control ϩ ATP 10Ϫ4 M), in the presence of different concentrations of drug solutions
enriched with ATP (10Ϫ4 M) or in the absence of nucleotide. A, in the presence of internal ATP both compounds, at 10Ϫ8 M concentration, enhanced
the KATP currents in respect with the current levels recorded in the presence of ATP alone. Concentration-response data were fitted by a single
stimulatory site function (continuous lines). B, in the absence of ATP, both compounds failed to inhibit the KATP currents. Concentration-response
relationships analysis showed that the 2-isopropyl 1,4-benzoxazine, in the range of concentrations tested, was capable to enhance the KATP current
even in the absence of internal nucleotide, whereas the 2-isobutyl 1,4-benzoxazine derivative did not affects the KATP current. Concentration-response
data of the 2-isopropyl analog were fitted by a single stimulatory site function at concentration (continuous lines). Each experimental point represents
the mean Ϯ S.E. of the percentage activation of the KATP currents versus the drug concentrations of a minimum of three and a maximum of six
macropatches.

56
Tricarico et al.
the activating/blocking actions of these compounds. The 2-isopropyl ϭ 2-n-butyl Ն 2-phenyl Ն 2-benzyl ϭ 2-isobutyl
strategy used was to replace the H atom at position 2 of the analogs.
2H-1,4-benzoxazine nucleus with different substitutes, such
as linear and branched alkyl chains or groups containing were effective as KATP channel blockers in the nanomolar
aromatic or aliphatic rings. concentration range. The 2-phenyl 9, 2-n-hexyl 3, and 2-n-
In the absence of internal ATP, some of these compounds
We showed here that the compounds most effective at butyl 5 analogs were the most effective and potent com-
activating the KATP channels in the presence of ATP were the pounds with respect to the other structurally related analogs.
2-n-hexyl and 2-cyclohexylmethyl Ϫ1,4-benzoxazine deriva- The rank order of efficacy of the blockers expressed as Imax
tives, which produced 105% and 88% activation of the chan- was 2-phenyl Ն 2-n-hexyl Ͼ 2-n-butyl Ͼ 2-cyclohexylmethyl2,
nels in excised patches, respectively. Less effective com- whereas the 2-arylalkyl analogs 7 and 8 and 2-branched
pounds were the analog 2 with shorter linear alkyl chain, the alkyl chain analogs 4 and 5 were not effective as blockers. We
2-branched alkyl chain analogs 4 and 5, and the 2-cyclic were surprised to find that the 2-isopropyl analog 4 was
aromatic analogs 7 and 9; the 2-phenylethyl analog 8 was not capable of opening the KATP channels even in the absence of
effective. In conclusion, the rank order of efficacy expressed nucleotide.
as Amax of the openers was 2-n-hexyl Ն2-cyclohexylmethyl Ͼ
These findings indicate that, first, the lengthening of the
1
Fig. 4. Effects of the introduction of the aliphatic or aromatic rings at position 2 of the 1,4-benzoxazine nucleus on the opening/blocking actions of the
2H-1,4-benzoxazine derivatives on the muscle KATP channels. The effects of the 2-phenylethyl, 2-benzyl, 2-phenyl and 2-cyclohexylmethyl 1,4-
benzoxazine derivatives were tested on KATP currents recorded in the excised macropatches during voltage step going from 0 mV of holding potential
to Ϫ60 mV (Vm), in the presence of KCl 150 mM on both sides of the membrane at 20°C. C and O in the traces indicated closed and open channel levels,
respectively. The drug solutions were applied on the internal side of the patches in the presence or absence of ATP. No more than two drug
concentrations were applied on the same patches. Sample traces of KATP currents recorded in control condition (Control) in the presence of internal
ATP (Control ϩ ATP 10Ϫ4 M), in the presence of different concentrations of drug solutions enriched with ATP (10Ϫ4 M) or in the absence of nucleotide.
A, in the presence of internal ATP all compounds, at 10Ϫ9 M concentration, enhanced the KATP current in respect with the current levels recorded in
the presence of ATP alone however with different efficacy. The 2-ciclohexylmethyl 1,4-benzoxazine was the most effective compound in activating the
KATP channel. At concentrations Ͼ10Ϫ6 M all compounds showed slight inhibitory responses. Concentration-response data were fitted by the sum of
a single stimulatory and an inhibitory sites function (continuous lines). B, in the absence of ATP, the compounds under investigations reduced the KATP
currents in respect with the current levels recorded in control condition but with different efficacy. The 2-phenyl 1,4-benzoxazine was the most effective
compound in inhibiting the KATP currents in respect with the other analogs. Concentration-response data of the 2-cyclohexylmethyl, 2-phenyl, and
2-phenylethyl-1,4-benzoxazine derivatives were fitted by a single inhibitory site function. Each experimental point represents the mean Ϯ S.E. of the
percentage activation or inhibition of the KATP channels versus the drug concentrations of a minimum of three and a maximum of six macropatches.

Potassium Channel Modulators and Skeletal Muscle
57
linear alkyl chain at position 2 of the 1,4-benzoxazine nucleus Although the substitutes at the position 2 of the 1,4-benzox-
determines the efficacy and potency of these molecules as azine nucleus could adopt conformations similar to that of
openers and blockers. This is demonstrated by the calculated the phosphate group of nucleotides determining the actions
Amax /Imax and DE50 /IC50 ratios, which are close to the of these compounds as blockers and openers.
1
2
1
unity for both 2-n-hexyl an1d–22-n-butyl analogs 3 and 2. Sec-
The opening action of the 2H-1,4-benzoxazine derivatives
ond, the molecular switch from blocking to opening actions could be mediated by the nucleotide-binding-folds of SUR2,
for the 2H-1,4-benzoxazine derivatives can be achieved by and it may be also dependent on the binding of these com-
replacing the linear alkyl chain with 2-branched alkyl chain pounds to the TMD13–17/NBD2 regions of this subunit. This
substitutes as observed for the analogs 4 and 5. Third, the is supported by the previously reported tissue-selective open-
introduction of an aromatic cycle in place of the linear alkyl ing action of the 2-n-propyl-1,4-benzoxazine derivative,
chain at position 2 of the 1,4-benzoxazine nucleus conferred which is capable of activating the skeletal muscle KATP chan-
pronounced blocking action to the molecule in the absence of nels but in contrast fails to activate the pancreatic channel
ATP, as in the case of the analog 9. An additional factor is the type, which is composed by the SUR1 subunit (Rolland et al.,
intramolecular distance of the substitutes from the 1,4-ben- 2006). The TMD13–17/NBD2 regions indeed distinguish the
zoxazine nucleus, which is inversely related to the effective- muscle type SUR2 from the pancreatic SUR1 subunit and are
ness of the molecules as blockers/openers as observed with involved in the binding/actions of several KCO.
the 2-arylalkyl analogs 7 and 8 and with the 2-branched
alkyl chain analog 5.
The reduced efficacy of the 2H-1,4-benzoxazine derivatives
in activating the KATP channels observed in the presence of
The observed differences in the molecular requisites re- internal ATP at micromolar concentrations has been de-
sponsible for the action of these compounds as openers and scribed also for other KCO and ADP molecules that are
blockers would suggest that distinct high-affinity binding capable to activate the recombinant and native KATP chan-
sites modulate their dual actions on the KATP channels. Pos- nels at low concentrations and to inhibit it at high concen-
sible sites of interactions for these compounds can be located trations. This generates bell-shaped concentration-response
on the nucleotide binding sites on the KATP channel subunits. relationships for these drugs and ADP molecules as also
Recognition sites for ATP and ADP are located on the Kir6.2 observed in our experiments. This phenomenon has been
subunit and on the SUR subunits such as the nucleotide- associated with several mechanisms including interaction
binding -fold (NBD1 and NBD2) of the KATP channel com-
plexes (Nichols, 2006; Haider et al., 2007). This is supported
by the observed structural similarities of the 2H-1,4-benzox-
azine derivatives with the ATP and ADP molecules. Prelim-
inary conformational analysis would indeed suggest that the
planar area of the 2H-1,4-benzoxazines overlaps with that of
the adenine nucleotide tri- and diphosphates. The electronic
distribution profile of this area is also similar with that of the
adenine nucleotides being electrons reached suggesting that
these compounds share a common area of interaction with
ATP and ADP on the receptor sites. We found that the best
overlay with the ATP conformer, which interacts with the
inhibitory site on Kir6.2, is observed with the linear alkyl
chain analogs, and particularly with the 2-n-hexyl analog 3,
explaining the blocking action of these compounds in the
absence and in the presence of ATP (Fig. 5). This is corrobo-
rated by the finding that the IC50 and IC50 of the 2-n-hexyl
1
and 2-n-butyl analogs 3 and 2, respectively,2evaluated in the
Fig. 5. Conformational analysis of the 2H-1,4-benzoxazine derivatives
and comparison with nucleotide triphosphate. (A and B) Stick and balls
presence and absence of ATP, did not differ significantly representations of the lower energy conformers, from left to right, of ATP
indicating a common site of interaction possibly on Kir6.2
subunit. Furthermore, we have shown that the 2-n-propyl-
anion, 2-n-hexyl, 2-phenyl, and 2-isopropyl 1,4-benzoxazine derivatives.
Conformers representation were selected on the basis of their energy
levels not exceeding 2 to 3 kcal/mol for the 2H-1,4-benzoxazine deriva-
1,4-benzoxazine derivative, in the absence of ATP, is capable
of inhibiting the truncated Kir6.2 channel expressed in
Hek293 cell line (Rolland et al., 2006). Conformational anal-
ysis also shows that molecules with a reduced three-dimen-
sional area of superimposition with the nucleotide may loose
the capability to inhibit the Kir6.2 subunit as in the case of
the 2-isopropyl analog 4 or may show a different inhibitory
profile as in the case of the 2-phenyl analog 9, which potently
inhibit the KATP channel, but only in the presence of ATP.
Therefore, it is likely that the hydrophobic planar area of the
2H-1,4-benzoxazine derivatives may fits into the hydrophobic
pocket of the Kir6.2, which is the area in which the ATP
binds. Ligand docking investigations indeed showed that the
ATP site is located into a hydrophobic pocket at the interface
tives and 7 kcal/mol for the ATP molecule. Moreover, within the low-
energy conformers, we identified those showing a conformation matching
with that adopted by the ATP molecule into the Kir6.2 task (Haider et al.,
2007). The low calculated differences in the energy levels between con-
formers for each molecule suggest that the interconversion between con-
formers is favored in physiological condition. C, optimized molecular
superimposition of 2-n-hexyl, 2-phenyl, and 2-isopropyl 1,4-benzoxazine
derivatives with ATP. Two different orientations of the molecules on the
z-axis are represented. The planar area of 2-n-hexyl (blue), 2-phenyl
(green), and of the 2-isopropyl (red), 1,4-benzoxazine derivatives matches
with that of the adenine ring of the ATP (violet) molecule. In the case of
2-hexyl and 2-isopropyl analogs, the 3-amino-pyridine substitute over-
laps with the ribose ring of the nucleotide, however in the 2-phenyl analog
9 does not. Moreover, the 2-linear alkyl chain of the 2-n-hexyl analog 3
may occupy the same area of the phosphate group of ATP, whereas this
is not observed for the substitutes of the 2-phenyl and 2-isopropyl analogs
9 and 4. Therefore, the best molecular overlay is observed with the
2-n-hexyl analog 3 followed by the 2-isopropyl and 2-phenyl analogs 4
between the N and C domain of the Kir6.2 (Nichols, 2006). and 9.

58
Tricarico et al.
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Address correspondence to: Domenico Tricarico, Dept. of Pharmacobiology,
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