Potassium channel activators based on the benzopyran substructure: Synthesis and activity of the C-8 substituent

Article abstract of DOI:10.1016/S0968-0896(03)00058-0

The synthesis of a series of methoxy bearing 2,2-dimethyl-2H-1-benzopyrans have been achieved for testing as potassium channel activators. The synthesis involves formation of 6-cyano-8-methoxy-2,2-dimethyl-2H-1-benzopyran from vanillin, epoxidation, then ring opening of the epoxide with nitrogen nucleophiles to produce the new benzopyrans. Biological testing showed a dramatic decrease in activity thus revealing an important site of activity in this class of compounds.

Full text of DOI:10.1016/S0968-0896(03)00058-0

Bioorganic & Medicinal Chemistry 11 (2003) 1663–1668
Potassium Channel Activators Based on the Benzopyran
Substructure: Synthesis and Activity of the C-8 Substituent
Rona Thompson,a Sheila Doggrellb,* and John O. Hoberga,*
aSchool of Chemical and Physical Sciences, Victoria University of Wellington, Box 600, Wellington, New Zealand
bDepartment of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072,
Australia
Received 30 September 2002; accepted 7January 2003
Abstract—The synthesis of a series of methoxy bearing 2,2-dimethyl-2H-1-benzopyrans have been achieved for testing as potassium
channel activators. The synthesis involves formation of 6-cyano-8-methoxy-2,2-dimethyl-2H-1-benzopyran from vanillin, epoxida-
tion, then ring opening of the epoxide with nitrogen nucleophiles to produce the new benzopyrans. Biological testing showed a
dramatic decrease in activity thus revealing an important site of activity in this class of compounds.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
These modified benzopyrans are the most extensively
studied chemical structures associated with the opening of
KATP channels,10 and structure–activity studies of benzo-
pyrans have focused mainly on modifications of the 4- and
6-positions.11 Small and highly electronegative moieties at
the 6-position have shown to be most effective, while lac-
tam groups appear to provide the most effective sub-
stituents at the 4-position. The contribution of the 6-
position to receptor affinity has been postulated to
involve two possible mechanisms. One mechanism
involves a withdrawing of electrons from the benzene
moiety of the benzopyran nucleus thus increasing charge-
transfer interactions of the aromatic moiety with the
receptor, or alternatively, these substituents can contribute
by direct interaction with the receptor site.
The benzopyran ring system 1 is found in over 4000
natural and designed products, exhibiting an extensive
range of biological activities.1 It also serves as the
foundation of a range of tannins, which can be found in
teas, red wines and fruits all which are important for
their health-related effects.2 Modifications of the pyran
olefin adds a further dimension to the biological prop-
erties often not observed with the olefinic complement.
For example, 4-benzylpiperazinobenzopyran (2) has
been shown to modulate multidrug resistance activity,
while benzopyran 3 has been shown to act as a highly
selective inhibitor of phosphodiesterase IV (Fig. 1).3,4
Cromakalin5 (4) and bimakalim6 (5) are potent activa-
tors of ATP-sensitive potassium channels. Also in this
class of potassium channel activators (KCAs), is pina-
cidil (6) which, like 4 and 5, is a smooth muscle relaxant
used intermittently in the treatment of hypertension and
asthma.7 The action of KCAs has been shown to be
through interaction with the ß-subunit of KATP chan-
nels8,9 thus increasing the outward movement of potas-
sium ions through vascular smooth muscle membrane
channels. This movement hyperpolarizes the cell mem-
brane, closing voltage-dependent calcium channels, and
relaxes the smooth muscle.
Although numerous studies on singly substituted benzo-
pyrans have been reported, the synthesis and biological
effects of doubly substituted benzopyrans have been lar-
gely ignored. To our knowledge, only the disubstituted
benzopyran 7, which combines the electron-withdrawing
cyano group at the 6-position with an electron-donating
amino function at C-7, has been reported, and also
revealed to give enhanced potency.12 We have therefore
synthesized a new series of doubly substituted benzopyrans
supporting an electron-donating group at the 8-position
and evaluated the relaxant activity of these compounds on
the rat aorta and portal vein. The starting material for the
synthesis is vanillin, which also has the added benefit of
being obtained from renewable biomass.13
*Corresponding authors. Fax: +64-4-463-5237; e-mail: john.hoberg@
vuw.ac.nz
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00058-0

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R. Thompson et al. / Bioorg. Med. Chem. 11 (2003) 1663–1668
Figure 1. Benzopyrans.
Results
The biological activity of 11a–12c have been evaluated
and compared to (Æ)-cromakalim and (Æ)-pinacidil
(Fig. 2). In tests with the isolated spontaneously con-
tracting rat portal vein, cromakalim at 3 Â 10À8–3 Â
Scheme 1 outlines our synthesis starting with the con-
version of vanillin (8a) into 4-hydroxy-3-methoxy-
benzonitrile (8b) in 91% yield.14 Etherification with
3-chloro-3-methyl-1-butyne and cyclization of the crude
material by Claisen rearrangement in boiling xylenes
produces chromene 9 in 95% yield for the two steps.
Epoxidation of 9 was attempted using a variety of con-
ditions. We initially attempted to use Jacobsen’s cata-
lyst15 and NaOCl as this would lead to the optically
active epoxide, however only trace amounts of 10 were
formed. Alternatively, the use of catalytic amounts of
methyltrioxorhenium16 (MTO) and hydrogen peroxide
also failed to provide significant amounts of the epox-
ide. Epoxidation using mCPBA does produce 10, but
isolation of pure product is difficult. We therefore used
a hydrobromination–epoxidation strategy to give race-
mic epoxide 10 in 93% yield. Final transformation into
the required racemic benzopyrans was accomplished
with several strategies. Formation of 11a was accom-
plished in 67% yield by reaction of 10 with pyrrolidine
in boiling ethanol. Similarly, 11b was formed in 56%
yield, only using 2-pyridinol and toluene as the solvent.
Attempted synthesis of 12a and 12b by heating in a
variety of solvents failed to produce any of the desired
benzopyrans. We therefore heated epoxide 10 with 2-
pyrrolidinone or 2-piperidone in the presence of NaH
thus producing 12a and 12b in 40 and 49%, respectively.
Finally, 12c was obtained in 43% yield by treating 10
with 2-pyridinol followed by elimination with KHSO4
in THF.
10À6 M, pinacidil at 10À7–3 Â 10À6 M and 11a at 10À5
–
10À4 M relaxed the vein. In relaxing the rat portal vein,
11a [pIC50=4.11Æ0.11, number of determinations
(nd=6)] was about 1000 times less potent than
(Æ)-cromakalim (pIC50=6.99Æ0.07, nd=10) or (Æ)-
pinacidil (pIC50=6.73Æ0.03, nd=11). The incor-
poration of a pyrrolidinone moiety, as in cromakalim,
as compared to the pyrrolidine moiety in 11a, has been
shown to result in a 3-fold increase in potency.17 How-
ever, this alone does not explain the dramatic decrease
in activity for 11a. We therefore tested the remaining
compounds (11b, 12a, 12b, and 12c), and as can be seen
these gave comparable results or were even less active
than 11a, with 12b being the least potent.
Given this surprising result, we next tested 11a with the
isolated aorta from 18-month-old spontaneously hyper-
tensive rat (SHR). For vasodilators to be useful in the
treatment of hypertension, they must be effective in the
presence of hypertension-associated hypertrophy of blood
vessels, and the aorta of 18-month-old SHR have
hypertension-associated hypertrophy.18 This test would
give more revealing insight to the effectiveness and use-
fullness of one of our more active compounds. In tests
with the isolated aorta, 11a at 10À8–10À4 M had no effect
on the quiescent tissue (n=4), although at 3 Â 10À6
–
10À4 M it produced relaxation of the KCl-contracted

R. Thompson et al. / Bioorg. Med. Chem. 11 (2003) 1663–1668
1665
Scheme 1. Synthesis of disubstituted KCAs: (1) NH2OH HCl, AcOH; (2) ClMe2CCꢀCH, KI, K2CO3, MeCN; (3) boiling xylenes; (4) NBS, H2O; (5)
NaH; (6) 11a=pyrrolidine, EtOH; 11b=2-pyridinol, toluene; (7) 12a=2-pyrrolidinone, NaH, DMF; 12b=2-piperidone, NaH, DMF; 12c=2-pyr-
idinol, NaH, DMF then KHSO4, THF.
Figure 2. Activity tests on the spontaneous contractions of the isolated portal vein: cromakalim (filled squares), pinacidil (open triangles), 11a (filled
diamonds), 11b (star), 12a (filled triangle), 12b (filled circle) and 12c (empty diamond). Each value is the meanÆSEM of 6–11 determinations.
Activity using KCl-contracted rat aorta: 11a (open circles).

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R. Thompson et al. / Bioorg. Med. Chem. 11 (2003) 1663–1668
aorta with a pIC50=4.54Æ0.11, nd=7( Fig. 2). There-
fore, 11a remains only slightly effective in tissue from
hypertensive animals.
solids with acetone. The solvents were removed, xylenes
(30 mL) were added and the mixture refluxed overnight
(ꢁ16 h). The reaction mixture was concentrated in
vacuo and the residue purified by flash chromatography
hexanes/EtOAc (1:1) (95% yield) or by recrystallization
using hexanes/Et2O (86% yield) to give benzopyran 9:
1H NMR (300 MHz, CDCl3) d 6.97(s, 1H, H-7), 6.95 (s,
1H, H-5), 6.27(d, J=10.0 Hz, 1H, H-4), 5.70 (d,
J=10.0 Hz, 1H, H-3), 3.87(s, 3H, MeO), 1.50 (s, 6H,
gem-Me2); 13C NMR (75 MHz, CDCl3) d 148.4, 146.1,
132.0, 123.3, 122.1, 120.8, 119.3, 114.9, 103.1, 78.0, 56.3,
Conclusion
In considering these results, it appears that the place-
ment of an electron-donating group at the C-8 position
of the benzopyran can have a significant effect on the
activity of this class of substituted benzopyrans. In line
with the postulated mechanisms, charge-transfer inter-
actions of the aromatic moiety with the receptor could
be affected by the inclusion of the methoxy unit. Alter-
natively, direct interaction of the methoxy group with
the receptor could also be occurring. In view of benzo-
pyran 7 exhibiting increased activity, a molecule that
also contains an electron donating group on the aro-
matic ring, we believe that latter explanation is more
likely. Since only high concentrations of 11a relaxed
blood vessels, the mechanism underlying these relaxant
effects was not further investigated. However, these
results show that C-8 substitution represents part of the
benzopyran pharmacophore and this may enable the
activity to be further tuned. We are therefore currently
undertaking studies on an array of substitution at C-8
and these results will be reported in due course.
28.1, 28.1. IR (neat) 2977, 2225, 1477, 1148, 1088 cmÀ1
;
MS m/z calcd for C13H13NO2 (M+1) 216.10191, found
216.10160.
3,4-Epoxy-3,4-dihydro-2,2-dimethyl-6-cyano-8-methoxy-
2H-1-benzopyran 10. To 9 (420 mg, 1.92 mmol) in THF
(6 mL) and H2O (4 mL) at 0 ꢂC was added solid NBS
(480, 2.69 mmol) over 10 min. The mixture was stirred
for 5 h at room temperature in the dark and then con-
centrated in vacuo. The residual solution was extracted
twice with ether (10 mL) and dried over MgSO4.
To a solution of NaH (69.1 mg, 2.88 mmol) in DMF
(1 mL) at 0 ꢂC was added the bromohydrin in DMF
(2 mL) drop wise over 5 min, complete transfer of the
bromohydrin was ensured by washing the flask with an
additional 1 mL of DMF. The mixture was allowed to
stir for 1 h at room temperature and then poured into
ether. The ether solution was washed twice with satu-
rated NaHCO3 (10 mL) once with water (10 mL) and
dried (MgSO4). Recrystallization from toluene/hexanes
gave 10 as a creme colored solid in 93% yield. 1H NMR
(300 MHz, CDCl3) d 7.30 (d, J=1.9 Hz, 1H, H-7), 7.06
(d, J=1.9 Hz, 1H, H-5), 3.88 (d, J=4.4 Hz, 1H, H-4),
3.83 (s, 3H, MeO), 3.51 (d, J=4.4 Hz, 1H, H-3), 1.65 (s,
3H, gem-Me), 1.30 (s, 3H, gem-Me); 13C NMR
(75 MHz, CDCl3) d 149.3, 146.0, 126.1, 121.4, 118.7,
115.6, 103.6, 74.8, 62.3, 56.2, 49.7, 25.4, 22.7. IR (neat)
Experimental
General
1H and 13C NMR spectra were recorded on a Varian
spectrometer at 300 and 75 MHz with chemical shifts
reported relative to CDCl3 (7.23 and 77.0, respectively).
IR spectra were measured on a FT-IR spectrometer.
Elemental analyses were obtained from Huffman
Laboratories, Inc., Golden, CO and HR-MS was
obtained using a Mariner TOF spectrometer. All sol-
vents were distilled from appropriate drying agents
prior to use. Standard syringe techniques were
employed for handling air-sensitive reagents and all
reactions were carried out under argon.
2982, 2223, 1587, 1498, 1367, 1293, 1132 cmÀ1
.
trans-3,4-Dihydro-6-cyano-3-hydroxy-8-methoxy-4-pyr-
rolidine-2,2-dimethyl-2H-1-benzopyran 11a. To epoxide
10 (242 mg, 1.046 mmol) in EtOH (3 mL) was added
pyrrolidine (0.184 mL, 2.20 mmol) and the mixture was
brought to a boil. After refluxing for 24 h, the solution
was cooled, concentrated and the residue purified by
flash chromatography with hexanes/ethyl acetate (1:1)
4-Hydroxy-3-methoxybenzonitrile 8. A mixture of vanil-
lin 8a (3.04 g, 20.0 mmol) and NH2OH HCl (2.09 g,
30.0 mmol) in acetic acid (16 mL) was refluxed for 1 h.
The solution was cooled, poured into Et2O (50 mL) and
washed once with H2O (25 mL) and twice with 5%
NaOH (25 mL). The combined aqueous layers were
extracted once with Et2O (25 mL) and the ether layers
dried over MgSO4. Recrystallization from toluene gave
2.70 g of 8b as a white solid (91% yield).
1
to give 210 mg (67%) of 11a as a white solid. H NMR
(300 MHz, CDCl3) d 7.22 (s, 1H, H-7), 6.95 (s, 1H, H-
5), 3.97(d, J=10.0 Hz, 1H, H-4), 3.86 (s, 3H, MeO),
3.60 (d, J=10.0 Hz, 1H, H-3), 3.10 (brs, 1H, OH), 3.01
(m, 2H, NCH2), 2.89 (m, 2H, NCH2), 1.89 (m, 4H,
NCH2CH2), 1.58 (s, 3H, gem-Me), 1.26 (s, 3H, gem-
Me); 13C NMR (75 MHz, CDCl3) d 149.2, 147.5, 124.7,
123.6, 119.5, 112.7, 102.4, 79.7, 70.5, 58.2, 56.1, 48.5,
26.8, 24.6, 18.6. IR (neat) 3479, 2972, 2225, 1477, 1142,
1094 cmÀ1; elemental analysis calcd for C17H22N2O3
(302.37): C 67.53, H 7.33; found: C 67.70, H 7.68.
6-Cyano-8-methoxy-2,2-dimethyl-2H-1-benzopyran 9.
To a 100 mL flask equipped with reflux condenser and
side-arm was added 4-hydroxy-3-methoxybenzonitrile
8b (2.60 g, 17.4 mmol), K2CO3 (2.89 g, 20.9 mmol) and
KI (4.63 g, 27.9 mmol). The flask was purged with argon
then MeCN (40 mL) and 3-chloro-3-methyl-1-butyne
(5.37g, 5.88 mL, 52.4 mmol) were added. The mixture
was refluxed for 16 h, cooled and filtered, washing the
trans-3,4-Dihydro-6-cyano-4-(1,2-dihydro-2-oxo-1-pyri-
dyl)-3-hydroxy-8-methoxy-2,2-dimethyl-2H-1-benzopyran
11b. To epoxide 10 (218 mg, 0.931 mmol) in toluene

R. Thompson et al. / Bioorg. Med. Chem. 11 (2003) 1663–1668
1667
(5 mL) was added 2-pyridinol (133 mg, 1.40 mmol) and
K2CO3 (515 mg, 3.72 mmol) and the mixture was
brought to a boil. After refluxing for 3 h, the solution
was cooled and filtered with the aid of EtOAc. Recrys-
tallization (two crops) from EtOAc gave 210 mg (68%)
6-Cyano-8-methoxy-4-(1,2-dihydro-2-oxo-1-pyridyl)-2,2-
dimethyl-2H-1-benzopyran 12c. To NaH (29 mg,
1.20 mmol, washed with hexanes and dried), was added
DMF (1.0 mL). A mixture of 2-pyridinol (85.3 mg,
0.896 mmol) and epoxide 10 (140 mg, 0.598 mmol) in
DMF (2 mL) was canulated into the NaH mixture at
room temperature, washing the flask with additional
DMF (1 mL). The mixture was stirred 0.5 h at room
temperature then heated to 80 ꢂC for 4 h. The mixture
was cooled and poured into H2O (5 mL), then extracted
three times with Et2O (20 mL) and dried (MgSO4). To
the white solid was added THF (3 mL) and KHSO4
(150 mg) and the mixture heated at reflux for 12 h. The
solution was poured into EtOAc (20 mL), washed twice
with H2O (5 mL) and dried (MgSO4). Recrystallization
with EtOAc gave 80 mg (43%) of 12c as a white solid.:
1H NMR (300 MHz, CDCl3) d 7.46 (t, J=9.0 Hz, 1H,
H-16), 7.14 (d, J=6.8 Hz, 1H, H-18), 7.04 (s, 1H, H-7),
6.66 (d, J=8.0 Hz, 1H, H-17), 6.65 (s, 1H, H-5), 6.27 (t,
J=6.8 Hz, 1H, H-15), 5.81 (s, 1H, H-3), 3.90 (s, 3H,
MeO), 1.61 (s, 3H, gem-Me), 1.58 (s, 3H, gem-Me); 13C
NMR (75 MHz, CDCl3) d 161.6, 148.8, 146.3, 140.6,
137.5, 133.6, 129.4, 121.5, 119.5, 119.2, 118.7, 115.6,
106.5, 103.4, 78.6, 56.3, 27.8, 27.7. IR (neat) 2985, 2230,
1671, 1588, 1470, 1380, 1277, 1148 cmÀ1. MS m/e calcd
for C18H17N2O3 (M+1): 309.12337, found 309.12427.
1
of 11b as a white solid. H NMR (300 MHz, CDCl3) d
7.39 (t, J=8.4 Hz, 1H, H-16), 6.90 (s, 1H, H-7), 6.84 (d,
J=6.9 Hz, 1H, H-18), 6.70 (s, 1H, H-5), 6.66 (d,
J=9.3 Hz, 1H, H-15), 6.32 (d, J=9.8 Hz, 1H, H-4), 6.24
(t, J=6.9 Hz, 1H, H-17), 4.18 (brs, 1H, OH), 3.88 (s,
3H, MeO), 3.85 (d, J=10.3 Hz, 1H, H-3), 1.60 (s, 3H,
gem-Me), 1.36 (s, 3H, gem-Me); 13C NMR (75 MHz,
CDCl3) d 165.2, 149.9, 147.7, 140.1, 133.9, 124.4, 121.4,
120.3, 118.6, 113.6, 108.2, 104.3, 81.6, 75.5, 56.3, 55.3,
26.2, 18.2. IR (neat) 3328, 2224, 1661, 1580, 1488,
1096 cmÀ1; MS m/z calcd for C18H17N2O4 (M-1):
325.11938, found 325.11900.
6-Cyano-8-methoxy-4-(pyrrolidin-2-one)-2,2-dimethyl-
2H-1-benzopyran 12a. To NaH (60 mg, 2.50 mmol,
washed with hexanes and dried), was added DMF
(1.0 mL).
A mixture of 2-pyrrolidinone (173 mg,
2.03 mmol) and epoxide 10 (238 mg, 1.02 mmol) in
DMF (2 mL) was canulated into the NaH mixture at
room temperature, washing the flask with additional
DMF (1 mL). The mixture was stirred 0.5 h at room
temperature then heated to 75 ꢂC for 4 h. The mixture
was cooled and poured into H2O (5 mL), then extracted
three times with Et2O (20 mL) and dried (MgSO4).
Flash chromatography with EtOAc/hexanes (3:1) gave
121 mg (40%) of 12a as a white solid. 1H NMR
(300 MHz, CDCl3) d 6.98 (s, 1H, H-7), 6.87 (s, 1H, H-
5), 5.65 (s, 1H, H-3), 3.82 (s, 3H, MeO), 3.56 (t,
J=7.0 Hz, 2H, H-17), 2.52 (t, J=8.0 Hz, 2H, H-15),
2.17(p, 2H, H-16), 1.49 (s, 6H, gem-Me2); 13C NMR
(75 MHz, CDCl3) d=174.9, 148.9, 146.8, 130.0, 127.8,
119.8, 119.6, 119.0, 115.2, 103.1, 78.4, 56.3, 49.8, 31.0,
27.7 (2C), 18.7. IR (neat) 2970, 2226, 1698, 1478,
1380 cmÀ1. MS m/z calcd for C17H19N2O3 (M+1):
299.13902, found 299.14022.
Biological methods: relaxant responses in rat portal vein
and aorta
A comparison of the effects of 11a, (Æ)-cromakalim
and (Æ)-pinacidil on the spontaneous contractile activ-
ity of the portal vein 20-week-old Wistar rats was made.
The effects of 11a on the quiescent and KCl-contracted
aorta of 18-month-old Spontaneously Hypertensive rats
(SHRs) were determined. 11a at 5 Â 10À2 M was dis-
solved in absolute ethanol, (Æ)-cromakalim and
(Æ)-pinacidil at 10À2 M were dissolved in 70% ethanol.
Dilutions were made in distilled water. Parallel experi-
ments showed that the ethanol vehicle has no effect
alone on the portal veins or aortae.
6-Cyano-8-methoxy-4-(piperidin-2-one)-2,2-dimethyl-2H-
1-benzopyran 12b. To NaH (24 mg, 1.00 mmol, washed
with hexanes and dried), was added DMF (1.0 mL). A
mixture of 2-piperidone (68 mg, 0.683 mmol) and epox-
ide 10 (80 mg, 0.342 mmol) in DMF (2 mL) was canu-
lated into the NaH mixture at room temperature,
washing the flask with additional DMF (1 mL). The
mixture was stirred 0.5 h at room temperature then
heated to 85 ꢂC for 5 h. The mixture was cooled and
poured into H2O (5 mL), then extracted three times with
Et2O (20 mL) and dried (MgSO4). Recrystallization
with Et2O gave 52 mg (49%) of 12b as a white solid. 1H
NMR (300 MHz, CDCl3) d 6.99 (d, J=1.7Hz, 1H, H-
7), 6.81 (d, J=1.7Hz, 1H, H-5), 5.64 (s, 1H, H-3), 3.84
(s, 3H, MeO), 3.41 (m, 2H, H-18), 2.52 (m, 2H, H-15),
1.92 (m, 4H, H-16, H-17), 1.54 (s, 3H, gem-Me), 1.51 (s,
3H, gem-Me); 13C NMR (75 MHz, CDCl3) d 169.9,
148.9, 147.0, 134.6, 128.4, 119.7, 119.3, 119.2, 115.3,
103.3, 78.6, 56.4, 50.7, 32.4, 28.2, 27.7, 23.2, 21.3. IR
(neat) 2952, 2224, 1651, 1466, 1376, 1285 cmÀ1. MS m/z
calcd for C18H21N2O3 (M+1): 313.15467, found
313.15568.
Rats were stunned and exsanguinated. The portal vein
or aorta was removed and placed in Krebs solution
saturated with 5% carbon dioxide in oxygen. All
experiments were performed in the presence of a mod-
ified Krebs solution (composition in mM: NaCl, 116;
KCl, 5.4: CaCl2, 2.5; MgCl2, 1.2; NaH2PO4, 1.2;
NaHCO3, 22.0; d-glucose, 11.2) that was being bubbled
with 5% CO2 in O2 at 37 ꢂC. An unstretched 10–12 mm
length of portal vein was mounted longitudinally in a
5 mL organ bath under 10 mN tension. Each endothe-
lium-intact thoracic aorta of about 3 mm in length was
suspended in an organ bath under 15 mN tension. Con-
tractile responses were measured isometrically with
force displacement transducers (Grass model FTO3.C)
and displayed on a polygraph (Grass model 79B). Por-
tal veins and aortae were equilibrated for 60 min during
which 250 mL Krebs superfused the tissues. Superfusion
of the portal veins was stopped, and the contractions
were allowed to stabilize over 20–30 min. Cumulative
challenges to 11a, (Æ)-cromakalim and (Æ)-pinacidil on
6 min cycle, or until a maximum response was obtained,

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R. Thompson et al. / Bioorg. Med. Chem. 11 (2003) 1663–1668
were determined. Superfusion of aortic rings was stop-
ped, and some rings remained quiescent whereas others
were contracted by the addition of 15 mM KCl. When a
plateau response to KCl had been reached, quiescent
and contracted aortae were challenged with 11a on
6 min cycle, or until a maximum response was obtained.
Picard, S.; Dawodu, A. A.; Criniti, A.; Ruvolo, G.; Campa,
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Allergy 1997, 27, 705. (c) Arch, J. R. S.; Bowring, N. E.;
Buckle, D. R. Pulm. Pharmacol. 1994, 7, 121. (d) Jan, L. Y.;
Jan, Y. N. Nature 1990, 345, 672. (e) Hoshi, T.; Zagotta, W. N.
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8. Noma, A. Nature 1983, 305, 147.
9. Ammala, C.; Moorhouse, A.; Ashcroft, F. M. J. Physiol.
1996, 494, 709.
The amplitudes of the final three contractions of the
portal veins before the addition of each concentration of
11a, (Æ)-cromakalim and (Æ)-pinacidil were measured
and averaged. The contraction amplitude induced by
KCl on the aorta prior to the addition of 11a was mea-
sured. Responses were calculated as% attenuation of
the spontaneous contractile activity of the portal vein or
the KCl contraction of the aorta. pIC50 values (the
negative logarithm of the molar concentration that
inhibits the contraction by 50%) were calculated for
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