Synthesis, molecular modeling, and K+ channel-blocking activity of dequalinium analogues having semirigid linkers

Article abstract of DOI:10.1021/jm950884a

Dequalinium [1,1'-(decane-l,10-diyl)bis(2-methyl-4-aminoquinolinium)] is an effective blocker of the small conductance Ca²⁺activated K⁺ channel. It has been shown that the number of methylene groups in the alkyl chain linking the two quinolinium rings of this type of molecule is not critical for activity. To further investigate the role of the linker, analogues of dequalinium have been synthesized, in which the alkyl chain has been replaced by CH₂XCH₂ where X is a rigid or semirigid group containing aromatic rings. The compounds have been tested for blockade of the slow after- hyperpolarization on rat sympathetic neurons. The most potent compounds have X = phenanthryl, fluorenyl, cis-stilbene, and C₆H₄(CH₂)(n)C₆H₄ where n = 0-4. The conformational preferences of the compounds were investigated using the XED/COSMIC molecular modeling system. Although there is some dependence of the potency of the analogue on the conformational properties of the linker (X), overall, X groups having substantial structural differences are tolerated. It seems that X provides a support for the two quinolinium groups and does not interact with the channel directly. The intramolecular separation between the quinolinium rings, which is provided by rigid groups X, is not critical for activity; this may be attributed to the residual conformational mobility of the heterocycles and to the extensive delocalization of the positive charge. These two factors may permit favorable contacts between the quinolinium groups and the channel over a range of intramolecular separations.

Full text of DOI:10.1021/jm950884a

J . Med. Chem. 1996, 39, 4247-4254
4247
Syn th esis, Molecu la r Mod elin g, a n d K+ Ch a n n el-Block in g Activity of
Dequ a lin iu m An a logu es Ha vin g Sem ir igid Lin k er s
J oaquin Campos Rosa,†,§ Dimitrios Galanakis,†,∇ C. Robin Ganellin,*,† and Philip M. Dunn‡
Departments of Chemistry and Pharmacology, University College London, Gower Street, London WC1E 6BT, U.K.
Received December 5, 1995X
Dequalinium [1,1′-(decane-1,10-diyl)bis(2-methyl-4-aminoquinolinium)] is an effective blocker
of the small conductance Ca2+-activated K+ channel. It has been shown that the number of
methylene groups in the alkyl chain linking the two quinolinium rings of this type of molecule
is not critical for activity. To further investigate the role of the linker, analogues of dequalinium
have been synthesized, in which the alkyl chain has been replaced by CH2XCH2 where X is a
rigid or semirigid group containing aromatic rings. The compounds have been tested for
blockade of the slow after-hyperpolarization on rat sympathetic neurons. The most potent
compounds have X ) phenanthryl, fluorenyl, cis-stilbene, and C6H4(CH2)nC6H4, where n ) 0-4.
The conformational preferences of the compounds were investigated using the XED/COSMIC
molecular modeling system. Although there is some dependence of the potency of the analogue
on the conformational properties of the linker (X), overall, X groups having substantial structural
differences are tolerated. It seems that X provides a support for the two quinolinium groups
and does not interact with the channel directly. The intramolecular separation between the
quinolinium rings, which is provided by rigid groups X, is not critical for activity; this may be
attributed to the residual conformational mobility of the heterocycles and to the extensive
delocalization of the positive charge. These two factors may permit favorable contacts between
the quinolinium groups and the channel over a range of intramolecular separations.
In tr od u ction
The most potent SKCa channel blockers known are the
natural peptidic toxins apamin,17-19 leiurotoxin I,20 and
PO5.21 Due to the limited availability of the three
peptides, research has been initiated toward the dis-
Compounds that modulate K+ channels have at-
tracted considerable attention in recent years.1,2 K+
channels form the most diverse family of ion channels,3
and potent and selective modulators are available only
for a few subtypes. The availability of modulators for
the less well-studied subtypes will enable the better
pharmacological and physiological characterization of
these K+ channels and may lead to novel therapeutic
approaches for disease states.1,2 Small conductance
Ca2+-activated K+ (SKCa) channels are activated by an
increase in the intracellular concentration of Ca2+ and
have a unitary conductance of 6-14 pS.4,5 They are
found in many tissues, but in general their physiological
role is not fully understood, although it has been
identified in some cases.6-11 It has been suggested that
these channels have a physiological role in the central
nervous system, since an endogenous ligand with SKCa
channel-blocking activity has been isolated from pig
brain.12 Such an endogenous modulator has also been
characterized in pheochromocytoma cells.13 Further-
more, SKCa channels have been suggested to play a role
in myotonic muscular dystrophy.14,15 There is evidence
that SKCa blockade in this disease can lead to a
reduction in basal muscle electrical activity and sup-
pression of myotonic discharges.16
covery of small, synthetic SKCa channel blockers.
A
starting point was the discovery that simple bis-charged
molecules such as tubocurarine are active.22-24 Dequal-
inium (1a , Chart 1) was then identified as the most
potent and selective available nonpeptidic bis-quater-
nary blocker of the SKCa channel.25,26 The stereoelec-
tronic requirements for optimal SKCa channel blockade
have since been probed in various series of dequalinium
analogues.27-31 Thus, it has been found that the quino-
linium groups can be replaced by other charged hetero-
cycles to provide effective blockers.28 The substituents
of the quinolinium group in molecules of the general
structure 1b and 2a have been suggested to modulate
the blocking activity of the compound by altering the
energy of the LUMO.30,31 Moreover, it has been estab-
lished that, surprisingly, the number of methylene
groups in the alkylene chain of dequalinium is not
critical for activity.32 Thus, the potency of the compound
is not significantly altered when reducing the number
of C atoms in the chain from 12 to 5, though a small
decrease is observed on further descending to 4 and 3
methylene groups. The remarkably low sensitivity of
potency to the length of the linker has led to the
suggestion that the molecule may adopt a folded con-
formation at the binding site.32 In the present study,
the flexible alkyl chain of dequalinium has been re-
placed with rigid or semirigid groups to further probe
the role of the linker. The choice of the type of linking
group in the present analogues was based upon the
previously established structure-activity relationships
of series 1b and 2a (Chart 1). Thus, optimal activity
had been obtained with N-benzyl substituents as in 1c
and 2b. Hence, it seemed reasonable to replace the
Presented in part at the 211th American Chemical Society
National Meeting, New Orleans, LA, March 24, 1996; MEDI 002.
* Address for correspondence: University College London, Depart-
ment of Chemistry, Christopher Ingold Laboratories, 20 Gordon Str.,
London WC1H 0AJ , U.K.
† Department of Chemistry.
‡ Department of Pharmacology.
§ On leave from the Universidad de Granada, Departamento de
Quimica Organica, Facultad de Farmacia, Campus de Cartuja, s/n
18071-Granada, Spain.
∇ Present address: Department of Pharmaceutical Chemistry,
School of Pharmacy, Aristotelian University of Thessaloniki, Thessa-
loniki 54006, Greece.
X Abstract published in Advance ACS Abstracts, September 1, 1996.
S0022-2623(95)00884-3 CCC: $12.00 © 1996 American Chemical Society

4248 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Campos Rosa et al.
Ch a r t 1
Sch em e 1
Sch em e 2
alkylene chain of dequalinium with benzylic groups to
give compounds of the general structure 3 (Chart 1). In
these analogues, X can be a mono-, bi-, or tricyclic
aromatic moiety. The use of aromatic groups as more
rigid substitutes of the alkane chain of dequalinium has
the additional advantage of maintaining a high lipo-
philic character in the linker. The 2-Me group of
dequalinium was omitted in this series, since it is known
to contribute little to potency while causing problems
in the synthesis by hindering the ring N atom and
making it difficult for the latter to undergo alkylation.30
in DMSO-d6 equilibrated on standing for 25 days to a
mixture of the cis- (10) to trans- (11) isomers in the ratio
of 1:3, respectively (data not shown). To avoid any
complications arising from this slow isomerization, fresh
solutions of the compounds were used in the biological
testing.
Ch em istr y
Compounds 4-19 (Table 1) were prepared via reac-
tion of 4-aminoquinoline33 with the appropriate dibro-
mide as detailed in Scheme 1. The synthesis of all
dibromides has been reported in the literature (see the
Experimental Section for each individual one). How-
ever, different routes from the published ones were used
for the preparation of 6c, 10c, 11c, 17c (Scheme 3), and
12f (Scheme 4). Thus, 6c, 10c, 11c, and 17c were
conveniently synthesized via LiAlH4 reduction of the
corresponding methyl esters of the diacids to give the
respective diols, which were brominated using PBr3. The
methyl ester 6a was prepared via photocyclization of
the trans-(E)-stilbene diester 11a (Scheme 2). The
synthesis of 12f (Scheme 4) involved bromination of 12c
to the meso dibromide 12d , double dehydrobromination
to the diphenylacetylene 12e, and free radical bromi-
nation of this to give the desired compound. The
purification of the final compounds was achieved through
recrystallization except in the case of 5 and 7, where
reverse phase preparative HPLC had to be used.
Biologica l Testin g
The SKCa-blocking action of the compounds was
assessed from their ability to inhibit the after-hyper-
polarization (AHP) in cultured rat sympathetic neu-
rons26 (see the Experimental Section). Briefly, each
compound was tested at two to four concentrations on
at least three cells. Between one and four compounds
were examined at a time, and in each such series of
experiments, dequalinium was also included as a refer-
ence compound. The Hill equation was fitted to the data
to obtain estimates of the IC50. However, because there
was some variation in the potency of dequalinium
during the course of the study, equieffective molar ratios
(EMR: relative to dequalinium) were also obtained by
simultaneous nonlinear least-squares fitting of the data
with the Hill equation. These are also listed in Table
1, and it is these values which have been used for the
comparison between compounds. It should be noted
that the compounds were applied in a continuously
An interesting isomerization was observed in the case
of the cis-(Z)-stilbene 10. A solution of this compound

Dequalinium Analogues with Semirigid Linkers
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4249
Sch em e 3
extension of the linker is similarly reflected in the 6-fold
potency difference between the cis- and trans-stilbenes
10 and 11. Finally, reducing the separation between
the quinolinium groups by attaching them to p- or
m-xylene (18, 19) markedly reduces potency. Thus,
although considerable variation of X is tolerated, there
appears to be an optimum between the limits repre-
sented by X ) benzene-1,4-diyl (18) and 1,1′:4′,1′′-
terphenyl-4,4′′-diyl (16).
We previously reported that for dequalinium homo-
logues there is little dependence of the SKCa channel-
blocking potency on the length of the aliphatic chain
(above (CH2)4) connecting the two quinolinium groups.32
This observation, however, does not of itself provide a
convincing answer to the question of whether the alkyl
chain binds to the channel. Any contribution to binding
from the hydrocarbon linker would be expected to be
hydrophobic in nature. The putative binding of the
flexible chain to the channel would also be associated
with an entropy loss resulting from the restriction of
the rotatable bonds of the chain. Thus, if the energy
gain from the hydrophobic binding were approximately
equal to the entropy loss due to conformational restric-
tion, then little change in the free energy of binding
would be expected on varying the chain length. Such a
compensation between favorable and unfavorable com-
ponents of the free energy of binding has been observed
in carbonic anhydrase inhibitors bearing chains of
variable length.34,35 The chains in these molecules
consisted of glycine units which appeared to make only
hydrophobic contacts with the enzyme, and there was
little dependence of the dissociation constants of the
inhibitors on the number of glycine units.
If the linker X of the present series of analogues were
binding to the channel, then it would be expected that
restriction of rotatable bonds within X, without signifi-
cant drop in the lipophilicity or change in the confor-
mational preferences of the group, would result in less
entropy loss upon binding and, hence, higher potency.
However, on comparing various pairs of compounds, e.g.,
4-6, 5-7, and 6-10, it is clear that the freezing of
degrees of freedom of X has no effect on potency. This
constitutes strong evidence that the linking chain of
dequalinium analogues does not bind directly to the
channel, and therefore, any change in the potency of
the compound as a result of alteration in X should be
attributed to modulation of other molecular properties
such as the conformation.
flowing solution to isolated cells, so that differences in
depletion as a consequence of variation in lipophilicity
are unlikely to have been a complicating factor.
Resu lts a n d Discu ssion
Structures and biological results for the compounds
are given in Table 1. It can be seen that the alkyl chain
of dequalinium can be replaced effectively with various
aromatic groups, with a modest increase in activity in
about one-half of the examples. The variation in the
structure of the linker is considerable, yet the maximum
difference in potency observed in the series is only
approximately 1 order of magnitude (cf. compounds 10
and 18), and this has important implications for the role
of the linker. Certain features do stand out, however.
The meta-disubstituted biphenyl 4 is similar in
potency to rigid analogues of 4 such as the phenan-
threne 6 and the cis-stilbene 10. Separation of the two
benzene rings of the para-disubstituted biphenyl 5 by
a poly(methylene) chain, (CH2)n, where n ) 1-4 (8, 9,
13, 15), has very little influence on potency, but rigidi-
fication to maintain the chain in an extended form (11,
12, 16) substantially reduces potency. The effect of
Sch em e 4

4250 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Campos Rosa et al.
Ta ble 1. Structures, Biological Results, and Distances between the Benzylic C Atoms for the Compounds
a
The interatomic distance between the two benzylic C atoms connected to the quinolinium ring N atom, in the lowest energy
conformation, using a dielectric constant of 1.0. As D1 but using a dielectric constant of 4.0. c Equieffective molar ratio: the ratio of the
b
concentrations of the test compound and dequalinium that cause 50% inhibition of the AHP, as determined in the same experiment.
d
Number of neurons tested. e Compound 4 can also formally be represented in an anti-conformation, but this is of higher energy (=2
kcal/mol); the distances D1 and D4 are 8.54 and 8.53 Å, respectively; in either case, the benzene rings are not coplanar but form a dihedral
angle of 50.5-53.6°.
To gain insight into the conformational behavior of
compounds 4-19, molecular modeling studies were
performed using the XEDMININ routine within the
XED/COSMIC package.36,37 Partial atomic charges
were obtained from a semiempirical AM138 calculation.
Initially, a conformational search was performed for

Dequalinium Analogues with Semirigid Linkers
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4251
F igu r e 1. Two of the low-energy conformations of the cis-stilbene derivative 10. Hydrogen atoms are omitted for clarity. The
conformers were obtained as detailed in the Experimental Section, using a dielectric constant of 4.0.
each of the compounds of Table 1 using a dielectric
constant of 1.0. Since the molecules possess two posi-
tively charged quinolinium groups, their conformational
preferences may depend upon the dielectric constant
used, and under vacuum conditions, the repulsions
between the quinolinium groups may be overestimated.
Since nothing is known with certainty about the biologi-
cal milieu in which these compounds act, the choice of
a biologically relevant dielectric constant is problematic.
Nevertheless, the conformational search was repeated
for each of the compounds using the higher dielectric
constant of 4.0, to investigate whether the behavior of
the molecule would be altered. The value of 4 is
commonly used in modeling studies to simulate a
protein environment.39
These results are in accord with the previously
suggested “folded” pharmacophore for the dequalinium
analogues.32 Further support for this argument is
provided by the drop in activity observed when the
ability of X to fold is removed (cf. compounds 10-11,
10-12, and 15-16). In rigid analogues in which the
benzylic C atoms occupy well-defined positions in space
(5-8, 10) distances between these atoms ranging from
≈7 to ≈10 Å have little effect on potency. This tolerance
may be explained by the relative mobility of the quino-
linium rings. Figure 1 shows two representative low-
energy conformations of the cis-stilbene 10, one with the
quinolinium groups in an “extended” arrangement and
the other with these groups in a “folded” conformation.
Although the distance between the benzylic C atoms in
both conformers is essentially the same, the distance
between the exocyclic N atoms varies from 19 Å in the
extended conformer to 4.5 Å in the folded one.
It has been established that the quinoline rings have
to be charged for potent blockade of the SKCa channel
in dequalinium analogues.31 In addition, it has been
shown that the charge is extensively delocalized in the
quinolinium group resulting in a large, ring-shaped
positive electrostatic field around this group.28 Hence,
the mobility of the quinolinium rings and the large
positive field around them may permit favorable inter-
actions with the channel in compounds with signifi-
cantly different benzylic C-C distances. This assumes
binding to an anionic site, but a similar argument can
be developed for binding to an aromatic site.28
The results of the modeling studies demonstrate that
the position in space of the quinolinium group, with
respect to the aromatic ring to which it is attached
through the benzylic C atom, is not well defined. This
is due to the presence of two single bonds between each
of the quinolinium ring N atoms and the aryl ring which
give rise to several low-energy conformations. The
quinolinium group, being always connected to a benzylic
C atom, adopts similar conformations for all the ana-
logues and so the analysis has to focus on differences
in the conformational behavior of the linker X in order
to explain changes in blocking activity.
In compounds with relatively flexible linkers, such as
9, 13, and 15, the conformation of X is dependent upon
the dielectric constant used. As the dielectric is in-
creased, the repulsions between the charged quinolinum
groups are attenuated and the molecule tends to adopt
partially folded conformations more easily. This is
exemplified by 9. The lowest energy vacuum conforma-
tion of X in this molecule has the ethylene bridge in a
trans-arrangement, giving a distance between the two
benzylic C atoms of 12.19 Å (Table 1). However,
increasing the dielectric to 4.0 causes the ethylene group
to adopt a gauche conformation which reduces the
distance by one-half. This conformation is stabilized
through π-stacking between the two phenyl rings as well
as between the two quinolinium groups. The lowest
energy conformations of 13 and 15 have the propylene
and butylene chains, respectively, in the trans-arrange-
ment, both when using a dielectric of 1.0 or 4.0.
Nevertheless, low-energy partially folded conformers do
exist in the latter case, and it would be expected that,
in an aqueous environment, folding would be promoted
even more by hydrophobic effects. Thus, the ability of
9, 13, and 15 to fold may enable them to simulate the
conformation of 10, and this may explain their potency.
Con clu sion
The alkyl chain of dequalinium can be replaced
effectively with rigid or semirigid linkers containing aryl
rings to give analogues with slightly increased potency
in many cases. The linker does not seem to interact
directly with the channel, but rather, its role seems to
be that of a “scaffold” for the two quinolinium rings.
Although the conformational properties of the linker are
of some importance, the dependence of the blocking
activity of the analogue on the distance between the
“anchoring” benzylic C atoms is not critical. This may
be due to a combination of two factors: the conforma-
tional mobility of the quinolinium groups and the
extensive delocalization of the positive charge, which
gives rise to a large positive field around these groups.
Exp er im en ta l Section
(a ) Ch em istr y. Melting points (mp) were obtained either
on an Electrothermal melting point apparatus or on a Kofler
apparatus equipped with a microscope RCH and are uncor-

4252 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Campos Rosa et al.
Ta ble 2. Yields, Melting Points, and Analytical Data for
rected. Nuclear magnetic resonance (NMR) spectra were
recorded on a Varian XL-200 (200 MHz) or VXR-400 (400 MHz)
spectrometer. The H NMR spectra of all compounds were in
Compounds 4-19a
1
yield
mol formulab
accord with the structures. Mass spectra were run on a ZAB
SE or VG 7070H spectrometer, using FAB in a matrix of
m-nitrobenzyl alcohol or thioglycerol (5, 7-10). All compounds
showed mass numbers corresponding to [M - H]+ except 6
[M]+. Analytical high-performance liquid chromatography
(HPLC) was performed on a Shimadzu HPLC apparatus with
a UV detector and a Kromasil C18 5 µm column. Solvent
mixtures of A ) water + 0.1% TFA and B ) MeOH + 0.1%
TFA were used at a flow rate of 1 mL/min. The ratio A:B was
1:1 except for compounds 6 (45:55), 15 (40:60), and 17 (55:45),
and in every case the absorption was >97% of the total, using
a detector set at λ 215 nm (4, 5, 13, 16), 254 nm (6, 10-12,
15, 17-19), 300 nm (7-9), or 315 nm (14). For preparative
HPLC a Gilson apparatus was used with a UV detector and a
Kromasil C18 5 µm column. The solvent mixtures were as
above, and the flow rate was 20 mL/min. All compounds were
dried at 60 °C and 0.1 mmHg for 15 h, but many held on
tenaciously to solvent molecules, especially water which ap-
pears to be a solvate. 3,3′-Bis(bromomethyl)biphenyl,40 4,4′-
bis(bromomethyl)biphenyl,41,42 2,7-bis(bromomethyl)fluorene,43
bis[p-(bromomethyl)diphenyl]methane,44 bis-p-(bromomethyl)-
bibenzyl,44 1,3-bis[4-(bromomethyl)phenyl]propane,44 2,6-bis-
[4-(bromomethyl)phenyl]pyridine,45 4-bis[4-(bromomethyl)-
phenyl]butane,44 and 4,4′′-bis(bromomethyl)-1,1′:4′,1′′-terphenyl46
were synthesized according to literature procedures, and R,R′-
dibromo-p-xylene and R,R′-dibromo-m-xylene were obtained
from Aldrich.
Gen er a l Exp er im en ta l P r oced u r e for th e P r ep a r a tion
of Com p ou n d s 1,1′-[Bip h en yl-3,3′-d iylbis(m eth ylen e)]-
bis(4-a m in oqu in olin iu m ) Dibr om id e Hyd r a te (4), 1,1′-
[Bip h en yl-4,4′-d iylbis(m eth ylen e)]bis(4-a m in oqu in olin -
iu m ) Dit r iflu or oa cet a t e (5), 1,1′-(P h en a n t h r en e-3,6--
d iylbis(m eth ylen e)]bis(4-a m in oqu in olin iu m ) Dibr om id e
Hyd r a te Eth a n oa te (6), 1,1′-[F lu or en e-2,7-d iylbis(m eth -
ylen e)]bis(4-a m in oqu in olin iu m ) Ditr iflu or oa ceta te (7),
1,1′-[Meth ylen ebis(ben zen e-1,4-diylm eth ylen e)]bis(4-am i-
n oqu in olin iu m ) Dibr om ide Hydr ate (8), 1,1′-[Eth ylen ebis-
(ben zen e-1,4-diylm eth ylen e)]bis(4-am in oqu in olin iu m ) Di-
br om id e Hyd r a te (9), (Z)-1,1′-[Stilben e-4,4′-d iylbis(m eth -
ylen e)]bis(4-a m in oqu in olin iu m ) Dibr om id e Sesqu ih y-
d r a te (10), (E)-1,1′-[Stilben e-4,4′-d iylbis(m eth ylen e)]bis-
(4-a m in oqu in olin iu m ) Dibr om id e Dih yd r a te (11), 1,1′-
[E t h yn e-1,2-d iylb is(b en zen e-1,4-d iylm et h ylen e)]b is(4-
a m in oqu in olin iu m ) Dibr om id e Sesqu ih yd r a te (12), 1,1′-
[P r op a n e-1,3-d iylbis(ben zen e-1,4-d iylm eth ylen e)]bis(4-
a m in oqu in olin iu m ) Dibr om id e Hem ih yd r a te Eth a n oa te
(13), 1,1′-[P yr id in e-2,6-d iylb is(b en zen e-1,4-d iylm et h yl-
en e)]bis(4-a m in oqu in olin iu m ) Dibr om id e Hyd r a te (14),
1,1′-[Bu ta n e-1,4-d iylbis(ben zen e-1,4-d iylm eth ylen e)]bis-
(4-a m in oqu in olin iu m ) Dibr om id e Hyd r a te (15), 1,1′-[1,1′:
4′,1′′-Ter ph en yl-4,4′′-diylbis(m eth ylen e)]bis(4-am in oqu in -
olin iu m ) Dibr om id e Tr ih yd r a te (16), 1,1′-[Na p h th a len e-
2,6-d i y l(b i s (m e t h y le n e )]b i s (4-a m i n o q u i n o li n i u m )
Dibr om id e Hyd r a te (17), 1,1′-[Ben zen e-1,4-d iylbis(m eth -
ylen e)]bis(4-a m in oq u in olin iu m ) Dib r om id e Dih yd r a t e
(18), a n d 1,1′-[Ben zen e-1,3-d iylbis(m eth ylen e)]bis(4-a m i-
n oqu in olin iu m ) Dibr om id e Hem ih yd r a te (19). 4-Amino-
quinoline33 (1.58 mmol), the corresponding bis(bromomethyl)
compound BrCH2XCH2Br (0.79 mmol), and MEK (25 mL) were
heated under reflux for 72 h. After filtration and washing
thoroughly with MEK and CHCl3, the solid product was
purified by crystallization from EtOH or EtOH/MeOH. In the
case of 5 and 7, the compounds were purified by reverse phase
preparative HPLC and obtained as poly(trifluoroacetate)s.
Yields, melting points, and analytical data are given in Table
2.
compd
(%)
mp (°C)
4
5
6
7
8
64
38
52
31
69
77
61
84
77
46
63
67
80
62
82
43
289-291
235-237
266-268
C32H28N42+‚2Br-‚H2O
C32H28N42+‚2CF3CO2-‚3CF3CO2H
C34H28N42+‚2Br-‚H2O‚C2H5OH
227-228 dec C33H30N42+‚2CF3CO2-‚2.3CF3CO2H
248-250
207-209
218-220
264-266
299-301
209-212
C33H30N42+‚2Br-‚H2O
9
C34H32N42+‚2Br-‚H2O
10
11
12
13
14
15
16
17
18
19
C34H30N42+‚2Br-‚1.5H2O
C34H30N42+‚2Br-‚2H2O
C34H28N42+‚2Br-‚1.5H2O
C35H34N42+‚2Br-‚C2H5OH‚0.5H2O
293-295 dec C37H31N52+‚2Br-‚H2O
228-230
243-245
240-242
319-321
321-322
C36H36N42+‚2Br-‚H2O
C38H32N42+‚2Br-‚3H2O
C30H26N42+‚2Br-‚H2Oc
C26H24N42+‚2Br-‚2H2O
C26H24N42+‚2Br-‚0.5H2O
a
Compounds were recrystallized from EtOH except for 4 and
14 (MeOH/EtOH) and 5 and 7 which were purified by reverse
phase preparative HPLC as detailed in the Experimental Section
b
and obtained as poly(trifluoroacetates). All compounds had CHN
analyses within (0.4% of the values calculated for the indicated
molecular formulae. c N: calcd, 9.03; found, 8.48.
of MeOH precipitated a white solid (0.96 g) which, on recrys-
tallization (CH2Cl2/MeOH mixture), gave 0.93 g (48%) of white
prisms: mp 208-210 °C. Anal. (C18H14O4) C, H.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
6b, 10b, 11b, a n d 17b. A solution (suspension in the case of
11a ) of the corresponding methyl diester in dry THF was
added dropwise with stirring to a solution of LiAlH4 (2.8 mol
excess) in dry THF held at room temperature under a N2
atmosphere. When the addition was complete, the mixture
was boiled under reflux for 1 h and then decomposed with
saturated aqueous Na2SO4 (a white precipitate formed). The
products were obtained after filtration, drying of the organic
layer, and concentration.
3,6-Bis(h yd r oxym eth yl)p h en a n th r en e (6b): mp 157-
158 °C (CHCl3) (lit.47 mp 159-160 °C).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
6c, 10c, 11c, a n d 17c. To a suspension of 6b, 10b, 11b, or
17b (2.06-2.87 mmol) in CHCl3 (20 mL) was added PBr3 (2.96
mmol) dropwise with stirring. After the end of the addition,
the suspension was stirred for 15 h. The CHCl3 layer was
washed with water (20 mL), dried (Na2SO4), and concentrated
to give the products.
3,6-Bis(br om om eth yl)p h en a n th r en e (6c): mp 173-175
°C (CH2Cl2/hexane) (lit.47 mp 173-175 °C).
(Z)-4,4′-Bis(h yd r oxym eth yl)stilben e (10b): mp 110-113
°C; lit.48 mp 110-113 °C).
(Z)-4,4′-Bis(br om om eth yl)stilben e (10c): mp 71-73 °C
(lit.48 mp 69-69.5 °C).
(E)-4,4′-Bis(h yd r oxym eth yl)stilben e (11b): mp 266-267
°C (lit.49 mp 268-270 °C).
(E)-4,4′-Bis(br om om eth yl)stilben e (11c): mp 191-193
°C (lit.50 mp 192-193 °C).
(E)-4,4′-Dim eth ylstilben e (12c): mp 179-180 °C (CCl4)
(lit.51 mp 175-177 °C).
Meso-1,2-dibr om o-1,2-bis(4-m eth ylph en yl)eth an e (12d).
A suspension of 12c (2 g, 9.6 mmol) and pyridinium hydro-
bromide perbromide52 (4 g, 12.5 mmol) in glacial AcOH (40
mL) was stirred at room temperature. After 1.5 h the reagent
was consumed and the precipitate of colorless, crystalline
product was collected, washed with MeOH, and dried (3.6 g,
88.2%): mp 208-210 °C. Anal. (C16H16Br2) C, H.
1,2-Bis(4-m eth ylp h en yl)eth yn e (12e): mp 138-140 °C
(EtOH) (lit.50 mp 136 °C).
Dim eth yl 3,6-P h en a n th r en ed ica r boxyla te (6a ). A solu-
tion of dimethyl 4,4′-stilbene-(E)-dicarboxylate (11a ; 1.95 g,
6.58 mmol) in benzene (700 mL) containing I2 (0.362 mmol)
was irradiated for 5 days using a medium-pressure mercury
lamp (450 W, Hanovia) in a quartz well, while O2 was slowly
bubbled through the reaction mixture. After removal of the
solvent in vacuo, the residue was taken up in CHCl3. Addition
1,2-Bis[4-(br om om eth yl)p h en yl]eth yn e (12f): mp 190-
192 °C (CH2Cl2/hexane) (lit.49 mp 187-190 °C).
2,6-Bis(h yd r oxym eth yl)n a p h th a len e (17b): mp 172-
174 °C (lit.53 mp 170-170.5 °C).
2,6-Bis(br om om eth yl)n a p h th a len e (17c): mp 178-179
°C (lit.54 mp 182-184 °C).

Dequalinium Analogues with Semirigid Linkers
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4253
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(b) Molecu la r Mod elin g. The structures of all compounds
were first built and minimized (with no charges on them) using
the XED module in the XED/COSMIC36,37 system running on
a Silicon Graphics Indigo/2 workstation. The minimized
structures were saved and the “.dat” files were converted to
Sybyl 6.0355 “.mol” structure files. The MOPAC package was
then used interfaced with Sybyl to perform a point semiem-
pirical MO calculation on the molecules using the AM1
Hamiltonian38 and Mulliken population analysis. The Mul-
liken charges were loaded and the molecules saved as “.mol”
files. These were converted back to “.dat” COSMIC files which
were read into XED and minimized. The minimized structures
were then subjected to conformational searching using the
XEDMININ routine. Typically, the rotational increment of
single bonds was between 30° and 90° depending on the
number of such bonds in the structure, and 200 randomiza-
tions of the molecule were performed. The dielectric constant
was set to either 1.0 or 4.0, and structures within 10 kcal/mol
(upper search limit) of the global minimum were stored. The
structures from this initial search were subsequently fully
minimized using the COSMIC force field. Visualization and
manipulation of the resultant conformers were achieved using
the XEDA routine.
(c) P h a r m a cology. Stock solutions (2 mM) of all com-
pounds were prepared in MeOH. Superior cervical ganglia
from 17-day-old rats were treated with collagenase and trypsin
and then dissociated using a fire-polished pipet. The resultant
cell suspension was plated onto laminin-coated plastic dishes
and maintained in tissue culture for 3-10 days. Culture
dishes were placed on the stage of an inverted microscope and
perfused with physiological salt solution of the following
composition (in mM): NaCl, 118; KCl, 4.8; CaCl2, 2.5; MgSO4,
1.19; NaHCO3, 25; KH2PO4, 1.18; and glucose, 11; they were
then warmed to 30 °C and equilibrated with 95% O2:5% CO2.
Intracellular recordings were made with conventional micro-
electrodes filled with 1 M KCl (resistance 80-120 MΩ),
connected to the headstage of a Neurolog NL 102 amplifier.
Action potentials were evoked by injection of 30 ms depolar-
izing current pulses every 5 s. Data acquisition and analysis
were performed on a microcomputer using the pClamp suite
of programs (Axon Instruments). Inhibition of the AHP was
measured by digitally subtracting records obtained in the
presence of blocker from controls and expressing the peak
difference as a percentage of the control AHP at the same time
point.
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Ack n ow led gm en t. This work was partially sup-
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P.M.D. The award of a grant from the Spanish DGICYT
to J .C.R. is acknowledged. We are grateful to Professor
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