Synthesis and structure-activity relationships of cetiedil analogues as blockers of the Ca2+-activated K+ permeability of erythrocytes

Article abstract of DOI:10.1021/jm001113w

Cetiedil, [2-cyclohexyl-2-(3-thienyl)ethanoic acid 2-(hexahydro-1H-azepin-1-yl)ethyl ester], which blocks the intermediate calcium-activated potassium ion permeability (IK_Ca) in red blood cells, was used as a lead for investigating structure-ac

Full text of DOI:10.1021/jm001113w

3244
J . Med. Chem. 2001, 44, 3244-3253
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of Cetied il An a logu es a s
Block er s of th e Ca 2+-Activa ted K+ P er m ea bility of Er yth r ocytes†
Craig J . Roxburgh,‡ C. Robin Ganellin,*,‡ Salah Athmani,‡ Alessandra Bisi,‡,§ Wilma Quaglia,‡,|
David C. H. Benton, Mark A. R. Shiner, Misbah Malik-Hall, Dennis G. Haylett, and Donald H. J enkinson
Departments of Chemistry and Pharmacology, University College London, Gower Street, London WC1E 6BT, U.K
Received November 9, 2000
Cetiedil, [2-cyclohexyl-2-(3-thienyl)ethanoic acid 2-(hexahydro-1H-azepin-1-yl)ethyl ester], which
blocks the intermediate calcium-activated potassium ion permeability (IKCa) in red blood cells,
was used as a lead for investigating structure-activity relationships with the aim of determining
the pharmacophore and of synthesizing agents of greater potency. A series of compounds having
structures related to cetiedil was made and tested on rabbit erythrocytes. Channel blocking
activity within the series was found to correlate well with octanol-water partition coefficients
but not with the specific chemical structure of the acid moiety. However, whereas log P for the
compounds spans a range of values over 4 orders of magnitude, potency only increases by 2
orders. This suggests that hydrophobic interactions with an active site on the channel are
probably not the main determinants of activity. It seems more likely that increased lipophilicity
enhances access to the channel, probably from within the cell membrane. In keeping with this
interpretation, cetiedil methoiodide was found to be inactive. Triphenylethanoic was found to
be a more effective acid grouping than 2-cyclohexyl-2-(3-thienyl)ethanoic, and its 2-(hexahydro-
1H-azepin-l-yl)ethyl ester (11) was approximately 3 times more potent than cetiedil. The
9-benzylfluoren-9-yl carboxylic acid ester (21) was found to be approximately 9 times more
active than cetiedil, and replacing -CO2- in 21 by an ethynyl (-CtC-) linkage (compound
26, UCL 1608) increased potency by some 15-fold over that of cetiedil.
In tr od u ction
of cells results in the formation of an intermediate
conductance Ca2+-activated K+ channel that has been
variously termed SK4, KCa4, IK1, and IKCa1. The same
channel occurs in T lymphocytes, where it is concerned
in the control of proliferation, raising the possibility that
selective IKCa channel blockers could provide a novel
means of immunosupression.10
K+ channels constitute a remarkably diverse family
of membrane-spanning proteins that have a wide range
of functions in electrically excitable and inexcitable cells.
One important class opens in response to an increase
in the concentration of calcium within the cytosol. This
was first demonstrated in red blood cells with which it
was found that treatments, such as metabolic inhibition,
which caused cytosolic Ca2+ to rise also resulted in a
large increase in K+ permeability (the Ga´rdos effect1).
The change in permeability was subsequently shown to
be mediated by the opening of Ca2+-activated K+ chan-
nels (KCa) in the membrane of the red cell. Later work,
based initially on pharmacological and electrophysi-
ological evidence (for reviews see refs 2-4) and more
recently based on structural evidence from cloning
studies,5 has established that there are several kinds
of Ca2+-activated K+ channels. Those in the red cell
belong to the intermediate conductance (IKCa) subtype,
so named because its single channel conductance (10-
Several compounds have been shown to block the
IKCa-mediated Ca2+-activated K+ permeability in red
blood cells. These include quinine and quinidine,11,12
some carbocyanine dyes,13 clotrimazole14,15 (Chart 1),
and some related compounds such as TRAM-3410b,c,15b
(Chart 1), charybdotoxin,16 dequalinium and some of its
derivatives17 (Chart 1), nifedipine,18 nitrendipine,19 and
20-23
cetiedil
(Chart 1), which is the starting point of
the present work. Cetiedil [(()-2-cyclohexyl-2-(3-thie-
nyl)ethanoic acid 2-(hexahydro-1H-azepin-1-yl)ethyl es-
ter, 1; Chart 1, Table 1] is one of a series of compounds
with spasmolytic, local anaesthetic, and analgesic ac-
tions24 and is of particular interest because it has been
suggested,20 though by no means proven, that the ability
of cetiedil to block the Ca2+-activated K+ permeability
in red blood cells may contribute to its reported efficacy
in the treatment of sickle-cell disease (e.g., see ref 25).
It is argued that cetiedil’s ability to reduce the loss of
K+ from the cells may diminish the reduction in cell
volume that can trigger the sickling of erythrocytes (see
refs 26 and 27 for reviews). Cetiedil is also of interest
because it blocks other K+ channels that are involved
in cell volume regulation in hepatocytes28 and lympho-
cytes.29,30 As part of a broader investigation of the
medicinal chemistry of nonpeptidic K+ channel blocking
30 pS) lies between that of the small conductance (SKCa
)
and high conductance (BKCa) subtypes (reviewed in refs
2 and 3). The gene (KCNN4) coding for the IKCa channel
has now been cloned6-9 and its expression in a variety
* Address for correspondence: Department of Chemistry, Christo-
pher Ingold Laboratories, University College London, 20 Gordon St.,
London WC1 OAJ , U.K. Tel: 44-207-380-7459. Fax: 44-207-380-7463.
E-mail: c.r.ganellin@ucl.ac.uk.
† Presented in part at the 15th European Federation of Medicinal
Chemistry Symposium in Edinburgh, Scotland, September 9, 1998.
‡ Department of Chemistry.
§ On leave from the University of Bologna.
| On leave from the University of Camerino.
Department of Pharmacology.
10.1021/jm001113w CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/28/2001

Blockers of the Ca2+-Activated K+ Permeability
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3245
Ch a r t 1
Ta ble 1. Cetiedil Analogues: Molecular Formulas, Melting Points, and Solvent for Crystallation
R1
R2
R3
n
A
m
formulaa
mp, °C
oilc
crystn solvent
HPLC
MeCN:EtOH (10:1)
EtOAc:EtOH
EtOAc:EtOH (10:1)
MeOH:Et2O (1:3)
EtOAc:EtOH (5:1)
EtOAc:EtOH (10:1)
EtOAc:EtOH (5:1)
EtOAc:MeOH (5:1)
washed EtOH
EtOAc:EtOH (10:1)
ppd from EtOH
EtOH:Et2O (6:1)
HPLC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
C6H11
C6H11
H
C6H11
C6H11
C6H11
C6H11
Ph
Ph
Ph
Ph
Ph
3-thienyl
H
H
H
H
OH
OH
H
H
H
H
H
Ph
Ph
Ph
H
0
0
0
0
0
0
0
0
1
0
0
1
0
0
2
COO
COO
COO
COO
COO
COO
COO
COO
COO
COO
COO
COO
COO
CONH
O
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
C20H31NO2S‚2CF3CO2Hb
C16H29NO2·1.1HCl‚0.5H2O
C14H21NO2S‚HCl
C20H31NO3 S‚HCld
C20H21NO4‚C2H2O4f
C22H39NO2‚1.1HCl‚0.2H2O
C22H33NO2‚HCl
22H27NO2‚HCl
23H29NO2‚0.7C2H2O4
20H25NO2S‚C2H2O4
158-159
141.5-142.5
176-177e
142-144
222-224
170-172g
156-157h
183-185
179-180i
220-221
204-205
214-215
oil
3-thienyl
3-thienyl
3-furyl
C6H11
Ph
Ph
Ph
C
C
C
C
C
C
3-thienyl
Ph
28H31NO2‚HCl‚0.5H2Oj
29H33NO2‚C2H2O4‚0.1H2Ok
29H33NO2‚HCl‚0.25H2O
Ph
Ph
Ph
C6H11
Ph
3-thieny l
Ph
C20H32N2OS‚CF3CO2H‚1.5H2Ol
Ph
C
31H37NO5‚1.25C2H2O4
180-183
EtOH
a
All compounds had C, H, N analyses within (0.4% for the formula shown. All hydrochloride salts also had Cl analysis within (0.4%,
in accord with the formula indicated. Cetiedil trifluoroacetate. c Citrate mp 115°, hydrochloride mp 152°. UCL 1269. e Hydrochloride24
b
d
mp 156°. f C2H2O4 ) oxalic acid. Hydrochloride43 mp 178°. Hydrochloride44 mp 142-143°. Hydrochloride45 mp 124°. UCL 1274.
g
h
i
j
k
l
Hygroscopic. 95% purity according to HPLC analysis; N analysis 0.6% low (calcd, 5.7; found, 5.1).
agents,31-34 we have initiated a study of the structure-
dro-1H-azepine (Scheme 2.) The requisite acids for
esters 1, 4, and 5, and amide 14 were prepared as
outlined in Scheme 3 using the published procedures.
For compound 4, the requisite carboxylic acid, 2-cyclo-
hexyl-2-hydroxy-2-(3-thienyl)ethanoic acid, was synthe-
sized as previously described;23 for 5, the synthesis is
given in the Experimental Section. For 10, 2-phenyl-2-
(3-thienyl)ethanoic acid was prepared according to the
method of ref 36. For 14, 2-cyclohexyl-2-(3-thienyl)-
ethanoic acid was prepared as published.23 Other acids
were obtained commercially. The respective acids for
esters 21-25 were made from 9-carboxyfluorene by
alkylation of the disodium salt in liquid ammonia37
using the appropriate p-substituted benzyl halide
(Scheme 4).
activity relationships and mechanism of action23,35 of
cetiedil and its congeners as inhibitors of the Ca2+
-
activated K+ permeability in mammalian erythrocytes.
Our aims are to characterize the pharmacophore for
cetiedil and to develop more potent and selective block-
ers of the intermediate conductance Ca2+-activated K+
channel.
Ch em istr y
The final products 1-27 are listed together with
melting points and crystallization solvents in Tables l
and 2.
The esters 2-12 and 17-25 were synthesized by
heating the sodium salt of the acid (generated from
sodium in 2-PrOH) in 2-PrOH under reflux or in DMF
(generated with NaH) with 1-(chloroalkyl)hexahydro-
1H-azepine, as exemplified for compound 5 (Scheme 1).
Esters 13 and 16 were made by treating the acid
chloride with the appropriate 1-(hydroxyalkyl)hexahy-
The amide 14 was prepared by coupling the acid with
1-(2-aminoethyl)hexahydro-1H-azepine using n-butyl
chloroformate (Scheme 5). The ether 15 was obtained
by a Williamson synthesis from triphenylpropanol and
1-(2-chloroethyl)hexahydro-1H-azepine using sodium

3246 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Roxburgh et al.
Ta ble 2. Cetiedil Analogues: Molecular Formulas, Melting Points, and Solvent for Crystallization
R
n
A
m
formulaa
19H31NO2Cl
19H23NO2Cl‚0.25H2O
19H23NO2Cl‚0.25H2O
23H25NO2‚Cl‚0.5H2O
22H25NO2‚C2H2O4‚0.75H2O
29H31NO2‚C2H2O4‚0.25H2O
30H33NO3‚C2H2O4
29H30FNO2‚C2H2O4
29H30ClNO2‚C2H2O
29H30N2O4‚C2H2O4·0.5H2O
30H31N‚0.8C2H2O4·0.25C3H7OH
21H34NO2SI
mp, °C
crystn solvent
16
17
18
19
20
21
22
23
24
25
26
27
1-adamantyl
1-naphthyl
2-naphthyl
9-anthryl
0
0
0
0
0
0
0
0
0
0
1
COO
COO
COO
COO
COO
COO
COO
COO
COO
COO
CtC
2
2
2
2
2
2
2
2
2
2
1
C
C
C
C
C
C
C
C
C
C
C
C
203-205
199-200
196-198
181-182
169-170
160-170
130-132
161-162
140-141
170-172
153-155
144-145
EtOAc:EtOH (5:1)
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
2-PrOH
MeOH
9-fluorenyl
9-benzylfluoren-9-yl
9-(p-methoxybenzyl)fluoren-9-yl
9-(p-fluorobenzyl)fluoren-9-yl
9-(p-chlorobenzyl)fluoren-9-yl
9-(p-nitrobenzyl)fluoren-9-yl
9-benzylfluoren-9-yl
2-PrOH
EtOAc:EtOH (1:1)
cetiedil methoiodide
Sch em e 1
Sch em e 5
Sch em e 2
Sch em e 6
Sch em e 3
Sch em e 7
Sch em e 4
Biologica l Testin g
The potency of the compounds was assessed from
their ability to inhibit the net loss of cell K+ that follows
the application of the Ca2+ ionophore A23187 to rabbit
erythrocytes in suspension. The ionophore increases the
cytosolic concentration of Ca2+, with the consequences
that the Ca2+-activated K+ channels in the membrane
open and K+ leaves the cells. The amount of K+ lost was
measured using a K+-sensitive electrode placed in the
suspension. This provides a convenient, if indirect,
measure of changes in the number of open K+ channels.
The method has recently been used to study the action
of a range of IKCa blocking agents, including cetiedil,
charybdotoxin, and clotrimazole.17,35c As before, the
hydride in DMF (Scheme 6). The acetylene derivative
26 was synthesized by alkylation of 9-benzylfluorene
anion (generated from NaH in DMSO) with 1-chloro-4-
(hexahydro-1H-azepin-1-yl)but-2-yne (Scheme 7), which
was made by the action of thionyl chloride on the
corresponding carbinol. The latter was synthesized via
a Mannich reaction between propargyl alcohol, formal-
dehyde, and hexahydroazepine catalyzed with cuprous
chloride.38 Cetiedil methoiodide (27) was prepared from
cetiedil base using methyl iodide in ether.

Blockers of the Ca2+-Activated K+ Permeability
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3247
experiments have been done with a low K+ (0.1 mM)
solution because cetiedil then becomes approximately
3-fold more active.35c This has the additional advantage
that small changes in the concentration of K+ in the cell
suspension are more readily measured using the K+-
sensitive electrode technique. The relation between the
concentration of each test compound and its inhibitory
action was analyzed by using the Hill equation to obtain
an estimate (( the approximate standard deviation) of
the concentration (IC50) that causes 50% inhibition of
the K+ loss initiated by A23187. In preliminary analy-
ses, the Hill coefficient (nH) was allowed to vary and
was consistently observed to be in the range 2-3.5, as
in earlier work.35c A common value of nH (usually ∼2.5)
was assumed when fitting sets of assays done at the
same time, and the maximum inhibition was taken to
be 100%, in keeping with the observations. Fitting was
done using a least-squares minimization curve fitting
program (CVFIT, written by Prof. D. Colquhoun, Uni-
versity College London, and available at www.ucl.ac.uk/
pharmacol/dc.html).
Similar results were obtained (ref 35d; unpublished
observations by Babaoglu, Benton, and Haylett) when
the response measured was the A23187-initiated in-
crease in the influx of 86Rb in erythrocytes rather than
the net loss of K+ as assessed using a K+-sensitive
electrode. This provides a more direct indication of IKCa
activation. In the same experiments, the inhibitory
action of cetiedil analogues on 86Rb influx was unaf-
fected by simultaneous blockade of Na+K+ ATPase and
the Na+K+ 2Cl- cotransporter.
Ta ble 3. Cetiedil Analogues: IKCa Channel Blocking Activities
and Octanol-Water Partition Estimates
compd
IC50 ( SD,a µM
log Pb
CLOG Pc
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
25 ( 1
290 ( 30
142 ( 36
155 ( 10
200 ( 10
14 ( 1
6.59
5.18
3.45
4.94
4.41
8.33
6.88
5.44
5.96
5.15
7.14
7.65
7.65
5.15
7.83
5.60
5.16
5.16
6.45
5.47
7.68
7.75
7.92
8.41
7.44
9.10
5.98
4.93
3.55
5.22
4.75
6.86
5.93
5.25
5.67
4.90
6.69
7.11
6.40
5.11
7.35
4.38
5.18
5.18
6.36
5.24
7.18
7.09
7.32
7.89
6.92
7.90
18 ( 3
164 ( 42
43 ( 4
47 ( 4
8.2 ( 0.3
9.3 ( 1.2
9.2 ( 0.8
75 ( 3
5.5 ( 0.3
178 ( 12
222 ( 26
285 ( 16
28 ( 1
42 ( 9
4.0 ( 0.2
2.8 ( 0.3
4.1 ( 0.8
2.9 ( 0.3
4.6 ( 0.6
1.5 ( 0.1
.200d
a
Concentration of the test compound that causes 50% inhibition
of K+ loss from rabbit blood cells treated with the Ca2+ ionophore
b
A23187. log P calculated for the free base using the revised
hydrophobic fragmental values (f) of Mannhold and Rekker et al.,39
where CH2 ) 0.519, CH ) 0.315, C ) 0.110, C6H5 ) 1.903, thienyl
(C4H3S) ) 1.613, furyl (C4H3O) ) 1.086, naphthyl ) 3.191, anthryl
) 4.478, fluorenyl ) 4.149, adamantyl ) 4.170, aliphatic -CO2
)
-1.200, -N(CH2)6 ) 1.442, aliphatic -CONH ) -2.435, aliphatic
-O- ) -1.545, aliphatic-OH ) -1.448, aryl OMe ) 0.274, aryl
F ) 0.444, aryl Cl ) 0.933, aryl NO2 ) -0.039. A correction of +
2CM ) +0.438 was applied to each of the hydrocarbon residues
C6H11 and N(CH2)6. No proximity effect corrections were applied.
Resu lts a n d Discu ssion
To determine the pharmacophore of cetiedil (1) we
have to ask how critical are the various chemical
features encompassed by cetiedil’s chemical structure.
We noted the presence of a cyclohexane ring, a 3-thienyl
group, an ester functionality, and a two-carbon chain
connecting to a cyclic tertiary amine in a seven-
membered aliphatic ring. The molecule is also chiral.
In our initial approach to this problem we synthesized
five analogues in which the hexahydroazepinylethyl
ester was maintained as a constant feature. Removing
the thienyl ring (2) or the cyclohexyl residue (3) reduced
potency by approximately 6-12-fold (Table 3). Replacing
3-thienyl by phenyl (7) did not change activity, thus
establishing that the point of substitution of thiophene
or even the presence of the S atom were apparently not
of importance. The carbinol (4), an intermediate in the
synthesis of cetiedil that now has three substituting
groups on the acidic functionality, was about 6-fold less
active. On the other hand, the ester with three phenyl
substituting groups (11) showed a 3-fold potency in-
crease.
Is there a pattern in these results? It seemed that the
introduction of OH, a polar group that reduced activity,
in contrast to the introduction of a phenyl group that
increased activity, was particularly informative, and
this led us to seek for a possible correlation with
lipophilicity. Estimates for the octanol-water partition
coefficients by the use of fragmental constants (f) of
Rekker39 and by the CLOG P program40 are listed in
Table 3.
d
c ref 40. Insufficiently active to allow accurate measurement
according to the estimate of log P by using f values or
CLOG P, respectively, are given below:
-log IC50 ) 0.26 log P + 2.61
r ) 0.86, n ) 6, F ) 11.0
(1)
(2)
-log IC50 ) 0.38 CLOG P + 2.10
r ) 0.79, n ) 6, F ) 6.7
These correlations suggest that activity is determined
predominantly by the lipophilicity of the compound:
there appears to be little (if any) structural requirement
for channel interaction encoded in the carboxylic acid
part of the molecule. In keeping with this conclusion,
the synthesis and testing of the two chiral enantiomers
of cetiedil showed them to be similarly active as
erythrocyte IKCa channel blockers.23
Cetiedil is estimated to be a very lipophilic molecule
(log P for the free base ) 6.59 by f values, or 5.98 by
CLOG P) and there is little prospect of obtaining more
potent and clinically useful agents by merely increasing
lipophilicity. Even so, it seemed possible that interesting
and potentially valuable pharmacological tools might be
obtained by this approach. Also, some other structural
features of the molecules that might affect potency were
thought worth investigating. Thus, 3-furyl was intro-
duced in place of 3-thienyl (4), giving compound 5, which
The correlation equations (1 and 2) for the activities
of cetiedil plus the five analogues, (i.e. 1-4, 7, 11)

3248 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Roxburgh et al.
was slightly less active than 4, indicating that there was
no advantage in increasing the hydrogen-bond acceptor
property of the heterocyclic ring. Incorporating two
cyclohexyl groups (6) (log P ) 8.33 by f values) increased
the potency about 2-fold relative to cetiedil, in keeping
with the higher lipophilicity. Homologous esters were
also investigated: the ester (9) of diphenylpropionic acid
was 3-4 times more potent than the lower homologue
8, but this accords with its higher lipophilicity. The
homologous esters 12 and 13 were not significantly more
potent than 11. Thus the distances between the aryl
groups and the amine do not appear to be critical.
Changing the polarity of the ester also does not seem
to be of importance for activity; the more polar amide
(14) isostere of cetiedil (1) was approximately 3 times
less active, but it is also less lipophilic. The less polar
ether (15) isostere of 12 was slightly more active. The
influence of bulk was explored with the adamantyl ester
16; it was less active than cetiedil and less lipophilic.
Some fused-ring structures were also examined to
explore the possibility that a larger aromatic area might
be advantageous for activity. Thus, 1-naphthyl (17) and
2-naphthyl (18) were less active than the thienyl (3)
carboxylic ester, implying that extension of the ring
area, which might have improved binding by stacking
interactions, was not effective. Fusing a third ring,
however, as in 9-anthryl (19) and 9-fluorenyl (20)
carboxylic esters, did increase potency; they can be
considered as ring-fused analogues of the diphenyl
analogue 8 and are some 5 times more potent than 8.
For 19, the increased potency accords with the increase
in lipophilicity (extra CH2 group), but 20 is of interest
since it has similar lipophilicity to 8, suggesting that
there may be something special about fluorenyl. This
ring system was therefore explored further. The 9-ben-
zyl analogue (21) was approximately 10 times more
potent than 20; however, when this fluorenyl compound
is compared with the triphenyl analogue 11, the im-
provement is seen to be smaller. Nevertheless, fluorene
has an important advantage for synthesis, and the
benzyl group was used to probe for possible electronic
contributions to binding interaction with the channel.
Substituents (MeO, F, Cl, NO2) were therefore intro-
duced into the para position of the benzyl group of the
9-benzyl-9-fluorenyl ester. There was only a small range
of potencies (IC50 ) 2.8-4.6 µM), offering little indica-
tion of an electronic contribution to binding. Since there
was a strong possibility that the ester grouping might
be subject to hydrolytic cleavage in vivo, the ester group
of 21 was also replaced by the electron rich, but
hydrolytically more stable, ethynyl group to give the
aminohydrocarbon 26 (UCL 1608), which was actually
the most active of this series of compounds (IC50 ) 1.5
µM), being approximately 15 times more potent than
cetiedil. The activities of all of these compounds, how-
ever, appear to correlate reasonably well with lipophi-
licity so that no specific structural features stand out.
Considering all 26 compounds (1-26) gives correlation
eqs 3 and 4 (relating to log P values and CLOG P,
respectively):
F igu r e 1. The relationship between the log IC50 (M) values
(Table 3) for compounds 1-26 and the calculated values of
log P (upper) and CLOG P (lower). The lines have been drawn
according to eqs 3 and 4, respectively.
-log IC50 ) 0.574 CLOG P + 1.128
r ) 0.92, n ) 26, F ) 124
(4)
The correlation between log P (determined from f
values) and CLOG P for these structures is reasonably
good as indicated by eq 5. It is interesting to note,
however, that the coefficient for log P is not 1; it appears
that for this group of compounds CLOG P is generally
less than the value of log P estimated by summing
fragmental constants.
CLOG P ) 0.76((0.05) log P + 1.09((0.30) (5)
r ) 0.960, n ) 26
Inspection of the plot of activity versus lipophilicity
in Figure 1 shows that, whereas log P increases over 4
orders of magnitude, potency only increases by 2 orders.
This is energetically very unfavorable. Such a poor
activity yield from the increased lipophilicity suggests
that hydrophobic interactions with the active site on the
channel are probably not the main determinants of
potency. Rather, it seems likely that lipophilicity is
important for access to the active site, presumably
because the compounds have to enter the cell mem-
brane. This is in keeping with earlier work on the
mechanism of action of cetiedil and its congeners.35
A
study35c of how the block varied with the time for which
the cells had been preincubated with the agent showed
that cetiedil reached its maximum action after 20-30
min as compared with 60-120 min for the less lipophilic
hydroxycetiedil (4, UCL 1269). Consistent with this, the
more lipophilic triphenylethanoic ester (11, UCL 1274)
acted rapidly, producing a maximal effect within 3
min.35c This and other evidence35b,c suggested that
cetiedil, in contrast to clotrimazole, acts on the channel
(probably in its open state35c) from within the membrane
rather than at its outer or inner surfaces.
-log IC50 ) 0.455 log P + 1.638
r ) 0.91, n ) 26, F ) 121
(3)

Blockers of the Ca2+-Activated K+ Permeability
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3249
In accord with this, the most likely explanation of the
lack of activity of the methoiodide derivative (27) of
cetiedil is the inability of this permanently charged
compound to enter the membrane and hence to reach
the site of action. However, cetiedil methoiodide differs
from cetiedil in another important way, namely, the
absence of a dissociable proton. This raises the possibil-
ity that the N+-H group in cetiedil could either act as
a hydrogen-bond donor or become deprotonated to give
the conjugate base, which might be imagined to be the
active form, or essential for access to the site of action.
Recent work on a related type of IKCa blocker suggests
that the latter mechanisms are unlikely. It was found17
that certain bisquinolino and bisquinolinium derivatives
of the SKCa blocker dequalinium (Chart 1) are potent
inhibitors of the IKCa-mediated loss of K+ from rabbit
red cells, studied by the same methods as used in the
present work. One of the most active of the series is the
dioxy analogue33d 1,10-bis(quinolin-4-yloxy)decane (28)
(Chart 1), which had an IC50 of 1.22 ( 0.04 µM. The
calculated log P for this compound is 7.93 and eq 3
predicts a corresponding IC50 of 5.2 µM. Since there are
two basic nitrogen centers in this symmetrical molecule,
which on statistical grounds would make a log difference
of 0.3 if it is assumed that either center can interact
with the channel, the result is in fair agreement.
Furthermore, the characteristics of the blocking action
of these bisquinolino compounds closely resemble those
of cetiedil but not clotrimazole (Chart 1). Thus, although
28 has a very different chemical structure from cetiedil,
it appears to act in the same way. The N,N-dimethyl
quaternary ammonium salt (29) of this compound was
also active,17 but much less so (IC50 of 78 ( 15 µM);
however, it still has approximately one-third of the
potency of cetiedil, and since it does not possess an NH+
group, this finding shows that the molecule does not
have to be a hydrogen-bond donor or become depro-
tonated in order to be able to block the channel. By
implication, neither does cetiedil. The potency difference
between compounds 28 and 29 (a factor of approxi-
mately 65) accords with the idea that a molecule that
is permanently doubly charged would be much less able
to penetrate the cell membrane. In contrast, the cetiedil
cation can dissociate a proton to give the uncharged and
highly lipophilic, and therefore membrane permeable,
conjugate base. The base becomes reprotonated when
it leaves the lipophilic region to act in its positively
charged form on the channel.
in developing IKCa channel blockers that act in the same
way as clotrimazole but with greater potency and
selectivity,10b,c the potential value of having several
different pharmacological classes of IKCa blocker avail-
able for immunosupression, as well as for the possible
treatment of sickle cell disease, makes this a goal worth
pursuing.
Exp er im en ta l Section
Melting points were determined in open glass capillary
tubes with an Electrothermal electrically heated copper block
apparatus and are uncorrected. IR spectra were recorded on
a Perkin-Elmer 983 spectrometer. 1H NMR spectra were
recorded at 200 MHZ on a Varian VXR 400 spectrometer in
solutions of CDCl3 using TMS as an internal reference.
Multiplicities are reported as (s) singlet, (d) doublet, (t) triplet,
(q) quartet, and (m) multiplet. Assignments of hydroxyl and
ammonium protons were checked by deuterium exchange.
Mass spectra were recorded on a VG 7070H double focusing
mass spectometer interfaced with a Finnagen INCOS data
system. Thin-layer chromatography was carried out on glass
plates (Merck Kiesegel 60 F254) and column chromatography
used Merck 7734 silica gel (63-20 mm). Preparative and
analytical HPLC was performed using a Gilson binary grading
system with an LB diode array detector, set at either 215, 240,
or 254 nm. Elemental analyses were determined in house by
A. A. T. Stones and were within (0.4% unless otherwise
indicated.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
2-12 (Sch em e 1). The appropriately substituted carboxylic
acid (9 mmol) was added to dry DMF (50 mL) containing a
stirred suspension of NaH (0.4 g, 10 mmol as a 60% dispersion
in mineral oil). 1-(2-Chloroethyl)-hexahydro-1H-azepine hy-
drochloride (2.0 g, 10 mmol from Aldrich) in dry DMF (50 mL)
containing NaH (0.4 g, 10 mmol) as a 60% dispersion in
mineral oil was added. The mixture was stirred at room
temperature overnight (approximately 20 °C) and then filtered
and concentrated under reduced pressure. Water (30 mL) was
added to the resulting oil and the mixture was extracted (3×)
with ethyl acetate. The combined extracts were dried (Na2-
SO4) and concentrated, and the resulting oil was dissolved in
methanol, treated with oxalic acid, and diluted with 5 volumes
of ethyl acetate to yield the product as a hydrogen oxalate,
which was washed well with ether and then recrystallized
(solvent indicated in Table 1). Alternatively, the oily residue
was dissolved in chloroform and treated with anhydrous HCl
gas; the chloroform was removed under reduced pressure and
the resulting oil was crystallized from EtOAc:EtOH (Table 1)
to afford the product as hydrochloride. Yields 40-50%.
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl cycloh exyleth a n o-
a te h yd r och lor id e h em ih yd r a te (2): 1H NMR (200 MHz,
CDCl3) δ 4.6 (t, J ) 3.2 Hz, 2H, OCH2), 3.6-3.0 (m, 4H, NCH2),
2.2 (d, J ) 7.6 Hz, 2H, CH2CO), 2.15-0.8 (m, 19H, CCHC).
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl 3-th ien yleth a n oa te
h yd r och lor id e (3): 1H NMR (200 MHz, CDC13) δ 12.5 (s,
1H, NH), 7.25-7.0 (m, 2H, thienyl), 7.1 (s, 1H, thienyl) 4.5 (t,
J ) 5.8 Hz, 2H, OCH2), 3.75 (s, 2H,CH2CO), 3.25-2.8 (m, 4H,
NCH2).
The findings of the present work together with these
mechanistic considerations suggest that cetiedil and its
congeners act in the charged form but reach their site
of action from within the membrane. Viewed in this
light, cetiedil methoiodide is ineffective because, when
tested at practicable concentrations, not enough of it can
reach the membrane region from which the compounds
gain access to the channel. Nevertheless, the effect of
the positive charge can be offset if the rest of the
molecule is sufficiently lipophilic. More generally, our
work also shows that designing compounds that act
specifically at the cetiedil site associated with the IKCa
channel is a challenging task because of the conflicting
requirements of lipophilicity (with its concomitant
reduction in aqueous solubility) and the presence of a
positive charge. Although much progress has been made
2-(Hexah ydr o-1H-azepin -1-yl)eth yl cycloh exylh ydr oxy-
3-th ien yleth a n oa te h yd r och lor id e (4): 1H NMR (400 MHz,
CD3OD) δ 7.41 (s, 1H, thienyl-2H), 7.31 (m, 1H, thienyl-4H),
7.20 (dd, 1H, thienyl-4H) 4.44 (t, J ) 2.5 Hz, 2H, OCH2), 3.47-
3.31 (m, 6H, NCH2), 2.1-1.0 (m, 19H, CCH2C); IR (Nujol mull)
ν 3226 (OH), 1736 (CdO), 732 (thienyl) cm-1
.
2-(Hexa h ydr o-1H-a zep in -1-yl)eth yl Cycloh exyl-3-fu r yl-
h yd r oxyeth a n oa te Hyd r ogen Oxa la te (5). 2-Cyclohexyl-
2-(3-furyl)ethanoic acid was synthesized according to the
procedure described23 for 2-cyclohexyl-2-(3-thienyl)ethanoic
acid. Thus 3-lithiofuran was prepared from 3-bromofuran (4.85
g, 33 mmol) in anhydrous Et2O (80 mL) at -78 °C in a nitrogen
atmosphere and n-butyllithium in hexane (6.6 mL, 60 mmol.
Then, 2-cyclohexyl-2-ketoethanoic acid23 (3.68 g, 24 mmol) in

3250 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Roxburgh et al.
dry Et2O (40 mL) at -78 °C was added and the mixture was
stirred for 5 h and then warmed to -10 °C and hydrolyzed
with ice. The aqueous layer was separated off, acidified with
2 N HCl, and extracted with Et2O. The combined extracts were
dried and evaporated and the residue was crystallized from
petroleum spirits to give 2-cyclohexyl-2-(3-furyl)ethanoic acid,
mp 131-133 °C; yield 1.8 g (33%). The corresponding amino-
Et3N (0.63 g, 6.2 mmol) was treated with n-butyl chloroformate
(0.9 mL, 7.1 mmol) with stirring for 30 min. Then 1-(2-
aminoethyl)hexahydro-1H-azepine (0.89 g, 6.2 mmol, prepared
from 1-(2-chloroethyl)-hexahydro-1H-azepine and 0.880 am-
monia in EtOH) was added and the mixture was stirred at
-20 °C for 5 h and then allowed to warm to 0 °C and basified.
The THF layer was separated, dried, and concentrated to yield
the product as an oil which was chromatographed to 95%
purity by preparative HPLC and isolated as an oily trifluoro-
acetate (yield 0.49 g, 23%): 1H NMR (400 MHz, CDCl3) δ 8.3
(s, 1H, NH), 7.2-7.05 (m, 3H, thienyl), 3.5-3.39 (m, 4H,
NCH2), 3.3 (s, 1H, NH), 3.21 (d, J ) 10.4 Hz, 1H, thienyl
CHCO), 3.2-2.67 (m, 4H, NCH2), 1.9-0.7 (m, 19H, CCH2C);
IR (CDCl3) ν 3607 (NH), 3293 (N+H), 1665 (CdO), 734 (thienyl)
cm-1. Anal. (C20H32N2OS‚CF3CO2H‚1.5H2O) C, H; N: calcd,
5.7; found, 5.1.
1
ethyl ester had H NMR (400 MHz, CDCl3) δ 7.5 (s, 1H, furyl-
5H), 7.4 (s, 1H, furyl-2H), 6.4 (s, 1H, furyl-4H), 4.55-3.20 (m,
8H, OCH2, NCH2), 2.0-1.1 (m, 19H, CCH2C); IR (KBr) ν 3439
(OH), 1734 (CdO), 1639 (furyl) cm-1
.
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl d icycloh exyleth -
a n oa te h yd r och lor id e (6): 1H NMR (200 MHz, CDC13) δ
4.6 (t, 2H, OCH2), 3.6-3.1 (m, 6H, NCH2), 2.2-2.1 (m, 1H,
CHCO), 2.0-0.8 (m, 30H, CCH2C).
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl cycloh exylp h en yl-
eth a n oa te h yd r och lor id e (7): 1H NMR (400 MHz, CDCl3)
δ 7.3 (m, 5H, Ph), 4.6 (m, 2H, OCH2), 3.3 (m, 2H, NCH2) 3.25
(d, J ) 10.6 Hz, 1H, COCH), 3.15 (m, 2H, NCH2), 2.7-2.5 (m,
2H, NCH2), 2.05-0.7 (m, 19H, CCH2C).
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl d ip h en yleth a n oa te
h yd r och lor id e (8): 1H NMR (400 MHz, CDCl3) δ 7.5-7.3 (m,
10H, Ph), 5.2 (s, 1H, PhCHCO), 4.7 (t, 2H, OCH2), 3.3-2.6
(m, 6H, NCH2), 2.0-1.5 (m, 8H, CCH2C).
2-(Hexa h yd r o-1H-a zep in -1-yl)et h yl 3,3-d ip h en ylp r o-
p ion a te h yd r ogen oxa la te (9): 1H NMR (400 MHz, DMSO-
d6) δ 7.3-7.1 (m, 10H, Ph), 4.4 (t, J ) 7.9 Hz, 1H, PhCH), 4.2
(t, J ) 5.0 Hz, 2H, OCH2), 3.2 (t, J ) 5.2 Hz, 2H, CH2N), 3.15
(d, J ) 8.1 Hz, 2H, CH2CO), 3.1 (m, 4H, NCH2), 1.7-1.0 (m,
8H, CCH2C).
1-[2-(6,6,6-Tr ip h en yl-3-oxa -1-h exa n yl)]h exa h yd r o-1H-
a zep in e Hyd r ogen Oxa la te (15) (Sch em e 6). Ethyl 3,3,3-
triphenylpropionate (1 g, 3.16 mmol) in dry THF (10 mL) was
added during 20 min to a stirred suspension of LiA1H4 (0.48
g, 12 mmol) in dry THF (20 mL) at 0 °C. The mixture was
stirred at 20 °C for 5 h and then excess LiA1H4 was decom-
posed by the cautious addition of aqueous KOH (5 mL of 20%
w/v). The mixture was filtered and the organic layer was dried
(MgSO4) and evaporated. The resulting oil was crystallized
from hexane to afford 0.6 g (66% yield) of 3,3,3-triphenylpro-
pan-1-ol, mp 104-106 °C (lit.41 mp 106-108 °C).
1-(2-Choroethyl)-hexahydro-1H-azepine hydrochloride (0.27
g, 1.39 mmol) in dry DMF (15 mL) was added dropwise to a
stirred suspension of NaH (0.066 g, 2.77 mmol) in dry DMF
(15 mL) at 20 °C. After 10 min, a solution of 3,3,3-triphenyl-
propan-1-ol (0.40 g, 1.39 mmol) in dry DMF (10 mL) was added
slowly and the stirred mixture was heated at 115 °C for 20 h
in a nitrogen atmosphere. The mixture was then filtered, the
solvent was removed under reduced pressure, and the result-
ing oil was purified by column chromatography using ethanol
to give the amine product. The latter was treated with oxalic
acid in ethanol and then with ether to form the insoluble
hydrogen oxalate; this was collected, washed with ether, and
recrystallized twice from ethanol to afford the pure product
(0.6 g, 66% yield): mp 180-183 °C; 1H NMR (200 MHz, CD3-
OD) δ 7.21-7.12 (m, 15H, Ph), 3.32 (t, 2H, PhCCH2), 3.13 (dd,
2H, OCH2), 2.91 (dd, 2H, OCH2), 2.63 (m, 6H, NCH2), 1.57 (s,
8H, CCH2C). Anal. (C28H36NO‚1.25C2H2O4) C, H, N.
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl 1-Ad a m a n tyleth -
a n oa te Hyd r och lor id e (16) (Sch em e 2). 1-(2-Hydroxyethyl)-
hexahydro-1H-azepine (1.44 g, 10 mmol) in dry CHCl3 (20 mL)
was added dropwise over l h to 1-adamantylcarboxyl chloride
(2 g, 10 mmol, from Aldrich) in dry CHCl3 (100 mL) at 0 °C
with stirring. The mixture was evaporated under reduced
pressure and the resulting oil was taken into EtOAc: EtOH
(5:1) and allowed to crystallize in a refrigerator. The product
was collected and recrystallized to yield 16 (0.88 g, 26%
yield): mp 203-205 °C; 1H NMR (400 MHz, CDCl3) δ 12.5 (s,
1H, NH), 4.6 (t, J ) 5.1 Hz, 2H, OCH2), 3.55-3.0 (m, 6H,
NCH2), 2.3-1.5 (m, 23H, CH). Anal. (C19H31NO2‚HCl) C, H,
N, Cl.
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl p h en yl-3-th ien yl-
eth a n oa te h yd r ogen oxa la te (10): 1H NMR (400 MHz,
CDCl3) δ 7.5-7.0 (m, 8H, Ph, thienyl), 5.3 (s, 1H, PhCHCO),
4.4 (m, 2H, OCH2), 3.3-3.03 (m, 6H, NCH2), 1.65-1.5 (m, 8H,
CCH2); IR (Nujol mull) ν 3445 (N+H), 1733 (CdO), 712
(thienyl) cm-1
.
2-(Hexah ydr o-1H-azepin -1-yl) 2,2,2-tr iph en yleth an oate
h yd r och lor id e h em ih yd r a te (11): 1H NMR (200 MHz,
CDCl3) δ 7.4-7.2 (m, l5H, Ph), 4.8 (bs, 1H, NH), 4.3 (t, 2H,
OCH2), 2.7 (t, 2H, CH2N), 2.5 (m, 4H, NCH2), 1.6-1.4 (m, 8H,
CCH2).
2-(Hexa h yd r o-1H-a zep in -1-yl)et h yl 3,3-d ip h en ylp r o-
p ion a te h yd r ogen oxa la te (12): 1H NMR (400 MHz, DMSO-
d6) δ 7.3-7.2 (m, l5H, Ph), 4.0 (t, 2H, OCH2), 3.8 (s, 2H,
CH2CO), 3.0 (m, 6H, NCH2), 1.7-1.5 (m, 8H, CCH2C); IR
(Nujol mull) ν 3440 (N+H), 1744 (CdO) cm-1
.
3-(Hexa h yd r o-1H-a zep in -1-yl)p r op yl 2,2,2-Tr ip h en yl-
eth a n oa te Hyd r och lor id e (13) (Sch em e 2). 3-Chloropro-
panol (1.5 g, 16 mmol) was heated with hexahydroazepine (1.6
g, 16 mmol) in DMF (50 mL) with anhydrous K2CO3 (3 g) under
reflux for 72 h, filtered, cooled, treated with decolorizing
charcoal, and evaporated under reduced pressure to afford 1-(3-
hydroxypropyl)hexahydro-1H-azepine, which was purified by
column chromatography using a EtOAc:MeOH (1:4) mixture.
Triphenylethanoic acid (1.2 g, 4.2 mmol) in dry THF (40 mL)
was treated with freshly distilled thionyl chloride (10 mL) and
then heated under reflux for 6 h. The mixture was evaporated
under reduced pressure and the residue was crystallized from
cyclohexane to afford triphenylethanoyl chloride, mp 129-131
°C (yield 0.41 g, 32%). The latter (13 mmol), in dry Et2O (20
mL), was added with stirring to 1-(3-hydroxypropyl)hexahydro-
1H-azepine (0.2 g, 13 mmol) in dry Et2O (20 mL), and the
mixture was heated under reflux for 2 h and then cooled and
filtered to afford the hydrochloride 13 (0.46 g), after being
recrystallized from EtOH:Et2O (6:1) mixture: mp 214-215 °C
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
17-20. 1-(2-Chloroethyl)-hexahydro-1H-azepine hydrochloride
(2.3 g, 11 mmol) was added to a solution of Na (0.26 g, 11
mmol) in dry 2-propanol (30 mL) and the mixture was stirred
for 10 min. The carboxylic acid (5.8 mmol) was added and the
mixture was heated under reflux for 24 h. The mixture was
then cooled, filtered, and evaporated under reduced pressure;
the resulting residue was dissolved in 2 N NaOH (20 mL) and
extracted with CHCl3 (4 × 20 mL). The combined extracts were
concentrated and the resulting oil was purified by column
chromatography eluting with CHCl3:MeOH (100:1). The re-
sulting purified oil in EtOH was treated with anhydrous HCl,
and the resulting hydrochloride salt was collected and recrys-
tallized from EtOH. The fluorenyl derivative (20) was too
hygroscopic and was therefore converted into the hydrogen
oxalate and recrystallized from EtOH.
1
(dec); yield 0.14 g (25%); H NMR (400 MHz, CDCl3) δ 7.3-
7.2 (m, 15H, Ph), 4.3 (t, 2H, OCH2), 2.6-2.1 (m, 6H, NCH2),
2.0-1.5 (m, 10H, CCH2C). Anal. (C29H33NO2‚HCl‚0.25H2O) C,
H, N, Cl.
N-[2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl]-2-cycloh exyl-2-
(3-th ien yl)eth a n oa m id e Tr iflu or oa ceta te Sesqu ih yd r a te
(14) (Sch em e 5). 2-Cyclohexyl-2-(3-thienyl)ethanoic acid23 (1.5
g, 6.2 mmol) in freshly distilled THF (30 mL) at -20 °C with

Blockers of the Ca2+-Activated K+ Permeability
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3251
2-(Hexa h yd r o-1H-a zep in -l-yl)eth yl 1-n a p h th oa te h y-
d r och lor id e (17): 1H NMR (400 MHz, CD3OD) δ 8.92-7.55
(m, 7H, naphthyl), 4.77 (t, 2H, OCH2), 3.68-3.48 (m, 6H,
CH2N), 1.94-1.74 (m, 8H, CCH2C).
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl 2-n a p h th oa te h y-
d r och lor id e (18): 1H NMR (400 MHz, CD3OD) δ 8.71 (s, 1H,
naphthyl-1H), 8.05-7.77 (m, 6H, naphthyl 4.74 (t, 2H, OCH2),
3.69-3.05 (m, 6H, NCH2), 1.96-1.75 (br s, 8H, CCH2C).
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl 9-a n th r a cen eca r -
boxyla te h yd r och lor id e h em ih yd r a te (19): 1H NMR (400
MHz, DMSO-d6) δ 8.72 (s, 1H, anthracenyl-10H), 8.15 (m, 5H,
Ar-H), 7.58 (m, 5H, Ar-H), 4.64 (t, J ) 6.0 Hz, 2H, OCH2),
2.87 (br t, 2H, NCH2) 2.72 (br t, J ) 5.7 Hz, 4H, NCH2), 1.64-
1.56 (m, 8H, CCH2C).
1-[(9-Ben zyl)flu or en -9-yl]-4-(h exa h yd r o-1H -a zep in -1-
yl)bu t-2-yn e Hyd r ogen Oxa la te (26). (Sch em e 7). Aqueous
propargyl alcohol (8.1 g of 39% solution, 143 mmol), formal-
dehyde (28 mL of 37% aqueous solution), hexahydroazepine
hydrochloride (21.8 g, 161 mmol), and CuCl were heated
together at 100 °C for 4 h. The mixture was then cooled,
acidified (2 N HCl), and washed with CHCl3 (3 × 20 mL). The
aqueous layer was basified (K2CO3 and 33% NH4OH) and
extracted with CHCl3 (10 × 20 mL). The combined organic
extracts were dried (MgSO4) and evaporated to an oil, which
was purified by column chromatography eluting with CHCl3:
MeOH (4:1) to afford 4-(hexahydro-1H-azepin-1-yl)but-2-yn-
1-ol (18 g, 75% yield).
To the above aminobutynol (5.15 g, 31 mmol) in CH2Cl2 (12
mL), cooled to 0 °C, was added dropwise thionyl chloride (4.0
g, 30 mmol) in CH2Cl2 (3 mL), and the mixture was left at 20
°C for 6 h. The mixture was then basified by cautious addition
of NH4OH and extracted with CHCl3 (4 × 20 mL). The
combined extracts were dried (MgSO4) and evaporated to an
oil, which was purified by column chromatography eluting with
Et2O to give 1-chloro-4-(hexahydro-1H-azepin-1-yl)but-2-yne
(3.61 g, 63% yield).
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl 9-flu or en ylca r box-
yla te h yd r ogen oxa la te (20): 1H NMR (400 MHz, DMSO-
d6) δ 7.89-7.36 (m, 8H, Ar-H), 5.13 (s, 1H, fluorenyl-9H), 4.38
(t, J ) 6 Hz, 2H, OCH2), 3.10 (br s, 6H, NCH2), 1.68-1.54 (m,
8H, CCH2C).
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
21-25 (Sch em e 4). 9-Carboxyfluorene (3.7 g, 17 mmol) was
added to a stirred suspension of Na metal (0.85 g, 37 mmol)
in liquid ammonia37 (100 mL) and then anhydrous Et2O (65
mL) was added. After 1 h the appropriate p-substituted benzyl
chloride (18 mmol) was added (for 23, p-fluorobenzyl bromide
was used) and stirring was continued for 4 h. The liquid
ammonia was removed, water (35 mL) was added, and the
aqueous phase was washed with Et2O (2 × 35 mL) and
acidified with 2 N HCl, and the resulting precipitate was
collected dried and crystallized from EtOAc:petroleum ether
mixture to give the 9-(p-substituted benzyl)-9-carboxyfluorene
in 21-62% yield. The following melting points were obtained
[substituent (% yield), mp (°C), analysis]: H (61%), 195-199
(lit.42 mp 204-205); OMe (21%), 200-202, C22H18O3; F (62%),
l95-196, C21H15FO2; Cl (37%), 190-191, C21H15ClO2; NO2
(51%), 201-203, C21H18NO4.
1-(2-Chloroethyl)hexahydro-1H-azepine hydrochloride (2.34
g, 11.6 mmol) was added to a solution of Na metal (0.27 g,
11.6 mmol) in anhydrous 2-propanol (35 mL) and the mixture
was stirred at 20 °C for 30 min. The 9-(p-substituted benzyl)-
9-carboxyfluorene (5.8 mmol) was added and the mixture was
heated with stirring under reflux for 36 h. The mixture was
then cooled, filtered, and evaporated under reduced pressure;
the resulting residue was dissolved in 2 N NaOH (20 mL) and
extracted with CHCl3 (4 × 20 mL). The combined extracts were
concentrated and the residual oil was purified by column
chromatography eluting with CHCl3:MeOH (100:1). The re-
sulting purified oil in Et2O (10 mL) was treated with anhy-
drous oxalic acid (1 mmol) in MeOH (0.5 mL) to give a
precipitate that was crystallized from the solvent indicated
in Table 2 to afford the corresponding product, 2-(hexahydro-
1H-azepin-1-yl)ethyl 9-(p-substituted benzyl) fluorene-9-car-
boxylate hydrogen oxalate (yield 30-45%).
9-Benzylfluorene (1.5 g, 5.7 mmol) was cautiously added in
portions to NaH (0.3 g, 7.5 mmol, of 60% dispersion in mineral
oil) in dry DMSO (4 mL) under a nitrogen atmosphere. The
mixture was stirred for 15 min at 20 °C and then the above
chlorobutyne (1.07 g, 5.7 mmol) in dry DMSO (1.5 mL) was
added, and the mixture was stirred for 1 h at 20 °C. Water
(10 mL) was then added and the mixture was extracted with
Et2O (5 × 10 mL). The combined ether extracts were washed
(5 × 10 mL), dried (MgSO4), and concentrated to afford an oil,
which was purified by column chromatography (SiO2 and Et2O:
petroleum ether, 3:2) to give the product (26, base, 1.0 g, 43%
yield). A portion of the latter (0.2 g) in Et2O (7 mL) was added
to a solution of anhydrous oxalic acid (0.053 g) in MeOH (0.4
mL), and the resulting precipitate was collected by filtration
and crystallized from 2-PrOH to give the product 26 hydrogen
oxalate: mp 153-155 °C (yield 0.17 g, 71% recovery); recrys-
tallization did not alter the melting point; 1H NMR (400 MHz,
DMSO-d6) δ 7.69-6.54 (m, 13H, Ar-H), 3.52 (s, 2H, PhCH2),
3.36 (s, 2H, CtCCH2) 3.08 (s, 2H, NCH2), 2.44 (m, 4H, NCH2),
1.49 (m, 8H, CCH2C); IR (Nujol mull) v 2248 (w, CtC) cm-1
Anal. (C30H31N‚0.8C2H2O4‚0.25C3H7OH) C, H, N.
.
2-(Hexa h yd r o-1H-a zep in -1-yl)eth yl 2-Cycloh exyl-2-(3-
th ien yl)eth a n oa te Meth oiod id e (Cetied il Meth oiod id e,
27). Cetiedil base (0.5 g, 1.43 mmol) in dry Et2O (20 mL) at
-20 °C was treated with methyl iodide (0.22 g, 1.55 mmol)
and then left for 12 h at 0 °C.
Methanol (20 mL) was added and the mixture was heated
at 50 °C for 4 h and then evaporated under reduced pressure.
The resulting solid was crystallized from EtOAc:EtOH (1:1)
to give the product (0.38 g, 54% yield): mp 144-145 °C; IR
(Nujol mull) ν 1732 (CdO), 728 (thienyl) cm-1. Anal. (C21H34
NO2S‚I) C, H, N, I.
-
21: 1H NMR (400 MHz, CD3OD) δ 7.63-6.68 (m, 13H, Ar-
H), 4.34 (t, 2H, OCH2), 3.46 (s, 2H, PhCH2), 3.30 (m, 4H,
NCH2), 2.21 (m, 2H, NCH2), 1.52 (m, 8H, CCH2C).
22: 1H NMR (400 MHz, DMSO-d6) δ 7.71-7.34 (m, 8H, Ar-
H), 6.52 (d, J ) 8.8 Hz, 2H, C6H4), 6.43 (d, J ) 8.8 Hz, 2H,
C6H4), 4.26 (t, J ) 4.0 Hz, 2H, OCH2), 3.61 (s, 2H, PhCH2)
3.53 (s, 3H, OCH3), 3.10 (t, J ) 4.0 Hz, 2H, NCH2), 1.49 (m,
8H, CCH2C).
23: 1H NMR (400 MHz, DMSO-d6) δ 7.68-7.35 (m, 8H, Ar-
H) 6.67 (t, 2H, C6H4), 6.57 (dd, 2H, C6H4), 4.27 (t, 2H, OCH2),
3.69 (s, 2H, PhCH2), 3.10 (t, 2H, NCH2) 2.77 (m, 4H, NCH2),
1.49 (m, 8H, CCH2C).
P h a r m a cology. Blood (2-5 mL) was drawn from the ear
vein of adult New Zealand white rabbits and mixed with
heparin (20 units/mL whole blood). After centrifugation (3 min
at 1600g) the supernatant and buffy coat were aspirated and
discarded, leaving a pellet of packed cells. This was then
resuspended in 5 volumes of a Ca2+-free solution containing
(in mM) NaC1, 145; KCl, 0.1; MgSO4, 1; EDTA, 1; TRIS, 10;
inosine, 10; pH adjusted to 7.4 by adding 1 M NaOH. The
suspension was centrifuged as before, and the pellet resus-
pended twice. After the final centrifugation, the cells were
stored as a pellet in this solution at 4 °C, for up to 4 days.
24: 1H NMR (400 MHz, DMSO-d6) δ 7.69-7.35 (m, 8H Ar-
H), 6.90 (d, J ) 8.4 Hz, 2H, C6H4), 6.55 (d, J ) 8.4 Hz, 2H,
C6H4), 4.27 (t, J ) 4.5 Hz, 2H, OCH2), 3.70 (s, 2H, PhCH2),
3.10 (t, J ) 4.5 Hz, 2H, NCH2) 2.77 (m, 4H, NCH2), 1.49 (m,
8H, CCH2C).
25: 1H NMR (400 MHz, DMSO-d6) δ 7.72-7.37 (m, 10H,
Ar-H + C6H4), 6.82 (d, J ) 8.8 Hz, 2H, C6H4), 4.28 (t, J ) 3.9
Hz, 2H, OCH2), 3.88 (s, 2H, PhCH2), 3.10 (t, J ) 4.3 Hz, 2H,
NCH2) 2.77 (m, 4H, NCH2), 1.49 (m, 8H, CCH2C).
The assays were done using a solution containing (mM)
NaCl, 145; KCl, 0.1; MgSO4 1; CaCl2 1; inosine 10; TRIS 10
(pH adjusted to 7.4 by the addition of 1 M NaOH). Because
earlier experiments35c had shown that the action of cetiedil
develops slowly (half-time 5 min), the cells were incubated with
the test compounds (with the exception of 21-26) for 40-60
min before the tests proper. This period was extended to 120
min in measurements with compound 4 (UCL 1269). The test
compounds were added as a small volume (typically e 4 µL)

3252 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
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mL of the low-K+ solution at 37 °C; hematocrit ∼1%). The cell
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