Soft drugs. 12. Design, synthesis, and evaluation of soft bufuralol analogues

Article abstract of DOI:10.1021/jm9904654

In the search for more potent but still short-acting β-blockers (BB), the methyl, ethyl, isopropyl, tert-butyl, cyclohexyl, 2-(1-adamantyl)ethyl, and methylthiomethyl esters of the acidic inactive metabolite of bufuralol were synthesized based on the 'ina

Full text of DOI:10.1021/jm9904654

J . Med. Chem. 2000, 43, 1525-1532
1525
Soft Dr u gs. 12. Design , Syn th esis, a n d Eva lu a tion of Soft Bu fu r a lol An a logu es
Sung-Kwan Hwang,† Attila J uhasz,‡ Sung-Hwa Yoon,† and Nicholas Bodor*,#
Department of Molecular Science and Technology, Ajou University, Suwon 442-749, Korea, 1st Department of Medicine,
University Medical School of Debrecen, Hungary, and Center for Drug Discovery, J . Hillis Miller Health Center, College of
Pharmacy, University of Florida, Gainesville, Florida 32611
Received September 13, 1999
In the search for more potent but still short-acting â-blockers (BB), the methyl, ethyl, isopropyl,
tert-butyl, cyclohexyl, 2-(1-adamantyl)ethyl, and methylthiomethyl esters of the acidic inactive
metabolite of bufuralol were synthesized based on the “inactive metabolite” approach. The
cleavage of the ester bond by blood and tissue esterases rapidly deactivates these compounds,
resulting in an ultrashort duration of action. The â-antagonist potencies and time courses of
actions of the new “soft” BBs were characterized by recording ECG and intra-arterial blood
pressure (BP) in rats. In the isoproterenol-induced tachycardia model, while bufuralol at an iv
dose of 1 mg/kg (3.8 µmol/kg) diminished heart rate (HR) for at least 2 h, the effects of the soft
drugs lasted for only 10-30 min at equimolar dose. The inactive metabolite did not decrease
HR significantly. The first four members of this series of compounds showed the highest
â-blocking potencies, ranging between 25% and 50% of that of bufuralol. Next, the effects of
these most active compounds on resting HR and BP were evaluated in comparison to esmolol.
Infused for 10 min at a rate of 20 µmol/kg/min, esmolol decreased HR and mean arterial pressure
(MAP) by 40% and 60%, respectively. The soft drugs at doses ranging only between 2 and 4
µmol/kg/min resulted in a 20-40% decrease in HR and a 30-50% reduction in MAP. However,
the time courses of both the bradycardic and hypotensive effects of the soft drugs were
superimposable to that of esmolol, diminishing within 60 min after the discontinuation of the
infusions.
In tr od u ction
â-Blockers are widely used in the treatment of various
cardiovascular diseases,1-7 including angina pectoris,
hypertension, and cardiac arrhythmias. Their efficacy
and safety have also been well-established in reducing
the risk of mortality and nonfatal reinfarction in
survivors of acute myocardial infarction.1 However, the
use of â-blockers in seriously ill patients is limited,
because of potentially adverse effects (i.e. bradycardia,
F igu r e 1. Chemical structures of esmolol and bufuralol.
hypotension, aggravation of heart failure, and broncho-
spasm). The ultrashort-acting â-antagonist esmolol is
often used to control acute supraventricular arrhyth-
mias, myocardial ischemia (acute myocardial infarction
and unstable angina), and perioperative and postopera-
tive hypertension in critically ill patients.8 In the
structure of esmolol (Figure 1), an ethylene-extended
methyl ester group is included, which makes the mol-
ecule susceptible to rapid hydrolysis by esterases.7,9,10
If unwanted side effects occur during esmolol treatment,
one can expect the rapid disappearance of adverse
reactions after the discontinuation of the infusion, as
the terminal half-life of esmolol is short, only 9.2 min.8
The use of â-blockers is often also complicated by their
oxidative metabolic transformation to products with
significant â-receptor-blocking activities but different
biological half-lives.11 For example, bufuraloll2,13 (Figure
1) is a potent, nonselective â-blocker14-16 that has
proven to be very effective in lowering blood pressure
and heart rate. It undergoes a complex series of meta-
bolic transformations in humans to alcohol and ketone
metabolites that also possess significant â-receptor-
blocking activities while having longer half-lives.17-22
The oxidative transformation of bufuralol by the hepatic
cytochrome P450 isozymes is under genetic control and
falls under the debrisoquine/spartein phenotype.23-26
A genetically determined defect of the hydroxylation
occurs in up to 10% of the Caucasian population (poor
metabolizers).26 This situation can further complicate
the pharmacokinetic profile by increasing drug bioavail-
ability and prolonging the elimination half-life so as to
produce more intense and sustained â-blockade27-29
that, in turn, can lead to severe hypotension and brady-
cardia.30
As the soft drug concept is particularly suitable for
addressing the above-mentioned therapeutic prob-
lems,31,32 we previously reported a series of soft â-block-
ers which revealed an ultrashort duration of action and
predictable metabolism by applying the “inactive me-
tabolite” approach.33-36 Continuing this search for more
potent and short-acting â-blockers, we selected the
* Corresponding author. Tel: 352-392-8186. Fax: 352-392-8589.
E-mail: bodor@cop.health.ufl.edu.
† Ajou University.
‡ University Medical School of Debrecen.
#
University of Florida.
10.1021/jm9904654 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/25/2000

1526 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Hwang et al.
Sch em e 1. Structural Features of the Soft Drugs and
Sch em e 3a
Their Metabolic Pathways
Sch em e 2a
a
Reagents: (a) (i) SOCl2, CH2Cl2, reflux, 2 h, (ii) ROH; (b) Br2,
CHCl3, rt, 30 min; (c) B2H6, THF, 0 °C, 30 min; (d) (i) 10 N NaOH,
THF, rt, 1 h, (ii) tert-butylamine, 2-propanol, rt, 36 h.
which was converted into the 7-allyl-2-acetylbenzofuran
(3) by treating of chloroacetone in ethanol.12,38 The
oxidative cleavage of 3 with potassium permanganate/
sodium periodate39 afforded the substituted acetic acid
4 in moderate yield.
After esterification of 4 with methanol, the resulting
methyl ester 5 was reacted with bromine in chloroform40
to give 6, which was then reduced with diborane in
tetrahydrofuran40 to alcohol 7. Epoxidation of 7 followed
by opening of the resulting epoxide with tert-butylamine
in 2-propanol produced 8b.41 Finally, basic hydrolysis
of the methyl ester followed by neutralization provided
the acid 8a .
From 8a we tried to prepare all of the designed
analogues by esterification with the corresponding
alcohols. However, we failed to prepare the bulkier
esters (8e-8i) under various esterification conditions,42-45
and only the ethyl (8c) and isopropyl (8d ) analogues
were obtained in moderate yields. Thus, the bulkier
esters 8e-8i were prepared from 4 (Scheme 3). Since
the benzofuranylacetic acid has low reactivity toward
the acid-catalyzed esterification with the bulky alcohols,
the acid 4 was first converted into the acid chloride with
thionyl chloride and then treated with the corresponding
alcohols to give the desired alkyl esters 9. Subsequently,
the methods used for 8b were applied for the syntheses
of 8e-8g. The methylthiomethyl ester (8h ) was pre-
pared from the reaction of the potassium salt, generated
by base hydrolysis of 8b with equimolar KOH in ethanol
and water,46,47 with methylthiomethyl chloride in ben-
zene45 (Scheme 4).
a
Reagents: (a) tributylamine, SnCl4, (CH2O)n, benzene, 100 °C,
12 h; (b) KOH, ClCH2COCH3, EtOH, reflux, 1 h; (c) KMnO4/NaIO4,
t-BuOH, 70 °C, 24 h; (d) MeOH, H2SO4, reflux, 4 h; (e) Br2, CHCl3,
rt, 30 min; (f) B2H6, THF, 0 °C, 30 min; (g) (i) 10 N NaOH, THF,
rt, 1 h, (ii) tert-butylamine, 2-propanol, rt, 36 h; (h) (i) 2 N KOH,
THF, rt, 2 h, (ii) HCl-ether, obtained as HCl salt form; (i) H2SO4,
ROH, rt, 4 h.
inactive metabolite of bufuralol to design a new series
of short-acting â-blockers in the present study. Seven
different sized alkyl moieties were selected to serve as
the ester functionalities to reactivate the inactive
metabolite of bufuralol. This design strategy considered
the observations that not only the rate of hydrolytic
deactivation can be controlled by the ester structure but
also esterases ubiquitously present in blood and tissues
should quickly hydrolyze the labile ester functionality
to produce the corresponding inactive acetic acid deriva-
tive as shown in Scheme 1. Here, we report the
syntheses, stabilities, and biological evaluations of seven
soft bufuralol analogues (8).
Sta bilities of th e Soft An a logu es in Aqu eou s
Bu ffer Solu tion s. The hydrolysis rates of the soft drugs
at physiological pH ) 7.4 and under basic conditions
pH ) 12 were investigated in order to assess their
chemical stabilities. At physiological pH, the soft ana-
logues 8b-8h did not undergo significant hydrolysis
within 24 h. Alternatively, at pH ) 12 the esters 8b-
8h were readily hydrolyzed to the acidic metabolite 8a .
Resu lts a n d Discu ssion
Ch em istr y. The key intermediate 8a for the synthe-
sis of soft analogues was prepared according to the
methods depicted in Scheme 2. Formylation of 2-allyl-
phenol (1) with paraformaldehyde37 in the presence of
tributylamine and Sn(IV) chloride gave aldehyde 2,

Soft Drugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1527
Sch em e 4a
a
Reagents: (a) KOH, EtOH/H2O (1:1), rt, 2 h; (b) NaI, 18-crown-
6, ClCH2SCH3, benzene, 70 °C, reflux.
Ta ble 1. Chemical Hydrolysis of â-Blocker Estersa
compd
k (min-1
)
t1/2 (min)
C0 (M)
8b (methyl)
8c (ethyl)
8d (isopropyl)
8e (tert-butyl)
8f (cyclohexyl)
8g (2-(1-adamantyl)ethyl)
8h (methylthiomethyl)
0.096
0.084
0.015
0.091
0.045
0.0016
0.027
7.2
8.2
1.2 × 10-5
1.3 × 10-5
1.3 × 10-5
1.4 × 10-5
1.3 × 10-5
1.1 × 10-5
1.1 × 10-5
45.7
75.7
15.2
150.0
25
F igu r e 2. Effects of vehicle, bufuralol, and the soft analogues
of bufuralol on isoproterenol-induced tachycardia in the rat.
Bufuralol (1 mg/kg ) 3.8 µmol/kg) and the soft analogues of
bufuralol (3.8 µmol/kg) were all dissolved in 10% DMSO in
30% HPBCD. The symbols represent the mean values of at
least three animals; error bars are omitted for better visibility.
The cluster analysis of heart rate data established three
different clusters. Between 0 and 5 min the cluster for vehicle
and 8a was significantly different from the clusters of bufuralol
and 8b-8h , while the cluster for bufuralol was different
significantly from those of vehicle and 8a -8h from 10 to 120
min (p < 0.05).
a
Apparent pseudo-first-order hydrolytic rate constants (k), half-
lives (t1/2), and initial concentrations (C0) in 0.01 N sodium
hydroxide at pH ) 12.0 and 37.0 ( 0.1 °C.
Ta ble 2. Enzymatic Hydrolysis of â-Blocker Estersa
compd
k (min-1
)
t1/2 (min)
C0 (M)
8b (methyl)
b
1.2 × 10-5
1.3 × 10-5
1.3 × 10-5
1.4 × 10-5
1.3 × 10-5
1.1 × 10-5
1.1 × 10-5
8c (ethyl)
b
8d (isopropyl)
8e (tert-butyl)
8f (cyclohexyl)
8g (2-(1-adamantyl)ethyl)
8h (methylthiomethyl)
0.0417
0.007
0.111
16.6
99.0
6.2
b
40% decrease in heart rate (Figure 2.). The maximal
bradycardic action of the drug developed after 30 min
and remained stable for the total course of the experi-
ment (up to 120 min) after the bolus injection. The bolus
injection of the soft bufuralol analogues 8b-8h at
equimolar doses (3.8 µmol/kg) resulted in a significant
but temporary decrease (10-20%) in heart rate. The
most active compounds in this model were 8d , 8e, 8b,
and 8c, while 8f-8h (the compounds with the bulkier
ester groups) showed weaker bardycardic actions against
the isoproterenol-induced tachycardia. Bufuralol and
the soft analogues were all administered as an iv bolus
injection, so the kinetics of their effects included first a
distribution and second an elimination phase. While the
primary aim of this study was not a pharmacokinetic
evaluation, one can assume that the disappearance of
the effects of the soft analogues was primarily the result
of the enzymatic inactivation by esterases present in
blood and tissues. In accordance with the soft nature of
these compounds, their bradycardic action lasted for
only 10-30 min. After 30 min, as shown by cluster
analysis, the effects of the soft drugs did not differ
significantly any more from those of either the vehicle
or the inactive metabolite 8a (Figure 2), which even at
a dose of 10 µmol/kg did not decrease heart rate
significantly. On the contrary, while the distribution of
bufuralol had to be very similar after the bolus injection,
its effect lasted for up to 120 min, mainly because of
the differences in its metabolic and elimination proper-
ties.
b
a
Apparent pseudo-first-order hydrolytic rate constants (k), half-
lives (t1/2), and initial concentrations (C0) in rat blood at 37.0 (
0.1 °C. b A half-life is less than 0.15 min, which indicates that more
than 95% of the compound was metabolized in 0.15 min.
The hydrolytic half-lives generally increased with in-
creasing steric hindrance of the alkyl group within each
ester (Table 1). Thus, the 2-(1-adamantyl)ethyl and tert-
butyl analogues showed longer half-lives with 150 and
75.7 min, respectively, while the methyl ester hydro-
lyzed with an ultrashort half-life of 7.3 min.
Meta bolism of th e Soft An a logu es in Biologica l
Med ia . The stability and the metabolic pathway of the
soft drugs in biological media were examined in rat
blood. The metabolism of the soft drugs in rat blood
follows a pseudo-first-order kinetics, and all soft drugs
were converted to the corresponding acid metabolite 8a
without formation of other metabolites. As expected,
compounds 8b, 8c, and 8h which have less sterically
hindered alkyl groups show shorter half-lives then the
bulkier 8d -8f esters (Table 2). These results can be
explained with the facile attack by hydrolytic enzymes
on the less hindered ester linkage. However, similar
differences between chemical and enzymatic cleavage
rate for an 2-(1-adamantyl)ethyl ester 8g were observed
for the soft â-blockers derived from metotrolol.35
Biologica l Eva lu a tion . 1. Effects on Isop r oter -
en ol-In d u ced Ta ch yca r d ia . There were no statisti-
cally significant differences in the baseline heart rate
and blood pressure parameters between the different
groups of rats receiving the different compounds. The
average heart rate increased by 23.68% (from 355.26 (
23.49/min to 439.4 ( 20.59/min) after the sc injection
of 50 µg/kg isoproterenol. Relative to this isoproterenol-
induced tachycardia, the iv bolus injection of bufuralol
at a dose of 1 mg/kg (3.8 µmol/kg) resulted in a 35-
2. E ffect s on R est in g H ea r t R a t e a n d Blood
P r essu r e. Esmolol is an ultrashort-acting â-blocker
that is available as an iv injection formulation. In the
clinical setting esmolol is usually applied at a 500 µg/
kg/min loading dose for 1 min, followed by a titration
phase with stepwise increments of dose between 50 and
300 µg/kg/min to reach the necessary decrease in heart
rate and/or blood pressure. Following this, esmolol is

1528 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Hwang et al.
F igu r e 3. Effects of 0.9% NaCl, compounds 8b-8e, and
esmolol-HCl infusions on resting heart rate. Drugs were
dissolved in 0.9% NaCl and were infused for 10 min. Infusion
rates were 2 µmol/kg/min for 8b, 8d , and 8e, 4 µmol/kg/min
for 8c, and 20 µmol/kg/min for esmolol-HCl. The symbols
represent the mean values of at least three animals; error bars
are omitted for better visibility. Cluster analysis demonstrated
that there was a significant difference between the active drugs
and vehicle for up to 60 min (p < 0.05), while there was no
difference between esmolol-HCl and compounds 8b-8e.
F igu r e 4. Effects of 0.9% NaCl, compounds 8b-8e, and
esmolol-HCl infusions on resting blood pressure (mean arterial
pressure ) MAP). Drugs were dissolved in 0.9% NaCl and were
infused for 10 min. Infusion rates were 2 µmol/kg/min for 8b,
8d , and 8e, 4 µmol/kg/min for 8c, and 20 µmol/kg/min for
esmolol-HCl. The symbols represent the mean values of at
least three animals; error bars are omitted for better visibility.
Cluster analysis demonstrated that there was a significant
difference between the active drugs and vehicle for up to 40
min (p < 0.05), while there was no difference between esmolol-
HCl and compounds 8b-8e.
administered as a continuous infusion of a dose, which
maintains the desired heart rate and blood pressure
control. Because of time limitations in the present
experiments, only a shorter single-dose infusion was
used, resulting in significant heart rate and blood
pressure reductions. However, this period was not long
enough to reach “steady state”. It was expected, how-
ever, that the effects of the soft bufuralol analogues
infused for 10 min with a pump would be similar to that
of esmolol applied in the same way. The most active
compounds 8b-8e, converted into their corresponding
HCl salt forms, were investigated on the resting heart
rate and blood pressure in comparison to esmolol-HCl
and the vehicle (0.9% NaCl solution), respectively. First,
the doses to reach similar heart rate and blood pressure
reductions by the end of the 10-min infusion periods
were determined. It was found that compounds 8b-8e
at a dose of 2 µmol/kg/min and compound 8c at 4 µmol/
kg/min were approximately equipotent with esmolol at
a dose of 20 µmol/kg/min. All the soft bufuralol ana-
logues and esmolol decreased heart rate (by 20-40% on
average) during the course of the infusion (i.e. 10 min).
After the discontinuation of the drug administration,
heart rates gradually returned to baseline values,
mostly within 60 min, and were not significantly dif-
ferent any more from the values of normal saline (Figure
3). Blood pressure changes were similar, but the effect
of esmolol at a dose of 20 µmol/kg/min was more
pronounced, resulting in a 60% decrease in mean
arterial pressure (MAP). Although 8b-8d only de-
creased MAP by 30-40% on average, and 8e resulted
in more than 50% decrease in MAP, these MAP reduc-
tions were obtained at smaller doses (i.e. 2-4 µmol/kg/
min). Again, after the cessation of the infusions, blood
pressure values returned to baseline values (Figure 4).
The kinetics of the heart rate and blood pressure
changes were superimposable on the changes evoked by
esmolol, as the cluster analysis demonstrated that only
the effects of the vehicle (0.9% NaCl) were significantly
different from the active compounds. The soft bufuralol
analogues resulted in a comparable heart rate decrease
at doses of 1/5 to 1/10 that of esmolol. With respect to
their MAP-reducing capabilities they still showed some-
what higher intrinsic activity than esmolol, although
their MAP reduction effect did not reach the extent of
esmolol. The bufuralol analogues apparently possess
â-blocking efficacy at lower doses than esmolol, but only
30-40% decreases in MAP were observed for all, except
one (8e, over 50%), of the new analogues compared to
the 60% decrease obtained with esmolol at a dose of 20
µmol/kg/min.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Deutrated and nondeutrated
solvents were obtained from Aldrich Chemical Co. The solvents
used for HPLC were spectral grade. Melting points were
measured on Fisher J ohns melting point appratus and were
uncorrected. Proton nuclear magnetic resonance spectra were
recorded on a Varian EM 390 spectrophotometer (1H NMR at
200 MHz, 13C NMR at 50 MHz). Chemical shifts are reported
in parts per million units (ppm) on the δ scale downfield from
tetramethylsilane which was used as an internal standard.
The solvents used are given in parentheses for each spectrum
reported. Multiplicities of protons are designated as singlet
(s), double (d), triplet (t), quartet (q) or multiplet (m). Infrared
(IR) spectra were recorded on a Perkin-Elmer 240 spectropho-
tometer. Solid samples were run as either a KBr pellet or a
Nujol mull; liquid samples were analyzed neat as a thin film
between NaCl plates. Mass spectra and elemental analyses
were performed by the Department of Environmental Engi-
neering, Ajou University, Suwon, Korea.
An a lytica l Meth od . The HPLC system used consisted of
a Waters 600 pump, a Rheodyne 7125 injector with a 20-µL
loop, a Spectroflow variable-wavelength UV/VIS detector and
HP 3396 integrator. The soft analogues and the acid metabolite
were analyzed by using Waters normal-phase µ Bondpack
amine column (30-cm × 3.9-mm i.d.) which provided complete
separation of acidic metabolite peak from the ester peak. The
mobile phase included two solvent systems: the isocratic
system of methanol for the chemical hydrolysis and the
gradient system of methanol-water (9:1) for the enzymatic
hydrolysis. The detector wavelength was set at 254 nm. The

Soft Drugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1529
flow rate of the mobile phase was 1 mL/min. The retention
times of the ester compounds were between 2.3 and 2.7 min,
while the retention time of the common acidic metabolite was
6.1 min. The calibration curve was linear (r ) 0.993-0.998)
for the compounds injected over the range of 10-120 µM.
was added Br2 (2.40 mL, 47.5 mmol) dropwise. The reaction
mixture was refluxed for 30 min at room temperature. When
the reddish solution had turned yellow, 50 mL of NaHCO3 was
added. The resulting mixture was stirred for 30 min and then
H2O (20 mL) was added. The reaction mixture was extracted
with chloroform (2 × 50 mL). The organic layer was dried over
anhydrous magnesium sulfate, filtered and evaporated. The
residue was purified by column chromatography on silica gel
(hexane:EtOAc, 4:1) to afford 8.7 g (65%) of 6 as a white solid:
3-Allylsa licyla ld eh yd e (2). To a solution of 2-allylphenol
(100 g, 740 mmol) in benzene (2 L) were added tin(IV) chloride
(16.6 g, 60.0 mmol) and tributylamine (17.4 g, 240 mmol). The
mixture was stirred for 20 min at room temperature, and then
paraformaldehyde (36.0 g, 1.20 mol) was added. The resulting
mixture was heated at 100 °C for 8 h. After cooling, the
reaction mixture was poured into cold water (300 mL) and
acidified to pH 2 with 2 N hydrochloric acid. The aqueous layer
was extracted with ether (3 × 50 mL). The organic layer was
washed with brine, dried over anhydrous sodium sulfate,
filtered and evaporated to give the residue, which was purified
by column chromatography on silica gel (hexane:EtOAc, 4:1)
to afford 61.0 g (50%) of 2 as a yellow oil: mp 45 °C; IR (neat)
3098, 2844, 1684 cm-1; 1H NMR (CDCl3) 3.41 (d, 2H, CH2CHd
CH2, J ) 6.4 Hz), 4.04-5.15 (m, 2H, CHdCH2), 5.90-6.15 (m,
1H, CHdCH2), 6.92-7.00 (m, 1H, ArH), 7.39-7.44 (m, 2H,
ArH), 9.85 (s, 1H, CHO), 11.32 (s, 1H, OH); 13C NMR (CDCl3)
33.0, 116.2, 119.5, 131.8, 135.7, 137.1, 196.6. Anal. (C10H10O2)
C, H.
2-Acetyl-7-a llylben zofu r a n (3). To a solution of 2 (60.0
g, 369 mmol) in absolute ethanol (100 mL) was added pellet
KOH (3.0 g). The resulting mixture was warmed until the
suspension turned into a clear solution. To this was added
chloroacetone (5.00 g, 63.0 mmol) dropwise and the resulting
dark solution was refluxed for 15 min. After the reaction
mixture was poured into 200 mL of ice-water, the ethanol
was removed under reduced pressure. The aqueous layer was
extracted with ether (3 × 50 mL). The organic layer was dried
over anhydrous magnesium sulfate, filtered and concentrated
to give a reddish residue, which was purified by column
chromatography on silica gel (hexane:EtOAc, 20:1) to afford
44.1 g (60%) of 3 as a white solid: mp 43 °C; IR (KBr) 3000,
1683 cm-1; 1H NMR (CDCl3) 2.60 (s, 3H, COCH3), 3.71 (d, 2H,
J ) 6.6 Hz, CH2CHdCH2), 5.1-5.23 (m, 2H, CHdCH2), 6.01-
6.15 (m, 1H, CHdCH2), 7.28-7.31 (m, 2H, ArH), 7.52 (s, 1H,
furan-H) 7.54-7.59 (m, 1H, ArH); 13C NMR (CDCl3) 26.1, 33.3,
112.7, 116.3, 152.3, 188.1. Anal. (C13H12O2) C, H.
mp 93 °C; IR (KBr) 3124, 2953, 1729, 1676 cm-1 1H NMR
;
(CDCl3) 3.75 (s, 3H, OCH3), 4.0 (s, 2H,CH2COO), 4.4 (s, 2H,
COCH2Br), 7.32-7.43 (m, 2H, ArH), 7.65-7.67 (m, 1H, ArH),
7.66 (s, 1H, furan-H); 13C NMR (CDCl3) 30.6, 34.6, 52.1, 114.7,
119.0, 122.5, 124.4, 126.8, 129.6, 150.1, 154.4, 170,7, 182.0.
Anal. (C13H11O4 Br) C, H.
Meth yl [2-(1-Hyd r oxy-2-br om oeth yl)ben zofu r a n -7-yl]-
a ceta te (7). To a solution of diborane (50 mL, 1 M solution in
THF) was added compound 6 (8.10 g, 26.0 mmol) in THF (40
mL) dropwise at 0 °C. The reaction mixture was stirred for 10
min at 0 °C. The reaction was quenched with 10 mL of
methanol, acidified with 1 N HCl, and then extracted with
ether (50 mL). The organic layer was dried over anhydrous
magnesium sulfate, filtered and evaporated. The residue was
purified by column chromatography on silica gel (hexane:
EtOAc, 1:4) to afford 6.5 g (80%) of 7 as a white solid: mp
1
80 °C; IR (KBr) 3499, 3124, 2960, 1736, 1439 cm-1; H NMR
(CDCl3) 3.67-3.84 (m, 2H, CH2Br), 3.69 (s, 3H, OCH3), 3.91
(s, 2H, CH2COO), 5.01-5.06 (m, 1H, CH(OH)), 6.70 (s, 1H,
furan-H), 7.16-7.19 (m, 2H, ArH), 7.43-7.48 (m, 1H, ArH);
13C NMR (CDCl3) 34.8, 35.5, 52.0, 67.8, 104.2, 117.3, 120.6,
122.9, 125.2, 127.6, 153.1, 155.8, 171.4. Anal. (C13H13O4Br)
C, H.
Meth yl [2-[1-Hyd r oxy-2-(ter t-bu tyla m in o)eth yl]ben zo-
fu r a n -7-yl]a ceta te (8b). To a solution of 7 (6.40 g, 20.4 mmol)
in THF (150 mL) was added 10 N NaOH (1.0 mL). After the
reaction mixture was stirred for 2 h, the solvent was evapo-
rated under reduced pressure. The residue was dissolved in
methylene chloride (50 mL) and the resulting solution was
dried over anhydrous magnesium sulfate, filtered and evapo-
rated to give the crude epoxide which was used in the next
reaction without further purification. The crude epoxide was
dissolved in 2-propanol (5 mL) and added to tert-butylamine
(3.02 mL, 28.2 mmol). The reaction mixture was stirred for
36 h and evaporated. The residue was purified by column
chromatography on silica gel (CH2Cl2:MeOH:NH4OH, 120:10:
1) to afford 4.3 g (69%) of 8b as a white solid: mp 82 °C; IR
[2-(Acetyl)ben zofu r a n -7-yl]a cetic Acid (4). To a solution
of 7-allyl-2-acetylbenzofuran (40.0 g, 200 mmol) in t-BuOH
(100 mL) was added dropwise the mixture of KMnO4 (32.0 mg,
0.20 mmol), NaIO4 (225 g, 1.05 mol), and K2CO3 (29.0 g, 209
mmol) in t-BuOH-H2O (7:3, 4 L). After the reaction mixture
was stirred at 70 °C for 20 h, the solvent mixture was
evaporated. The residue was purified by column chromatog-
raphy on silica gel (methylene chloride:methanol, 4:1) to afford
26.2 g (60%) of 4 as a white solid: mp 143 °C; IR (KBr) 3377,
2933, 1699, 1666 cm-1; 1H NMR (CDCl3) 2.54 (s, 3H, COCH3),
3.86 (s, 2H, CH2COO), 7.25-7.35 (m, 2H, ArH), 7.61-7.65 (m,
1H, ArH), 7.63 (s, 1H, furan-H); 13C NMR (CDCl3) 24.8, 33.1,
112.2, 118.1, 120.5, 122.3, 125.2, 127.6, 150.6, 152.5, 170.1,
186.1. Anal. (C12H10O4) C, H.
(KBr) 2968-3400, 1732 cm-1 1H NMR (CDCl3) 1.25 (s, 9H,
;
C(CH3)3), 3.09-3.24 (m, 2H, CH2 NH), 3.70 (s, 3H, OCH3), 3.91
(s, 2H, CH2COO), 4.41 (br, 1H, NH) 5.15-5.18 (m, 1H, CHOH),
6.72 (s, 1H, furan-H), 7.14-7.20 (m, 2H, ArH), 7.42-7.45 (m,
1H, ArH); 13C NMR (CDCl3) 28.7, 34.8, 46.2, 50.6, 51.9, 66.1,
103.1, 117.4, 119.9, 122.8, 124.7, 128.0, 153.2, 158.8, 171.3.
Anal. (C17H23NO4) C, H, N.
[2-[1-Hyd r oxy-2-(ter t-bu tyla m in o)eth yl]ben zofu r a n -7-
yl]a cetic Acid Hyd r och lor id e (8a ). To a solution of 8b (3.76
g, 12.3 mmol) in THF (10 mL) was added 2 N KOH (10 mL).
After the reaction mixture was stirred for 2 h, it was neutral-
ized with 1 N HCl solution. After most of solvent was
evaporated, the resulting solid residue was dissolved again in
THF (100 mL) and then filtered. The filtrate was dried over
anhydrous magnesium sulfate, filtered and evaporated. The
residue was dissolved in methylene chloride followed by
addition with ethereal HCl to afford 3.3 g (82%) of 8a as a
Meth yl [2-(Acetyl)ben zofu r a n -7-yl]a ceta te (5). To a
solution of 4 (18.0 g, 82.5 mmol) in MeOH (100 mL) was added
concentrated H2SO4 (4 mL) at 0 °C. After the reaction mixture
was heated at 70 °C for 1 h, it was diluted with 50 mL of H2O.
The methanol was evaporated, and the aqueous layer was
extracted with chloroform (3 × 20 mL). The organic layer was
dried over anhydrous magnesium sulfate, filtered and evapo-
rated. The residue was purified by column chromatography
on silica gel (hexane:EtOAc, 1:1) to afford 17.4 g (91%) of 5 as
1
hydrogen chloride salt: IR (KBr) 2968-3400, 1720 cm-1; H
NMR (D2O) 1.45 (s, 9H, C(CH3)3), 3.42-3.61 (m, 2H, CH2NH),
3.85 (s, 2H,CH2COO), 5.12-5.23 (s, 1H, CHOH), 6.90 (s, 1H,
furan-H), 7.11-7.32 (m, 2H, ArH), 7.52-7.60 (m, 1H, ArH);
13C NMR (D2O) 27.6, 47.2, 47.8, 60.8, 66.5, 107.7, 122.9, 123.9,
126.3, 128.8, 130.3, 140.0, 156.4, 157.3, 182.7. Anal. (C16H22O4-
NCl) C, H, N.
a white solid: mp 84 °C; IR (KBr) 3139, 2958, 1735, 1680 cm-1
;
1H NMR (CDCl3) 2.58 (s, 3H, COCH3), 3.71 (s, 2H, OCH3), 3.90
(s, 2H, CH2COO) 7.33 (dd, 1H, ArH, J ) 7.7 Hz, 7.6 Hz), 7.37
(d, 1H, ArH, J ) 7.7 Hz), 7.49 (s, 1H, furan-H), 7.61 (d, 1H,
ArH, J ) 7.6 Hz); 13C NMR (CDCl3) 26.4, 34.6, 52.1, 112.9,
118.9, 122.3,124.0, 127.0, 129.0, 152.7, 154.1, 170.9, 188.5.
Anal. (C13H12O4 ) C, H.
Eth yl [2-[1-Hyd r oxy-2-(ter t-bu tyla m in o)eth yl]ben zo-
fu r a n -7-yl]a ceta te (8c). To a solution of 8a (1.00 g, 3.00
mmol) in EtOH (10 mL) was added concentrated H2SO4 (0.2
mL). After the reaction mixture was stirred for 1 h at 60 °C,
Meth yl [2-(Br om oa cetyl)ben zofu r a n -7-yl]a ceta te (6).
To a solution of 5 (10.0 g, 43.0 mmol) in chloroform (250 mL)

1530 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Hwang et al.
the solvent was evaporated. The residue was purified by
column chromatography on silica gel (CH2Cl2:MeOH:NH4OH,
120:10:1) to afford 0.41 g (42%) of 8c as a yellow solid: mp 82
From 9b (1.60 g, 5.49 mmol), 1.31 g (62%) of 10b was obtained
as a white solid: mp 81 °C; IR (KBr) 2923, 1701, 1690 cm-1
;
1H NMR(CDCl3) 1.40-1.85 (s, 10H, (CH2)5), 3.90 (s, 2H, CH2
COO), 4.40 (s, 2H, CH2Br), 4.80-4.84 (m, 1H, OCH), 7.31-
7.37 (m, 2H, ArH), 7.50 (s, 1H, furan-H), 7.59-7.64 (s, 1H,
ArH); 13C NMR (CDCl3) 23.5, 24.5, 30.4, 31.4, 34.3, 72.3, 114.0,
119.4, 122.1, 124.0, 126.9, 129.1, 150.2, 154.1, 170.0, 182.1.
Anal. (C18H19O4Br) C, H.
1
°C; IR (KBr) 2968-3460, 1739 cm-1 ; H NMR (CDCl3) 1.25
(s, 9H, C(CH3)3), 1.35 (s, 3H, OCH2CH3), 3.12 (m, 2H, CH2-
NH), 3.92 (s, 2H, CH2COO), 4.27 (q, 2H, OCH2CH3), 5.01-
5.08 (m, 1H, CHOH), 6.80 (s, 1H, furan-H), 7.15-7.21 (m, 2H,
ArH), 7.52-7.60 (m, 1H, ArH); 13C NMR (CDCl3) 14.7, 28.7,
34.9, 46.2, 50.4, 60.7, 66.1, 102.9, 117.5, 119.8, 122.7, 124.6,
128.0, 128.1, 153.2, 158.9, 170.8. Anal. (C18H25NO4) C, H, N.
Isop r op yl [2-[1-Hyd r oxy-2-(ter t-bu tyla m in o)eth yl]ben -
zofu r a n -7-yl]a ceta te (8d ). To a solution of 8a (1.00 g, 3.0
mmol) in i-PrOH (5 mL) was added concentrated H2SO4 (0. 1
mL). After the reaction mixture was stirred for 1 h at 60 °C,
the solvent was evaporated. The residue was purified by
column chromatography on silica gel (CH2Cl2:MeOH:NH4OH,
120:10:1) to afford 0.42 g (41%) of 8d as a white solid: mp 83
°C; IR (KBr) 2970-3460, 1732 cm-1; 1H NMR (CDCl3) 1.12 (s,
9H, C(CH3)3), 1.23 (d, 6H, J ) 6. 3 Hz, CH(CH3)2 ), 2.80-3.12
(m, 2H, CH2NH), 3.87 (s, 2H, CH2COO), 4.78-4.82 (m, 1H,
CHOH), 5.01-5.08 (m, 1H, CH(CH3)2), 6.67 (s, 1H, furan-H),
7.15-7.1 7 (m, 2H, ArH), 7.42-7.44 (m, 1H, ArH); 13C NMR
(CDCl3) 21.6, 28.8, 35.4, 46.1, 50.4, 66.2, 68.2, 103.1, 117.8,
2-(1-Ad a m a n tyl)eth yl [2-(Br om oa cetyl)ben zofu r a n -7-
yl]a ceta te (10c). This compound was prepared in the same
fashion as 6. From 9c (1.51 g, 3.9 mmol), 1.20 g (66%) of 10c
was obtained as a yellow oil: mp 83 °C; IR (neat) 2958, 1737,
1
1690 cm-1; H NMR (CDCl3) 1.34-1.87 (m, 17H, -adaman-
tylethyl), 3.96 (s, 2H, CH2COO), 4.16 (t, 2H, COOCH2), 4.45
(s, 2H, CH2Br), 7.31-7.37 (m, 2H, ArH), 7.60 (s, 1H, furan-
H), 7.59-7.64 (s, 1H, ArH); 13C NMR (CDCl3) 28.3, 30.2, 31.5,
35.0, 36.7, 42.2, 61.6, 114.8, 119.1, 122.4, 124.3, 126.7, 129.7,
150.0, 154.3, 170.3, 182.0. Anal. (C24H27O4Br) C, H.
ter t-Bu tyl [2-(1-Hyd r oxy-2-br om oeth yl)ben zofu r a n -7-
yl]a ceta te (11a ). This compound was prepared in the same
fashion as 7. From 10a (1.60 g, 5.8 mmol), 1.10 g (91%) of 11a
was obtained as a white solid: mp 75 °C; IR (KBr) 3402, 2918,
1
1720 cm-1; H NMR (CDCl3) 1.46 (s, 9H, C(CH3)3), 2.91 (br,
119.8, 122.8, 124.7, 128.0, 153.3, 158.7, 170.4. Anal. (C19H27
NO4) C, H, N.
-
1H, OH), 3.80-3.88 (m, 2H, CH2Br), 3.90 (s, 2H, CH2COO)
5.08-5.10 (m, 1H, CHOH), 6.71 (s, 1H, furan-H) 7.20-7.25
(m, 2H, ArH), 7.44-7.48 (s, 1H, ArH); 13C NMR (CDCl3) 28.0,
36.4, 68.1, 81.08, 104.4, 118.5, 120.1, 123.1, 125.5, 127.7, 150.6,
155.4, 170.2. Anal. (C16H19O4Br) C, H.
ter t-Bu tyl [2-(Acetyl)ben zofu r a n -7-yl]a ceta te (9a ). To
a solution of 4 (2.00 g, 9.10 mmol) in CH2Cl2 (20 mL) was added
SOCl2 (2.18 g, 18.3 mmol). After the reaction mixture was
stirred for 1 h at 60 °C, the solution was concentrated to
dryness. The residue was dissolved in t-BuOH (1.01 g, 13.6
mmol) and the resulting solution was stirred for 2 h at room
temperature. After the reaction mixture was diluted with 50
mL of H2O, it was extracted with chloroform (2 × 20 mL). The
organic layer was dried over anhydrous magnesium sulfate,
filtered and evaporated. The residue was purified by column
chromatography on silica gel with hexanes-EtOAc (1:1) to
afford 1.6 g (63%) of 9a as a white solid: mp 85 °C; IR (KBr)
Cycloh exyl [2-(1-Hyd r oxy-2-br om oeth yl)ben zofu r a n -
7-yl]a ceta te (11b). This compound was prepared in the same
fashion as 7. From 10b (1.30 g, 3.5 mmol), 1.12 g (84%) of 11b
was obtained as a white solid: mp 81 °C; IR (KBr) 3419, 2923,
1
1722 cm-1; H NMR (CDCl3) 1.40-1.85 (s, 10H, (CH2)5), 3.90
(s, 2H, CH2COO), 3.70-3.80 (s, 2H, CH2Br), 4.80-4.84 (m, 1H,
OCH), 5.11-5.19 (m, 1H, CHOH), 6.71 (s, 1H, furan-H), 7.31-
7.37 (m, 2H, ArH), 7.59-7.63 (s, 1H, ArH); 13C NMR (CDCl3)
23.5, 25.3, 31.4, 35.6, 36.4, 68.1, 73.2, 104.4, 118.2, 120.2, 123.1,
125.4, 127.7, 153.0, 155.5, 170.3. Anal. (C18H21O4 Br) C, H.
2-(1-Ad a m a n t yl)et h yl [2-(1-H yd r oxy-2-b r om oet h yl)-
ben zofu r a n -7-yl]a ceta te (11c). This compound was prepared
in the same fashion as 7. From 10c (1.20 g, 2.6 mmol), 1.02 g
(85%) of 11c was obtained as a yellow oil: IR (neat) 3404, 2972,
3120, 2976, 1722, 1674 cm-1 1H NMR (CDCl3) 1.46 (s, 9H,
;
C(CH3)3), 2.61 (s, 3H, COCH3), 3.90 (s, 2H, CH2COO), 7.31-
7.37 (m, 2H, ArH), 7.50 (s, 1H, furan-H), 7.59-7.61 (s, 1H,
ArH); 13C NMR (CDCl3) 26.4, 28.3, 36.1, 81.2, 113.0, 119.7,
122.0,123.8, 126.9, 128.9, 152.6, 154.3, 169.8, 188.6. Anal.
(C16H18O4) C, H.
1
1739 cm-1; H NMR (CDCl3) 1.26-1.89 (m, 17H, -adaman-
Cycloh exyl [2-(Acet yl)b en zofu r a n -7-yl]a cet a t e (9b ).
This compound was prepared in the same fashion as 9a . From
compound 4 (2.00 g, 9.1 mmol), 1.6 g (58%) of 9b was obtained
tylethyl), 3.41(br, 1H, OH), 3.41-3.44 (m, 2H, CH2Br), 3.89
(s, 2H, CH2COO), 4.16 (t, 2H, COOCH2), 5.10-5.19 (m, 1H,
CHOH), 6.73 (s, 1H, furan-H), 7.31-7.37 (m, 2H, ArH), 7.44-
7.46 (m, 1H, ArH); 13C NMR (CDCl3) 28.4, 31.6, 35.3, 36.1,
36.8, 42.1, 61.6, 68.0, 104.3, 117.7, 120.2, 123.0, 125.4, 127.7,
153.3, 155.6, 171.1. Anal. (C24H29O4Br) C, H.
as a white solid: mp 81 °C; IR (KBr) 2923, 1728, 1684 cm-1
;
1H NMR (CDCl3) 1.40-1.85 (s, 10H, (CH2)5), 2.61(s, 3H,
COCH3), 3.90 (s, 2H, CH2COO), 4.80-4.84 (m, 1H, OCH),
7.31-7.37 (m, 2H, ArH), 7.50 (s, 1H, furan-H), 7.59-7.64 (s,
1H, ArH); 13C NMR (CDCl3) 23.5, 25.2, 26.6, 31.4, 35.4, 76.3,
113.0, 119.4, 122.1, 124.0, 126.9, 128.9, 152.6, 154.2, 170.0,
188.6. Anal. (C18H20O4) C, H.
ter t-Bu tyl [2-[1-Hyd r oxy-2-(ter t-bu tyla m in o)eth yl]ben -
zofu r a n -7-yl]a ceta te (8e). This compound was prepared in
the same fashion as 8b. From 11a (1.00 g, 2.8 mmol), 1.10 g
(33%) of 8e was obtained as a yellow oil: IR (neat) 3200-3460,
1737 cm-1; 1H NMR (CDCl3) 1.12 (s, 9H, C(CH3)3), 1.44 (s, 9H,
OC(CH3)3), 2.35 (br, 1H, NH) 3.00-3.04 (m, 2H, CH2NH), 3.82
(s, 2H, CH2COO), 4.80-4.84 (m, 1H, CHOH), 6.68 (s, 1H,
furan-H), 7.15-7.18 (m, 2H, ArH), 7.42-7.45 (m, 1H, ArH);
13C NMR (CDCl3) 27.9, 28.8, 36.4, 46.1, 50.6, 66.2, 68.2, 103.1,
118.2, 119.7, 122.7, 124.7, 127.9, 153.3, 158.7, 170.2. Anal.
(C20H29NO4) C, H, N.
Cycloh exyl [2-[1-Hyd r oxy-2-(ter t-b u t yla m in o)et h yl]-
ben zofu r a n -7-yl]a ceta te (8f). This compound was prepared
in the same fashion 8b. From 11b (1.10 g, 2.80 mmol), 0.35 g
(32%) of 8f was obtained as a white solid: mp 83 °C; IR (KBr)
3200-3460, 1743 cm-1; 1H NMR (CDCl3) 1.18 (s, 9H, C(CH3)3),
1.19-1.79 (m, 10H, (CH2)5), 2.98-3.04 (m, 2H, CH2NH), 3.62
(br, 1H, NH), 3.89 (s, 2H, CH2COO), 4.79-4.84 (m, 1H, OCH),
4.85-4.91 (m, 1H, CHOH), 6.66 (s, 1H, furan-H), 7.14-7.17
(m, 2H, ArH), 7.40-7.45 (m, 1H, ArH); 13C NMR (CDCl3) 23.3,
25.1, 28.5, 31.3, 35.3, 46.2, 50.4, 66.2, 72.9, 102.9, 117.8, 119.7,
122.6, 124.6, 127.9, 153.2, 158.8, 170.2. Anal. (C22H31NO4) C,
H, N.
2-(1-Ad a m a n th yl)eth yl [2-(Acetyl)ben zofu r a n -7-yl]a c-
eta te (9c). This compound was prepared in the same fashion
as 9a . From 4 (2.00 g, 9.17 mmol), 1.5 g (43%) of 9c was
obtained as a yellow oil: IR (neat) 2058, 1730, 1670 cm-1; 1H
NMR (CDCl3) 1.34-1.87 (m, 17H, -adamantylethyl), 2.59 (s,
3H, COCH3), 3.96 (s, 2H, CH2COO), 4.16 (t, 2H, COOCH2),
7.31-7.37 (m, 2H, ArH), 7.50 (s, 1H, furan-H), 7.59-7.64 (s,
1H, ArH); 13C NMR (CDCl3) 26.1, 28.2, 30.8, 34.5, 37.2, 42.0,
62.1, 113.1, 119.5, 122.0, 124.0, 127.2, 129.8, 152.2, 154.0,
171.6, 188.1. Anal. (C24H28O4 ) C, H.
ter t-Bu tyl 2-[2-(Br om oa cetyl)ben zofu r a n -7-yl]a ceta te
(10a ). This compound was prepared in the same fashion as 6.
From 9a (1.60 g, 5.83 mmol), 1.21 g (58%) of 10a was obtained
as a white solid: mp 83 °C; IR (KBr) 3039, 2979, 1718, 1676
cm-1; 1H NMR (CDCl3) 1.46 (s, 9H, C(CH3)3), 4.41(s, 2H, CH2-
Br), 3.90 (s, 2H, CH2COO), 7.31-7.61 (m, 3H, ArH), 7.70 (s,
1H, furan-H); 13C NMR (CDCl3) 28.0, 30.1, 36.1, 81.2, 114.9,
119.8, 122.2, 124.3, 126.2, 130.1, 128.9, 150.6, 154.3, 169.8,
182.6. Anal. (C16H17O4Br) C, H.
Cycloh exyl [2-(Br om oa cetyl)ben zofu r a n -7-yl]a ceta te
(10b). This compound was prepared in the same fashion as 6.
2-(1-Ad a m a n tyl)eth yl [2-[1-Hyd r oxy-2-(ter t-bu tyla m i-
n o)eth yl]ben zofu r a n -7-yl]a ceta te (8g). This compound was

Soft Drugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1531
prepared in the same fashion as 8b. From 11c (1.02 g, 2.20
mmol), 0.33 g (85%) of 8g was obtained as a slick oil: IR (neat)
3200-3460, 1732 cm-1; 1H NMR (CDCl3) 1.18 (s, 9H, C(CH3)3),
1.19-1.79 (m, 17H, -adamantylethyl) 2.98-3.04 (m, 2H, CH2-
NH), 3.62 (br, 1H, NH), 3.89 (s, 2H, CH2COO), 4.15 (t, 2H,
OCH2), 4.80-4.92 (m, 1H, CHOH), 6.66 (s, 1H, furan-H), 7.14-
7.17 (m, 2H, ArH), 7.40-7.45 (m, 1H, ArH); 13C NMR (CDCl3)
28.4, 28.8, 31.5, 35.2, 36.8, 42.1, 46.2, 50.5, 61.3, 66.1, 103.0,
117.5, 119.8, 122.7, 124.7, 128.0, 153.3, 158.8, 170.9. Anal.
(C28H39NO4) C, H, N.
pressure. Needle electrodes were inserted sc and together with
the pressure transducer were joined to a Gould TA 2000
recorder (Gould Inc., Cleveland, OH). Leads II, aVF and intra-
arterial blood pressure were monitored simultaneously through-
out the experiments and recorded at certain intervals at 50
mm/s paper speed. Baseline heart rate and blood pressure
parameters were recorded for 25 or 30 min, at every 5 min,
before any drugs were given. Drug administrations were
carried out as either bolus injections into the jugular veins or
as infusions for 10 min through a plastic catheter (Terumo 24
GA *3/4′′, Terumo Medical Corp., Elkton, MD) inserted into
the jugular vein on either side. During the first series of
experiments 50 µg/kg isoproterenol (Sigma, St. Louis, MO) was
injected sc at -5 min. Heart rate and blood pressure were
recorded at -5, -4 and -2 min. At 0 min the soft bufuralol
analogues as free base forms dissolved in 10% DMSO (Fisher
Scientific, Fair Lawn, NJ ) in 30% hydroxypropyl-â-cyclodextrin
(HPBCD) (Pharmos Inc., Alachua, FL) or bufuralol (R-(tert-
butylamino)methyl-7-ethyl-2-benzofuranmethanol; Roche Prod-
ucts Ltd., Welwyn Garden City, U.K.) dissolved in the same
vehicle or vehicle (i.e. 10% DMSO in 30% HPBCD) was injected
into the jugular vein as bolus injections. Heart rate and blood
pressure were registered at 1, 3, 5, 10, 15, 20, 25, 30, 40, 45,
50, 60, 70, 80, 90, 105 and 120 min. The percent changes in
heart rate were calculated as follows: % change in HR ) (HRt
- HR-2)/HR-2 × 100, relative to the maximal isoproterenol-
induced heart rate increase registered at -2 min, where t )
0, 1, 3, ... 120 min. During the second series of experiments
the methyl (8b), ethyl (8c), isopropyl (8d ), and tert-butyl (8e)
analogues of bufuralol were converted to their corresponding
HCl salt forms and were dissolved in 0.9% NaCl. Baseline
heart rate and blood pressure values were registered again at
every 5 min for 30 min. At 0 min the four bufuralol analogues,
or esmolol-HCl diluted with 0.9% NaCl (Brevibloc, 2.5 g/10 mL,
Ohmeda Pharmaceutical Products Division Inc., Liberty Cor-
ner, NJ ), were infused into the jugular vein for 10 min with a
syringe pump (Sage Instruments, model 341B, Orion Res. Inc.,
Boston, MA). The doses were 2 µmol/kg/min for 8b, 8d , and
8e, 4 µmol/kg/min for 8c, and 20 µmol/kg/min for esmolol. The
percent changes in heart rate and blood pressure values (mean
arterial pressure: MAP) were calculated relative to the
average baseline values recorded between -30 and 0 min,
i.e.: % change in HR ) (HRt - HRav)/HRav × 100, or % change
in MAP ) (MAPt - MAPav)/MAPav × 100, where t ) 1, 3, ...
120 min. Each compound and vehicle were administered to at
least three different animals, and the average and the stan-
dard deviation of all heart rate and blood pressure data were
calculated. All data were subjected to cluster analysis and to
multivariate analysis of variance for repeated measures (GLM
Repeated Measures of ANOVA) with the SPSS for Windows
7.5 program. Statistically significant difference between the
effects of the different compounds was accepted at p < 0.05.
In Figures 2-4, the average values are represented without
error bars.
Meth ylth iom eth yl [2-[1-Hyd r oxy-2-(ter t-bu tyla m in o)-
eth yl]ben zofu r a n -7-yl]a ceta te (8h ). The potassium salt of
8a was prepared as follows: The solution of 8b (1.20 g, 3.93
mmol) and KOH (0.22 g, 3.93 mmol) in ethanol-water (1:1,
100 mL) was refluxed for 10 h. After the ethanol was removed
under reduced pressure, the aqueous layer was washed with
methylene chloride (30 mL) and then concentrated to give the
product in quantitative yield, which was used for next reaction
without further purification. Potassium [2-[1-hydroxy-2-(tert-
butylamino)ethyl]benzofuran-7-yl]acetate (1.00 g, 3.0 mmol),
sodium iodide (0.12 g, 0.8 mmol) and 18-crown-6 (0.27 g, 1.0
mmol) were dried over phosphorus pentoxide under reduced
pressure and then suspended in dried benzene (100 mL).
Chloromethylmethyl sulfide (0.75 g, 7.7 mmol) was then added
to the suspension, and the reaction mixture was refluxed for
15 h under nitrogen atmosphere. The reaction mixture was
cooled to room temperature and washed with saturated
aqueous sodium carbonate solution (3 × 80 mL) followed by
brine (2 × 80 mL). The organic layer was dried and evaporated
to give a crude oil product, which was purified by column
chromatography on silica gel (CH2Cl2:MeOH:NH4OH, 120:10:
1) to afford 0.45 g (46%) of 8h as a yellow oil: IR (KBr) 3200-
3460, 1732 cm-1; 1H NMR (CDCl3) 1.26 (s, 9H, C(CH3)3), 2.17
(s, 3H, SCH3) 3.12-3.18 (m, 2H, CH2NH), 3.96 (s, 1H, CH2-
COO), 4.20 (br, 1H, NH) 5.11-5.13 (m, 1H, CHOH) 6.73 (s,
1H, furan-H), 7.17-7.20 (m, 2H, ArH), 7.44-7.48 (m, 1H,
ArH); 13C NMR (CDCl3) 15.2, 28.1, 35.2, 46.2, 53.0, 65.3, 68.6,
103.8, 117.2, 120.3, 123.0, 125.0, 128.0, 153.3, 157.6, 170.5.
Anal. (C18H25NO4S) C, H, N, S.
Sta bility in Aqu eou s Bu ffer Solu tion s. Isotonic phos-
phate buffer solutions (0.5 mM) of pH ) 7.4 and 12 at 37 °C
were used. Solutions of the bufuralol analogues were added
to the buffered solutions to result an initial concentration of
120 µM. Samples (0.1 mL) were withdrawn at selected time
intervals, diluted with cold methanol (0.9 mL), vortexed and
placed in a freezer. The final sample concentration was 12 µM.
Samples (8a -8h ) were kept at 0 °C until analyzed by HPLC.
The pseudo-first-order rate constant for hydrolysis of 8a -8h
was determined by linear regression from the plot of natural
logarithm of the HPLC peak area versus time.
Meta bolism a n d Sta bility in Biologica l Med ia . The
stability of soft analogues was determined in freshly collected
Sprague-Dawley male rat blood. The heparinized blood was
used within 30 min. Compound solutions in methanol (0.1 mL)
were mixed with blood (0.9 mL) at 37 °C. Samples (0.1 mL)
were withdrawn at selected time intervals, mixed with cold
methanol (0.9 mL), vortexed, and placed in a freezer. When
all samples had been collected, they were centrifuged at 24 000
rpm for 3 min. The final sample concentration was 12 µM. The
supernatant was analyzed for the compounds and their
metabolite 8a by HPLC as described earlier.
Ack n ow led gm en t. The authors are indebted to
Mrs. Kery and Ildiko Fulop for their help in the
statistical analysis and to Dr. Katalin Prokai-Tatrai for
assistance in preparation of the salts used in the in vivo
studies.
Refer en ces
An im a l Stu d ies. Male Sprague-Dawley rats (Harlan
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were anesthetized with Na-pentobarbital (50 mg/kg ∼ 0.1 mL/
100 g) ip. Both jugular veins and the left carotid artery were
isolated, and the latter was tied up cranially with a surgical
silk (Ethicon 4-0, Ethicon Inc., Australia). A plastic catheter
(Intracath 19GA, Becton Dickinson, Sandy, UT) containing
10% Na-heparin (Elkins-Sinn Inc., Cherry Hill, NJ ) in normal
saline (100 U/mL Na-heparin) was introduced into the artery
and fixed with surgical silk. The catheter was connected to a
pressure transducer (Ohmeda P23-XL, Ohmeda Medical De-
vices Division Inc., Madison, WI) filled with the same hep-
arinized 0.9% NaCl solution to register beat-to-beat arterial
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