Design, Pharmacokinetic, and Pharmacodynamic Evaluation of a New Class of Soft Anticholinergics

Article abstract of DOI:10.1023/A:1026160023030

Purpose. To design and evaluate a new class of soft anticholinergics with subtype selectivity. Methods. A new class of soft anticholinergics was designed based on the "inactive metabolite" approach. Four compounds were synthesized. The potency and soft na

Full text of DOI:10.1023/A:1026160023030

Pharmaceutical Research, Vol. 20, No. 10, October 2003 (© 2003)
Research Paper
age of the anticholinergics into the systemic circulation. For
example, at least six deaths have been attributed to the ocular
administration of atropine (6). Various psychic disturbances
have been reported to the application of atropine (7), scopol-
amine (8), and cyclopentolate (9) as mydriatic/cycloplegic
agents. Severe cardiac dysfunction has been reported with the
use of atropine as a perioperative mydriatic/cycoplegic agent
(10,11). That application is particularly dangerous to the el-
derly because older people are more likely to suffer from
cardiac diseases (11,12). Anticholinergics have been evalu-
ated for their use as antiperspirants for a long time (13–15);
however, because of the risk of systemic toxicity when over-
dosed, it was suggested that these agents should be used un-
der direct physician supervision and not be available for over-
the-counter sale (16).
Design, Pharmacokinetic, and
Pharmacodynamic Evaluation of a
New Class of Soft Anticholinergics
Fenglei Huang,1 Clinton E. Browne,1 Whei-Mei Wu,1
Attila Juha´sz,1,2 Fubao Ji,1 and Nicholas Bodor1,3
Received February 19, 2003; accepted June 9, 2003
Purpose. To design and evaluate a new class of soft anticholinergics
with subtype selectivity.
Methods. A new class of soft anticholinergics was designed based on
the “inactive metabolite” approach. Four compounds were synthe-
sized. The potency and soft nature of the compounds were evaluated
by receptor binding, cardiac, and mydriatic studies. Stability and
pharmacokinetic studies were also performed on these newly synthe-
sized soft anticholinergics.
Results. Receptor binding studies of the soft anticholinergics on
cloned muscarinic receptors indicated pKi values in the range of 7.5
to 8.9. Two compounds, 9a and 13a, of the series showed muscarinic
subtype receptor selectivity (M3/M2). In mydriatic studies, 13a and
13b showed shorter duration of action in the treated eyes than tro-
picamide. In the control eyes, significant dilation of pupils was found
only in rabbits treated with atropine and tropicamide, indicating that
the soft anticholinergics lack systemic effects because of their facile
hydrolytic deactivation. Consistent with their soft nature, this new
class of soft anticholinergics displayed much shorter cardiovascular
effects in the carbachol-induced bradycardia (10 to 15 min) in rats
than atropine (> 60 min). Stability and pharmacokinetic studies sug-
gested that the new soft anticholinergics were rapidly eliminated from
plasma (systemic circulation) after i.v. administration.
In order to separate the desired therapeutic effects of
anticholinergics from their undesired toxic effects, two major
approaches have been used. The first of these is the pharma-
cokinetic (retrometabolic) approach, which includes the de-
velopment of “soft” anticholinergics to minimize systemic
toxicity of anticholinergics (17–20). Briefly, based on specific
design concepts, structural analogues of known anticholiner-
gics are designed to include an easily metabolizable moiety in
the structure that assures facile deactivation of the com-
pounds when they reach the circulatory system, after they
have achieved their therapeutic role. Soft anticholinergics are
primarily designed for topical application. Two distinctly dif-
ferent design approaches have been applied: the soft ana-
logue (20,21) and the inactive metabolite approaches (22–26).
And one of these compounds, tematropium methyl sulfate,
the methyl sulfate of the ethyl ester of [( )-(carboxyl)-8-
methyl-8-azabicycl (3.2.1)oct-3-yl-benzene acetate], based on
the inactive metabolite approach, has successfully reached
phase II human trials as a short-acting mydriatic agent.
The second approach is the pharmacodynamic approach:
development of muscarinic receptor subtype-selective an-
tagonists to minimize toxicity. In parallel with our efforts di-
rected toward the development of safer anticholinergics by
soft drug approaches, considerable research has been directed
toward the delineation of muscarinic receptors and receptor
subtypes in the past 20 years. This was done in the hope that
by understanding the function of muscarinic receptors and
receptor subtypes, muscarinic antagonists selectively targeted
to particular symptom(s) can be made, and therefore, the
therapeutic index can be improved (27). Three mammalian
muscarinic receptor subtypes have been located and charac-
terized by biochemical and functional studies. Of the three
major subtypes (M , M , M ), each one has its differential
Conclusions. A new class of anticholinergics was designed and syn-
thesized, and the PK/PD evaluation confirmed they were potent
“soft” anticholinergics; two of them showed muscarinic receptor sub-
type selectivity (M3/M2).
KEY WORDS: soft drugs; anticholinergic agents; receptor binding;
muscarinic receptor subtypes; metabolism.
INTRODUCTION
Anticholinergics, such as atropine and scopolamine, have
been used clinically for the treatment of a variety of disorders
(1), predominantly to inhibit the parasympathetic nervous
system. Anticholinergics find use in the management of pep-
tic ulcer, as mydriatic/cycloplegic agents, as adjuncts to anes-
thetic medications, and as antiperspirants. However, the use-
fulness of anticholinergics is limited by their unwanted toxic
effects. For instance, the use of anticholinergics as premedi-
cation in anesthetic practice often results in cardiac side ef-
fects (2–5); even topical application of anticholinergics can
lead to systemic toxicity because of the absorption and drain-
2
3
location (e.g., M in1brain and M in heart) and is responsible
1
for a specific action; for exampl2e, M relates to central ner-
1
vous system (CNS) function, M triggers cardiac effects, and
2
M3 stimulates smooth muscle contraction (27). With advances
in molecular pharmacology, five muscarinic receptor subtypes
(m1, m2, m3, m4, and m5) have been cloned from human tissue
(28–30) and have been readily available since the early 1990s.
The cloned human muscarinic receptor subtypes (m1 to m3)
have been shown to correlate very well to the ones derived
from mammalian tissues (31,32).
1Center for Drug Discovery, College of Pharmacy, University of
Florida, Gainesville, Florida 32610. 2 On leave of absence from the
First Department of Medicine, University Medical School of Debre-
cen, Hungary.
3 To whom correspondence should be addressed. (email: bodor@
cop.ufl.edu)
Cloned receptors have been used to determine the po-
tency of novel compounds and to determine the subtype se-
ABBREVIATIONS: SASS: soft anticholinergics with muscarinic re-
ceptor subtype selectivity; [3H]NMS, N-[3H]methylscopolamine.
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1682
Huang et al.
lectivity of antimuscarinic agents (33–35). With the help of
modern receptor pharmacology in the past 15 years, signifi-
cant progress has been made in the search for muscarinic
antagonists with subtype selectivity. Currently, several con-
ventional (“hard”) muscarinic receptor subtype-selective
agents are in advanced clinical trials (36,37), and a few of
them such as pirenzepine and AF-DX 116 have been ap-
proved for clinical use (37). In order to take advantage of the
latest development in receptor pharmacology, a validated re-
ceptor-binding method based on commercially available
cloned human receptor subtypes has been specifically devel-
oped for the fast screening of soft anticholinergics in our labo-
ratory, and a quantitative structure–activity relationship
(QSAR) has been established based on the affinity of the soft
anticholinergics toward the muscarinic receptor subtypes
(38).
The main objective of the present study is to apply the
molecular receptor pharmacology technique to our search for
a new class of anticholinergics: soft anticholinergics with mus-
carinic receptor subtype selectivity (SASS). Because both
pharmacokinetic and pharmacodynamic approaches are used
in the design of SASS, it is expected that this new class of soft
anticholinergics is safer than the ones derived from either
approach alone. In addition to being used topically as safer
mydriatics, antiperspirants, or antiulcer agents, SASS can be
used intravenously as a safer preoperative medication for in-
hibiting excessive secretion. The M2-mediated cardiac side
effects that prevent the conventional anticholinergics such as
atropine and glycopyrrolate from being safely administered to
cardiac patients (2,39,40) are expected to be minimal because
of the subtype selectivity (M3/M2) of SASS. And the soft
nature of the compounds will allow us to determine the
needed dose during the anesthetic procedure.
Fig. 1. Design of a new class of soft anticholinergics.
nucleophilic groups are shown to be present at the muscarinic
receptor site (44), the zwitterionic metabolites (II or IIa) that
result from the hydrolysis of the soft drugs will have an un-
favorable interaction with the receptor site; thus, they are
expected to be less active than the parent soft drugs (22,23).
Design
MATERIALS AND METHODS
N-Alkyl-nortropine esters of 2-phenyl-2-cyclohex-
enecarboxylic acid (I) (Fig. 1) were originally synthesized as
bronchodilator agents, some of which showed high anticho-
linergic activity (41). Receptor binding and functional studies
indicated that one of these compounds, ( )-3{[(2-phenyl-
cyclohexene-1-yl)carbonyl]oxy}-8,8-diethyl-8-azoniabi-
cyclo[3.2.1]octane iodide, is a potent and selective antagonist
All chemicals used were reagent grade. N-[3H]Methyl-
scopolamine was obtained from Dupont NEN Research (Bos-
ton, MA). Atropine was from Sigma Chemicals Co. (St.
Louis, MO). Tropicamide was from Schein Pharmaceutical
(Florham Park, NJ). All other chemicals were from Aldrich
Chemical Company (Milwaukee, WI). Scintiverse BD and
other solvents were from Fisher Scientific Co. (Pittsburgh,
PA). All melting points were recorded using Fisher-Johns
melting point apparatus and were uncorrected. NMR data
were recorded with Varian 300 NMR spectrometer and were
reported in parts per million (␦) relative to tetramethylsilane.
All compounds were dissolved in CDCl3. Elemental analysis
was carried out at Atlantic Microlab Inc. (Atlanta, GA).
Thin-layer chromatography was carried out using an EM Sci-
ence DC-Plastic foil plate coated to a thickness of 0.2 mm
with silica gel 60 containing fluorescent (254) indicator. Col-
umn chromatography was performed with silica gel (70–230)
on an appropriate mobile phase. All the animal studies were
conducted in accordance with the guidelines set forth in the
Declaration of Helsinki and the “Guiding Principles in the
Care and Use of Animals” (DHEW Publication, NIH 80-23).
of the M -receptor subtype (42). In order to design the SASS
3
based on this group of agents (I), the inactive metabolite
approach (20,43) for the design of soft drugs was adapted. A
hypothetical metabolite II (Fig. 1) was chosen as the lead
compound for the design of the soft drugs; maintaining the
positively charged quaternary nitrogen would minimize the
central nervous system (CNS) toxic effects because of lack of
penetration of the blood–brain barrier. The soft drugs (III)
designed are expected to be rapidly hydrolyzed to II in vivo
by ubiquitous esterases. Another hypothetical metabolite,
IIa, is the positional isomer of II. It is known that the smaller
size of the equatorial N-substituent of N-alkyl-nortropine es-
ters of 2-phenyl-2-cyclohexenecarboxylic acids is associated
with higher potency (41). Thus, it is expected that the soft
drugs based on IIa as the lead will be more potent than those
based on II. The hypothetical metabolites are expected to be
highly polar and ionized at physiologic pH and thus be subject
to facile elimination from the systemic circulation either di-
Synthesis
The pathways of synthesis of soft drugs are presented in
rectly or after conjugation. On the other hand, because strong Fig. 2 and 3. and Syntheses of 1– 4 were performed by fol-

A New Class of Soft Anticholinergics
1683
storage in a −20°C freezer overnight, the above mixture was
filtered to give 7 as a white solid (0.75g, 83%).
1H-NMR (CDCl3): 1.71–2.56 (6H, m, 3CH2, cyclo-
hexyl), 3.69 (1H, br, CHCO2), 6.22 (1H, t, 3-H), 7.20-
7.33 (5H, m, ph) ppm.
Synthesis of 8 (8-aza-8-methylbicyclo [3.2.1]oct-3-yl
2-phenylcyclohex-2-enecarboxylate (a colorless liquid) was
achieved through appropriate modifications of the method of
Juhasz et al. (25).
1H-NMR (CDCl3): 1.20-2.27 (14H, m, 3CH2 cyclohexe-
nyl, 4CH2, tropyl), 2.26 (3H, s, NCH3), 2.98-3.10 (2H,
brd, tropyl 1, 5-H), 3.70 (1H, s, CHCO2), 4.87 (1H,
tropyl 3-H CHO), 6.22 (1H, t, CHסCC6H5), 7.25–7.34
(5H, m, ph) ppm.
The synthesis of methyl-2-[8-aza-endo-8-methyl-3-(2-
phenylcyclohex-2-enylcarbonyloxy)bicyclo[3.2.1]oct-8-
yl]acetate, bromide (9a) and ethyl 2-[8-aza-endo-8-methyl-3-
(2-phenylcyclohex-2-enylcarbonyloxy)bicyclo[3.2.1]oct-8-
yl]acetate, bromide (9b) was done as follows: To 2 g (6.14
mmol) of 8 in 20 ml of anhydrous acetonitrile, 15.36 mmol of
methyl bromoacetate or ethyl bromoacetate was added. The
above mixture was stirred under argon for 19 h, which evap-
orated acetonitrile to generate an oily substance, which was
further purified by precipitation with CH2Cl2/ethyl ether to
give pure 9a or 9b as a white solid.
Fig. 2. Synthesis of 2-phenylcyclohex-2-enecarboxylic acid.
lowing the method of Hitchcock et al. (45), and the syntheses
of 5–6 were accomplished by appropriate modifications of the
method of Zimmerman et al. (46). The synthesis of compound
7 (2-phenylcyclohex-2-enecarboxylic acid) is done as follows
(41): 1 g of 6 (4.5 mmol) was added to a mixture of sulfuric
acid (1.5 ml) and acetic acid (8.5 ml) with stirring at room
temperature. The mixture was stirred for another 15 min to
dissolve all solids and then poured into 100 ml of ice water
with stirring. After white crystallized solids appeared, the
mixture was continuously stirred for another 30 min. After
9a: (2.4 g, 81%). M.P. 170-171°C. 1H-NMR (CDCl3):
1.26–2.28 (6H, m, 3CH2, cyclohexenyl), 2.63–2.69 (8H,
m, 4CH2, tropyl), 3.57 (3H, s, NCH3), 3.78 (4H, s,
OCH3 and 1-H of cyclohexenyl), 4.59–4.69 (2H, br d,
tropyl’s 1,5-H), 4.84 (2H,s, NCH2), 5.05 (1H, t, CHO),
6.28 (1H, t, 2-H of cyclohexenyl), 7.23–7.33 (5H, m, Ph)
ppm. Elemental analysis (C24H32O4NBr): calculated/
found (%); C, 60.25/60.06; H, 6.74/6.81; N, 2.92/2.87.
9b: (2.2 g, 73%). M.P.186–187°C. 1H NMR (CDCl3):
1.26–2.60 (14H, m, 3CH2 cyclohexenyl, 4CH2 tropyl),
1.31 (3H, t, CH2CH3), 3.37 (3H, s, NCH3), 3.76 (1H, t,
1-H of cyclohexenyl), 4.22 (2H, q, CH2CH3), 4.58, 4.67
(2H, br d, tropyl’s 1,5-H), 4.82 (2H, s, NCH2), 4.95 (1H,
t, CHO), 6.22 (1H, t, 2-H of cyclohexenyl), 7.20–7.31
(5H, m, Ph) ppm. Elemental analysis (C25H34O4NBr):
calculated/found (%); C, 60.97/61.10; H, 6.55/6.96; N,
2.84/2.79.
The synthesis of 11, 8-azabicyclo[3.2.1]oct-3-yl 2-phenyl-
cyclohex-2-enecarboxylate, proceeded as follows. To 1 g (3.07
mmol) of 8 in 10 ml of 1,2-dichloroethane, 1.09 g (7.63 mmol)
of 1-choroethyl chloroformate in 5 ml of 1,2-dichoroethane
was added dropwise at 0°C. The mixture was then refluxed
for 1 h. Residue from evaporation of the solvent of the reac-
tion mixture was then refluxed in methanol for 45 min. Re-
moval of methanol under reduced pressure gave 11 as a solid
(0.93g, 97%).
1H NMR (CDCl3): 1.46–2.25(6H, m, 3CH2, cyclohexe-
nyl), 2.66–2.90 (8H, m, 4CH2, tropyl), 3.71 (1H, s, 1-H
of cyclohexenyl), 3.80–3.85 (2H, br d, tropyl’s 1,5-H),
4.95 (1H, t, CHO), 6.23 (1H, t, cyclohexenyl’s 3-H),
7.23–7.36 (5H, m, Ph) ppm.
The synthesis of methyl-2-[8-aza-3-(2-phenylcyclohex-2-
enylcarbonyloxy)bicyclo[3.2.1]oct-8-yl]acetate (12a) and
ethyl-2-[8-aza-3-(2-phenylcyclohex-2-enylcarbo-
nyloxy)bicyclo[3.2.1]oct-8-yl]acetate (12b) was done as fol-
lows: To well-stirred compound 11 (1 g, 3.21 mmol) in 20 ml
of N,N-dimethyl formamide (DMF) with 1 g of K2CO3 was
added 3.21 mmol of methyl bromoacetate or ethyl bromoac-
Fig. 3. Synthesis of 9a, 9b, 13a, and 13b.

1684
Huang et al.
etate. The mixture was stirred under argon for 20 h. Then the Stability Studies
DMF was removed under reduced pressure. The residue was
added with 5 ml of saturated NaHCO3 solution, which was
then extracted three times with ethyl ether to give crude 12a
or 12b. The above crude products were further purified by
flash chromatography on ethyl acetate to give pure 12a or 12b
as colorless oil.
Analytic Methods
A high-performance liquid chromatographic method was
developed to detect the soft drugs. The system consisted of a
Spectra Physics (San Jose, CA) SP 8810 isocratic pump, a SP
8450 uv/vis detector with wavelength set to 254 nm, and SP
4290 integrator. A Supelcosil LC ABZ column (Supelco, Bel-
lofonte, PA) was used. The mobile phase consisted of aceto-
nitrile and water (40:60), including 0.1% octanesulfonic acid,
0.2% acetic acid, and 0.1% tetrahydrofuran (THF). At a flow
rate of 1.5 ml/min, the retention times of 9a, 9b, 13a, and 13b
were 3.63, 5.14, 3.91, and 5.14 min, respectively. With an in-
jection volume of 20 ␮l, the detection limits of the compounds
were 1 ␮g/ml.
12a (0.95g, 75%). 1H NMR (CDCl3): 1.20–1.80 (6H, m,
3CH2, cyclohexenyl), 1.81–2.20 (8H, m, 4CH2, tropyl),
3.01–3.06 (2H, br d, tropyl’s 1,5-H), 3.06 (2H, s, NH2)
3.63 (4H, s, OCH3 and 1-H of cyclohexenyl), 4.82 (1H,
m, OCH), 7.12–7.28 (5H, m, Ph) ppm.
12b (0.97g, 76%). 1H-NMR (CDCl3): 1.23–1.70 (6H, m,
3CH2, cyclohexenyl), 1.71–2.27 (8H, m, 4CH2, tropyl),
1.25 (3H, t, CH3), 3.01-3.15 (2H, br d, tropyl’s 1,5-H),
3.72 (1H, t, 1-H of cyclohexenyl), 4.15–4.17 (2H, q,
CH2CH3), 4.89 (1H, m, OCH), 7.25–7.35 (5H, m, Ph)
ppm.
Stability in Biologic Media
Methods were adapted from Huang et al. (47).
The synthesis of methyl 2-[8-aza-exo-8-methyl-3-(2-
phenylcyclohex-2-enylcarbonyloxy)bicyclo[3.2.1]oct-8-
yl]acetate, methyl hydroxysulfonate (13a) and ethyl 2-[8-aza-
exo-8-methyl-3-(2-phenylcyclohex-2-enylcarbonyloxy)
bicyclo[3.2.1]oct-8-yl]acetate, methyl hydroxysulfonate (13b)
was carried out as follows: To compound 12 (a or b, 2.50
mmol) in 10 ml of anhydrous acetonitrile was added dimethyl
sulfate (0.788 g, 6.25 mmol). The mixture was stirred at room
temperature for 15 h, and then acetonitrile was removed un-
der reduced pressure. The residue was purified by precipita-
tion (methylene chloride/ethyl ether) to give 13a or 13b as
white solids.
Pharmacokinetic Studies
Rats (300 to 400 g) were anesthetized by intraperitoneal
injection of sodium pentobarbital (30 mg/kg). 13a (dissolved
in 20% (w/w) of 2-hydroxypropyl-␤-cyclodextrin solution)
was injected into the jugular vein, over 1 min, at doses of 5, 10,
and 15 mg/kg and a dosing volume of 1 ml/kg. For the data
treatment (as bolus injection), the midtime of the injection
was used as 0 time. Blood samples, 0.1 ml, were collected
through the jugular vein at appropriate time intervals for 150
min. The samples were mixed immediately with 0.2 ml of
acetonitrile containing 5% dimethyl sulfoxide solution to halt
further enzymatic hydrolysis in the blood, and then centri-
fuged. Subsequently, the samples were injected in the HPLC
for the determination the concentrations of soft drugs. The
concentration of 13a was determined using a calibration curve
developed by adding known amounts of the compound to the
blood (r ס 0.995) and prepared as samples. Noncompartmen-
tal and compartmental pharmacokinetic analysis: in noncom-
partmental analysis, the area under the curve, AUC, of the
blood concentration vs. time was calculated using the trap-
ezoidal rule. The area from the last measurement, Ct, to in-
finity was calculated as Ct/␤, where ␤ was the terminal dis-
position rate constant. The total body clearance, Cltot, was
calculated as dose/AUC. Mean resident time, MRT, was cal-
culated as AUMC/AUC, where AUMC, the area under the
first moment curve, was calculated using the trapezoidal rule
from the curve of blood concentration × time vs. time, and the
area from the last time point, t, to infinity was calculated as
Ct/␤ + Ct/␤2. The volume of distribution at the steady state,
Vdss, was calculated as Cltot multiplied by MRT. For com-
partmental analysis, a pharmacokinetic analysis program
(PK-Analyst, Micromath, Salt Lake City, UT) was used. The
results were best fitted into a two-compartment model, C ס
Ae−␣t + Be−␤t, where C is the drug concentration in plasma,
A and B are the exponential multipliers, and ␣ and ␤ are the
hybrid constant in the central compartment and peripheral
13a (1.15 g, 90%): M.P.150–151°C. 1H NMR (CDCl3):
1.25–2.70 (14H, m, 3CH2 cyclohexenyl, 4CH2 tropyl),
3.18 (3H, s, NCH3), 3.66 (3H, s, CH3SO4), 3.71(1H, t,
1-H of cyclohexenyl), 3.77 (3H, s, OCH3), 4.35–4.45
(2H, br d, tropyl’s 1,5-H), 4.70 (2H, s, NCH2), 5.03 (1H,
t, OCH), 7.26–7.31 (5H, m, Ph) ppm. Elemental analy-
sis (C25H35O8NS): calculated/found (%); C, 58.92/
58.97; H, 6.92/6.86; N, 2.75/2.45.
13b (1.19 g, 91%): M.P.169–170°C. 1H NMR (CDCl3):
1.25–2.60 (14H, m, 3CH2 cyclohexenyl, 4CH2, tropyl),
3.24(3H, s, NCH3), 3.72 (4H,s, CH3SO4 and 1-H of cy-
clohexenyl), 4.24 (2H, q, CH3CH2O), 4.40–4.54 (2H, br
d, tropyl’s 1,5-H), 4.60 (2H, s, NCH2), 5.01 (1H, m,
OCH), 6.30 (1H, t, cyclohexenyl 1-H), 7.21–7.40 (5H,
m, Ph) ppm. Elemental analysis (C26H37O8NS): calcu-
lated/found (%); C, 58.82/58.77; H, 7.02/7.08; N, 2.64/
2.60.
Receptor Binding Studies
Binding studies and data analyses were performed by
following previously published methods (38).
Mydriatic Studies
Mydriatic studies were conducted by adapting the previ-
ously published methods (47).
compartment, respectively. The AUC was calculated as A/␣ +
1
B/␤, and the half-life of the terminal phase, t
⁄2
, was calculated
Cardiac Studies
as ln2/␤. The volume of distribution of the central compart-
Studies were performed by appropriate modification of ment, Vdc was calculated as dose/(A + B); the volume of
previously published methods (25). distribution during the elimination phase, Vdarea was cacu-

A New Class of Soft Anticholinergics
1685
Table I. Receptor Binding Values* for 9(a,b) and 13(a,b)
lated as CLtot/␤, and the elimination rate constant, Kel, was
calculated as CLtot/Vdc.
Receptor subtype
RESULTS
Synthesis
Compound
Atropine
m1
m2
m3
m4
9.50 0.04
9.08 0.12
9.04 0.20
9.28 0.07
(0.98 0.03) (1.01 0.02) (0.96 0.02) 0.101 0.04)
7.86 0.03 7.73 0.10 8.99 0.01 8.43 0.07
(0.78 0.05) (0.91 0.10) (0.81 0.02) (0.90 0.02)
7.93 0.04 7.97 0.03 8.64 0.05 8.20 0.06
(0.86 0.01) (0.88 0.03) (0.87 0.06) (0.91 0.07)
7.89 0.07 7.38 0.07 8.49 0.02 8.11 0.06
(0.83 0.05) (1.07 0.07) (0.81 0.04) (1.07 0.02)
7.98 0.04 7.70 0.06 8.62 0.05 8.17 0.03
(0.91 0.05) (0.80 0.06) (0.87 0.05) (0.82 0.05)
8.20 7.47 8.64 N/A
9a
The synthesis of the 2-phenylcyclohex-2-enecarboxylic
acid (7) intermediate is shown in Fig. 2; the subsequent syn-
theses of the soft drugs 9a, 9b, 13a, 13b are presented in Fig.
3. 8-Aza-8-methylbicyclo [3.2.1]oct-3-yl 2-phenylcyclohex-2-
enecarboxylate (8) was synthesized based on the methods of
Crowther et al. (48) and Juhasz et al. (25) by reacting
2-phenylcyclohex-2-enecarboxylic chloride with the lithium
salt of tropine. The syntheses of methyl-2-[8-aza -8-methyl-3-
(2-phenylcyclohex-2-enylcarbonyloxy)bicyclo[3.2.1]oct-8-
yl]acetate, bromide (9a) and ethyl 2-[8-aza-endo-8-methyl-3-
(2-phenylcyclohex-2-enylcarbonyloxy)bicyclo[3.2.1]oct-8-
yl]acetate, bromide (9b) were accomplished by quaternizing 8
with the corresponding bromoacetyl ester. It is well estab-
lished that the configuration at the ring nitrogen atom in the
quaternary tropane derivatives depends on the sequence in
which the individual alkyl groups are introduced (49,50). The
alkyl group that entered last has been shown to enter the
equatorial position. The syntheses of the isomeric methyl
2-[8-aza-3-(2-phenylcyclohex-2-enylcarbonyloxy)bi-
cyclo[3.2.1]oct-8-yl]acetate (12a) and ethyl 2-[8-aza-3-(2-
phenylcyclohex-2-enylcarbonyloxy)bicyclo[3.2.1]oct-8-
yl]acetate (12b) were less straightforward. First, we attempted
to react 2-phenylcyclohex-2-enecarboxylic chloride with the
lithium salt of alkoxyl 2-[8-aza-3-bicyclo[3.2.1]oct-8-
yl]acetate. However, because of the lability of the alkoxyl
2-[8-aza-3-bicyclo[3.2.1]oct-8-yl]acetate group, the lithium
salt of alkoxyl 2-[8-aza-3-bicyclo[3.2.1]oct-8-yl]acetate could
not be made. The syntheses of 12a and 12b were eventually
accomplished by adapting the method of Koreeda and Lueng
(51) through the initial demethylation of 8 followed by the
introduction of the alkyoxycarbonylmethyl group. Quater-
nization of 12a and 12b with dimethyl sulfate produced the
soft drugs methyl 2-[8-aza-exo-8-methyl-3-(2-phenylcyclohex-
2-enylcarbonyloxy)bicyclo[3.2.1]oct-8-yl]acetate, methyl hy-
droxysulfonate (13a) and ethyl 2-[8-aza-exo-8-methyl-3-(2-
phenylcyclohex-2-enylcarbonyloxy)bicyclo[3.2.1]oct-8-
yl]acetate, methyl hydroxysulfonate (13b).
9b
13a
13b
Lead†
* The affinity estimates were derived from [3H]NMS displacement
experiments and represented the mean ( SEM, n ס 3–5) for the
negative logarithm of Ki. The Hill coefficients are given in paren-
theses. To ensure that the experimental conditions are consistent,
the receptor binding values of atropine were determined simulta-
neously with the representative soft anticholinergics at each experi-
ment.
† Data were adapted from D’Agostino et al.42
tween the methyl- or ethylester soft anticholinergics. Also,
there was no significant difference between the potency of the
endo and exo isomers.
In Vivo Pharmacodynamic Evaluation
Mydriatic Studies
First, the dose and mydriatic response relationship was
established by administering increasing concentrations of the
compounds until the maximum dilation was achieved. The
lowest dose that produces the maximum achievable dilation
was used for comparison. Accordingly, atropine (0.3% w/v),
tropicamide (0.33% w/v), 13a (0.3% w/v), 13b (0.5% w/v),
and 9a (0.5% w/v) produced equivalent mydriasis. Compound
9b was not studied because of its low solubility. The maximal
dilation was observed within 1 h after administration without
any significant differences among soft drugs, atropine, and
tropicamide. In order to adequately compare the duration of
the mydriatic action of soft anticholinergics with those of at-
ropine and tropicamide, the area under the response curve in
24 h (AUC24h) was calculated with trapezoidal rules for each
compound in each trial. The results are shown in upper panel
of Fig. 4. The AUC24h of 13a was significantly smaller than
that of tropicamide (p < 0.05). The AUC24hs of 13a and 13b
were significantly smaller than that of atropine sulfate (p <
0.05). The time courses of mydriatic activity of the treated
eyes are depicted in middle panel of Fig. 4. The recovery time
for atropine, tropicamide, 9a, 13a, and 13b are 24 h, 10 h, 20
h, 6.5 h, and 8.0 h, respectively.
In Vitro Pharmacodynamic Evaluation: Receptor Binding
Studies
Receptor binding studies using m1, m2, m3, and m4 sub-
types were performed on the soft anticholinergics 9(a, b) and
13(a, b). The results are summarized in Table I. It is shown
that the pKi values ranged from 7.5 to 8.9, indicating compa-
rable potency to the lead compound. The results demon-
strated that compounds 9a and 13a displayed muscarinic sub-
type selectivity (M3/M2). In addition, compound 9a also
showed M3/M1 selectivity. Based on QSAR studies, Turbanti
et al. (41) established that smaller size of the equatorial N-
substituent of an N-alkyl-nortropine esters of 2-phenyl-2- cy-
Cardiac Studies
The cardiac effects of the soft anticholinergics were as-
clohexenecarboxylic acids, was favored for higher potency. sessed by measuring the extent and duration of their brady-
By their arguments, 13a of our series should be more potent cardia protective activities. Intravenous injection of carbachol
than the other soft anticholinergics. However, this was not the at a dose of 5–8 ␮M (27–44 pmol/kg) to male Sprague-Dawley
case. There was no significant difference in the potency be- rats produced the temporary development of sinus bradycar-

1686
Huang et al.
Fig. 5. Bradycardia protective effects of 9a, 13a, and atropine sulfate
as illustrated by the percentage change in the heart rate.
1
used to calculate their t
⁄
2
values. As shown in Table II, al-
though it was not significant in some cases, the methyl esters
were relatively less stable than the ethyl esters. Also, there
was no apparent stability relationship between the endo and
exo isomers. Curiously, the hydrolysis was much faster in
plasma than in blood or liver homogenate, probably indicat-
ing that erythrocyte binding of the compounds was signifi-
cant. The overall results of the in vitro biotransformation
studies of the compounds demonstrated that the “soft” qua-
ternary ammonium acid ester linkage provided a metaboli-
cally sensitive spot that allowed the facile inactivation of mol-
ecules.
Pharmacokinetic Studies
Pharmacokinetic studies on the selected 13a were per-
Fig. 4. Mydiatic studies of soft drugs, atropine, and tropicamide. Top
panel, Comparison of the mydriatic activity of soft drug with atropine formed in rats. The concentrations of the compound in the
and tropicamide by AUC24h method (treated eye) at equieffective
doses. Middle panel, Time course of mydriatic response (treatment
eye) after administration of atropine, tropicamide, 13a, 13b, and 9a.
Bottom panel, Time course of mydriatic response (control eye) after
administration of atropine, tropicamide, 13a, 13b, and 9a.
blood samples were determined by HPLC. Because of the low
detection limit (1 ␮g/ml) of the HPLC system, relatively high
doses were used in the pharmacokinetic studies. As shown on
the blood concentration curves in Fig. 6, 13a was eliminated
from the blood in a biphasic manner. The data were analyzed
by noncompartmental and compartmental methods, and the
resulting pharmacokinetic parameters are listed in Table III.
The concentration–time curves were very well described by
an i.v. bolus two-compartmental model according to a biex-
ponential equation, Cס Ae−␣t + Be−␤t. The statistics on the
correlation coefficents of variation, >0.995, and the model
selection criterion, range 3.8–5.7, indicating the goodness of
fit. As Table III displays, at doses of 5, 10, and 15 mg/kg, the
half-lives (15, 48, and 60 min) and elimination constants (0.23,
0.12, and 0.11 min−1) showed dose dependence. The total
body clearance, although similar at the doses of 15 mg/kg
(11.72 0.35 ml/min/kg) and 10 mg/kg (11.01 0.69 ml/min/
kg), was clearly higher at the dose of 5 mg/kg (16.31 0.63
dia and Mobitz II A-V block, safely and reproducibly. This
effect can be fully prevented by prior administration of an
anticholinergic agent such as atropine or scopolamine. The
bradycardia protective effects of the different anticholinergics
differed greatly in terms of their extent and duration of ac-
tion. In order to compare the duration of action of the soft
anticholinergics and atropine, the approximate pharmacody-
namic equivalent doses were administered to Sprague-
Dawley rats. After the administration of two compounds, 9a
(2 ␮M) or 13a (2 ␮M), the Mobitz II A-V block appeared in
about 15–20 min (Fig. 5). On the other hand, atropine methyl
bromide displayed protective effects against Mobitz II A-V
block for at least 60 min. Actually, in preliminary experi-
ments, duration of action longer than 2 h was found for at-
ropine methyl bromide.
Table II. In Vitro Stability of Compounds 9a, 9b, 13a, and 13b*
Rat liver
Compound
Rat plasma
Rat blood
homogenate
In Vitro Stability Studies
9a
9b
13a
13b
15.21 8.5
21.88 8.21
30.26 2.09
34.29 4.80
150 15.97
185.30 15.92
105.30 20.66
190.80 25.32
111.68 7.10
182.29 15.69
94.61 5.41
99.89 18.72
The enzymatic target of the soft drugs is the ester func-
tion, which is expected to undergo facile, one-step hydrolysis
by blood and tissue esterases. The stability and metabolic
pathways of the present soft drugs in rat plasma, blood, and
liver homogenate were investigated. The pseudomonomo-
lecular rates of disappearance of the drugs in the media were
* Half-lives (min) of the compounds in biological media. Data are the
mean ( SD) of three determinations.

A New Class of Soft Anticholinergics
1687
the higher TI. With the advance of molecular biology, mus-
carinic subtype receptors were cloned (28,29) and became
readily available. This has facilitated the drug development
efforts to find anticholinergics with muscarinic receptor sub-
type selectivity: a few of the compounds already discovered
have reached clinical trials (36). In the present study, we were
trying to improve therapeutic index for anticholinergics by
combining these two approaches: to develop soft anticholin-
ergics with muscarnic receptor subtype selectivity (M3/M2).
Soft anticholinergics with subtype selectivity (SASS) can be
used not only as safer antiperspirants, mydriatics, and antiul-
cer agents but also as safer preoperative medications for in-
hibiting excessive secretion. Anticholinergics, such as atro-
pine and glycopyrrolate, are frequently used for premedica-
tion to reduce oral and respiratory secretion and prevent
bradycardia (1). They are also administered with neostigmine
for the reversal of nondeploarizing neuromuscular blockade
(3–5). However, such application has been complicated by the
cardiac side effects of anticholinergics (2–5), particularly dan-
gerous to the patients with preexisting cardiac diseases (40).
SASS (M3/M2) will assist the safer administration in anesthe-
sia, either as premedication for reducing excessive salivation
and secretion of the respiratory tract induced by administra-
tion of general anesthetic agents or as an agent to reverse the
nondeplorizing neromuscular blockade. Because of their
muscarinic receptor subtype selectivity (M3/M2), the occa-
sional serious arrhythmias associated with the conventional
anticholinergics are expected to be greatly reduced when
SASS are used in an anesthetic procedures, and the soft na-
ture of these compounds will allow us to titrate the needed
dose during these procedures.
In the mydriatic studies, it was shown that at pharmaco-
dynamic dose-equivalent doses, two of the soft anticholiner-
gics, 13a and 13b, displayed shorter duration than tropic-
amide, the most frequently used short-term mydriatic agent in
the market; all soft anticholinergics tested, except 9a, showed
significantly shorter duration of mydriatic action in the
treated eye than atropine sulfate. In our studies, the recovery
time of tropicamide was 10 h, which is different from the
published data of 6 h (22,53). It might be caused by physi-
ologic differences between the groups of rabbits. At the
equieffective dose, all soft anticholinergics tested, except 9a,
showed significantly shorter duration of mydriatic action in
the treated eye than atropine sulfate. In the present study, the
methyl esters (soft anticholinergics) displayed shorter dura-
tion than did the ethyl esters, which was inconsistent with the
previous findings in the literature (55). In our studies, the
lowest concentration needed to achieve the maximum pupil
dilation was generally in agreement with the receptor binding
data. For 9a, 13a, and 13b, the m3 receptor binding values
were almost the same (9a ס 8.99, 13a ס 8.49, 13b ס 8.62),
the lowest concentrations required to reach maximum pupil
Fig. 6. Mean plasma concentration-time profiles after intravenous
injection at a dose of 5 mg/kg (᭡), 10 mg/kg (᭹), or 15 mg/kg (᭿).
ml/min/kg). A pharmacokinetic study was also performed at
low doses such as 1 and 0.5 mg/kg. The results indicated a
rapid disappearance of 13a from the blood. However, because
of the detection limit in the HPLC, the data were not used for
pharmacokinetic analysis.
DISCUSSION
Muscarinic receptors are involved in the control of func-
tions of many organs in the body. Three major muscarinic
receptor subtypes—M1, M2, and M3—mediate a variety of
basic functions of the body (27). These receptors have specific
locations and control particular physiologic activities. How-
ever, most of the currently available anticholinergic agents
are not subtype selective and inhibit all muscarinic subtype
receptors in the body equally once they are in the systemic
circulation. Thus, therapeutics for an intended symptom usu-
ally results in many undesired effects.
To improve the therapeutic index (TI) of anticholiner-
gics, the soft drug design principles were applied to anticho-
linergics from the early 1980s (52). The safer anticholinergics
were primarily designed for topical application, such as anti-
perspirants (52), mydriatics (53,54), and antiulcer drugs (21),
and their facile, predictable hydrolytic deactivation assured
Table III. Pharmacokinetic Parameters of 13a After Intravenous Bo-
lus Administration to Rats*
Dose (mg/kg)
AUC (␮g и min/ml) 307.0 11.9
Cltot (ml/min/kg)
MRT (min)
Vdss (ml/kg)
A (␮g/ml)
B (␮g/ml)
␣ (1/min)
␤ (1/min)
t1/2 (␤) (min)
Vdc (ml/kg)
Vdarea (ml/kg)
kel (l/min)
5 (n ס 3)
10 (n ס 4)
945.0 77.4
11.0 0.7
15 (n ס 4)
1278.5 54.8
11.7 0.4
16.3 0.6
16.9 0.9
274.6 4.6
60.3 15.8
11.5 0.4
0.95 0.18
56.9 6.6
72.7 12.5
577.5 54.7
115.6 30.6
12.1 1.3
896.5 205.2 dilation were virtually the same (9a ס 0.5%, 13a ס 0.3%, 13b
117.3 12.4
12.9 2.3
0.68 0.09
0.012 0.001
59.5 9.0
116.6 13.1
1045.2 167.7
0.11 0.00
ס 0.3%). For 9a, even though its binding value (m3, pKi ס
8.99) was a little bit higher than that of 13a and 13b, it needed
a slightly higher concentration to achieve the maximum pupil
dilation. This could be related to its endo isomer structure,
which may be unfavorable to bind to the receptor in iris-
ciliary body.
The soft anticholinergics were found to cause no or mild
irritation on topical administration into the eye. Reports in
the literature suggested a relationship between the length of
0.73 0.24
0.045 0.005 0.014 0.001
14.6 1.0
73.1 15.6
365.5 26.6
0.23 0.04
47.5 4.2
90.2 21.6
698.3 69.0
0.12 0.03
* Each value represents mean SD of 3–4 trials.

1688
Huang et al.
hydrophobic side chains and irritation potential of pharma- studies suggest that the new soft anticholinergics are rapidly
ceutical compounds (6). As the soft anticholinergics tested eliminated from plasma (systemic circulation) after i.v. ad-
have generally shorter side chains (methyl and ethyl group), ministration.
they may cause less irritation. The time courses of mydriatic
activity in the untreated eyes (at the equieffective dose) are
depicted in the bottom panel of Fig. 4. Significant dilation of
the untreated eyes was observed with atropine sulfate and
tropicamide, but not with the soft drugs. It suggested that the
“hard drugs” atropine and tropicamide were able to dilate the
untreated eye after systemic absorption. Soft drugs, however,
during and after absorption into the systemic circulation, un-
derwent a one-step deactivation to the inactive metabolites,
and therefore, the systemic toxicities were expected to be
significantly reduced.
In cardiac studies, compared with atropine, the shorter
cardiac protective effect of equipotent soft drug doses was
consistent with the facile hydrolysis of the soft anticholiner-
gics to inactive metabolite. Juhasz et al. (25) reported studies
on duration of antagonism of carbachol-induced brandycardia
of several other types of soft anticholinergics, finding that
methoxycarbonylphenylcyclopentylacetyl-N,N-dimethyl-
tropinium methyl sulfate (PCMS-II), a different type of hin-
dered ester, also had a duration time of 15–30 min. The pres-
ent 9a and 13a, although different type of structures, pos-
sessed similar duration of action. However, tematropium sul-
fate, which lacked a bulky group in the moiety, had duration
of cardiac protective action of only 3 min (43).
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