Pyridophens: Binary pyridostigmine-aprophen prodrugs with differential inhibition of acetylcholinesterase, butyrylcholinesterase, and muscarinic receptors

Article abstract of DOI:10.1021/jm010196t

A series of "binary prodrugs" called carbaphens, carbamylated derivatives on one or both of the aromatic rings of the muscarinic receptor antagonist aprophen [(N,N-diethylamino)ethyl 2,2-diphenylpropionate], were synthesized to develop binary prophylactic

Full text of DOI:10.1021/jm010196t

902
J . Med. Chem. 2002, 45, 902-910
P yr id op h en s: Bin a r y P yr id ostigm in e-Ap r op h en P r od r u gs w ith Differ en tia l
In h ibition of Acetylch olin ester a se, Bu tyr ylch olin ester a se, a n d Mu sca r in ic
Recep tor s
Haim Leader,† Alan David Wolfe,‡ Peter K. Chiang,‡ and Richard K. Gordon*,‡
Division of Biochemistry, Walter Reed Army Institute of Research, 503 Robert Grant Road,
Silver Spring, Maryland 20910-7500
Received May 4, 2001
A series of “binary prodrugs” called carbaphens,1 carbamylated derivatives on one or both of
the aromatic rings of the muscarinic receptor antagonist aprophen [(N,N-diethylamino)ethyl
2,2-diphenylpropionate], were synthesized to develop binary prophylactic agents against
organophosphorus intoxication. As a group, the carbaphens retained the muscarinic receptor
antagonist properties of aprophen but also preferentially inhibited butyrylcholinesterase (BChE)
in contrast to acetylcholinesterase (AChE). Therefore, a new series of compounds named
pyridophens were designed and synthesized to achieve binary prodrugs to preferentially inhibit
AChE over BChE, while still retaining the muscarinic receptor antagonism of aprophen. The
pyridophens consist of the basic pyridostigmine skeleton combined with the 2,2-diphenylpro-
pionate portion of aprophen by replacement of the diethylamino group. Three compounds, 9 (a
tertiary pyridine), 10 (a quaternary pyridine), and 12 (a tertiary tetrahydropyridine), were
found to be effective inhibitors of both BChE and AChE. However, 10, N-methyl-3-[[(dimethyl-
amino)carbonyl]oxy]-2-(2′2′-diphenylpropionoxy-methyl)pyridinium iodide, inhibited AChE
selectively over BChE, with a bimolecular rate constant similar to pyridostigmine. In contrast
to their potent cholinesterase inhibitory activity, all of the pyridophen analogues were less
potent antagonists of the muscarinic receptor than aprophen.
In tr od u ction
were designed and synthesized to achieve preferential
inhibition of AChE over BChE, while retaining anti-
muscarinic activity. We describe here (i) the synthesis
of pyridophens, (ii) their anticholinesterase properties,
and (iii) their antimuscarinic activity.
Current prophylactic and therapeutic regimens for the
protection against organophosphate (OP) poisoning
include a pretreatment regimen consisting of (i) pyri-
dostigmine, the reversible, covalent acetylcholinesterase
(EC 3.1.1.7; AChE) inhibitor,2-4 (ii) the muscarinic
receptor antagonist atropine (ATR),5 and (iii) the AChE
reactivator pralidoxime chloride.3,6 Additional treatment
with diazepam has proven advantageous in attempts
to prevent convulsions.7-9 These regimens provide
extensive protection against OP intoxication in animals.
However, treatment might be considerably simplified
if drugs that possessed multiple protective functions
were used. For example, a single drug that exhibited
both reversible antimuscarinic (in place of ATR) and
reversible anticholinesterase (in place of pyridostigmine)
properties might be of unusual utility and efficacy,
simultaneously protecting AChE from an irreversible
OP inhibition and the occupation of muscarinic recep-
tors by accumulated acetylcholine. To test this hypoth-
esis, a group of experimental binary prodrugs, the car-
baphens, were synthesized and evaluated.1 These com-
pounds, which contain a carbamyl substituent on one
or the other aromatic rings of aprophen, a muscarinic
receptor antagonist,11 retained the antimuscarinic activ-
ity of aprophen but inhibited butyrylcholinesterase (EC
3.1.1.8; BChE) preferentially in comparison with AChE.10
In view of these findings, the pyridophen analogues
Ch em istr y
To synthesize the pyridophens, the pyridostigmine
skeleton was introduced and combined with the basic
structure of aprophen, leading to the quaternary com-
pound 10 and to the tertiary tetrahydropyridine ana-
logue 12. The synthesis is outlined in Figure 1. The key
intermediate, 2-hydroxymethyl-3-dimethylaminocarbo-
nyl-oxypyridine (2), was isolated by column chromatog-
raphy as the major product from the carbamylation of
3-hydroxy-2-hydroxymethylpyridine HCl (1) with di-
methylcarbamyl chloride. The other two products iso-
lated from the reaction mixture were found to be the
isomeric carbamate 3 and the bis-carbamate 4. Struc-
tural elucidation of the isomers 2 and 3 was based on
1
the H nuclear magnetic resonance (NMR) spectrum of
these two compounds.
Acylation of 2 with 2,2-diphenylpropionyl chloride (6)
afforded the carbamyl-ester (9) in moderate yield.
However, 9 could be obtained through an alternative
pathway by acylation of 1 with the acyl chloride 6,
followed by carbamylation of the hydroxy ester 7 by
dimethylcarbamyl chloride; this alternative route was
found to give a higher yield. Carbamylation of 7
with methylisocyanate afforded the monomethylcar-
bamate 8.
* Corresponding author. Tel: 301-319-9987. Fax: 301-319-9571.
E-mail: Richard.Gordon@na.amedd.army.mil.
The quaternary carbamate 10 (X ) I-) was obtained
by methylation of 9 with methyl iodide in dry acetone.
† Israel Institute for Biological Research, POB 19, Ness Ziona, Israel.
‡ Walter Reed Army Institute of Research.
10.1021/jm010196t CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/11/2002

Binary Pyridostigmine-Aprophen Prodrugs
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 903
F igu r e 1. Synthetic pathways for the pyridophen analogues. Key: (a) (CH3)2NCOCl. (b) SOCl2. (c) 1, triethylamine, benzene. (d)
CH3NCO, benzene. (e) 2, pyridine. (f) CH3I or (CH3O)2SO2, acetone. (g) CH3I, acetone. (h) 6, pyridine. (i) NaBH4, methanol. (j)
(CH3)2SO2, acetone, X- is I- or [CH3SO4]-.
The same quaternary compound could be obtained
directly from the methyl pyridinium carbamate 11
(obtained by methylation of 2 with methyl iodide) with
the acyl chloride 6 in pyridine. The two products were
identical, thus verifying the structural elucidation of the
carbamates 2, 9, and 11. Also, the quaternary carbam-
ate 10 (X ) -OSO2OCH3) was obtained by methylation
of 9 with (CH3O)2SO2 in dry acetone. The latter product
was identical in all biological assays and offered two
advantages: the quaternization was faster, and the
product was more stable in solution.
Reduction of the carbamylated methylpyridinium
iodide 10 with NaBH4 in methanol afforded the tet-
1
rahydropyridine analogue 12. Here again, the H NMR
spectrum, including homodecoupling irradiation tech-
niques, was the major basis for the elucidation of the
structure and assignment for the location of the double
bond in the tetrahydropyridine structure.
Biologica l Activities
F igu r e 2. Pyridophen inhibition of BChE and AChE. This
histogram compares the inhibitory activity of the pyridophens
with AChE (hatched) and BChE (solid). The ordinate repre-
sents the percent residual cholinesterase activity. Assay
conditions were as follows: compound concentrations, 10-4 M;
enzyme concentrations, AChE ) 10 U and BChE ) 15 U;
incubation, 1 h at 25 °C in a final volume of 100 µL of 0.05 M
phosphate buffer, pH 8.0. Each value represents the mean of
3-5 independent assays ( SEM.
I. ACh E a n d BCh E In h ibition . A comparison of the
inhibitory potency of the respective compounds (Figure
1) with AChE (hatched bar) and BChE (solid bar) is
illustrated in Figure 2. In this comparison, each enzyme
was incubated with a concentration of 10-4 M of the
appropriate compound for 1 h at 2 °C. AChE was
inhibited most strongly by 10, while BChE was inhibited
most strongly by 9 and 12. Compounds 2, 3, and 11 also
inhibited AChE preferentially in comparison with BChE,
although such inhibition was considerably less than that
exhibited by 10 (Figure 2). Compounds 7 and 13, which
lack the pyridostigmine skeleton, were without effect
at these concentrations. Table 1 presents a summary
of the kinetic and affinity constants derived from (i)
time-dependent enzyme incubation with graded con-
centrations of 2, 9, 10, 12, and pyridostigmine (i.e.,

904 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Leader et al.
Ta ble 1. Kinetic and Equilibrium Constants for AChE and
BChEa
compound
Ki (M)
k2′ (M-1 min-1
)
k2c (min-1
)
AChE
2
9
10
12
5.21 × 10-5
3.88 × 10-5
5.62 × 10-6
4.59 × 10-5
1.97 × 10-5
1.25 × 103
2.78 × 104
2.28 × 105
8.45 × 103
2.91 × 105
0.07
1.08
1.28
0.39
5.78
pyridostigmineb
BChE
2
9
10
12
1.38 × 10-3
8.51 × 10-6
3.14 × 10-6
5.53 × 10-6
1.14 × 10-4
7.87 × 102
3.43 × 105
9.38 × 103
2.55 × 105
4.56 × 103
1.08
2.91
0.03
1.41
0.52
pyridostigmine
a
Each constant represents the mean of 3-5 independent
experiments, and r2 g 0.95 for all primary and secondary plots
from which the constants were derived (SEM e 13%). Data from
b
ref 11.
F igu r e 4. Typical secondary plots of pseudo-first reaction
curves for doses of 10 (upper curve) and pyridostigmine12
(lower curve); linear regression coefficients were g0.99. Kinetic
and affinity constants derived from eq 2 are reported in Table
1. Y-axis, reciprocal of the reaction velocity × the concentration
of the inhibitor; X-axis, reciprocal of the apparent equilibrium
constant (molarity). AChE concentration for 10 and pyridostig-
mine was 11 and 40 U/mL, respectively.
F igu r e 3. Time-dependent inhibition of AChE by 10. Y-axis,
log percent residual activity; X-axis, time in minutes. The
linear regression coefficients for the six doses in this typical
experiment were g0.99.
Figure 3 illustrates the inhibition of AChE by 10) and
(ii) secondary plots11 of such data (i.e., Figure 4 shows
the effect of 10 and pyridostigmine on AChE). Com-
pound 10 preferentially inhibited AChE in comparison
with BChE and inhibited both cholinesterases at least
as strongly as pyridostigmine.12 A comparison of the
affinity constant (Ki) values for the initial reaction (eq
1, Table 1) of 10 and pyridostigmine with both enzymes
shows 10 to bind more tightly to these enzymes than
pyridostigmine, whereas differences in inhibition occur
through differences in the carbamylation rates (k2c;
release of the alcoholic moieties from the enzyme-
inhibitor [E‚I] complexes). The rate of carbamylation of
AChE by 10 was 44 times greater than observed with
BChE.
Bimolecular rate constants (k2′ ) affinity constant/
carbamylation rate; eq 1, Table 1) for 9 and 12 revealed
that these compounds react 10 and 3 times more
strongly with BChE than with AChE, respectively. The
affinity of 9 was 20 times greater for BChE than for
AChE, while its carbamylation rate with BChE was
twice its carbamylation rate for AChE. The Ki of 12 was
10-fold greater for BChE than for AChE, while its
carbamylation rate was 3.5-fold greater with BChE than
AChE.
F igu r e 5. Dose response inhibition curve of BChE by 10 (left)
and aprophen (right). The reaction was initiated by the
addition of 15 U of BChE to Ellman assay mixtures. Y-axis,
percent residual enzyme activity; X-axis, log of the molar
concentration of 10 or aprophen. The representative experi-
ment yielded linear regression coefficients of g0.98.
To detect noncovalent, reversible enzyme inhibition
by the compounds, they were evaluated by inclusion in
the colorimetric assay solution,13 rather than by prein-
cubation with enzyme. Aprophen is a BChE inhibitor
of this type;14 it lacks a carbamyl function; therefore,
its ability to inhibit BChE depends on ionic and hydro-
phobic interactions. BChE was completely inhibited by
10 in this assay, whereas 9 and 12 inhibited BChE to
the same extent as aprophen. Figure 5 compares dose
response curves for reversible BChE inhibition by 10
and aprophen: the 50% inhibitory concentration (I
)
50
of 10 was 1.7 × 10-7 M, while the I50 of aprophen was
6.7 × 10-6 M.14 Thus, the apparent hydrophobic and

Binary Pyridostigmine-Aprophen Prodrugs
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 905
Ta ble 2. Antimuscarinic Activity of Pyridophen Analogues and
Standard Compounds
ionic affinity of 10 for BChE was 40-fold greater than
that of aprophen, and the observed I50 for aprophen was
in agreement with a previous report.14 For these com-
pounds, only results with BChE are shown, since AChE
failed to undergo this type of inhibition.
compound
Ki (M) ( SDa
compound
Ki (M) ( SDa
7
8
9
10
11
7.5 ( 1.1 × 10-5 12
8.1 ( 1.8 × 10-6
2.9 ( 1.6 × 10-5 13
2.6 ( 0.9 × 10-6
4.2 ( 1.7 × 10-5 pyridostigmine >10-4
Strong, preferential inhibition of AChE occurred with
compound 10, and in structure and activity, 10 more
closely resembled its progenitors, pyridostigmine and
aprophen, than other compounds in this series. Com-
pound 10 binds strongly to AChE through a time-
dependent carbamylation reaction similar in magnitude
to the one between AChE and pyridostigmine. Also, 10
binds strongly to BChE both covalently, as does pyri-
dostigmine, and noncovalently through hydrophobic and
ionic interactions similar to the binding of aprophen to
BChE. Likewise, 9 and 12 inhibited BChE nonco-
valently (not shown), with affinities similar to aprophen.
4.7 ( 1.4 × 10-6 aprophenb
5.1 ( 1.0 × 10-8
>10-4
ATRb
2.4 ( 0.7 × 10-9
[3H]-NMS binding to cerebral cortex membranes. Each inhibi-
tion constant represents the mean of 3-6 independent experiments
( SD of the mean. The Hill slopes for all compounds were not
a
b
significantly different from 1. From ref 1.
The basis for preferential BChE inhibition by ap-
rophen and its analogues now appears explicable based
on the structure and properties of chimeric AChE
mutants.15-17 In one group of mutants, the conversion
of TYR337 (mouse AChE) or PHE330 (Torpedo californica
AChE) to ALA was accompanied by a concomitant
increase in the affinity of acridines and phenothiazines
to that which occurs with BChE.16 In other mutants,
replacement of PHE295 and PHE297 with aliphatic resi-
dues releases constraints upon the acyl pocket, bestow-
ing BChE-like properties upon the chimeras.15-17 Such
mutants provide the basis for an experimental resolu-
tion of the preferential inhibition of BChE as opposed
to AChE by aprophen and other benzilic acid esters.
Compound 10 contains both the N-methyl pyridinium
ring and the dimethylaminocarbamyl substituent of
pyridostigmine in contrast to 8, which contains a
monomethylamino carbamyl substituent but lacks a
quaternary ring nitrogen, while 9 lacks a quaternary
ring nitrogen. Replacement of the pyridinium ring of 10
with a partially saturated structure in 12 also resulted
in the absence of a quaternary ring nitrogen. These
changes indicate that both the quaternary ring nitrogen
and the presence and position of the dimethylaminocar-
bamyl substituent of pyridostigmine found in 10 are
necessary for a strong inhibition of AChE, and each
change in this structure reduces activity against AChE
(Figure 2). Thus, the presence in 11 of a hydroxymethyl
group in the 2-position of pyridostigmine reduced activ-
ity. While 11 preferentially inhibited AChE, this inhibi-
tion was very weak in comparison with 10. The presence
of the 2,2-diphenylpropionic acid substituent of ap-
rophen in 8-10 and 12 also caused a significant
increase in hydrophobic, noncovalent reversible inhibi-
tion of BChE.
F igu r e 6. Displacement of [3H]-NMS bound to cortex mAChR
by pyridophes 10 (top) and 12 (bottom). Y-axis, percent control
[3H]-NMS bound; X-axis, log concentration (M) of pyridophen
10 or 12. Each point represents the mean ( SD of three
replicates.
II. Mu sca r in ic Recep tor In h ibition . Table 2 lists
the Ki values obtained for the inhibition of [3H]-NMS
(N-[3H]methylscopolamine) binding to mAChR by com-
pounds 7-10, 12, and 13; the Ki values were 75, 29,
42, 4.7, 8.1, and 2.6 µM, respectively. The Hill slopes
for the inhibition curves were not significantly different
from 1, as shown in Figure 6 for 10 and 12. In contrast
to these pyridophen analogues, both 11 and pyridostig-
mine at a dose of 100 µM minimally inhibited [3H]-NMS
binding to mAChR (less than 20%). Also shown for
reference in Table 2 are the Ki values for the inhibition
of mAChR obtained for aprophen and the muscarinic
antagonist ATR.1,10 Both the iodide and the methyl
sulfate of 10 and 13 yielded similar Ki values and are
not reported separately.
Next, the possible effects of 10 and 12 on the dis-
sociation kinetics of [3H]-NMS binding to the mAChR
in cortex tissue were evaluated. A decrease in the rate
of dissociation of [3H]-NMS from mAChR would indicate
an allosteric interaction on mAChR by these compounds
in addition to inhibition at the antagonist binding
site.18-20 The dissociation of bound [3H]-NMS in the

906 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Leader et al.
Ta ble 3. Dissociation Constants for [3H]-NMS Binding to
Guinea Pig Cerebral Cortex Muscarinic Receptorsa
dissociation rate constants (min-1
)
compd(s) µM
ATR
k-1
k-2
A fraction
0
0.095 ( 0.011
0.082 ( 0.005
0.079 ( 0.007
0.089 ( 0.013
0.108 ( 0.002
0.102 ( 0.006
0.096 ( 0.010
0.108 ( 0.006
0.018 ( 0.005
0.015 ( 0.007
0.013 ( 0.009
0.020 ( 0.009
0.025 ( 0.008
0.021 ( 0.003
0.019 ( 0.004
0.022 ( 0.009
0.44 ( 0.07
0.47 ( 0.10
0.49 ( 0.09
0.49 ( 0.09
0.51 ( 0.05
0.51 ( 0.09
0.47 ( 0.07
0.48 ( 0.07
ATR + 10 12
ATR + 10 25
ATR + 10 50
ATR
0
ATR + 12 12
ATR + 12 25
ATR + 12 50
a
After a 90 min incubation with 2 nM [3H]-NMS, 1 µM ATR
was added to each sample along with the indicated concentration
(0, 12, 25, or 50 µM) of the pyridophen analogue (10 or 12). The
means ( SD are listed for two experiments, each value in
triplicate. As described in the Experimental Section, the dissocia-
tion constants were determined using the equation:18 (T) ) Ae-k
-
+ Be-k T. The A fraction is that portion of the bound ligand-
1T
-2
receptor complex with a fast dissociating constant, k-1; B is the
fraction with a slow dissociation constant, k-2 (i.e., 1 - A ) B).
analogues contrast with ATR and aprophen, which have
Ki values of 2.4 and 51 nM, respectively (Table 2). Thus,
the pyridophens as a group were all more than 50-fold
less potent than the parent compound aprophen. In
contrast, pyridostigmine, which carbamylates cholinest-
erases, had a minimal effect on [3H]-NMS binding to
mAChR at 100 µM (Table 2).
We have previously demonstrated that monomethyl-
carbaphen (â-(N,N-diethylamino)ethyl 2-phenyl-2-[4-
[[(methyl-amino)carbonyl]oxy]phenyl]propionate), where
a carbamyl group is substituted at the para position of
the phenyl ring of aprophen at the bulky hydrophobic
portion of the molecule, retained antimuscarinic potency
similar to the parent compound aprophen (28 vs 51 nM,
respectively).1 Unlike the carbaphen analogues, all of
the pyridophens were significantly less potent than
aprophen. The most potent compound in the series with
a Ki of 2.6 µM was 13, which represents the leaving
group after 10 carbamylates the cholinesterases. Struc-
ture-activity relationship data suggest that the ionic
binding by the mAChR to the protonated nitrogen of
aprophen, ATR, or the carbaphens must be significantly
different than found in the pyridophens. The location
of the added carbamate moiety in the pyridophen
analogues, e.g., 10, might prevent a proper interaction
of its quaternary nitrogen with the anionic site of the
mAChR.21,22 However, 13 contains a hydroxyl group in
place of the carbamate moiety and is only about 2-fold
more potent. The mechanism by which the carbamate
and/or hydroxyl moiety in the pyridophens prevented
high mAChR binding is not clear. Several possible
explanations include (i) the carbamate ester positioned
the pyridophen in a location normally reserved for the
ester of the 2,2-diphenylpropionate group, (ii) the OH
or carbamate disrupted ionic bonding to the protonated
nitrogen, and (iii) the size of the OH or carbamate group
excluded proper alignment within the antagonist bind-
ing site. There is evidence for carbamate-induced mis-
alignment within the hydrophobic portion of antimus-
carinic compounds. A single carbamate, but not two
carbamate groups, added to the phenyl moiety of 2,2-
diphenylpropionate is accommodated into the large
hydrophobic pocket of the receptor without altering
potency.1 However, note that in the latter case, the
carbamate(s) were added to the bulky phenyl groups
F igu r e 7. Time course for the dissociation of bound [3H]-NMS
from cortex mAChR. Dissociation was measured in the pres-
ence of 1 µM ATR alone or 1 µM ATR and indicated doses of
10 or 12. Each point represents the mean ( SD of duplicate
experiments each in triplicate; (top) 10, (bottom) 12. Y-axis,
fraction of control [3H]-NMS bound; X-axis, time in minutes.
presence of ATR alone or ATR combined with graded
doses of 10 or 12 are shown in Figure 7, top and bottom,
respectively. The dissociation rate constants obtained
from these figures by nonlinear curve fitting are re-
ported in Table 3. The dissociation of the [3H]-NMS-
receptor complex in the presence of ATR alone was
biphasic in the cortex membrane (Table 3; the portion
of muscarinic receptors possessing a fast dissociation
constant is the A fraction, as previously observed).20
However, the dissociation curves obtained when 10 or
12 were added together with 1 µM ATR remained
unaltered (Figure 7, Table 3) since the curves did not
shift. An upward shift would indicate a slowing of the
dissociation of [3H]-NMS from the muscarinic recep-
tor.18,20 Because neither 10 nor 12 had an affect on the
k-1 or k-2 dissociation rate constants (Table 3) and the
Hill slopes (Figure 6) were not significantly different
from 1, these compounds did not allosterically modulate
[3H]-NMS binding in cortex but rather interacted only
at the [3H]-NMS antagonist binding site.
The potency order of the six pyridophen analogues as
antagonists of mAChR was as follows: 13 ∼ 10 ∼ 12 >
8 ∼ 9 ∼ 7 (Table 2). The potency of the pyridophen

Binary Pyridostigmine-Aprophen Prodrugs
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 907
3-h ydr oxypyr idin e (3), an d 2-[[(Dim eth ylam in o)car bon yl]-
oxy]m eth yl-3-[[(d im eth yla m in o)ca r bon yl]oxy]p yr id in e
(4). Dimethylcarbamyl chloride (5 mL) was added to a warm
solution (50 °C) of 3-hydroxy-2-(hydroxymethyl)pyridine hy-
drochloride (1, 3.2 g, 0.02 mol) in 50 mL of dry pyridine. The
mixture was heated (50 °C) with stirring for another 30 min
and cooled to 20 °C, and the pyridine was evaporated under
vacuum. The crude reaction mixture was dissolved in H2O (100
mL), excess K2CO3 was added, and the basic aqueous solution
was extracted with ethyl acetate (3 × 50 mL). The combined
extracts were washed with brine (2 × 50 mL) and dried
(MgSO4), and the solvent was evaporated to give 3.1 g of a
brown viscous oil, which was found to be a mixture of 2-4
rather than to the opposite end of the compound
containing the quaternary amine.
An alternate explanation for the decreased potency
of the pyridophens in comparison with aprophen might
be the result of the rigidity of the pyridostigmine moiety
replacing the very flexible diethylaminoethanol found
in aprophen.21,22 The flexibility or rigidity of an antago-
nist has been proposed as a mechanism by which a com-
pound can attain the conformation necessary to interact
with mAChRs in general and subtypes of mAChRs
specifically.21 In this study, aliphatic and therefore less
rigid compounds with the quaternary nitrogen three
carbon atoms distant from the ester group of the 2,2-
diphenylpropionate yielded Ki values of about 140 and
500 nM for N-methyl-2-piperidinemethyl-2,2-diphenyl-
propionate and N-ethyl-3-piperidyl-2,2-diphenylpropi-
onate, respectively. These results suggest that the
rigidity of the pyridinium ring containing either a
hydroxyl or a carbamate moiety as found in 13 or 10
decreases potency 5-25-fold over the aliphatic ring
conformation.
1
(60:30:10) according to TLC and H NMR analysis. Separation
and purification of 2-4 were achieved by column chromatog-
raphy (silica, 5% MeOH-CHCl3).
Com p ou n d 2. Yielded 2.10 g, viscous oil, solidified on
standing, mp 55 °C, TLC (silica, 5% MeOH-CHCl3) Rf
)
0.50. 1H NMR (CDCl3): δ 8.42 (d.d, 1H, J ) 4.8, 1.3 Hz),
7.55 (d.d, 1H, J ) 8.1, 1.3 Hz), 7.28 (d.d, 1H, J ) 8.1, 4.8 Hz),
4.73 (s, 2H), 3.12 and 3.03 (2 s, 6H, Me2N-). Anal. (C9H12N2O3)
C, H, N.
Com p ou n d 3. Yielded 0.66 g, mp 118-119 °C (dec, from
ethyl acetate; ether), TLC (silica, 5% MeOH-CHCl3) Rf ) 0.65.
1H NMR (CDCl3): δ 8.16 (d.d, H, J ) 4.4, 1.5 Hz), 7.28 (d.d,
1H, J ) 6.4, 1.5 Hz), 7.23 (d.d, 1H J ) 8.3, 4.4 Hz), 5.22 (s,
2H), 2.94 (s, 6H, Me2N-). Anal. (C9H12N2O3) C, H, N.
Com p ou n d 4. Yielded 0.15 g of viscous oil, TLC (Silica, 5%
Con clu sion s
The series of pyridophens resulted in compounds,
notably 9 and 12, which inhibited BChE specifically,
similar to the previously synthesized carbaphens.1 More
important, the pyridophens 10 and to a lesser extent 2,
3, and 11 preferentially inhibited AChE over BChE
and contrasts with the previously described carbaphens
that showed no AChE specificity.1 With respect to
muscarinic receptors, all of the pyridophens displayed
competitive antagonism to the binding of [3H]-NMS to
cortex receptors based on Hill values and dissociation
kinetics. The pyridophens, however, were less potent
muscarinic receptor inhibitors than the parent com-
pound aprophen.
1
MeOH-CHCl3) Rf ) 0.58. H NMR (CDCl3): δ 8.41 (d.d, 1H,
J ) 4.6, 1.5 Hz), 7.5 (d.d, 1H, J ) 8.2, 1.5 Hz), 7.26 (d.d, 1H,
J ) 8.2, 4.8), 5.22 (s, 2H), 3.08 and 2.98 (2s, 6H, -N(CH3)2),
2.87 (s, 6H, -N(CH3)2). Anal. (C12H17N3O4) C, H, N.
2,2-Dip h en ylp r op ion yl Ch lor id e, (6). 2,2-Diphenylpro-
pionic acid (5, 3.0 g) was dispersed in freshly distilled thionyl
chloride (20 mL), and the mixture was refluxed for 2 h. The
thionyl chloride was removed under vacuum, and the residue
was dissolved in n-hexane and filtered, and the solvent was
evaporated to leave 3.0 g of colorless oil.
1
Com p ou n d 6. H NMR (CDCl3): δ 7.27 (m, 6H), 7.17 (m,
4H), 2.01 (s, 3H). This acid chloride was used without any
further purification.
3-H yd r oxy-2-(2′,2′-d ip h en ylp r op ion oxym et h yl)p yr i-
d in e (7). A solution of the acid chloride 6 (1.22 g, 0.005 mol)
in dry benzene (10 mL) was added to a solution of 3-hydroxy-
2-(hydroxymethyl)pyridine hydrochloride (0.9 g, 0.006 mol) in
dry pyridine (30 mL). The mixture was warmed (55 °C) with
stirring for 3 h and cooled to room temperature, and the
benzene and pyridine were evaporated under reduced pres-
sure. The semisolid residue was suspended in H2O (50 mL)
and extracted with ethyl acetate (3 × 50 mL). The combined
extracts were washed with brine and dried (MgSO4), and the
solvent was evaporated under vacuum to give 1.5 g of white
solid. During the ethyl acetate extraction, some solid (100-
200 mg) was found in the interphase between the organic and
the aqueous phases. This solid was filtered and found to be
identical to the major solid product.
Ideally, the pyridophens (10, 2, 3, and 11) should act
minimally in the periphery and penetrate the blood
brain barrier. There, the pyridophens would carbamy-
late AChE, which is consequently sequestered and
protected from OP binding. Also upon carbamylation,
the release of the mAChR antagonist metabolite occurs.
This metabolite of cholinesterase carbamylation would
be in the ideal location to function as a muscarinic
antagonist to protect these receptors from elevated ACh
levels. High potency mAChR antagonism would not be
desirable before interaction with AChE but rather only
after AChE protection. Therefore, the decrease in mus-
carinic potency observed for the pyridophens is compat-
ible with the goal of protecting AChE first and then the
mAChR.
Com p ou n d 7. The compound was purified by chromatog-
raphy (silica, CHCl3) to give 1.35 g (80% yield) of white
crystals, mp 164-165 °C. 1H NMR (CDCl3 + 1%CD3OD): δ
8.06 (d.d, 1H, J ) 4.92 Hz, 1.76 Hz), 7.16 (m, 12H), 5.24 (s,
2H), 1.87 (s, 3H). Anal. (C21H19NO3) C, H, N.
Exp er im en ta l Section
3-[[(Meth yla m in o)ca r bon yl]oxy]-2-(2′,2′-d ip h en ylp r o-
p ion oxym eth yl)p yr id in e (8). A small piece of sodium (∼5
mg) was added to a solution of 7 (0.2 g, 0.6 mmol) and
methylisocyanate (0.25 g, excess) in dry benzene (100 mL). The
reaction mixture was stirred for 72 h at room temperature.
The solvent was removed under reduced pressure, and the
viscous residue was chromatographed (silica, 2% MeOH-
CHCl3) to give 0.18 g (77% yield) of 8 as a colorless viscous
oil.
Com p ou n d 8. 1H NMR (CDCl3): δ 8.39 (d, J ) 4.4 Hz, 1H),
7.58 (d, J ) 8.1 Hz, 1H), 7.26 (m, 11H), 5.24 (s, 2H), 4.6 (bs,
1H, -NH), 2.66 (d, J ) 4.4 Hz, 3H), 1.84 (s, 3H). Anal.
(C23H22N2O4) C, H, N.
I. Ch em istr y. Melting points were determined on a Tho-
mas-Hoover melting point apparatus and are uncorrected. 1H
NMR spectra were obtained on a Varian XL 300 (Me4Si). Mass
spectra (MS) were obtained on a Finnigen 1015 mass spec-
trometer (chemical ionization-NH3). For purity tests, thin-layer
chromatography (TLC) was performed on fluorescent silica gel
plates (Polygram Sil G/UV254), and for each of the compounds,
only one spot (visualized by UV light and I2 vapor) was
obtained. All new compounds gave satisfactory microanalyses
for C, H, and N within (0.4% and/or mass spectra consistent
with the assigned structures.
2-Hyd r oxym eth yl-3-[[(d im eth yla m in o)ca r bon yl]oxy]-
p yr id in e (2), 2-[[(Dim eth yla m in o)ca r bon yl]oxy]m eth yl-

908 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Leader et al.
3-[[(Dim et h yla m in o)ca r b on yl]oxy]-2-(2′,2′-d ip h en yl-
p r op ion oxym eth yl)p yr id in e (9). a . By Ca r ba m yla tion of
th e Hyd r oxyp yr id in e (7). Dimethylcarbamyl chloride (3 mL,
excess) was added to a solution of the hydroxypyridine 7 (0.33
g, 0.001 mol) in dry pyridine (20 mL). The solution was
warmed with stirring to 55 °C for 30 min and left overnight
at room temperature. The pyridine was removed under
vacuum, and the residue was dissolved in ethyl acetate (50
mL). The ethyl acetate solution was washed with brine (2 ×
20 mL) and dried (MgSO4), and the solvent was evaporated to
give a pale brown viscous oil, which was purified by column
chromatography (silica, 2% MeOH-CHCl3), to yield a colorless
oil, 0.28 g (70% yield).
(10, X ) [CH3SO4]-). A solution of 9 (100 mg) and dimethyl
sulfate (120 mg, excess) in deuterated acetone ((CD3)2CO, 2
mL) was kept at room temperature. The reaction progress was
monitored by 1H NMR. After 5 days, ∼90% of the tertiary
pyridine compound was converted to the quaternary meth-
ylpyridinium methyl sulfate 10, X ) [CH3SO4]-. The deuter-
ated acetone was removed under vacuum, and the residual
viscous oil was triturated 3 times with hot dry ether (reflux,
3 × 50 mL) and dried under vacuum for 24 h at room
temperature.
Com p ou n d 10, X ) [CH3SO4]-. 1H NMR (1% CD3OD-
CDCL3): δ 8.73 (d, 1H, J ) 6.0), 8.23 (d, 1H, J ) 8.7), 7.92
(d.d, J ) 8.7, 6.0), 7.23-7.05 (m, 10H), 5.58 (s, 2H), 4.12 (s,
3H, N + -CH3), 3.57 (s, 3H, -OCH3), 3.05 and 2.94 (2s, 6H,
-N(CH3)2), 1.86 (s, 3H, C-CH3). Anal. calcd for ([C25H27N2O4]+-
[CH3SO4]-): C, 58.86; H, 5.66; N, 5.28. Found: C, 58.05; H,
5.50; N, 5.05. According to the 1H NMR spectrum, this
compound contained a small amount (∼3%) of dimethyl sulfate.
MS (FAB): 419 ([intact cation]+).
N-Met h yl-3-[[(d im et h yla m in o)ca r b on yl]oxy]-2-(2′2′-
d ip h e n ylp r op ion oxym e t h yl)-1,2,5,6-t e t r a h yd r op yr i-
d in e (12). Sodium borohydride (75 mg, 1.8 mmol) was added
in small portions to a cold solution (5 °C) of 10 (250 mg,
0.45 mmol) in methanol (high-performance liquid chrom-
atography (HPLC) grade). Stirring was continued for 6 h at
5-10 °C. Water was added (∼10 mL), and the mixture was
extracted with ethyl acetate (3 × 20 mL). After it was dried,
the solvent was evaporated under reduced pressure, and the
pale yellow viscous oil residue was chromatographed (silica,
ethyl acetate) to give 12 as a colorless viscous oil (120 mg, 62%
yield).
1
Com p ou n d 9. H NMR (CDCl3): δ 8.37 (d.d, 1H, J ) 4.6,
1.53 Hz), 7.51 (d.d, 1H, J ) 8.24, 1.22 Hz), 7.20 (m, 11H), 5.26
(s, 2H), 2.89, 2.82 (2s, Me2N-), 1.87 (s, 3H). Anal. (C24H24N2O4)
C, H, N.
b. By Ester ifica tion of th e Ca r ba m a te 3. A solution of
acyl chloride 6 (0.244 g, 0.001 mol) in dry benzene (5 mL) was
added dropwise to a stirred solution of the carbamate 3 (0.196
g, 0.001 mol) and triethylamine (0.2 g, 0.002 mol) in dry
benzene (15 mL). The reaction mixture was refluxed for 3 h,
the triethylamine HCl was filtered, and the organic solution
was washed with brine (2 × 20 mL), dried (MgSO4), and
evaporated to leave a yellow viscous oil; according to TLC and
1H NMR analysis, the oil was a mixture of 9 and starting
materials. The desired compound 9 (0.1 g, 25% yield) was
isolated and purified by column chromatography (silica, 5%
MeOH-CHCl3). 1H NMR was identical to the 1H NMR
spectrum of 9 obtained by method a, by carbamylation of the
hydroxypyridine 7.
N-Meth yl-2-h yd r oxym eth yl-3-[[(d im eth la m in o)ca r bo-
n yl]oxy]p yr id in iu m Iod id e (11). A solution of 3 (70 mg) and
methyl iodide (100 mg) in dry acetone was warmed to 50 °C
for 10 h. The methyl pyridinium iodide 11 that formed
separated out from the acetone solution as a pale brown
viscous oil (110 mg, 90% yield).
Com p ou n d 12. 1H NMR (CDCl3): δ 7.4-7.2 (m, 10 H), 5.51
(d.t, 1 H, J ) 4.0, 1.1 Hz), 4.41 (d.d, 1 H, J ) 12.1, 5.5 Hz),
4.32 (d.d, 1 H, J ) 12.1, 2.3 Hz), 3.28 (m, 1H), 2.90 and 2.86
(2s, 6H, -N(Me)2), 2.75 (m, 1H), 2.55 (m, 1H), 2.39 (s, 3H,
N-CH3), 2.25 (m, 1H), 2.0 (m, 1H), 1.92 (s, 3H). Anal.
(C25H30N2O4) C, H, N.
Com p ou n d 11. 1H NMR ((CD3)2CO): δ 9.17 (d, 1H, J )
5.7 Hz), 8.56 (d, 1H, 8.8 Hz), 8.18 (d.d, 1H, J ) 8.5, 6.1 Hz)
5.06 (s, 2H), 4.72 (s, 3H), 3.22, 3.01 (2s, 6H, Me2N-). Anal.
(C10H15N2O3I) C, H, N.
N-Meth yl-3-h yd r oxy-2-(2′2′-d ip h en ylp r op ion oxym eth -
yl)p yr id in iu m Iod id e (13, X ) I-). A solution of 7 (0.1 g)
and methyl iodide (0.1 g, excess) in deuterated acetone ((CD3)2-
CO, 4 mL) was kept at room temperature. Reaction progress
was monitored by 1H NMR. After 48 h, all of the tertiary
pyridine compound was converted to the quaternary methyl
pyridinium iodide 13, X ) I-. The acetone was removed under
reduced pressure, and the crude viscous brown oil was
triturated in dry ether to give yellow crystals (65 mg), mp 112-
114 °C (dec).
N-Met h yl-3-[[(Dim et h yla m in o)ca r b on yl]oxy]-2-(2′,2′-
d ip h en ylp r op ion oxym eth yl)p yr id in iu m Iod id e (10, X )
I-). a . By Ester ifica tion of th e Hyd r oxym eth ylp yr i-
d in iu m Iod id e (11). A solution of 11 (0.112 g, 0.3 mmol) and
the acid chloride 6 (0.140 g, 0.6 mmol) in 2 mL of deuterated
pyridine (C6D5N) was kept at room temperature for 4 days.
Progress of the reaction was monitored by 1H NMR. The
pyridine was removed under vacuum (10-3 mm Hg), and the
viscous residue was extracted with ether (3 × 10 mL) to
remove the excess of the acid chloride 7. The pale brown
viscous oil was purified by column chromatography (silica,
5-10% MeOH-CHCl3) to give a pale yellow viscous oil (85
mg, 60%).
Com p ou n d 13, X ) I-. 1H NMR ((CD3)2CO): δ (relative to
CD2 H ) 2.10 ppm) 8.50 (d, 1H, J ) 6.0 Hz), 8.41 (d, 1H, J )
8.7 Hz), 7.80 (d.d, 1H, J ) 8.7, 6.0 Hz), 7.4-7.2 (m, 10H), 5.77
(s, 2H), 4.31 (s, 3H, -N+CH3), 2.00 (s, 3H, C-CH3). Anal.
([C22H22NO3]+I-) C, H, N. MS (FAB): 348 ([intact cation]+).
N-Meth yl-3-h yd r oxy-2-(2′2′-d ip h en ylp r op ion oxym eth -
yl)p yr id in iu m Meth yl Su lfa te (13, X ) -OSO2OCH3). A
solution of 7 (0.1 g) and dimethyl sulfate (0.1 g) in deuterated
acetone ((CD3)2CO, 5 mL) was kept at room temperature. After
48 h, the quaternization of the tertiary pyridine compound was
completed (1H NMR). The acetone was removed under reduced
pressure, and the viscous residue was triturated 3 times with
dry ether and acetone to give white crystals, mp 164-166 °C.
1
Com p ou n d 10 (X ) I-). H NMR (C6D5N): δ 9.90 (d, 1H,
J ) 6.1 Hz), 8.86 (d, 1H, J ) 8.8 Hz), 8.41 (d.d, J ) 8.8, 6.1
Hz), 7.5-7.1 (m, 10H), 6.18 (s, 2H), 4.85 (s, 3H, N+-CH3), 2.94
and 2.87 (2s, 6H, -N(CH3)2), 2.01 (s, 3H). 1H NMR (CD3-
COCD3): δ 9.29 (d, 1H, J ) 6.1 Hz), 8.63 (d, 1H, J ) 8.8 Hz),
8.25 (d.d, 1H, J ) 8.8, 6.1 Hz), 7.4-7.2 (m, 10H), 5.81 (s, 2H),
4.50 (s, 3H, N+-CH3), 3.08 and 2.97 (2s, 6H, -N(CH3)), 1.94
(s, 3H). Anal. (C25H27N2O4I) C, H, N. MS fast atom bombard-
ment (FAB): 419 ([intact cation]+).
Com p ou n d 13 (X ) -OSO2OCH3). 1H NMR (D2O):
δ
(relative to DHO ) 4.8 ppm) 8.23 (d, 1H, J ) 6.0 Hz), 7.97 (d,
1H, J ) 8.7 Hz), 7.79 (d.d, 1H, J ) 8.7, 6.0 Hz), 5.65 (s, 2H),
4.84 (s, 3H, -OCH3), 4.04 (s, 3H, -N+CH3), 2.00 (s, 3H,
C-CH3). Anal. ([C22H22NO3]+[CH3SO4]-) C, H, N. MS (FAB):
348 ([intact cation]+).
b. By Meth yla tion of th e Ca r ba m a te Ester 9 w ith
Meth yl Iod id e. A solution of 9 (100 mg) and methyl iodide
(100 mg, excess) in deuterated acetone ((CD3)2CO, 2 mL), was
kept at room temperature. The reaction progress was moni-
II. Biologica l Activities. Ch olin ester a se P u r ifica tion
a n d Kin etic Assa ys. Both fetal bovine serum (GIBCO, Grand
Island, NY) AChE and horse serum BChE (Sigma Chemical
Co., St. Louis, MO) were purified to electrophoretic homogene-
ity by procainamide affinity chromatography.23,24 Pyridostig-
mine was purchased from Sigma Chemical Co., and aprophen
was synthesized at Walter Reed using the method of Zuagg
and Horrom.25
1
tored by H NMR. After 7 days, ∼95% of the tertiary pyridine
compound was converted to the quaternary methyl pyridinium
1
iodide 10 (X ) I-). H NMR was identical with that of 10 (X
) I-) obtained in method a, by esterification of the hydroxy-
methylpyridinium iodide 11, as described above.
N-Met h yl-3-[[(Dim et h yla m in o)ca r b on yl]oxy]-2-(2′,2′-
d ip h en ylp r op ion oxym eth yl)p yr id in iu m Meth yl Su lfa te

Binary Pyridostigmine-Aprophen Prodrugs
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4 909
Both enzymes were assayed colorimetrically,13 using a
concentration of either 1 mM acetylthiocholine as substrate
for AChE or 1 mM butyrylthiocholine as substrate for BChE
in a 3 mL solution of 0.1 M phosphate buffer, pH 8.0,
containing 3.11 × 10-4 M 5,5′-dithio-bis-(2-nitrobenzoic acid).
Assays were conducted at 25 °C. The specific activity of AChE
was 6700 U/mg, and the specific activity of BChE was 700
U/mg. Time-dependent inhibition reactions contained 15 U
AChE or BChE in 100 µL of 0.05 M phosphate buffer, pH 8.0,
and graded carbamate concentrations. Aliquots of 5 or 10 µL
were withdrawn at the indicated time intervals, and enzyme
activity was assayed. Kinetic constants were calculated from
secondary plots of the slopes of pseudo-first-order progress
curves as functions of the inhibitor concentrations as described
by Main.12 In this treatment, the reaction between an inhibitor
and an enzyme is described by equation 1:
the authors and are not necessarily endorsed by the U.
S. Army. Research was conducted in compliance with
the Animal Welfare Act and other Federal statutes and
regulations relating to animals and experiments involv-
ing animals and adheres to principles stated in the
Guide for the Care and Use of Laboratory Animals, NRC
publication, 96-23, 1996 edition and Walter Reed Army
Institute of Research protocols and AR 70-18, 1 J un 84.
Refer en ces
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B. P.; Gordon, R. K.; Chiang, P. K. Binary antidotes for
organophosphate poisoning: aprophen analogues that are both
antimuscarinics and carbamates. J . Med. Chem. 1989, 32, 1522-
1528.
(2) Dirnhuber, P.; French, M. C.; Green, D. M.; Leadbeater, L.;
Stratton, J . A. The protection of primates against soman
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and 2-PAM against soman, tabun, sarin, and VX in the rabbit.
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k2c
k3
9
I + E {kk } E‚I 8 E′
8 E + carbamic acid
(1)
1
-1
and leads to a graphic solution for the desired kinetic constants
through the derived equation 2:
[I] ∆t/2.3 ∆log v ) [I]/k2c + 1/k2′
(2)
where the reciprocal of the slope yields k2c, the carbamylation
constant; the intercept on the X-axis yields (-Ki), the equilib-
rium affinity constant; and the reciprocal of the intercept on
the Y-axis yields 1/k2′, the bimolecular rate constant.
Gu in ea P ig Cor tex Mem br a n e P r ep a r a tion s. Brain
(male guinea pig, 200-400 g) was obtained after decapitation21
and processed at 4 °C. Briefly, the cortex was homogenized in
20 volumes of 50 mM potassium phosphate buffer, pH 7.4, and
centrifuged two times at 39 000g for 20 min. The brain was
then resuspended in 4 volumes of buffer and frozen (-20 °C)
in aliquots until use.
Bin d in g Assa ys. Equ ilibr iu m Bin d in g Assa ys. As previ-
ously described, equilibrium binding assays were performed
in triplicates containing 200-400 µg of protein and 1-2 nM
[3H]-NMS (72 Ci/mmol, NEN Life Science Products, Inc.,
Boston, MA) in 50 mM potassium phosphate buffer, pH 7.4,
at 25 °C.21 ATR (1 µM) was added to determine nonspecific
binding, which typically was about 10% of the total counts.
The assays were terminated after 1 h by collecting the samples
with a cell harvester (Brandel, Gaithersburg, MD) on to glass-
fiber filters.
(9) McDonough, J . H.; McMonagle, J .; Copeland, T.; Zoeffel, D.; Shih,
T. M. Comparative evaluation of benzodiazepines for control of
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1983, 32, 2979-2981.
(11) Wolfe, A. D.; Chiang, P. K.; Doctor, B. P.; Fryar, N. Rhee, J . P.;
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stants for the inhibition of acetylcholinesterase by physostigmine
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B. M. A new and rapid colorimetric determination of acetylcho-
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Biochem. Pharmacol. 1985, 34, 2063-2068.
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Three distinct domains in the cholinesterase molecule confer
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determined by subtracting the total from the nonspecific
counts. The Ki values were derived from the IC50 values by
correcting for receptor occupancy by the [3H]-NMS;20 the KD
value determined for cortex was 0.48 nM.
Dissocia tion Bin d in g Assa ys. Cortex membranes were
preincubated with 1-2 nM [3H]-NMS for 90 min at 25 EC with
gentle mixing.20 Then, 1 µM ATR was added simultaneously
with graded doses of 10 or 12 for time intervals between 0
and 180 min. Dissociation curve kinetic parameters were
determined using the equation18 where [3H]-NMS bound to
each tissue receptor at a measured time, T:
(17) Ariel, N.; Ordentlich, A.; Barak, D.; Bino, T.; Velan, B.; Shaf-
ferman, A. The ‘aromatic patch’ of three proximal residues in
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allosteric interactions on muscarinic receptors. Eur. J . Phar-
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-k-1
T
-k-2T
(T) ) Ae
+ Be
The portion of the bound ligand-receptor complex with a fast
dissociating constant k-1 is denoted the “A fraction”. B is the
fraction with a slow dissociating constant k-2. The parameters
were fitted to curves using nonlinear curve fitting; the r2 values
(goodness of curve fit) were all >0.97 (Prism version 3.02,
GraphPad Software, San Diego, CA).
(20) Gordon, R. K.; Chiang, P. K. Differential allosteric effects of
8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate-HCl (TMB-
8) on muscarinic receptor subtypes. FEBS Lett. 1989, 257, 383-
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(21) Gordon, R. K.; Breuer, E.; Padilla, F. N.; Smejkal, R. M.; Chiang,
P. K. Distance geometry of R-substituted 2,2-diphenylpropionate
antimuscarinics. Mol. Pharmacol. 1989, 36, 766-772.
Ack n ow led gm en t. This work was done while H.L.
held a National Research Council-WRAIR Research
Fellowship. We thank J oshua A. Lenavitt for expert
technical assistance and dedication. Opinions, interpre-
tations, conclusions, and recommendations are those of

910 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 4
Leader et al.
(22) Karle, J . M.; Karle, I. L.; Gordon, R. K.; Chiang, P. K. Relation-
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