Metaproterenol, isoproterenol, and their bisdimethylcarbamate derivatives as human cholinesterase inhibitors

Article abstract of DOI:10.1021/jm300289k

Metaproterenol and isoproterenol are bronchodilators that provide a structural basis for many other bronchodilators currently in use. One of these structurally related bronchodilators is terbutaline; it is administered as a prodrug, bambuterol, and is metabolized (bioconverted) into terbutaline by butyrylcholinesterase (BChE). The metabolism rate can be affected by BChE gene polymorphism in the human population and BChE stereoselectivity. The aim of our study was to investigate inhibition of human BChE and acetylcholinesterase (AChE) with metaproterenol, isoproterenol, and newly synthesized racemic bisdimethylcarbamate derivatives of metaproterenol (metacarb) and isoproterenol (isocarb) and their (R)-enantiomers to see if their bioconversion is affected by BChE inhibition in the same way as that for bambuterol. Metacarb and isocarb proved to be selective BChE inhibitors, as they progressively inhibited AChE 960 to 80 times more slowly than BChE_UU. All studied cholinesterases displayed poor affinity for metaproterenol and isoproterenol, yet BChE _UU had an affinity about five times higher than that of AChE.

Full text of DOI:10.1021/jm300289k

Subscriber access provided by UNIVERSITY OF SASKATCHEWAN LIBRARY
Article
Metaproterenol, isoproterenol and their bisdimethylcarbamate
derivatives as human cholinesterase inhibitors
ANITA BOSAK, Ivana Gazi# Smilovi#, Goran Šinko, Vladimir Vinkovi#, and Zrinka Kovarik
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300289k • Publication Date (Web): 20 Jul 2012
Downloaded from http://pubs.acs.org on July 23, 2012
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
Metaproterenol, isoproterenol and their bisdimethylcarbamate derivatives as
human cholinesterase inhibitors
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Anita Bosak†, Ivana Gazić Smilović≠, Goran Šinko†, Vladimir Vinković≠, Zrinka Kovarik†,*
†Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HRꢀ10000 Zagreb,
Croatia
≠Ruñer Bošković Institute, Bijenička 54, HRꢀ10000 Zagreb, Croatia
AUTHOR INFORMATION
Corresponding author: Zrinka Kovarik
Phone: +385 1 4682500. Fax: +385 1 4673303. Eꢀmail: zkovarik@imi.hr
KEY WORDS: butyrylcholinesterase variants, selectivity, stereoselectivity, prodrugs,
bronchodilators
ABBREVIATION LIST
AChE, acetylcholinesterase; BChE, butyrylcholinesterase; metacarb, bisdimethylcarbamate ester
of metaproterenol; isocarb, bisdimethylcarbamate ester of isoproterenol; ATCh, acetylthiocholine
iodide; PTCh, propionylthiocholine iodide; DTNB, 5,5´ꢀdithiobis(2ꢀnitrobenzoic acid); BChEUU,
usual BChE variant; BChEAA, atypical BChE variant; BChEFF, fluoride resistant BChE variant,
BChEUA heterozygote of usual and atypical BChE variants; BChEAF heterozygote of atypical and
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 34
1
2
3
4
5
6
fluoride resistant BChE variants; NMR, nuclear magnetic resonance; HPLC, high performance
liquid chromatography; TMS, tetramethylsilane
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PDB ID Codes: 2PM8 and 3DJY
2
ACS Paragon Plus Environment

Page 3 of 34
Journal of Medicinal Chemistry
1
2
3
4
ABSTRACT
5
6
7
8
Metaproterenol and isoproterenol are bronchodilators that provide a structural basis for
many other bronchodilators currently in use. One of these structurally related bronchodilators is
terbutaline; it is administered as a prodrug bambuterol and is metabolized (bioconverted) into
terbutaline by butyrylcholinesterase (BChE). The metabolism rate can be affected by BChE gene
polymorphism in human population and BChE stereoselectivity. The aim of our study was to
investigate inhibition of human BChE and acetylcholinesterase (AChE) with metaproterenol,
isoproterenol and newly synthesized racemic bisdimethylcarbamate derivatives of metaproterenol
(metacarb) and isoproterenol (isocarb), and their (R)ꢀenantiomers to see if their bioconversion is
affected by BChE inhibition in the same way as is bambuterol's. Metacarb and isocarb proved to
be selective BChE inhibitors, as they progressively inhibited AChE 960 to 80 times more slowly
than BChEUU. All studied cholinesterases displayed poor affinity for metaproterenol and
isoproterenol, yet BChEUU had about five times higher affinity than AChE.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 34
1
2
3
4
INTRODUCTION
5
6
7
8
Metaproterenol (also known as orciprenaline) is a bronchodilator currently used to treat
asthma, bronchitis, and emphysema. Isoproterenol (also known as isoprenaline) is mostly used to
treat bradycardia and heart block, while its use as a bronchodilator is rare. Metaproterenol and
isoproterenol are structurally related to terbutaline, a fastꢀacting bronchodilator and tocolytic for
delaying premature labor. All three compounds are βꢀaminoalcohol benzenediols that constitute
the elementary pattern for most bronchodilators in use or with a potential to become one.1 The
side effects of the marketed bronchodilators include tachycardia, hypertension, and tremor, and
their therapeutic effect is often short.1 To prolong the therapeutic effect some formulations use a
prodrug such as bambuterol, a bisdimethylcarbamate ester of terbutaline. 1,2 In vivo conversion of
bambuterol involves hydrolysis by butyrylcholinesterase (BChE¸ EC 3.1.1.8), which is present in
the organism in high concentrations and is capable of hydrolyzing a variety of esters.3 BChE and
the related enzyme acetylcholinesterase (AChE; EC 3.1.1.7) hydrolyze carbamates by forming a
covalent bond between the carbamic group of carbamate ester and the hydroxy group of catalytic
serine.4 However, due to a slow decarbamylation rate, carbamates represent progressive
inhibitors of cholinesterases.4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The high therapeutic index of bambuterol is the result of its inhibitory preference of BChE
over AChE.5ꢀ7 It inhibits usual human BChE about 20000 times faster than human AChE and
mouse BChE about 8600 times faster than mouse AChE.6,8,9 BChE is also involved in the
bioconversion of other esterꢀbased prodrugs.10 The efficacy of a drug or a prodrug that requires
BChE action is affected by mutations of the human BCHE gene. More than 56 mutations of
BCHE gene have been identified to date, and several BChE variants possess the different
catalytic properties or are expressed at lower levels than usual BChE (BChEUU).3,11 For example,
4
ACS Paragon Plus Environment

Page 5 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
people with atypical BChE (BChEAA) are not able to hydrolyze succynilcholine and can
experience prolonged apnea if this muscle relaxant is administered.12
7
8
9
Among the many chiral drugs, one enantiomer is often more active than the other in
treating a medical condition. The (R)ꢀenantiomer of the shortꢀacting bronchodilator albuterol is
approximately 80 times more potent as a β adrenoceptor agonist than the (S)ꢀenantiomer, and the
administration of pure (R)ꢀalbuterol improves the therapeutic effects and reduce side effects.13
Similar in vivo results were obtained with the enantiomers of terbutaline.14 Both AChE and BChE
are stereoselective in the interaction with various esters such as phosphonates, acetate derivatives
of quinuclidineꢀ3ꢀols, and carbamates such as physostigmine and bambuterol.7ꢀ9,15,16
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this paper, we describe the synthesis of racemic bisdimethylcarbamate derivatives of
metaproterenol (metacarb) and isoproterenol (isocarb) and their (R)ꢀenatiomers (Figure 1). The
aim was to determine the inhibition rates of five BChE variants, expected by analogy to
bambuterol to critically affect the rates of metacarb and isocarb bioconversion. We also
determined inhibition rates of AChE to establish metacarb and isocarb inhibition selectivity
between AChE and BChE, expecting to be similar to that of bambuterol. The stereoselectivity of
AChE and four BChE variants was estimated based on comparison of inhibition rate constants
determined for racemic and (R)ꢀenantiomers of bisdimethylcarbamates. Inhibition selectivity of
metacarb and isocarb, and stereoselectivity of BChEUU were analyzed by molecular modeling of
the transition state of carbamylation by (R)ꢀenantiomers.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 34
1
2
3
4
2. RESULTS AND DISCUSSION
5
6
Inhibition by metacarb and isocarb
7
8
9
Bisdimethylcarbamates, metacarb and isocarb, displayed timeꢀdependent inhibition of all
the tested BChE variants. Inhibition followed the firstꢀorder kinetics at any given inhibitor
concentration (Figure 2A), and the firstꢀorder rate constant (k[AB]) was a linear function of
metacarb and isocarb concentration (Figure 2B). The overall inhibition rate constant (ki)
expresses the first step in metacarb and isocarb hydrolysis (Table 1) and represents a measure of
metacarb and isocarb inhibition potency. The order of magnitude of ki constants for the tested
bisdimethylcarbamates and BChE variants was 106 Mꢀ1minꢀ1, which makes both compounds very
fast BChE inhibitors. The fastest inhibition by both metacarb and isocarb was obtained for
BChEUU, and the slowest for BChEAA. The difference in inhibition rates of BChEUU and BChEAA
is primarily the result of amino acid substitution at position 70, where acidic amino acid aspartate
is replaced with aliphatic glycine (D70G). This is in agreement with previous studies which
showed that charged inhibitors such as bambuterol, dibucaine, and Ro 02ꢀ0683 reduced their
inhibition potency toward the atypical BChE variant.6,8,17 Inhibition of fluorideꢀresistant BChE
(BChEFF) by metacarb or isocarb was about three times or about 30 % lower than the inhibition
of BChEUU. This drop could be attributed to fluorideꢀresistant variant mutations T243M or
G390V that, even when far from the enzyme active center, could cause conformational changes
resulting in lower inhibition potency of positively charged ligands.6,18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The rate of cholinesterase carbamylation is determined by the entrance of carbamate and
its orientation into the active site of the enzyme.4 Metacarb and isocarb differ in disposition of
carbamate groups on the benzene ring: metaꢀposition in metacarb vs. orthoꢀposition in isocarb
(Figure 3). Since metacarb was up to 11 times more potent BChE inhibitor than isocarb (Table 1),
it seems that metaꢀposition of carbamate groups on benzene ring is preferred. This is supported
6
ACS Paragon Plus Environment

Page 7 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
by the fact that the overall inhibition rate of metacarb and bambuterol, where carbamate groups
are also metaꢀpositioned (Figure 3), are within the same order of magnitude.8,17 Two times slower
inhibition of usual BChE by metacarb compared to inhibition by bambuterol could be attributed
to the different size of alcohol moiety of these two biscarbamates: isoꢀpropyl group of metacarb
vs. tertꢀbutyl group of bambuterol.8 To elucidate the inhibition potency of studied biscarbamates,
we used molecular modeling to study the transition state of carbamylation reaction. Fig. 4
presents a simulation of the transition state between the hydroxyl group of the active site serine
(Ser198) of human BChEUU (PDB cod 2PM8) and the carbamic group on the C2 atom of the
benzene ring of (R)ꢀmetacarb (Figure 4A), (R)ꢀisocarb (Figure 4B) and (R)ꢀbambuterol (Figure
4C). The transition states of carbamylation by three carbamates were stabilized by the cation–π
interaction between protonated nitrogen of carbamates and the indole ring of Trp82, and by the
hydrogen bond between the oxygen of the carbamic group of carbamates and the amino group of
Gly117. Differences in carbamylation rate by metacarb, isocarb and bambuterol can be accounted
to additional stabilization typical for the each carbamate. The transition state of (R)ꢀmetacarb was
additionally stabilized by two hydrogen bonds between the hydroxyl group on the chiral center
and residues His438 and Glu197 (Figure 4A), and (R)ꢀisocarb by the hydrogen bond between
protonated nitrogen and Glu197. The transition state of (R)ꢀbambuterol was stabilized by the
cation–π interaction between protonated nitrogen and Tyr440, and by the hydrogen bond between
Glu197 and the hydroxyl group on the chiral center (Figure 4C).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Dimethylcarbamates, metacarb and isocarb, also inhibited AChE timeꢀdependently, but
relatively slowly. The order of magnitude of overall inhibition rate constants was 103 Mꢀ1minꢀ1,
which was about ten times higher than previously determined for bambuterol, classifying
metacarb and isocarb as inhibitors with low AChE inhibition potency.7,8 The relation between the
first order rate constant (k[AB]) and inhibitor concentration deviated from linearity, allowing
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 34
1
2
3
4
5
6
7
8
9
determination of the maximal firstꢀorder rate constant of carbamylation, kmax, and dissociation
constant, Ki, (Table 1). Although overall metacarb and isocarb inhibition rate constants of AChE
were very similar, intrinsic carbamylation constants differed. AChE had about two times higher
kmax for isocarb than metacarb, while due to high standard deviations of the Ki constants, higher
affinity (1/Ki) of AChE for metacarb than isocarb must be considered with a caution. Poor
inhibition of AChE may have to do with π−π interactions between the aromatic ring of
bisdimethylcarbamates and aromatic residues of the active site that, due to steric constrains,
disabled proper orientation of the carbamate group to catalytic serine, stabilizing the Michaelis
type of the bisdimethylcarbamate−AChE complex. The final result is the slow carbamylation
rate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The ratio of overall inhibition rate constants for BChE and AChE describes the inhibition
selectivity of metacarb and isocarb (Table 1). Both bisdimethylcarbamates showed distinct BChE
selectivity, as they inhibited AChE 80 to 960 times more slowly than BChEUU. The highest BChE
inhibition selectivity was determined for BChEUU while the lowest for BChEAA by both
bisdimethylcarbamates. In general, metacarb was more selective than isocarb. However, both
compounds were less selective inhibitors compared to bambuterol, which could affect their
possible future use as bronchodilators.
Inhibition by (R)-enantiomers of metacarb and isocarb
As metacarb and isocarb are chiral molecules with the chiral center on the alcohol part of
the molecule, stereoselectivity of cholinesterases could be expected as result of different
inhibition rates by enantiomers (Fig. 1). Due to difficulties in the synthesis of (S)ꢀenantiomers,
the stereoselectivity of cholinesterases was estimated from the ratio of the inhibition rate constant
for racemate and (R)ꢀenantiomer of metacarb and isocarb (Table 2). (R)ꢀmetacarb was a 1.3 ꢀ 2.0
8
ACS Paragon Plus Environment

Page 9 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
times more potent inhibitor than racemate. Inhibition by (R)ꢀisocarb was 1.3 ꢀ 1.7 times faster
than racemate for BChEUU, BChEAA, and BChEUA (heterozygote of usual and atypical BChE),
while no difference was shown for BChEFF, BChEUF (heterozygote of usual and fluoride resistant
BChE), and AChE (Table 2). Stereoselectivity of human cholinesterases for bambuterol was
determined earlier when all the studied BChE variants as well as AChE revealed four times
higher preference for (R)ꢀ over (S)ꢀbambuterol.8 A comparison of the ki ratio obtained for (R)ꢀ
bambuterol and the racemate showed that cholinesterase stereoselectivity for metacarb and
isocarb could be similar to those of bambuterol (Table 2).8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Molecular modeling study of the transition state of carbamylation reaction was done for
(R)ꢀmetacarb, (R)ꢀisocarb and (R)ꢀbambuterol (Fig. 4). Stabilization of the transition state of (S)ꢀ
enantiomer of bisdimethylcarbamates involves spatial reorientation of the hydroxyl group, which
could lead to disappearance of the hydroxyl group interaction that stabilized transition state of
(R)ꢀenantiomers.
Inhibition by metaproterenol and isoproterenol
Metaproterenol and isoproterenol are the final products of metacarb and isocarb
hydrolysis, respectively. Both reversibly inhibited BChE and AChE, forming noncovalent
interactions within the active site of the enzyme. To measure metaproterenol and isoproterenol
inhibition potency, we determined the dissociation constants of the enzymeꢀinhibitor complex
(K(I)) and dissociation constants of the enzymeꢀsubstrate complex (K(S)) (Table 3). K(I) constants
ranged from 0.5 mM to 6.0 mM, typical for lowꢀpotency inhibitors. Usual BChE showed the
highest affinity (1/K(I)) for both inhibitors, and atypical BChE the lowest. Furthermore, the
affinity of BChEUU, BChEUA and BChEFF was similar for metaproterenol and isoproterenol. The
only exception was BChEAA with four times higher affinity for isoproterenol. The affinity of
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 34
1
2
3
4
5
6
7
8
9
AChE to metaproterenol or isoproterenol was about four times lower than the affinity of the
tested BChE variants. A comparison with the affinity to terbutaline, the final product of
bambuterol hydrolysis, showed that usual BChE had about three times higher affinity to
terbutaline than to metaproterenol or isoproterenol, while BChEAA had the highest affinity to
isoproterenol, about two times higher than to terbutaline.17 The determined K(S) constants were in
good agreement with the constants corresponding to the Michaelis type complex or to the
enzymeꢀsubstrate dissociation constants for binding at the catalytic site of the enzyme published
earlier.17,19 This implies that at given substrate concentrations both inhibitors bind to the catalytic
site of the enzymes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4. CONCLUSION
A very desirable characteristic for an asthma drug is prolonged action, so that a patient
can have a whole night's sleep. Bambuterol, a bisdimethylcarbamate of terbutaline, ensures
release of terbutaline over 24 h. The high therapeutic index of bambuterol is associated with its
extremely selective inhibition of BChE compared to AChE. Dimethylcarbamates studied in this
paper, metacarb and isocarb, are structurally similar to bambuterol and we expected that they
should display a similar selective inhibition of BChE. However, both proved far less selective for
BChE than bambuterol, and therefore less likely to be used as prodrugs of bronchodilating
agents. Both bisdimethylcarbamates have about 50 times higher affinity for the BChEUU than for
BChEAA, as was earlier demonstrated for bambuterol.8,17 This study has also shown that
metaproterenol and isoproterenol are reversible cholinesterase inhibitors. The affinity of both for
any of the BChE variants was too low to interfere with metacarb and isocarb inhibition. Our
findings provide new information on the kinetics of bisdimethylcarbamates by BChE. It seems
10
ACS Paragon Plus Environment

Page 11 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
that the metaꢀposition of the two carbamate groups on the benzene ring allows easier entrance of
bisdimethylcarbamate compounds and its orientation into the active site of the BChE. In addition,
the tertꢀbutyl group on the alcohol moiety of the ester, as it is in bambuterol, ensures efficient
carbamylation of BChE by bisdimethycarbamates. BChE is stereoselective preferring (R)ꢀ
bisdimethylcarbamates over (S)ꢀenantiomer, probably because the stabilization of the transition
state of (S)ꢀenantiomer involves spatial reorientation of the hydroxyl group.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5. EXPERIMENTAL SECTION
5.1. Chemicals
Racemic isoproterenol bitartarate and metaproterenol hemisulfate were purchased from
SigmaꢀAldridge (Sigma Chimica, Italy) and converted to hydrochlorides by treating them with an
aqueous solution of Na2CO3 and by adding a solution of HCl in iꢀPrOH to form salts.
All chemicals, reagents and solvents for the preparation of metaproterenol and
isoproterenol bisdimethylcarbamate derivatives were purchased from commercial sources.
Acetylthiocholine iodide (ATCh), propionylthiocholine iodide (PTCh) and 5,5´ꢀ
dithiobis(2ꢀnitrobenzoic acid) (DTNB) were purchased from Sigma Chemical Co., USA. All
inhibitors and substrates were dissolved in water, while DTNB was dissolved in 0.1 M sodium
phosphate buffer (pH 7.4).
5.2. Synthesis
The racemic bisdimethylcarbamate derivate of metaproterenol (metacarb) was prepared
following slightly modified protocols as described in the patent US7495028B2 (Scheme 1).20 2'ꢀ
Bromoꢀ3,5ꢀdi(N,Nꢀdimethylcarbamyloxy)acetophenone (1) was prepared (80 % yield) by the
reaction of copper(II) bromide with 3,5ꢀdi(N,Nꢀdimethylcarbamyloxy)acetophenone, which was
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 34
1
2
3
4
5
6
7
8
9
prepared from commercially available 3,5ꢀdihydroxyacetophenone. The racemic
bisdimethylcarbamate derivate of isoproterenol (isocarb) was prepared in a similar way (Scheme
1), from the 2'ꢀchloroꢀ3,4ꢀdi(N,Nꢀdimethylcarbamyloxy)acetophenone (2), which was prepared
from commercially available 2'ꢀchloroꢀ3,4ꢀdihydroxyacetophenone. The substituted
acetophenones 1 and 2 were reduced with NaBH4 to respective alcohols 3 and 4, which were
further reacted with NaOH to turn into their epoxides 5 and 6, respectively (details in Supporting
Information).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3,5-bis(N,N-dimethylcarbamyloxy)-metaproterenol hydrochloride (7). 2ꢀ[3,5ꢀdi(N,Nꢀ
dimethylcarbamyloxy)phenyl]oxirane (5) (1.0 mmol) was treated with isopropylamine under
reflux overnight. The mixture was evaporated to dryness, diluted with water and extracted with
ethyl acetate (2 x 10 mL). The combined organic layers were washed with water, dried over
MgSO4, filtered off and evaporated to dryness. The residue was purified by column
chromatography (dichloromethane/methanol/triethylamine 20:1:0.04) to provide a free base in 75
% yield as an oil. Purity of compound was 98.79 % (determined by RP HPLC). 1H NMR (CDCl3,
600 MHz) δ = 1.12 (d, J = 6.3 Hz, 6H), 2.70 (dd, J = 12.3 and 9.0 Hz, 1H), 2.85ꢀ2.94 (m, 2H),
2.99 (s, 6H), 3.07 (s, 6H), 4.73 (dd, J = 8.9 and 5.6 Hz, 1H), 6.68 (t, 2.6 Hz, 1H), 7.01 (d, J = 2.3
13
Hz, 2H) ppm. C NMR (CDCl3, 100 MHz) δ = 25.1, 36.1, 36.5, 47.6, 56.4, 78.2, 113.9, 116.9,
141.3, 153.2, 157.4 ppm. A solution of the obtained base (1.0 mmol) in iꢀPrOH (3 mL) was
treated with a solution of HCl in iꢀPrOH (pH = 3) to give hydrochloride salt of the metaproterenol
bisdimethylcarbamate derivate.
3,4-bis(N,N-dimethylcarbamyloxy)-isoproterenol hydrochloride (8). 2ꢀ[3,4ꢀdi(N,Nꢀ
dimethylcarbamyloxy)phenyl]oxirane (6) (1.0 mmol) was treated with isopropylamine under
reflux overnight. The mixture was evaporated to dryness, diluted with water and extracted with
ethyl acetate (2 x 10 mL). The combined organic layers were washed with water, dried over
12
ACS Paragon Plus Environment

Page 13 of 34
Journal of Medicinal Chemistry
1
2
3
4
MgSO4, filtered off and evaporated to dryness. The residue was purified by column
5
6
7
8
chromatography (dichloromethane/methanol/triethylamine 20:1:0.04) to provide a free base in 71
% yield as an oil. Purity of compound was 98.65 % (determined by RP HPLC). 1H NMR (CDCl3,
600 MHz) δ = 1.08 (d, J = 6.3 Hz, 6H), 2.66ꢀ2.91 (m, 3H), 3.01 (s, 6H), 3.07 (s, 6H), 4.69 (dd, J
= 8.7 and 5.1 Hz, 1H), 7.20 (s, 2H), 7.25 (s, 1H) ppm. 13C NMR (CDCl3, 100 MHz) δ = 25.0,
36.4, 36.4, 48.1, 55.9, 78.1, 121.0, 121.4, 123.1, 136.2, 145.1, 154.6 ppm.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A solution of the obtained base (1.0 mmol) in iꢀPrOH (3 mL) was treated with a solution of HCl
in iꢀPrOH (pH = 3) to give hydrochloride salt of the isoproterenol bisdimethylcarbamate derivate.
Scheme 1. Synthesis of racemic metacarb (7) and isocarb (8)a
OH
O
X
X
Me2NOCO
Me2NOCO
a
OCONMe2
OCONMe2
1 (m-OCONMe2, X=Br)
2 (p-OCONMe2, X=Cl)
3 (m-OCONMe2, X=Br)
4 (p-OCONMe2, X=Cl)
OH
O
H
N
HCl
c,d
b
Me2NOCO
Me2NOCO
OCONMe2
OCONMe2
5 (m-OCONMe2)
6 (p-OCONMe2)
7 (m-OCONMe2)
8 (p-OCONMe2)
a (a) NaBH4, MeOH; (b) NaOH aq, EtOH; (c) isopropylamine; (d) HCl in iꢀPrOH.
(R)ꢀenantiomers of metacarb and isocarb were prepared following the above procedure.
Chiral center was introduced in the reduction step (Scheme 2) using the enantiomerically pure
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 34
1
2
3
4
reagent, (ꢀ)ꢀchlorodiisopinocamphenylborane ((ꢀ)ꢀDIPꢀchloride). This reagent provided the (R)ꢀ
5
6
absolute configuration of metacarb and isocarb.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Synthesis of (R)-metacarb (R-7) and (R)-isocarb (R-8)a
OH
OH
O
H
N
X
X
a
HCl
Me2NOCO
Me2NOCO
Me2NOCO
b
c,d
OCONMe2
OCONMe2
OCONMe2
R-3 (m-OCONMe2, X=Br)
R-4 (p-OCONMe2, X=Cl)
1 (m-OCONMe2, X=Br)
2 (p-OCONMe2, X=Cl)
R-7 (m-OCONMe2)
R-8 (p-OCONMe2)
a
(a) (ꢀ)ꢀDIPꢀchloride, THF, diethanolamine; (b) NaOH (aq), EtOH; (c) isopropylamine; (d) HCl
in iꢀPrOH.
(R)-3,5-bis(N,N-dimethylcarbamyloxy)-metaproterenol hydrochloride (R-7) was prepared
from (R)ꢀ2ꢀ[3,5ꢀdi(N,Nꢀdimethylcarbamyloxy)phenyl]oxirane (Rꢀ5) following the same
procedure as for the preparation of the racemic metaproterenol derivate in 80 % yield, 98.65 %
pure (determined by RP HPLC) and 99.54 % e.e. (determined by HPLC, column ChiralpakꢀAD).
NMR spectrums were the same as for the racemic product.
(R)-3,4-bis(N,N-dimethylcarbamyloxy)-isoproterenol hydrochloride (R-8) was prepared from
(R)ꢀ2ꢀ[3,4ꢀdi(N,Nꢀdimethylcarbamyloxy)phenyl]oxirane (Rꢀ6) following the same procedure as
for the preparation of the racemic isoproterenol derivate in 70 % yield, 98.48 % (determined by
RP HPLC) and 83.51 % e.e. (determined by HPLC, column ChiralcelꢀOD). NMR spectrums
were the same as for the racemic product.
14
ACS Paragon Plus Environment

Page 15 of 34
Journal of Medicinal Chemistry
1
2
3
4
5.3. Enzymes
5
6
7
8
The source of BChE was human native plasma and the source of AChE was human
hemolyzed erythrocytes. Blood from donors with the usual BChE variant were collected at the
Institute for Medical Research and Occupational Health, Zagreb, Croatia. BChE was obtained
from heparinized blood after centrifugation (20 min, 2500 rpm, 25 °C) as supernatant and was
phenotyped as described earlier.21 Sedimented erythrocytes were made up to the volume of whole
blood by phosphate buffer. Atypical BChE (BChEAA) was a gift from Dr. O. Lockridge
(University of Nebraska Medical Center, Eppley Institute, Omaha, USA). Specimens of fluoride
resistant BChE (BChEFF) and heterozygotes BChEUA and BChEAF were donated by Dr. R. T.
Evans (Cholinesterase Investigation Unit, St. James’s University Hospital, Leeds, Great Britain).
All enzymes were used without further purification, and all enzyme dilutions were done in
phosphate buffer.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5.4. Spectrophotometric measurements
Enzyme activities were measured spectrophotometrically by the Ellman method at 412 nm
using DTNB (final concentration 0.3 mM) as thiol reagent and ATCh (AChE activity) or PTCh
(BChE activity) as substrate.22,23 Erythrocytes were diluted 600 times, while plasma 150 ꢀ 300
times for the enzyme activity measurements. All experiments were done in 0.1 M phosphate
buffer, pH 7.4, at 25 °C on a CARY 300 spectrophotometer equipped with a temperature
controller (Varian Inc., Australia).
Progressive inhibition by carbamates proceeds according to Scheme 3 where E, AB, EAB, EA
and B stand for the free enzyme, carbamate, Michaelisꢀtype complex between enzyme and
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 34
1
2
3
4
5
6
carbamate, carbamylated enzyme and leaving group, respectively. k+1, kꢀ1 and kmax are rate
constants of the respective reactions, while ki is the overall inhibition rate constant.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Progressive inhibition of cholinesterases by carbamates.
k+1
kꢀ1
kmax
EAB
EA +B
E + AB
ki
Enzyme samples were incubated for ≤30 min with the carbamates. The inhibition reaction was
stopped by the addition of ATCh (1.0 mM final concentration) or PTCh (4.0 mM final
concentration), and the extent of inhibition was determined by measuring the residual activity. At
least three different concentrations of carbamates were used in at least two experiments. The firstꢀ
order rate constants (k[AB]) were calculated by linear regression analysis at any given carbamate
concentration [AB] (Figure 2A):24
v0
ln = k[AB] ⋅ ti
v
where v0 and vi stand for the enzyme activity in the absence and in the presence of carbamate at
the time of inhibition ti. When k[AB] was a linear function of [AB] (Figure 2B), the overall
inhibition rate constant ki was calculated by linear regression analysis from:
k[AB
]
ki =
[AB
]
16
ACS Paragon Plus Environment

Page 17 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The nonꢀlinear relationship between k[AB] and [AB] indicates the presence of a reversible
Michaelisꢀtype complex between enzyme and carbamate. Then, the maximal firstꢀorder rate
constant of carbamylation (kmax) and dissociation constant of the Michaelisꢀtype complex (Ki)
were calculated by:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
v0 kmax
⋅
[
[
AB
]
ln
=
⋅ti = k[AB] ⋅ti
v
Ki +
AB
]
The overall inhibition rate constant (ki) corresponds to the ratio kmax / Ki.
Reversible inhibition by metaproterenol and isoproterenol were measured at different
substrate concentrations ([S]; Table 3) in the absence (v0) and in the presence of the inhibitor (vi)
of a given inhibitor concentrations ([I]; 0.5 mM ꢀ 10 mM.). For each substrate concentration, the
apparent dissociation constant (Ki,app) was calculated by the HunterꢀDowns equation using linear
regression analysis:25
K(
vi
I
)
Ki,app
=
⋅
[I
]
= K(
+
)
⋅
[S]
I
v0 − vi
K(S)
Yꢀintercept determines the enzymeꢀinhibitor dissociation constants (Ki), while xꢀintercept
determines the enzymeꢀsubstrate dissociation constant, K(S) (Figure 5).
5.5. Molecular modeling
The studied carbamates were docked using the Accelrys Discovery Studio 2.1 CDOCKER
docking protocol and CHARMm force field.26 The crystal structure deposited in PDB (PDB ID
2PM8) was used for the structural model of BChE.27 Water molecules were removed from these
structure. Minimized structures of three studied carbamates were docked into the active site of
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 34
1
2
3
4
5
6
7
8
9
BChE. The protocol resulted in clusters of carbamate poses. Poses were selected based on the
orientation of a single carbamate group and its distance from the catalytic serine hydroxyl group.
The criterion was that after the nucleophilic attack of the catalytic serine on the carbon atom from
the carbonyl group, the newly formed asymmetrical carbon had to form the (S) configuration
because of stabilization of carbamyl oxygen in the oxyanion hole.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The transition state of BChE carbamylation was simulated using the crystal structure of
tabunꢀphosphorylated BChE (PDB ID 3DJY) as a template.28 Phosphorus atom was replaced with
the carbon atom, phosphorusꢀoxygen double bond was then converted into a carbonꢀoxygen
single bond and the charge on the oxygen was set to ꢀ1. The ethoxy group of the tabunꢁBChE
conjugate was also modified by deleting the ethyl part of the ethoxy group. Then, two crystal
structures of BChE (2PM8 and 3DJY) were superimposed with the purpose of translating the
carbamate molecule from the nonꢀphosphorylated 2PM8 structure into a phosphorylated 3DJY
structure. The resulting complex of 3DJY structure and carbamate was additionally modified by
the formation of a new bond between the oxygen atom from the ethoxy group and carbon from
the carbamate benzene ring. Prior to minimization, the position of the following atoms was fixed
to remain as they were in phosphorylated BChE: asymmetrical carbon with the surrounding
catalytic serine oxygen, negatively charged oxygen, and nitrogen and oxygen connected to
carbon from the benzene ring. The structure of this conjugate, a transition state analogue, was
minimized except for the fixed atoms. Upon minimization, the resulting structure was subjected
to a 10 ps NVT dynamic simulation including 13000 production steps with heating, equilibration
and dynamics simulation.26 This simulation was repeated and interactions between BChE
residues and the carbamyl part of the molecule were evaluated from the final structure. During
simulations, the CHARMM force field with full potential minimization was used, heating target
18
ACS Paragon Plus Environment

Page 19 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
temperature was 700 K, equilibration target temperature was 310 K and the target temperature for
dynamics simulation was 310 K.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Supporting Information: Detailed description of purity analysis of compounds, synthesis of
compounds 1ꢀ6, RP HPLC chromatograms of metacarb (7), isocarb (8), (R)ꢀmetacarb (Rꢀ7) and
(R)ꢀisocarb.
This material is available free of charge via Internet at http://pubs.acs.org.
Acknowledgment. This study was supported by the Ministry of Science, Education and Sports,
Republic of Croatia (Grants 022ꢀ0222148ꢀ2889 and 098ꢀ0982904ꢀ2910)
References
(1) Waldeck, B. βꢀAdrenoceptor agonists and asthmaꢀ100 years of development. Eur. J.
Pharmacol. 2002, 445, 1−12.
(2) Testa, B. Prodrug research: futile or fertile? Biochem. Pharmacol. 2004, 68, 2097−2106.
(3) Lockridge, O.; Masson, P. Pesticides and susceptible populations: people with
butyrylcholinesterase genetic variants may be at risk. Neurotoxicology 2000, 21, 113−126.
(4) Reiner, E.; Radić, Z. Mechanism of action of cholinesterase inhibitor. In Cholinesterases
and Cholinesterase Inhibitors. 1st ed.; Giacobini, E., Ed.; Martin Dunitz Ltd.: London,
2000; pp 103−121.
(5) Tunek, A.; Svensson, L.ꢀÅ. Bambuterol, a carbamate ester prodrug of terbutaline, as
inhibitor of cholinesterases in human blood. Drug Metab. Dispos. 1988, 16, 759−764.
(6) Kovarik, Z.; Radić, Z.; Grgas, B.; ŠkrinjarićꢀŠpoljarić, M.; Reiner, E.; SimeonꢀRudolf, V.
Amino acids residues involved in the interaction of acetylcholinesterase with carbamates
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 34
1
2
3
4
5
6
Ro 02ꢀ0683 and bambuterol, and with terbutaline. Biochim. Biophys. Acta 1999, 1433,
261−271.
7
8
9
(7) Bosak A.; Gazić I.; Vinković V.; Kovarik Z. Amino acids involved in stereoselective
inhibition of cholinesterases with bambuterol. Arch. Biochem. Biophys. 2008, 471, 72−76.
(8) Gazić, I.; Bosak, A.; Šinko, G.; Vinković, V.; Kovarik, Z. Preparative HPLC separation and
stereoselective inhibition of human cholinesterases. Anal. Bioanal. Chem. 2006, 385,
1513−1519.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(9) Bosak, A.; Gazić, I.; Vinković, V.; Kovarik, Z. Stereoselective inhibition of human, mouse,
and horse cholinesterases by bambuterol enantiomers. Chem.ꢀBiol. Interact. 2008, 175,
192−195.
(10) Liederer, B. M.; Borchardt, R. T. Enzymes involved in the bioconversion of esterꢀbased
prodrugs. J. Pharm. Sci. 2006, 95, 1177−1195.
(11) Souza, R. L. R.; Mikami, L. R.; Maegawa R. O. B.; ChautardꢀFreireꢀMaia, E. A. Four new
mutations in the BCHE gene of human butyrylcholinesterase in a Brazilian blood donor
sample. Mol. Genet. Metab. 2005, 84, 349−353.
(12) Jensen, F. S.; VibyꢀMogensen, J. Plasma cholinesterase and abnormal reaction to
succinylcholine: twenty years' experience with the Danish Cholinesterase Research Unit.
Acta Anaesthesiol. Scand. 1995, 39, 150−156.
(13) Hartley, D.; Middlemis, D. Absolute configuration of the optical isomers of salbutamol. J.
Med. Chem. 1971, 14, 895−896.
(14) Källström, B.ꢀL.; Sjöberg, J.; Waldeck, B. Steric aspects of formoterol and terbutalin: Is
there an adverse effect of the distomer on airway smooth muscle function? Chirality 1996,
8, 567−573.
20
ACS Paragon Plus Environment

Page 21 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(15) Kovarik, Z.; Radić, Z.; Berman, H. A.; SimeonꢀRudolf, V.; Reiner, E.; Taylor, P.
Acetylcholinesterase active centre and gorge conformation analysed by combinatorial
mutations and enantiomeric phosphonates. Biochem. J. 2003, 373, 33−40.
(16) Bosak, A.; Primožič, I.; Oršulić, M.; Tomić, S.; SimeonꢀRudolf, V. Enantiomers of
quinuclidinꢀ3ꢀol derivatives: Resolution and interactions with human cholinesterases.
Croat. Chem. Acta 2005, 78, 121−128.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(17) Kovarik, Z.; SimeonꢀRudolf, V. Interaction of human butyrylcholinesterase variants with
bambuterol and terbutaline. J. Enzym. Inhib. Med. Ch. 2004, 19, 113−117.
(18) Masson, P.; Adkins, S.; Gouet, P.; Lockridge, O. Recombinant human
butyrylcholinesterase G390V, the fluorideꢀ2 variant, expressed in Chinese hamster ovary
cells, is a low affinity variant. J. Biol. Chem. 1993, 268, 14329−14341.
(19) SimeonꢀRudolf, V.; Juršić, B. Heterogeneity of human serum cholinesterase revealed by
thiocholine substrates. Period. Biol. 1996, 98, 331−335.
(20) Tan, W.; Cheng, J. L., Rꢀbambuterol, its preparation and therapeutic uses. U.S. Patent
US7495028B2.
(21) Kovarik, Z.; SimeonꢀRudolf, V. An improvement in segregation of human
butyrylcholinesterase phenotypes having the fluorideꢀresistant. Arh. Hig. Rada Toksikol.
2003, 54, 239ꢀ244.
(22) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7,
88−95.
(23) Eyer, P.; Worek, F.; Kiderlen, D.; Sinko, G.; Stuglin, A.; SimeonꢀRudolf, V.; Reiner E.
Molar absorption coefficients for the reduced Ellman reagent: reassessment. Anal.
Biochem. 2003, 312, 224–227.
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 34
1
2
3
4
5
6
(24) R. Main, Affinity and phosphorylation constants for the inhibition of esterases by
organophosphates. Science 1964, 14, 992−993.
7
8
9
(25) Aldridge, W. N.; Reiner, E. Enzyme Inhibitors as Substrates. Interaction of Esterases with
Esters of Organophosphorus and Carbamic Acids. NorthꢀHolland: New York, 1975.
(26) Accelrys Software Inc.: San Diego, USA, 2005ꢀ2009.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(27) Ngamelue, M. N.; Homma, K.; Lockridge, O.; Asojoa, O. A., Crystallization and Xꢀray
structure of fullꢀlength recombinant human butyrylcholinesterase. Acta Crystallogr. F
2007, F63, 723ꢀ727.
(28) Carletti, E.; Li, H.; Li, B.; Ekström, F.; Nicolet, Y.; Loiodice, M.; Gillon, E.; Froment, M.
T.; Lockridge, O.; Schopfer, L. M.; Masson, P.; Nachon, F. Aging of cholinesterases
phosphylated by tabun proceeds through Oꢀdealkylation. J. Am. Chem. Soc. 2008, 130,
16011ꢀ16020.
22
ACS Paragon Plus Environment

Page 23 of 34
Journal of Medicinal Chemistry
1
2
3
4
Table 1 Progressive inhibition of human BChE and AChE by racemic metacarb and isocarb
5
6
7
8
9
ki (103 Mꢀ1minꢀ1)
Enzyme
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Metacarbrac
2200 ± 40
40 ± 8.8
Isocarbrac
200 ± 7.9
21 ± 0.48
160 ± 8.5
140 ± 2.6
87 ± 0.60
BChEUU
BChEAA
BChEUA
1400 ± 46
600 ± 16
440 ± 16
BChEFF
BChEAF
AChE
ki (103 Mꢀ1minꢀ1)
kmax (minꢀ1)
Ki (mM)
2.3 ± 0.51
1.6 ± 0.14
0.71 ± 0.14
2.5 ± 0.91
3.5 ± 0.57
1.4 ± 0.46
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 34
1
2
3
4
Table 2 Progressive inhibition of human BChE and AChE by Rꢀmetacarb and Rꢀisocarb
5
6
7
8
9
(R)ꢀmetacarb
(R)ꢀisocarb
Bambuterola
Enzyme
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ki (103 Mꢀ1minꢀ1) ki(R) / ki(rac) ki (103 Mꢀ1minꢀ1) ki(R) / ki(rac)
ki(R) / ki(rac)
1.5
BChEUU
BChEAA
3200 ± 300
76 ± 2.3
1.5
1.9
1.6
1.8
1.3
340 ± 8.7
31 ± 0.53
210 ± 8.6
150 ± 6.3
100 ± 3.0
1.7
1.5
1.3
1.1
1.1
1.3
BChEUA
2200 ± 77
1100 ± 42
550 ± 19
1.4
BChEFF
1.6
BChEAF
1.5
AChE
ki (103 Mꢀ1minꢀ1)
kmax (minꢀ1)
Ki (mM)
4.7 ± 1.2
1.9 ± 0.18
2.0
2.7 ± 0.78
3.1 ± 0.41
1.2 ± 0.31
1.1
2.0
0.39 ± 0.096
a Ref. 8
24
ACS Paragon Plus Environment

Page 25 of 34
Journal of Medicinal Chemistry
1
2
3
4
Table 3 Reversible inhibition of human AChE, BChE and four BChE variants by metaproterenol
5
6
and isoproterenol
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Metaproterenol
Isoproterenol
Enzyme
[s] (mM)
K(I) (mM)
K(S) (mM)
[s] (mM)
K(I) (mM)
K(S) (mM)
BChEUU 0.05 ꢀ 0.30 0.55 ± 0.11 0.098 ± 0.022 0.05ꢀ0.35 0.47 ± 0.21 0.095 ± 0.047
BChEAA 0.05 ꢀ 1.0 6.1 ± 0.71 0.26 ± 0.035 0.05ꢀ1.0 1.6 ± 0.25 0.21 ± 0.039
BChEFF 0.05 ꢀ 0.50 0.87 ± 0.18 0.14 ± 0.031 0.05ꢀ1.0 0.99 ± 0.12 0.42 ± 0.064
BChEUA 0.05 ꢀ 0.50 0.65 ± 0.11 0.12 ± 0.021 0.05ꢀ0.5 0.72 ± 0.082 0.42 ± 0.057
AChE 0.03 ꢀ 0.20 3.1 ± 1.2 0.035 ± 0.014 0.05ꢀ1.0
2.5 ± 1.0
0.54 ± 0.31
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 34
1
2
3
4
FIGURE CAPTIONS
5
6
7
8
9
Fig. 1. Chemical structures of tested inhibitors. Asterisk denotes a chiral center.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 2. Representative progressive inhibition of BChEUU by racemic metacarb. (A) points indicate
the logarithm of residual activity, while slope of the lines determines the first order rate constant
kobs. (B) kobs were plotted as a function of metacarbrac concentration wherefrom the second order
rate constant, ki, was calculated.
Fig. 3. Minimized structures of bambuterol, metacarb and isocarb. For better visibility, hydrogen
atoms are not displayed.
Fig. 4. Computational molecular modeling of the transition states of human BChE and (R)ꢀ
metacarb (A), (R)ꢀisocarb (B) and (R)ꢀbambuterol (C). Amino acid residues that stabilize the
transition state are displayed. Ser198, His438 and Glu325 represent the catalytic triad. The
structure was generated using the Accelrys Studio program,26 and the BChE structure (PDB ID
code 2PM8).27
Fig. 5. Reversible inhibition of human BChEFF by metaproterenol. Points indicate the average
apparent enzymeꢀinhibitor constant at a given substrate (PTCh) concentration, Ki,app (± standard
deviation).
26
ACS Paragon Plus Environment

Page 27 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 1
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 34
1
2
3
4
5
6
7
8
A
B
1.5
9
2.0
1.8
1.6
1.4
1.2
1.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
500 nM
200 nM
100 nM
50 nM
1.0
0.5
0.0
0
5
10
15
20
0
100
200
300
400
500
t / min
[metacarbrac] / nM
Fig. 2.
28
ACS Paragon Plus Environment

Page 29 of 34
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 3
29
ACS Paragon Plus Environment