3-Amidoquinuclidine derivatives: Synthesis of compounds and inhibition of butyrylcholinesterase

Article abstract of DOI:10.1016/j.bioorg.2006.01.004

The synthesis of racemic and enantiomerically pure 3- butanamidoquinuclidines ((±)-Bu, (R)-Bu and (S)-Bu), (1-3) and 3-benzamidoquinuclidines ((±)-Bz, (R)-Bz, and (S)-Bz), (4-6) is described. The N-quaternary derivatives, N-benzyl-3-butanamidoquinuclidinium bromides ((±)-BnlBu, (R)-BnlBu and (S)-BnlBu), (7-9) and N-benzyl-3-benzamidoquinuclidinium bromides ((±)-BnlBz, (R)-BnlBz and (S)-BnlBz), (10-12) were subsequently synthesized. The interaction of the four enantiomerically pure quaternary derivatives with horse serum butyrylcholinesterase (BChE) was tested. All tested compounds inhibited the enzyme. The best inhibitior of the enzyme was (S)-BnlBz with a K_i = 3.7 μM. The inhibitor potency decreases in order (S)-BnlBz > (R)-BnlBz ? (R)-BnlBu > (S)-BnlBu.

Full text of DOI:10.1016/j.bioorg.2006.01.004

Bioorganic Chemistry 34 (2006) 90–98
www.elsevier.com/locate/bioorg
3-Amidoquinuclidine derivatives: Synthesis
of compounds and inhibition
of butyrylcholinesterase
-
*
´
Renata Odzˇak , Srdanka Tomic
Laboratory of Organic Chemistry, Department of Chemistry, Faculty of Science, University of Zagreb,
Horvatovac 102A, HR-10 000 Zagreb, Croatia
Received 27 October 2005
Abstract
The synthesis of racemic and enantiomerically pure 3-butanamidoquinuclidines (( )-Bu, (R)-Bu
and (S)-Bu), (1–3) and 3-benzamidoquinuclidines (( )-Bz, (R)-Bz, and (S)-Bz), (4–6) is described.
The N-quaternary derivatives, N-benzyl-3-butanamidoquinuclidinium bromides (( )-BnlBu,
(R)-BnlBu and (S)-BnlBu), (7–9) and N-benzyl-3-benzamidoquinuclidinium bromides (( )-BnlBz,
(R)-BnlBz and (S)-BnlBz), (10–12) were subsequently synthesized. The interaction of the four enan-
tiomerically pure quaternary derivatives with horse serum butyrylcholinesterase (BChE) was tested.
All tested compounds inhibited the enzyme. The best inhibitior of the enzyme was (S)-BnlBz with a
Ki = 3.7 lM. The inhibitor potency decreases in order (S)-BnlBz > (R)-BnlBz ꢀ (R)-BnlBu > (S)-
BnlBu.
ꢀ 2006 Elsevier Inc. All rights reserved.
Keywords: 3-Amidoquinuclidines; Quaternary 3-amidoquinuclidines; Synthesis; Butyrylcholinesterase inhibition
1. Introduction
Quinuclidine (1-azabicyclo[2.2.2]octane) is a constituent of a large number of natural
compounds such as the Cinchona, Iboga, Voacanga, Ajmaline and Sarpagine alkaloids
*
Corresponding author. Fax: +385 1 4606401.
E-mail address: rodzak@chem.pmf.hr (R. Odzˇak).
0045-2068/$ - see front matter ꢀ 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2006.01.004

R. Odzˇak, S. Tomic´ / Bioorganic Chemistry 34 (2006) 90–98
91
a
NH2
NH2
N
N
. 2HCl
( )-
(R)-
(S)-
( )-
(R)-
(S)-
O
b
NHCR
N
R
( )-
(R)-
(S)-
3
1-6
-CH2CH2CH3
-C6H5
1
92%
90%
2
92%
84%
86%
93%
4
5
6
c
O
R
( )-
(R)-
(S)-
NHCR
+
N
-CH2CH2CH3
-C6H5
7
94%
8
92%
9
87%
Br-
10 80%
11 80%
12 91%
7-12
Scheme 1. (a) KOH, K2CO3, (b) (CH3CH2CH2CO)2O or (C6H5CO)2O, (c) C6H5CH2Br, dry diethyl ether.
CH3
O
C
H3C
H3C
+
N
BuCh
BzCh
O
CH3
CH3
O
H3C
H3C
+
N
C
O
O
+
N
C
N
H
R
R:-CH2CH2CH3;
-C6H5
Fig. 1. Comparison of structures of BuCh and BzCh with quaternary derivatives of 3-amidoquinuclidines.

92
R. Odzˇak, S. Tomic´ / Bioorganic Chemistry 34 (2006) 90–98
[1–3]. In addition, many synthetic physiologically active substances with antiarythmic and
antihistaminic activities contain the quinuclidine moiety [4]. Synthetic 3-substituted quinu-
clidine derivatives are of special interest due to their various pharmacological properties.
Many esters of quinuclidine-3-ol were also extensively studied and recognized as bioactive
substances, and as such some of them are commercially available as therapeutics such as
aceclidine [4]. 3-Amidoquinuclidine derivatives are classical 5-hydroxytryptamine3 (5-
HT3) receptor antagonists since they contain the generally recognized pharmacophore, a
basic nitrogen, carbonyl group and an aromatic ring. Some known examples which con-
tain the 3-amidoquinuclidine moiety are zacopride and RG 12915 [5]. Compounds con-
taining the 3-substituted quinuclidine moiety have also been found to be potential
antidotes for organophosphate poisons, among which warfare agents are of special inter-
est [6–10].
3-Substituted quinuclidines contain an asymmetric carbon atom and any classical syn-
thesis starting from quinuclidin-3-ol or 3-aminoquinuclidine leads to racemates. Race-
mates of pharmaceuticals are rarely acceptable in therapies since enantiomers of a given
bioactive compound can cause different biological effects ranging from lower activity of
the undesired enantiomer to increased toxicity. Therefore, many investigations concen-
trate on the resolution of racemic quinuclidin-3-ol derivatives using chemical [11,12]
and biocatalytic [13,14] methods. One of the enzymes tested as a biocatalyst in the resolu-
tion of esters of quinuclidin-3-ol was butyrylcholinesterase (BChE, EC 3.1.1.8) from horse
serum [13,15–18]. Only a few examples were reported concerning the resolution of 3-amid-
oquinuclidines using chemical [19,20] and biocatalytic methods [21].
In this work we report the synthesis of some 3-amidoquinuclidines in racemic and enan-
tiomerically pure forms as well as the synthesis of their quaternary salts (Scheme 1). Benzyl
bromide was used as quaternization agent because the kinetics of a reaction of quaternary
amides with BChE can be monitored by following the production of benzoic acid by UV
detector in HPLC.
The racemic non-quaternary and quaternary 3-amidoquinuclidines (1, 4, 7, and 10)
were tested as substrates of some hydrolytic enzymes including BChE but no hydrolysis
was observed. Instead, inhibition of BChE from horse serum by (R)- and (S)-enantiomers
of quaternary 3-amidoquinuclidines was observed. This finding is not surprising because
N-quaternary derivatives of 3-amidoquinuclidine may be looked upon as bicyclic ana-
logues of butyrylcholine (BuCh) and benzoylcholine (BzCh) which are good substrates
for BChE, and therefore their interaction with the enzyme could have been expected
(Fig. 1).
2. Experimental
2.1. General methods and chemicals
Melting points were determined in open capillaries using a Buchi B-540 apparatus and
¨
are uncorrected. Optical rotations (in degrees) were measured on an Optical Activity AA-
10 automatic polarimeter at ambient temperature in methanol. Elemental analyses were
performed with a Perkin-Elmer PE 2400 Series II CHNS/O Analyser. IR spectra were
recorded with a Perkin-Elmer FTIR 1725 X spectrometer. 1H and 13C 1D and 2D
NMR spectra were recorded on a Bruker AV300 or Bruker AV600 spectrometers at room
temperature. Chemical shifts are given in ppm downfield from TMS as internal standard.

R. Odzˇak, S. Tomic´ / Bioorganic Chemistry 34 (2006) 90–98
93
The ( )-, (R)-, and (S)-3-aminoquinuclidine dihydrochlorides (Aldrich) were used for the
synthesis of the appropriate 3-amidoquinuclidines with butyric (Aldrich) and benzoic
(Merck) acid anhydrides.
2.2. Synthesis of compounds
The ( )-, (R)-, and (S)-3-aminoquinuclidines were prepared in high yields (>90%) by
treating commercial ( )-, (R)-, and (S)-3-aminoquinuclidine dihydrochlorides (200 mg,
1.00 mmol) with saturated aqueous solutions of KOH (7.13 mmol in 0.4 mL of H2O).
The aqueous reaction solution was then extracted with chloroform (10 · 2 mL). The
extracts were dried over K2CO3, filtered and evaporated under reduced pressure. The sam-
ples were stored over CaCl2 under reduced pressure.
2.2.1. Synthesis of 3-amidoquinuclidines
2.2.1.1. 3-Butanamidoquinuclidines. A mixture of ( )-3-aminoquinuclidine (3.20 mmol)
and butyric acid anhydride (15.8 mmol) was heated with stirring for 2 h at 110–120 ꢁC.
Water (2 mL) was added to the reaction mixture after cooling and pH values were adjusted
to ꢁ10 with saturated aqueous K2CO3 followed by extraction with chloroform
(5 · 20 mL). The extracts were dried over Na2SO4, filtered and evaporated under reduced
pressure. The residue was distilled under reduced pressure (oil pump) to give:
( )-3-Butanamidoquinuclidine as white crystals (1, 92%), mp 120–122 ꢁC. IR (KBr):
~m = 3369, 2949, 2868, 1635, 1524, 1127, 768 cmꢂ1
.
1H NMR (600 MHz, DMSO-d6):
d = 0.85 (t, J = 7.38, 3H, CH3CH2CH2), 1.25–1.29 (m, 1H, H-8), 1.45–1.56 (m, 4H,
CH3CH2CH2 and H-5), 1.68–1.72 (m, 2H, H-4, and H-8), 2.06 (t, J = 7.29, 2H,
CH3CH2CH2), 2.37–2.41 (m, 1H, H-2), 2.57–2.68 (m, 3H, H-6, and H-7), 2.73–2.78
(m, 1H, H-7), 3.00–3.04 (m, 1H, H-2), 3.68–3.71 (m, 1H, H-3), 7.81 (d, J = 6.78,
1H, CONH) ppm. 13C NMR (150 MHz, DMSO-d6): d = 13.52 (CH3CH2CH2), 18.79
(C-5), 19.81 (C-8), 25.60 (CH3CH2CH2), 25.65 (C-4), 37.24 (CH3CH2CH2), 45.97 (C-
3), 46.25 (C-6), 46.85 (C-7), 54.46 (C-2), 171.82 (C@O) ppm. Anal. Calcd for
C11H20N2O (196.29): C, 67.31; H, 10.27; N, 14.27. Found: C, 67.05; H, 9.92; N,
13.92%.
(R)-3-butanamidoquinuclidine (2, 92%); ½aꢃ2D5 +28ꢁ (c = 0.99, MeOH); mp 89.3–90.8 ꢁC.
25
(S)-3-butanamidoquinuclidine (3, 86%); ½aꢃD ꢂ27ꢁ (c = 1.0, MeOH); mp 84.2–84.9 ꢁC.
2.2.1.2. 3-Benzamidoquinuclidines. The same reaction procedure as described for 1 was
followed using benzoic acid anhydride. Diethyl ether (2 mL) and water (2 mL) were added
to a cold reaction mixture. After extraction with diethyl ether, the pH value of the water
phase was adjusted to ꢁ10 with saturated aqueous K2CO3 and extracted with chloroform
(5 · 10 mL). The chloroform extracts were dried over Na2SO4 and evaporated under
reduced pressure to give:
( )-3-Benzamidoquinuclidine as white crystals (4, 90%ꢂ),1 mp 158.2–161.9 ꢁC. IR
.
1H NMR (300 MHz,
~
(KBr): m = 3287, 2934, 2866, 1630, 1539, 1153, 692 cm
CDCl3): d = 1.49–1.53 (m, 1H, H-5), 1.67–1.73 (m, 3H, H-5, and H-8), 2.03–2.06
(m, 1H, H-4), 2.63 (dd, J = 4.73, J = 14.13, 1H, H-2), 2.75–2.98 (m, 4H, H-6, and
H-7), 3.38–3.46 (m, 1H, H-2), 4.13–4.15 (m, 1H, H-3), 6.45 (d, J = 5.78, 1H, CONH),
7.47–7.49 (m, 3H, H-3 Bz, H-4 Bz, and H-5 Bz), 7.77 (d, J = 7.06, 2H, H-2 Bz, and
H-6 Bz) ppm. 13C NMR (75 MHz, CDCl3): d = 20.07 (C-5), 25.56 (C-4), 25.60 (C-8),

94
R. Odzˇak, S. Tomic´ / Bioorganic Chemistry 34 (2006) 90–98
46.49 (C-6), 46.97 (C-3), 47.23 (C-7), 55.81 (C-2), 126.72 (C-2 Bz and C-6 Bz),128.38
(C-3 Bz and C-5 Bz), 131.28 (C-4 Bz), 134.48 (C-1 Bz), 167.37 (C@O) ppm. Anal.
Calcd for C14H18N2O (230.31): C, 73.01; H, 7.88; N, 12.16. Found: C, 72.76; H,
7.71; N, 11.86%.
25
(R)-3-Benzamidoquinuclidine (5, 84%); ½aꢃD +40ꢁ (c = 0.99, MeOH); mp 152.4–
153.8 ꢁC.
25
(S)-3-Benzamidoquinuclidine (6, 93%); ½aꢃD ꢂ39ꢁ (c = 1.0, MeOH); mp 148.9–150.4 ꢁC.
2.2.2. Synthesis of N-quaternary derivatives
To the solution of the appropriate 3-butanamidoquinuclidine (1–3, 7.6 mmol) in dry
diethyl ether equimolar amounts of benzyl bromide were added at room temperature.
The reaction mixture was kept in the dark overnight to obtain a solid. Recrystallization
from acetonitrile gave:
( )-N-Benzyl-3-butanamidoquinuclidinium bromide as white crystals (7, 94%), mp
ꢂ1
.
1H
~
153.2–154.8 ꢁC. IR (KBr): m = 3426, 2961, 2868, 1656, 1538, 1143, 769 cm
NMR (300 MHz, DMSO-d6): d = 0.84 (t, J = 7.31, 3H, CH3CH2CH2), 1.44–1.56 (m,
4H, CH3CH2CH2 and H-5), 1.87–1.98 (m, 2H, H-8), 2.05–2.09 (m, 3H, CH3CH2CH2
and H-4), 3.05–3.15 (m, 1H, H-2), 3.35–3.39 (m, 4H, H-6 and H-7), 3.78–3.86 (m,
1H, H-2), 4.07–4.16 (m, 1H, H-3), 4.46 (s, 2H, CH2 Bnl), 7.52 (s, 5H, Bnl), 8.22 (d,
J = 5.57, 1H, CONH) ppm. 13C NMR (75 MHz, DMSO-d6): d = 13.57 (CH3CH2CH2),
18.25 (C-5), 18.58 (C-8), 22.26 (CH3CH2CH2), 24.68 (C-4), 36.94 (CH3CH2CH2), 44.29
(C-3), 53.36 (C-6 and C-7), 59.48 (C-2), 66.30 (CH2 Bnl), 127.46 (C-1 Bnl), 129.00 C-3
Bnl and C-5 (Bnl), 130.22 (C-4 Bnl), 133.09 (C-2 Bnl and C-6 Bnl), 172.42 (C@O) ppm.
Anal. Calcd for C18H27BrN2O (367.32): C, 58.86; H, 7.41; N 7.63. Found: C, 58.54; H,
7.35; N, 7.63%.
25
(R)-N-Benzyl-3-butanamidoquinuclidinium bromide (8, 92%); ½aꢃD +30ꢁ (c = 1,
MeOH); mp 179.4–180 ꢁC.
25
(S)-N-Benzyl-3-butanamidoquinuclidinium bromide (9, 87%); ½aꢃD ꢂ29ꢁ (c = 1.02,
MeOH); mp 168.2–169.5 ꢁC.
The same reaction procedure as described for 7 was followed using the appropriate 3-
benzamidoquinuclidine (4–6, 0.43 mmol).
( )-N-Benzyl-3-benzamidoquinuclidinium bromide as white crystals, (10, 80%) mp
~
243.3–244.7 ꢁC. IR (KBr): m = 3468, 3409, 3258, 2927, 2865, 1630, 1543, 1317, 1054,
1
696 cmꢂ1. H NMR (300 MHz, CDCl3): d = 1.75–1.77 (m, 1H, H-5), 1.91–2.14 (m, 2H,
H-8), 2.43–2.44 (m, 2H, H-4, and H-5), 3.28–3.30 (m, 1H, H-2), 3.34–3.47 (m, 4H, H-6,
and H-7), 3.85–3.89 (m, 1H, H-2), 4.30–4.31 (m, 1H, H-3), 4.45 (s, 2H, CH2, Bnl),
7.39–7.51 (m, 8H, Bnl, and H-3, H-4, and H-5 Bz), 7.80 (d, J = 7.16, 2H, H-2 and H-6
Bz), 8.58 (d, J = 5.93, 1H, CONH) ppm. 13C NMR (75 MHz, CDCl3): d = 18.39 (C-5),
22.33 (C-8), 24.88 (C-4), 45.38 (C-3), 53.37 (C-6), 53.56 (C-7), 58.60 (C-2), 66.38 (CH2Bnl),
124.82 (C-1 Bnl), 127.50 (C-3 Bnl and C-5 Bnl), 128.22 (C-3 Bz and C-5 Bz), 128.97 (C-2
Bnl and C-6 Bnl), 130.18 (C-4 Bnl), 131.51 (C-4 Bz), 133.10 (C-2 Bz and C-6 Bz), 133.82
(C-1 Bz), 166.91 (C@O) ppm. Anal. Calcd for C21H25BrN2O (401.34): C, 62.85; H, 6.28;
N, 6.98. Found: C, 63.26; H, 6.40; N, 7.05%.
25
(R)-N-Benzyl-3-benzamidoquinuclidinium bromide (11, 80%); ½aꢃD +7ꢁ (c = 1.04,
MeOH); mp 221.0–221.5 ꢁC.
25
(S)-N-Benzyl-3-benzamidoquinuclidinium bromide (12, 91%); ½aꢃD ꢂ5ꢁ (c = 1, MeOH);
mp 236.0–236.5 ꢁC.

R. Odzˇak, S. Tomic´ / Bioorganic Chemistry 34 (2006) 90–98
95
2.3. Inhibition of butyrylcholinesterase
BChE (EC, 3.1.1.8), type IV-S, lyophilized from horse serum (Sigma Chemical
Co.) was used without further purification. Benzoylcholine chloride (BDH Chemi-
cals Ltd.) was used as a substrate for BChE. HPLC analyses (Thermo Separation
Products, Spectra SYSTEM 2000) were performed on an RP-18 column (Waters,
SymmetryShield, 5 lm, 150 · 3.9 mm i.d.) at 40 ꢁC. The mobile phase used was
water–methanol–acetonitrile–acetic acid–triethylamine (60:25:15:0.33:0.2) at a flow-
rate of 1.0 mL minꢂ1. The reactions were carried out in a Heidolph UNIMAX
1100 shaker.
The kinetics of the reaction between enantiomers of N-quaternary quinuclidinamides 8,
9, 11, and 12 and BChE were monitored by following the production of benzoic acid at
230 nm using HPLC. All experiments were performed in a total volume of 1.0 mL and
an enzyme concentration of 1.5 · 10ꢂ9 M (19.8 U/mg solid, 0.016 mg mLꢂ1). The enzy-
matic reaction was stopped by addition of aliquots (20 lL) of the reaction mixture to
the HPLC mobile phase (200 lL). Two to three measurements were made with each sub-
strate concentration (0.1–0.5 mM). KM values were obtained by non-linear regression of
the experimental data to the Michaelis–Menten equation. The dissociation constant of
enzyme–inhibitor complex was determined from Hunter-Downs plot, using 0.1–0.5 mM
concentrations of the inhibitor.
3. Results and discussion
Racemic and chiral (R)- and (S)-3-amidoquinuclidine derivatives (1–6) were synthesized
by the classical reaction of the appropriate amine and anhydride. Their quaternary salts
were prepared as well (7–12) with benzyl bromide as quaternization agent. The structure
and purity of all compounds were determined by elemental analyses, IR, 1H and 13C NMR
and 2D NMR spectroscopy. Optical purities were checked by optical rotation
measurements.
Racemic non-quaternary and quaternary (1, 4, 7, and 10) 3-amidoderivatives were used
to evaluate selective hydrolysis catalyzed by some hydrolases such as PLE (an esterase),
subtilisin and chymotrypsin (proteases) and an amidase from human serum [22] but no
hydrolysis was observed. BChE was also used as a catalyst since it was previously shown
that BChE accepts as substrates and catalyzes the hydrolysis of compounds with diverse
structures such as heroin, aspirin and succinylcholine [23]. In the case of quinuclidines,
BChE has been previously used for the hydrolysis of the racemic quinuclidine-3-yl buty-
rate [14], quinuclidin-3-yl benzoates and their N-methyl and N-benzyl derivatives
[16,17]. Quaternary quinuclidinium benzoates were better substrates for BChE than their
non-quaternized analogues [17]. That is not surprising because N-quaternary derivatives
of quinuclidine esters and amides have structural similarities with BzCh (Fig. 1). However,
hydrolysis of the compounds 1, 4, 7 and 10 was not observed in the experimental condi-
tions used in this work.
It was shown that the enantiomers of N-benzyl derivatives (8, 9, 11, and 12) act as
reversible inhibitors of BChE. Inhibition was determined with benzoylcholine (BzCh) as
the enzyme’s substrate. The measured activity of BChE at different concentrations of
BzCh showed that in a given concentration range the Michaelis–Menten kinetics was fol-
lowed. The value of KM of 0.17 0.01 mM was obtained from Lineweaver–Burk plot

96
R. Odzˇak, S. Tomic´ / Bioorganic Chemistry 34 (2006) 90–98
(Fig. 2) for BzCh as substrates of BChE. This is in accord with previously published data
[17].
We started with the assumption that our compounds were competitive inhibitors,
i.e., that they compete for the same active site on the enzyme as does the substrate
(BzCh). In this case in the Lineweaver–Burk plot the same ordinal intercept (1/v) is
expected in the presence or absence of the inhibitor. The effect of the competitive
inhibitor, therefore, is to produce an apparent increase in KM values. The kinetics of
inhibition by the (R)-enantiomer (11) indicated that this compound is a competitive
inhibitor and therefore binds to the catalytic site of BChE. On the other hand the
(S)-enantiomer (12) does not have the same ordinal intercept (1/v) as the substrate
which indicates that this enantiomer was not exclusively bound to the catalytic site
of the enzyme (Fig. 2). The same results were obtained in the case of N-benzyl-3-but-
anamidoquinuclidines (8 and 9).
The concentrations of the inhibitor which were chosen inhibited the enzyme between 20
and 80%. The constants were evaluated from the kinetics of competition between BzCh
and inhibitors of BChE. Activities of the enzymes were measured at different substrate
concentrations (S) in the absence (v0) and presence (vi) of the given inhibitor concentration
(I). The concentration constant (Kapp) was calculated for each substrate. The enzyme-in-
hibitor dissociation constants Ki were evaluated from the Hunter-Downs equation using
linear regression analysis [24] where Kapp is the apparent enzyme–inhibitor dissociation
constant at a given substrate concentration (S) and KM is the Michaelis constant for the
substrate.
viI
v0 ꢂ vi
Ki
KM
Kapp
¼
¼ Ki þ
S
ð1Þ
300
1/v (mM-1min)
250
200
150
100
50
0
-8
-6
-4
-2
0
2
4
6
8
10
12
-50
1/S (mM-1)
Fig. 2. Lineweaver–Burk plot in the absence ( ) and presence of inhibitor 11 ( ) and 12 ( ).
d

R. Odzˇak, S. Tomic´ / Bioorganic Chemistry 34 (2006) 90–98
97
Table 1
Reversible inhibition of BChE measurement with benzoylcholine as a substrate in the presence of compounds as
inhibitors
a
b
Compound
Ki (lM)
KM (mM)
8
159.88 0.47
241.96 0.08
25.92 0.01
3.70 0.00
0.30
0.36
0.19
0.45
9
11
12
a
Ki is the enzyme–inhibitor dissociation constant calculated according to Eq. (1). Each measurement was
repeated two to three times.
b
KM were obtained from Lineweaver–Burk plot. Relative standard deviations of the KM values were on average
15%.
The Hunter-Downs plot of the reversible inhibition of BChE by the quaternary enan-
tiomers of 3-amidoquinuclidine derivatives displayed a linear relationship between the
apparent dissociation constant and the substrate concentration between 0.1 and
0.5 mM. The enzyme–inhibitor dissociation constant was obtained for all tested com-
pounds. The most potent inhibitor (with the lowest dissociation constant) was the (S)-
enantiomer of N-benzyl-3-benzamidoquinuclidine (12) with Ki = 3.7 0.0 lM. This could
have been expected since previously reported data showed that the (S)-enantiomer of N-
benzylquinuclidine-3-yl benzoate was the poorest substrate but an efficient inhibitor of all
tested enantiomers of N-methyl and N-benzylquinuclidine-3-yl benzoates, with
Ki = 3.3 0.2 lM [16]. The (R)-enantiomer of N-benzyl-3-benzamidoquinuclidine (11)
was a 7-fold weaker inhibitor than its (S)-enantiomer. The least potent inhibitor was
the (S)-enantiomer of the quaternary derivative of butyric acid which was 65-fold weaker
than the most potent inhibitor 12. Both enantiomers of quaternary 3-benzamidoquinucli-
dines (11 and 12) were more potent inhibitors of BChE than enantiomers of quaternary 3-
buatanamidoquinuclidines (8 and 9). The obtained kinetic constants for inhibitors are dis-
played in Table 1.
4. Conclusion
We have synthesized racemic and enantiomerically pure amides of 3-aminoquinuclidine
with butyric and benzoic acid anhydrides. Their quaternary N-benzyl derivatives were
prepared as well and tested as possible inhibitors of BChE. For all tested quaternary com-
pounds inhibition of BChE was observed. The most potent inhibitor was the (S)- enantio-
mer of N-benzyl derivatives of 3-benzamidoquinuclidine (12) which was 7-fold more
potent than the (R)-enantiomer (11). Both enantiomers of quaternary derivatives of 3-but-
anamidoquinuclidine were weaker inhibitors than enantiomers of quaternary derivatives
of 3-benzamidoquinuclidine. Thus, the (S)-N-benzyl derivatives of butyric acid (8) was
65-fold less potent than the compound 12.
Acknowledgments
Grateful thanks are due to Dr Ines Primozˇicˇ, Faculty of Science, University of Zagreb
for the useful advice regarding the kinetic study. We are very grateful to Dr Vera Simeon-
Rudolf, Institute for Medical Research and Occupational Health, Zagreb, for her kind

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help with chemicals and helpful comments. This work was supported by the Ministry of
Science, Education and Sports of the Republic of Croatia, Project No. 0119610.
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