Synthesis and in vitro muscarinic activities of a Series of 1,3-diazacycloalkyl carboxaldehyde oxime derivatives

Article abstract of DOI:10.1016/S0968-0896(01)00379-0

A series of 1,3-diazacycloalkyl carboxaldehyde oxime derivatives was synthesized and tested for muscarinic activity in receptor binding assays using [³H]-oxotremorine-M (OXO-M) and [³H]-pirenzepine (PZ) as ligands. Potential muscarinic agonistic or antagonistic properties of the compounds were determined using binding studies measuring their potencies to inhibit the binding of OXO-M and PZ. Preferential inhibition of OXO-M binding was used as an indicator for potential muscarinic agonistic properties; this potential was confirmed in functional studies on isolated organs. Copyright

Full text of DOI:10.1016/S0968-0896(01)00379-0

Bioorganic & Medicinal Chemistry 10 (2002) 1143–1152
Synthesis and In Vitro Muscarinic Activities of a Series of
1,3-Diazacycloalkyl Carboxaldehyde Oxime Derivatives
Ralf Plate,a,* Christan G.J.M. Jans,a Marc J.M. Plauma and Thijs de Boerb
aDepartment of Medicinal Chemistry, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands
bLead Discovery Unit, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands
Received 9 July 2001; accepted 26 October 2001
Abstract—A series of 1,3-diazacycloalkyl carboxaldehyde oxime derivatives was synthesized and tested for muscarinic activity in
receptor binding assays using [3H]-oxotremorine-M (OXO-M) and [3H]-pirenzepine (PZ) as ligands. Potential muscarinic agonistic
or antagonistic properties of the compounds were determined using binding studies measuring their potencies to inhibit the binding
of OXO-M and PZ. Preferential inhibition of OXO-M binding was used as an indicator for potential muscarinic agonistic proper-
ties; this potential was confirmed in functional studies on isolated organs. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
dose may improve certain cognitive functions without
severely compromising side effects,9,10 indicating the
potential of a cholinergic strategy for treatment of Alz-
heimer patients. Five muscarinic cholinergic receptor
subtypes are known and their structure and distribution
have been described.11 These receptors regulate a wide
range of peripheral and central functions12 and conse-
quently it can be expected that treatments with non-
selective agonists will in addition to their cognitive
activities produce many undesirable effects. Therefore,
attention has shifted to subtype selective muscarinic
agonists as a means to improve therapeutic and at the
same time to reduce cholinergic side effects. Despite
some potential problems with this approach,13 attention
has now focused on a pharmacochemical approach
aiming for compounds with preferential M1 or M1/M3
agonist properties.14 Animal studies indeed revealed
positive effects of such compounds in tests of learning
and memory,15,16 but more definite conclusions await
the outcome for these compounds with such profiles in
clinical trials.
The degeneration of forebrain muscarinic cholinergic
neurons of patients suffering from Alzheimer’s disease
has been associated with reduction in cognitive func-
tion.1 In particular, the presynaptic rather than post-
synaptic neurons are affected suggesting that
a
therapeutic strategy based on the cholinergic deficit
hypothesis2 might have potential for the improvement
of the cognitive aspects of Alzheimer disease. Treatment
of Alzheimer patients with acetylcholine esterase inhi-
bitors such as tacrine,3 to increase acetylcholine release,
indirectly resulted in improvement of only some aspects
of cognitive performance, and in association with severe
side effects.3ꢀ6 This poor therapeutic outcome after
treatment with these acetylcholine esterase inhibitors
might be due to stimulation of presynaptic inhibitory
autoreceptors following an increase in extracellular
acetylcholine, thus limitating the increase in the release
of accumulated acetylcholine.
We have concentrated on the stimulation of post-
synaptic receptors to correct the cholinergic deficit.
Unfortunately, studies involving direct stimulation of
cholinergic receptors with non-selective cholinergic
agonists have been marred by their side-effect profile
(e.g., oxotremorine,7 RS 868). Careful adjustment of the
*Corresponding author. Tel.: +31-412-661957; fax: +31-412-662546;
e-mail: ralf.plate@organon.com
Figure 1.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00379-0

1144
R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
The aim of the present study was to design and test a
series of compounds with selective effects at specific
receptor subtypes in vitro, and then to investigate the
central and peripheral effects in vivo including effects on
memory. The ultimate objective of these studies is to
arrive at compounds with improved efficacy, pre-
ferentially action in the central nervous system and
associated with a reduced incidence of peripheral side
effects (Fig. 1).
(approximately 0.5% w/w) solution of 2 in methanol
containing three equivalents of methanolic HCl.21
A
more concentrated reaction mixture or a lower amount
of catalyst caused the formation of the undesired
enamine A as byproduct.22 The desired methyl 3-amino-
2-(aminomethyl)propionate (3) was isolated as dihy-
drochloric acid salt in 58% after recrystallization. The
amino groups of 3 were protected using N-(benzyloxy-
carbonyloxy)succinimide as reagent. The corresponding
aldehyde 5 was obtained after reduction of the methyl
ester moiety of 4 with diisobutylaluminium hydride
(DIBAH) as reagent. Treatment with O-alkylhydrox-
ylamine derivatives gave the oxime derivatives 6a–b.
Deprotection by hydrogenation gave the diamino deri-
vatives 7a–b. Ring closure of 7a–b, using trimethyl-
orthoformate as reagent, gave the desired 1,4,5,6-
tetrahydro-5-pyrimidine carboxaldehyde O-alkyloximes
8a and 8b, respectively, either as pure E-isomers or Z/E
mixtures.
We chose arecoline (Fig. 2) as a useful starting point for
the design of the title series. arecoline has shown thera-
peutically beneficial effects, which are, however, of lim-
ited use due to its short duration of action.
In our search for a bioisosteric replacement of the ester
function of arecoline, we developed several series of
potent muscarinic agonists.17 Moreover, we included
the muscarinic agonist isoarecoline as a lead structure in
our study. Here, we report the synthesis of the related
series of 1,3-diazacycloalkyl carboxaldehyde and etha-
none oxime derivatives (Table 1) which was tested for
muscarinic activity using [3H]-oxotremorine-M (OXO-
M) and [3H]-pirenzepine (PZ) as ligands (Table 2) and
was further profiled in our in vitro (Table 3) test bat-
tery.18
After heating the Z/E-mixtures in ethanol, an equili-
brium ratio which was in favor of the E-isomer was
observed (for details, see Experimental). The mixtures,
which were not separable by HPLC, were tested.
This synthetic route turned out to be rather laborious.
Consequently, a new, more efficient route was devel-
oped. Ring closure of 3 using trimethylorthoformate as
reagent gave methyl 1,4,5,6-tetrahydro-5-pyrimidine
carboxylate (9). Treatment of this methyl ester 9 with
diisobutylaluminium hydride, at ꢀ78 ꢁC gave 1,4,5,6-
tetrahydro-5-pyrimidine carboxaldehyde (10). In the
final step, the aldehyde was converted in situ at ꢀ20 ꢁC
into the desired O-ethyl oxime derivative 8c.
Chemistry
The methods for three different novel approaches of
synthesizing of the hitherto unknown 1,4,5,6-tetrahydro-
5-pyrimidine carboxaldehyde O-alkyloximes, 1,4,5,6-
tetrahydro-4-pyrimidine carboxaldehyde O-alkyloximes,
4,5,6,7-tetrahydro-1H-1,3-diazepine-4-carboxaldehyde
O-alkyloximes and 1-(1,4,5,6-tetrahydro-5-pyrimidine)-
ethanone O-alkyloximes are summarized in Schemes 1–
3.
Synthesis of 1,4,5,6-tetrahydro-4-pyrimidine carbox-
aldehyde oximes and 4,5,6,7-tetrahydro-1H-1,3-diaze-
pine-4-carboxaldehyde oximes, 14a–j
The route for the synthesis of the desired 4-carbox-
aldehyde derivatives started with the ringclosure reaction
of the commercial available a-amino acid derivatives 11a
and 11b using trimethylorthoformate as reagent (Scheme
2). Esterification with thionylchloride in methanol gave
the corresponding methyl esters 12a–b. Subsequently,
reduction using diisobutylaluminium hydride, at ꢀ78 ꢁC
as reagent, as described above, gave 1,4,5,6-tetrahydro-
4-pyrimidine carboxaldehyde (13a) and the correspond-
ing 4,5,6,7-tetrahydro-1H-1,3-diazepine-4-carboxalde-
hyde (13b). Treatment of these aldehydes in situ with
O-alkyl hydroxylamine derivatives gave the desired oxi-
mes 14a–j as Z/E mixtures (approx. 1:1).
Synthesis of 1,4,5,6-tetrahydro-5-pyrimidine carbox-
aldehyde O-alkyloximes, 8a–c
For the synthesis 1,4,5,6-tetrahydro-5-pyrimidine car-
boxaldehyde, we selected the following strategy starting
from the raedily available malononitrile (1). Addition of
1 to methylchloroformate gave the potassium salt of
methyldicyanoacetate (2, Scheme 1).19 The conditions
for the reduction of the dinitrile 2 into the diamino
derivative 3 such as the concentration of the reagents
and the amount of the used catalyst (10% Pd on car-
bon) were found to be very important. The method of
20
choice was 2equivalents 10% Pd/C (w/w) in a diluted
Figure 2.

R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
Table 1. Physical properties 1,3-diazacycloalkyl carboxaldehyde oxime derivatives
1145
Compound
Position
n
R1
R2
Salta
Molecular formula
Z/E ratio
8a
8b
8c
5
5
5
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
1
1
1
1
1
1
1
1
Methyl
sec-Propyl
Ethyl
H
Methyl
Ethyl
2-Propynyl
sec-Propyl
(Z)-2-Butenyl
tert-Butyl
2-Hexenyl
H
H
H
H
H
H
H
H
H
H
H
H
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
mal
C6H11N3Oꢂ HCl
C8H15N3Oꢂ HCl
C7H13N3Oꢂ HCl
C5H9N3Oꢂ HCl
C6H11N3Oꢂ HCl
C7H13N3Oꢂ HCl
C8H11N3Oꢂ HCl
C8H15N3Oꢂ HCl
C9H15N3Oꢂ HCl
C9H17N3Oꢂ C4H4O4
C11H17N3Oꢂ HCl
6H11N3Oꢂ HCl
7H13N3Oꢂ C4H4O4
C7H13N3Oꢂ C4H4O4
C8H15N3Oꢂ C4H4O4
C9H13N3Oꢂ C4H4O4
C12H17N3Oꢂ C4H4O4
C6H11N3Oꢂ HCl
1:4
0:1
0:1
0:1
0:1
2:5
3:7
0:1
1:10
1:5
1:5
1:12
1:14
0:1
0:1
0:1
0:1
0:1
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
20a
20b
20c
20d
20e
1
1
1
2H
2Methyl
1
1
1
1
1
HCl
HCl
HCl
C
C
H
Methyl
Ethyl
2-Propynyl
Me
Me
Me
Me
Me
mal
mal
mal
mal
HCl
(E)-3-Methyl-2-penten-4-ynyl
H
amal, maleic acid.
After heating the Z/E mixtures in ethanol an equili-
brium ratio, which was in favor of the E-isomer, was
observed (see Experimental).
affinity in the PZ binding and have OXO-M/PZ binding
ratios exceeding 100. Several of these compounds were
tested in the three functional in vitro models and dis-
played muscarinic agonistic activity: 8a, 12b, 14b, 14j,
20a. In contrast, only modest affinities were found in the
OXO-M binding tests for the OH-oxime derivatives
14a, 14i, 20e with OXO-M/PZ ratios below 30; weak
antagonistic properties were found for these compounds
Synthesis of 1-(1,4,5,6-tetrahydro-5-pyrimidine)ethanone
O-alkyloximes, 20a–d. For the synthesis of the 1-(1,4,5,6-
tetrahydro-5-pyrimidine)ethanone oxime derivatives the
following strategy was selected (Scheme 3). Reaction of
ethyl acetoacetate 15 with N-(hydroxymethyl)-
phtalimide 16 in concentrated sulfuric acid gave 2-ace-
tyl-1,3-diphtaloylpropane (17).23 Treatment of the
acetyl derivative 17 with the desired O-alkylhydroxy-
lamine derivatives gave the corresponding (E)-oximes
18a–e. Deprotection of both amino functions using
hydroxylamine in the presence of sodium methanolate
as reagent gave the diamino derivatives 19a–e. Finally,
treatment of 19a–e with trimethylorthoformate gave
the desired 1-(1,4,5,6-tetrahydro-5-pyrimidine)ethanone
O-alkyloximes 20a–e.
Table 2. Receptor binding affinity and affinity ratios for assays using
agonist, oxotremorine-M (OXO-M), and antagonist pirenzepine (PZ)
ligandsa
Compound
3H-OXO-M
3H-PZ
Ratio
(Ki in mM)
(Ki in mM)
Arecoline
Isoarecoline
9
12a
12b
8a
8b
8c
14a
14b
14c
8.1
7.3
7.4
6.3
7.24.4
8.1
6.9
7.9
5.6
8.3
7.7
7.9
7.4
5.7
3.3
4.1
3.9
600
4.6
5.7
5.2500
4.4
4.5
5.2300
5.3
5.7
250
10,000
2000
225
3000
15
15
6000
Pharmacology
Results
14d
14e
400
50
The physical properties of the compounds are shown in
Table 1. The present series of muscarinic cholinergic
ligands was evaluated for potential muscarinic cholin-
ergic agonistic properties by assessing the PZ/OXO-M
binding ratio. This ratio of agonist and antagonist
binding affinities provides an indication of potential
muscarinic cholinergic agonistic properties.17e Together
with the methyl esters 9, 12a and 12b, which are isosters
of either arecoline or isoarecoline, the four O-methyl-
oxime derivatives 8a, 14b, 14j and 20a shared a rela-
tively high affinity in the OXO-M binding with a low
14f
8.1
7.1
7.3
6.1
7.25.1 0
7.5
6.7
6.9
6.3
5.8
5.8
5.8
5.1
12
4.7
5.8
6.1
200
20
30
10
14g
14h
14i
14j
20a
20b
20c
20d
20e
600
8
6
2
2.5
6.0
5.1
5.5
aData are mean values of 2–4 binding inhibition experiments. For
further details, see Experimental.

1146
R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
Table 3. Muscarinic cholinergic activity in guinea pig ileum (M3), rat left atrium (M2) and hippocampus (M1)
Compound
M3 (ileum)
M2 (left atrium)
M1 (hippocampus)
pD2
a
pA2
pD2
a
pA2
pD2
a
pA2
Arecoline
Isoarecoline
9
12a
12b
8a
6.5
5.0
5.8
nt
5.21.0
nt
—
5.6
5.6
5.6
5.9
5.1
5.1
—
4.8
5.3
5.4
1.0
1.3
1.1
—
—
—
6.9
—
6.1
1.0
—
1.0
—
—
—
5.4
—
4.7
0.72—
—
0.96
—
—
—
5.1
1.0
—
4.5
0.49
—
nt
4.6
5.6
5.6
5.7
5.9
—
5.0
5.0
4.7
4.4
4.6
5.3
5.3
5.6
0.9
<4.0
0.77
0.92—
0.75
0.9
0.15
0.07
0
—
0
—
8b
8c
—
—
—
—
—
—
—
4.6
—
—
1.0
1.0
1.0
1.0
—
—
—
—
—
—
—
—
1.0
1.3
1.7
1.0
1.1
0.6
—
0.6
1.2—
1.3
14b
14c
14d
14e
14f
14g
14h
14j
—
—
—
—
—
—
—
5.1
5.3
5.5
5.1
—
<3.5
4.8
—
0.11
5.7
1.0
—
4.0
0.6
—
20a
—
5.4
0.8
—
4.3
0.8
—
nt, not tested; pD2, agonist values; a, intrinsic activity; pA2, antagonist values.
(data not shown). Elongation of the O-substituted side
chain was studied further in series of compounds 14.
The methyl, ethyl and propargyl oxime derivatives (14b,
14c, 14d) showed OXO-M/PZ binding ratios of 300–
6000 in agreement with their full agonist properties for
all three muscarinic subtypes. With the compounds
where R1 is s-propyl, (Z)-2-butenyl or 2-hexenyl 14e,
14f, 14h exhibited intermediate OXO-M/PZ binding
ratio of approximately 20–50 and displayed differential
agonistic/antagonistic properties for the three subtypes.
All three compounds showed M2 antagonistic proper-
ties and displayed M1 agonistic properties with a low
intrinsic activity (a=0.1). In contrast, these compounds
remained quite active on M3 receptors with intrinsic
activities between 0.6 and 1.1. In these functional mod-
els, the compounds 14e, 14f and 14h showed equal affi-
nity or slight preference for the M1 receptor subtype in
contrast with the other analogues 14b, 14c, 14d. For the
latter three compounds, the pD2 values for M1 were
found to be 4- to 15-fold lower for M1 as compared to
M2 or M3. In agreement with these observations 14e and
14f showed a 15-fold higher affinity in the PZ binding test
than 14b. Only the oxime derivative 14g, having a bulky
t-butyl substituent with a low OXO-M/PZ binding ratio
of 20, lacked agonistic properties for all three subtypes
and instead exhibited antagonistic properties.
muscarinic cholinergic subtypes, while ratios of 30 or
lower predict muscarinic cholinerergic antagonistic
properties. Compounds showing intermediate ratios in
between 30 and 300 showed partial agonistic properties
with agonistic properties being most pronounced for the
M3 subtype. While these compounds shared a slight
preference for M1 receptors in functional models,
intrinsic activities in the present M1 model (measuring
Discussion
The present series of 1,3-diazacycloalkyl carbox-
aldehyde oxime derivatives was compared to the parent
compound arecoline in and evaluated for potential
muscarinic cholinergic agonistic properties by measur-
ing their affinity in OXOM and PZ binding tests. The
PZ/OXO-M binding ratio gives an acccurate prediction
of potential muscarinic agonistic properties.17 This was
shown by their characterization in functional models
revealing muscarinic subtype related agonistic activities.
Indeed OXOM/PZ ratios of 300 or higher are associated
Scheme 1. (A) (1) KOH; (2) methylchloroformate; (B) H2 Pd/C 10%,
MeOH/HCl; (C) Z-succinimide, DMF, triethylamine; (D) DIBAH,
CH2Cl2, ꢀ78 ꢁC; (E) O-alkyl hydroxylamine HCl, MeOH; (F) H2, Pd/C
10%, MeOH/HCl; (G) trimethylorthoformate, MeOH; (H) trimethy-
lorthoformate, MeOH; (I) DIBAH, CH2Cl2, ꢀ78 ꢁC; (J) O-alkyl
hydroxylamine HCl, MeOH.
with full muscarinic agonistic properties for all three

R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
1147
M1 mediated changes in hippocampal cell firing) are
rather low. However, in this series, the slight preference
for the M1 receptor subtype and the M2-antagonistic
properties are lost when the intrinsic activity becomes
high as for 8b, 12b, 14b and 14d.
and mixed with the matrix compounds on standard
stainless steel targets. Exact masses of the protonated
molecular ions were determined with the peak matching
technique at a mass resolution of >7500 (10% valley
definition) in the positive ion mode using reference
masses 369 and 461 from glycerol. Average exact masses
were calculated from at least 10 computer-controlled
measurements using the bracketing method. Proton
magnetic resonance spectra were measured on a Bruker
WP200 or AC200 instrument. Chemical shifts are
reported as d values (parts per million) relative to Me4Si
as an internal standard. Thin layer chromatography
(TLC) was carried out by using Merck precoated silicagel
F-254 plates (thickness 0.25 mm). Spots were visualized
with a UV hand lamp and Cl2/tetramethylbenzidine.
In conclusion, compounds 14e, 14f and 14h show an
improved profile compared to arecoline with a combi-
nation of M2 antagonistic and partial muscarinic M1
agonistic properties. While their affinity for the M1
receptor was maintained, the affinity for the M3 recep-
tor as measured in the ileum was 30-fold reduced and
the M2 agonistic properties were eliminated. However, a
satisfactory muscarinic profile with better in vivo
potential requires a further increase in M1/M3 selectivity
and intrinsic M1 activity. The present series offers an
interesting opportunity to develop such a profile.
Potassium salt of methyl dicyanoacetate (2). A solution
of methylchloroformate 1 (49.5 g, 0.53 mol) and malo-
dinitrile (33 g, 0.5 mol) in 75 mL THF was added in
30 min, with vigorous stirring, to a solution of potas-
sium hydroxide (56.1 g, 1.0 mol) in 50 mL water, keep-
ing the temperature below 40 ꢁC. After 2h stirring at
room temperature, the reaction mixture was cooled to
0 ꢁC. The precipitate was filtered off and washed with
ice-cold water and ethanol, to give a white crystalline
product 2 (68 g, 0.42mol, 84%). 1H NMR (D2O,
200 MHz) d 3.65 (s, 3H). 13C NMR (D2O, 50 MHz) d
176.5 (s, C¼O), 125.9 (s, 2 CN), 54.6 (q, CH3).
Experimental
Chemistry
Melting points were taken on a Buchi capillary melting
point apparatus and are uncorrected. Fast atom bom-
bardment (FAB) mass spectra were recorded with a
Finnigan MAT 90 mass spectrometer (Finnigan MAT,
Bremen, FRG). Samples were dissolved in methanol
Scheme 2. (A) (1) Trimethylorthoformate, MeOH, 18 h reflux; (2) SOCl2, MeOH 3 h reflux; (B) DIBAH, CH2Cl2, ꢀ78 ꢁC; (C) (1) O-alkyl
hydroxylamine HCl, MeOH; (2) ethanol reflux.
Scheme 3. (A) Concd H2SO4, 24 h rt; (B) O-alkyl hydroxylamine HCl; (C) sodium methanolate, hydroxylamine HCl, 3 h rt; (D) (1) trimethyl-
orthoformate, MeOH; (2) NaOH; (3) maleic acid.

1148
R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
1
Methyl 3-amino-2-(aminomethyl)propionate dihydro-
chloride (3). A suspension of 2 (12.8 g, 80 mmol) and
26 g 10% Pd/C in 4 L methanol (dry) and 3 equivalents
of HCl in methanol was treated with hydrogen. After
24 h, the catalyst was filtered off and the solvent was
removed in vacuo. Recrystallization of the crude pro-
a 1:4 Z/E isomer. H NMR (CD3OD) d 3.0 (m, 1H),
3.3–3.6 (m, 4H), 3.8 (s, 3H, E-isomer), 3.9 (s, 3H, Z-
isomer), 6.65 (d, 1H, Z-isomer), 7.4 (d, 1H (E-isomer)),
8.0 (s, 1H).
The following compounds were prepared in a similar
manner as described above.
duct from methanol/ethyl acetate gave
3 (9.5 g,
46 mmol, 58%). Mp 178 ꢁC. H NMR (D2O, 200 MHz)
d 3.85 (s, 3H), 3.50–3.20 (m, 5H). 13C NMR (D2O,
50 MHz) d 174.5 (s, C¼O), 56.4 (q, OCH3), 43.2(d,
1
(E)-1,4,5,6-Tetrahydro-5-pyrimidine carboxaldehyde O-(1-
methylethyl)oxime monohydrochloride (8b). Yield 27%.
Mp 133 ꢁC. H NMR (CD3OD) d 1.2(d, 6H), 3.0 (m,
0
1
CH), 41.1 (t, 4H, CH2 s).
1H), 3.4–3.7 (m, 4H), 4.3 (m, 1H), 7.35 (d, 1H), 8.0 (s,
1H). Exact mass calcd for [M+H]+ 170.1293, found
170.1289.
(Z:E 1:4) 1,4,5,6-Tetrahydro-5-pyrimidinecarboxalde-
hyde O-methyloxime monohydrochloride (8a). A solu-
tion of 3 (9.1 g, 44.6 mmol) in dry DMF (250 mL) and
triethylamine (12.5 mL, 89.2 mmol) was stirred for
20 min. Then triethylamine (12.5 mL, 89.2 mmol) was
Methyl-1,4,5,6-tetrahydro-5-pyrimidinecarboxylate mono-
hydrochloride (9). A solution of 3 (8.68 g, 42.3 mmol)
and trimethylorthoformate (50 mL) in dry methanol
(250 mL) was refluxed for 18 h and evaporated to dry-
ness. Crystallization from methanol/ethyl acetate gave 9
added
followed
by
N-(benzyloxycarbonyloxy)-
succinimide (22.2 g, 89.2 mmol) while keeping the tem-
perature at 20–25 ꢁC. After stirring the reaction mixture
at room temperature for 5 h, the precipitate was
removed by filtration and the filtrate was evaporated.
The residue was partitioned between water and ethyl
acetate. The organic layer was washed twice with small
portions of water. The dried (MgSO4) organic phase
was evaporated in vacuo. The residue was purified by
column chromatography on neutral Al2O3 using tolu-
ene/ethyl acetate (8/2v/v) as eluent, to give methyl
N,N0-(dibenzyloxycarbonyl)-3-amino-2-(aminomethyl)-
propionate 4 (11.0 g, 27.5 mmol, 62%) as an oil.
(5.5 g, 31 mmol, 73%) as a yellow solid. Mp 171 ꢁC. H
1
NMR (D2O, 200 MHz) d 7.95 (s, 1H), 3.80 (s, 3H), 3.70
(d, 4H), 3.40–3.25 (m, 1H). 13C NMR (D2O, 50 MHz) d
175.3 (s, 0C¼O) 154.2(N ¼C–N), 55.9 (q, CH3), 41.9 (t,
4H, CH2 s), 36.7 (d, CH)
1,4,5,6-Tetrahydro-5-pyrimidinecarboxaldehyde (10). To
a cooled (ꢀ78 ꢁC) suspension of 9 (1.5 g, 8.40 mmol) in
dry methylene chloride (100 mL) 2.5 equivalents of cold
(ꢀ78 ꢁC) DIBAH (21 mL, 21 mmol of a 1 M solution of
DIBAH in methylene chloride) was added dropwise.
The reaction mixture was stirred for 3 h and quenched
with cold (ꢀ78 ꢁC) methanol (5 mL). Then water (3 mL)
was added followed by methanol (20 mL), the reaction
mixture was allowed to warm to ꢀ20 ꢁC to give the
crude aldehyde 10, which was used directly for the fol-
lowing reaction step.
DIBAH (20.5 mmol, (17.1 mL of a 1.2 M solution in
toluene) was added to a cooled (ꢀ70 ꢁC) solution of 4
(6.8 g, 17.1 mmol) in dry methylene chloride (50 mL).
The reaction mixture was stirred for 3 h and then quen-
ched with methanol (3 mL). The mixture was allowed to
warm to room temperature, after which water (20 mL)
was added. The mixture was filtered and the organic
phase was separated and subsequently dried (MgSO4).
The solvents were evaporated in vacuo to give a residue
(7.45 g) containing a mixture of N,N 0-(dibenzyloxy-
(E)-1,4,5,6-Tetrahydro-5-pyrimidine carboxaldehyde O-
ethyloxime monohydrochloride (8c). To aldehyde 10
(8.40 mmol),
O-ethyl
hydroxylamine
(819 mg,
carbonyl)-3-amino2-(aminomethyl)propionaldehyde
5
8.40 mmol) was added at ꢀ20 ꢁC. The reaction mixture
was stirred for 20 h at room temperature. Then the alu-
minum salts were filtered and the filtrate was evapor-
ated.The crude mixture contained a small amount of
methylester 9, which was removed by treatment with
sodiumhydroxide in water. Subsequent extraction with
methylene chloride and treatment with methanolic
hydrochloride afforded 8c (500 mg, 2.6 mmol, 27%). Mp
and starting material 4 (30%). Methoxylamine hydro-
chloride (790 mg, 9.5 mmol) was added to a solution of
5 (3.7 g, crude) in dry methanol (125 mL). After stirring
of the reaction mixture for 3.5 h at 65 ꢁC the solvent was
evaporated in vacuo. Ethyl acetate was added to the
residue. Residual methoxylamine HCl was removed by
filtration. The filtrate was evaporated in vacuo to give a
residue which was chromatographed on silica (toluene/
ethyl acetate 8:2, v/v%) to give N,N 0-(dibenzyloxy-
carbonyl)-3-amino-2-methylaminopropionaldehyde O-
methyloxime 6a (390 mg, 0.98 mmol, 47%) as a 1:4 Z/E
isomer. Hydrogen was passed through a solution of 6a
(350 mg, 0.88 mmol) in dry methanol (15 mL) contain-
ing 2equivalents of hydrochloric acid and Pd/C 10%.
After 4 h, the reaction mixture was filtered and the filtrate
was evaporated in vacuo to give 3-amino-2-methyla-
mino-propionaldehyde O-methyloxime dihydrochloride:
7a in quantitative yield. A solution of 7a (170 mg,
0.84 mmol) and trimethylorthoformate (10 mL) in dry
methanol (25 mL) was refluxed for 24 h. The mixture
was evaporated to dryness (120 mg, 0.68 mmol, 80%) as
139 ꢁC. H NMR (CD3OD) d 1.25 (t, 3H), 3.0 (m, 1H),
1
3.4–3.7 (m, 4H), 4.1 (q, 2H), 7.4 (d, 1H), 8.05 (s, 1H).
13C NMR (MeOD, 50 MHz) d 152.8, 147.5, 70.5, 41.4,
30.7, 14.8. Exact mass calcd. for [M+H]+ 156.1137,
found 156.1108.
Methyl-1,4,5,6-tetrahydro-4-pyrimidinecarboxylate mono-
hydrochloride (12a). A solution of dl-2,4-diaminobutyric
acid 2HCl 11a (49.7 g, 0.26 mol) and trimethylortho-
formate (114 mL, 1.04 mol) in dry methanol (500 mL) was
refluxed. The crude compound was dissolved in dry
methanol (500 mL) and cooled to 0 ꢁC. Thionylchloride
(37.1 g, 0.31 mol) was added dropwise, then the solution
was refluxed for 3 h and evaporated to dryness. Toluene

R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
1149
was added and the suspension was evaporated to dry-
ness. The product 12a (46.4 g, 0.26 mol, 100%) was
obtained as a white solid. H NMR (CD3OD) d 2.1–2.4
(m, 2H), 3.3–3.6 (m, 2H), 3.8 (s, 3H), 4.4 (m, 1H), 8.1 (s,
1H).
(E)-1,4,5,6-Tetrahydro-4-pyrimidinecarboxaldehyde O-(1-
methyletꢁhyl)oxime monohydrochloride (14e). Yield 70%.
1
1
Mp 110 C. H NMR (CD3OD) d 1.2(d, 6H), 1.95–2.3
(m, 2H), 3.4–3.6 (m, 2H), 4.3 (m, 1H), 4.35 (m, 1H), 7.4
(d, 1H), 8.1 (s, 1H). 13C NMR (MeOD, 50 MHz) d
152.8, 147.1, 77.2, 37.3, 23.1, 21.7. Exact mass calcd for
[M+H]+ 170.1293, found 170.1252.
1,4,5,6-Tetrahydro-4-pyrimidinecarboxaldehyde
To
(13a).
a
cooled (ꢀ78 ꢁC) suspension of 12a (2.0 g,
11.2 mmol) in dry methylene chloride (125 mL) 2.5
equivalents of cold (ꢀ78 ꢁC) DIBAH (28 mL, 28 mmol
of a 1 M solution of DiBA1H in methylene chloride)
was added dropwise. The reaction mixture was stirred
for 3 h and then quenched with cold (ꢀ78 ꢁC) methanol
(5 mL). Then water (3 mL) was added followed by
methanol (20 mL), the reaction mixture was allowed to
warm to ꢀ20 ꢁC. to give the crude product 13a.
(Z:E 1:10) 1,4,5,6-Tetrahydro-4-pyrimidinecarboxalde-
hyde O-(2-(Z)-butenyl)oxime monohydrochloride (14f).
Yield 70%. Mp 111 ꢁC. H NMR (CD3OD) d 1.65 (d,
1
3H), 2.0–2.3 (m, 2H), 3.4–3.6 (m, 2H), 4.45 (m, 1H), 4.6
(d, 2H (E-isomer)), 4.7 (d, 2H (Z-isomer)), 5.5–5.8 (m,
2H), 6.85 (d, 1H (Z-isomer)), 7.45 (d, 1H (E-isomer)),
8.05 (s, 1H). Exact mass calcd for [M+H]+ 182.1293,
found 182.1269.
(E)-1,4,5,6-Tetrahydro-4-pyrimidinecarboxaldehyde O-me-
thyloxime monohydrochloride (14b). The cold (ꢀ20 ꢁC)
solution of 13a (11.2mmol) was added to a solution of
methoxylamine HCl (940 mg, 11.2mmol) in methanol
(25 mL). The resulting reaction mixture was stirred for
20 h at room temperature. Then the aluminum salts
were filtered off and the filtrate was evaporated to dry-
ness to give 14b (2.0 g, 100%) as a 1/1 Z/E-mixture. A
solution of 14b (2.0 g, 11.2 mmol) in ethanol was heated
to reflux for 20 h and evaporated to dryness. Crystal-
lization from methanol/ethyl acetate gave 14b (1.22 g,
(Z:E 1:5) 1,4,5,6-Tetrahydro-4-pyrimidinecarboxalde-
hyde O-(1,1-dimethylethyl)oxime (Z)-2-butenedioate
(14g). Yield 15%. Mp 122 ꢁC. H NMR (CD3OD) d
1
1.25 (s, 9H (E-isomer)), 1.3 (s, 9H (Z-isomer)), 2.0–2.3
(m, 2H), 3.5 (m, 2H), 4.4 (m, 1H), 6.25 (s, 2H), 6.8 (d,
1H (Z-isomer)), 7.4 (d, 1H (E-isomer)), 8.05 (s, 1H). 13C
NMR (MeOD, 50 MHz) d 170.8, 152.8, 146.5, 136.8,
80.3, 37.3, 27.7, 23.0. Exact mass calcd for [M+H]+
184.1450, found 184.1403.
(Z:E 1:5) 1,4,5,6-Tetrahydro-4-pyrimidinecarboxalde-
hyde O-(2-hexynyl)oxime monohydrochloride (14h).
ꢁ
7.42mmol, 61%) as pure E-isomer. Mp 149 C. 1H
1
NMR (CD3OD) d 1.9–2.3 (m, 2H), 3.45 (m, 2H), 3.85
(s, 3H), 4.35 (m, 1H), 7.45 (d, 1H), 8.1 s, 1H). 13C
NMR (MeOD, 50 MHz) d 152.8, 147.9, 62.5, 37.3, 23.0.
Exact mass calcd. for [M+H]+ 142.0980, found
142.0994.
Yield 37% oil. H NMR (CD3OD) d 1.0 (t, 3H), 1.5
(m, 2H), 2.0–2.3 (m, 4H), 3.5 (m, 2H), 4.4 (m, 1H), 4.65
(m, 2H (E-isomer)), 4.7 (m, 2H (Z-isomer)), 6.9 (d, 1H
(Z-isomer)), 7.5 (d, 1H (E-isomer)), 8.05 (s, 1H). Exact
mass calcd for [M+H]+ 208.1449, found 208.1437.
The following compounds were prepared in a similar
manner as described above.
Methyl-4,5,6,7-tetrahydro-1H-1,3-diazepine-4-carboxy-
late (12b). A solution of dl-ornithine HCl 11b (30.4 g,
0.18 mol) and trimethylorthoformate (59 mL, 0.54 mol)
in dry methanol (600 mL) was refluxed. After evapora-
tion the crude compound was dissolved in dry methanol
(400 mL) and cooled (0 ꢁC). Thionylchloride (53.7 g,
0.45 mol) was added dropwise, then the solution was
refluxed for 3 h and evaporated to dryness. Toluene was
added and the suspension was evaporated to dryness.
The product 2b (27.5 g, 0.14 mol, 79%) was obtained as
(E)-1,4,5,6-Tetrahydro-4-pyrimidinecarboxaldehyde oxime
monohydrochloride (14a). Mp 174 ꢁC. Yield 48%. 1H
NMR (CD3OD) d 1.9–2.3 (m, 2H), 3.35–3.6 (m, 2H),
4.45 (m, 1H), 7.4 (d, 1H), 8.05 (s, 1H). 13C NMR
(MeOD, 50 MHz) d 152.8, 147.6, 37.4, 23.2. Exact mass
calcd. for [M+H]+ 128.0824, found 128.0820.
1
(Z:E 2:5) 1,4,5,6-Tetrahydro-4-pyrimidinecarboxalde-
hyde O-ethyloxime monohydrochloride (14c). Yield
73%. Mp 96 ꢁC. 1H NMR (CD3OD) d 1.25 (t, 3H), 2.0–
2.3 (m, 2H), 3.45 (m, 2H), 4.1 (q, 2H), 4.4 (m, 1H), 6.85
(d, 1H (Z-isomer)), 7.45 (d, 1H (E-isomer)), 8.1 (s, 1H).
13C NMR (MeOD, 50 MHz) d 152.8, 148.4, 147.6, 71.4,
70.9, 37.7, 37.3, 23.1, 22.3, 14.8. Exact mass calcd for
[M+H]+ 156.1137, found 156.1113.
a white solid. H NMR (CD3OD) d 1.9–2.1 (m, 2H),
2.2–2.4 (m, 2H), 3.4–3.7 (m, 2H), 3.75 (s, 3H), 4.65 (m,
1H), 7.8 (s, 1H).
Methyl-4,5,6,7-tetrahydro-1H-1,3-diazepine-4-carbox-
aldehyde (13b). To a cooled (ꢀ78 ꢁC) suspension of 12b
(2.0 g, 10.4 mmol) in dry methylene chloride (125 mL)
2.5 equivalents of cold (ꢀ78 ꢁC) DiBA1H (26 mL,
26 mmol of a 1 M solution of DIBAH in methylene
chloride) was added dropwise. The reaction mixture was
stirred for 3 h and then quenched with cold (ꢀ78 ꢁC)
methanol (5 mL). Then water (3 mL) was added followed
by methanol (20 mL), the reaction mixture was allowed
to warm to ꢀ20 ꢁC. to give the crude product 13b.
(Z:E 3:7) 1,4,5,6-Tetrahydro-4-pyrimidinecarboxalde-
hyde O-2-propynyloxime monohydrochloride (14d).
Yield 50%. Mp 127 ꢁC. H NMR (CD3OD) d 2.0–2.3
1
(m, 2H), 3.0 (m, 1H), 3.4–3.6 (m, 2H), 4.4 (m, 1H), 4.65
(d, 2H (E-isomer)), 4.7 (d, 2H (Z-isomer)), 6.95 (d, 1H
(Z-isomer)), 7.55 (d, 1H (E-isomer)), 8.1 (s, 1H). 13C
NMR (MeOD, 50 MHz) d 152.9, 150.2, 149.6, 76.5,
76.2, 63.2, 62.8, 37.5, 37.1, 22.8, 22.3. Exact mass calcd
for [M+H]+ 166.0980, found 166.0984.
4,5,6,7-Tetrahydro-1H-1,3-diazepine-4-carboxaldehyde
oxime monohydrochloride (14i). The cold (ꢀ20 ꢁC) solu-
tion of 13b (10.4 mmol) was added to a solution of

1150
R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
hydroxylamine HCl (723 mg, 10.4 mmol) in methanol
(25 mL). The resulting reaction mixture was stirred for
20 h at room temperature. Then the aluminum salts
were filtered off and the filtrate was evaporated to dry-
ness to give 14i (1.7 g, 92%) as a 1/3 Z/E-mixture. A
solution of 14i (1.7 g, 9.6 mmol) in ethanol was heated
to reflux for 20 h and evaporated to dryness. Crystal-
lization from ethanol/ethyl acetate gave 14i (320 mg,
1.8 mmol, 18%) as a 1/12Z/E mixture.
3-(Methylphtaloyl)-4-phtaloyl-2-butanone O-(E)-3-methyl-
2-penten-4-ynyloxime (18d). Yield 70%. 1H NMR
(CDC13) d 1.65 (s, 3H), 1.9 (s, 3H), 3.3 (m, 1H), 3.7–4.0
(m, 4H), 4.35 (d, 2H), 5.8 (m, 1H), 7.6–7.9 (m, 8H).
3-(Methylphtaloyl)-4-phtaloyl-2-butanone oxime (18e).
Yield 100% (crude).
4-Amino-3-(methylamino)-2-butanone
O-methyloxime
(19a). To a solution of methanol (100 mL) sodium
(1.38 g, 60 mmol) and subsequently hydroxylamine HC1
(1.95 g, 28 mmol) were added. After stirring for 15 min,
18a (5.67 g, 14 mmol) dissolved in dry methylene chlor-
ide (150 mL) was added. The dark-red solution was
stirred for 3 h at room temperature. A methanolic
hydrochloride solution (60 mmol, 32mL of a 1.9 M
solution of hydrochloride in methanol) was added to the
reaction mixture. Sodium chloride was filtered from the
reaction mixture and the filtrate was evaporated to dry-
ness. Crystallization from methanol/ethyl acetate gave
Mp 173 ꢁC. H NMR (CD3OD) d 1.8–2.4 (m, 4H), 3.4
1
(m, 1H), 3.7 (m, 1H), 4.5 (m, 1H), 6.8 (d, 1H (Z-isomer)),
7.4 (d, 1H (E-isomer)), 7.65 (s, 1H). 13C NMR (MeOD,
50 MHz) d 154.8, 147.7, 57.4, 46.8, 32.0, 25.5, 17.4. Exact
mass calcd for [M+H]+ 142.0980, found 142.0992.
The following compounds were prepared in a similar
manner as described above.
4,5,6,7-Tetrahydro-1H-1,3-diazepine-4-carboxaldehyde
O-methyloxime monohydrochloride (14j). Yield 35%.
1
19a (2.2 g, 10.1 mmol, 72%) as a yellow solid. H NMR
(CD3OD) d 1.95 (s, 3H), 3.0 (m, 1H), 3.1–3.3 (m, 4H),
3.9 (s, 3H).
Mp 126 ꢁC. H NMR (CD3OD) d 1.8–2.3 (m, 4H), 3.4
1
(m, 1H), 3.7 (m, 1H), 3.8 (s, 3H (E-isomer)), 3.9 (s, 3H
(Z-isomer)), 4.5 (m, 1H), 6.85 (d, 1H (Z-isomer)), 7.4 (d,
1H (E-isomer)), 7.65 (s, 1H). Exact mass calcd for
[M+H]+ 156.1137, found 156.1109.
The following compounds were prepared in a similar
manner as described above.
2-Acetyl-1,3-diphtaloylpropane (17). Ethylacetoacetate
15 (14.7 g, 113 mmol) was added to cooled (0 ꢁC) con-
centrated sulfuric acid (240 mL), then N-(hydroxy-
methyl)phtalimide 16 (40 g, 226 mmol) was added. The
reaction mixture was stirred for 24 h at room tempera-
ture. The dark-red solution was poured on ice-water,
the yellow solid was filtered and washed with water. The
crude solid was stirred in hot acetone and after cooling
collected by filtration to give 17 (32g, 85 mmol, 75%;
yellow solid). Mp 170 ꢁC. 1H NMR (CDC13) d 2.35
(s, 3H), 3.65 (m, 1H), 3.8–4.2(m, 4H), 7.6–7.9 (m,
8H).
4-Amino-3-(methylamino)-2-butanone O-ethyloxime (19b).
1
Yield 100% (crude). H NMR (CD3OD) d 1.3 (t, 3H),
1.95 (s, 3H), 3.0 (m, 1H), 3.1–3.3 (m, 4H), 4.2(q, 2H).
4-Amino-3-(methylamino)-2-butanone O-2-propynylox-
1
ime (19c). Yield 100% (crude). H NMR (CD3OD) d
1.95 (s, 3H), 2.9 (m, 1H), 3.0 (m, 1H), 3.1–3.3 (m, 4H),
4.7 (m, 2H).
4-Amino-3-(methylamino)-2-butanone O-(E)-3-methyl-2-
penten-4-ynyloxime (19d). Yield 100% (crude). 1H
NMR (CD3OD) d 1.8 (s, 3H) 1.95 (s, 3H), 3.1 (m, 1H),
3.1–3.3 (m, 4H), 4.75 (d, 2H), 6.1 (m, 1H).
3-(Methylphtaloyl)-4-phtaloyl-2-butanone O-methylox-
ime (18a). To a solution of 17 (8.0 g, 21.3 mmol) in
methylene chloride (120 mL) methoxylamine HC1
(1.78 g, 21.3 mmol) and methanol (80 mL) were added.
The reaction mixture was stirred for 60 h at room tem-
perature and then evaporated to dryness. The crude
solid was stirred in hot acetone and after cooling col-
lected by filtration to give 18a (5.7 g, 14 mmol, 66%) as
4-Amino-3-(methylamino)-2-butanone oxime (19e). Yield
100% (crude).
(E)-1-(1,4,5,6-Tetrahydro-5-pyrimidine)ethanone O-me-
thyloxime (20a). A solution of 19a (2.2 g, 10.1 mmol)
and trimethylorthoformate (15 mL) in dry methanol
(60 mL) was refluxed for 20 h and concentrated to dry-
ness. The residue was partitioned between an aqueous
sodiumhydroxide brine solution (5 mL) and methylene
chloride (100 mL) and then the organic layer was con-
centrated. To a solution of the free base of 20a (960 mg,
6.2mmol) in methanol maleic acid (719 mg, 6.2mmol)
was added. Crystallization from methanol/ether gave
1
a yellow solid. H NMR (CDC13) d 1.9 (s, 3H), 3,3 (m,
1H), 3.5 (s, 3H), 3.7–4.0 (m, 4H), 7.6–7.9 (m, 8H).
The following compounds were prepared in a similar
manner as described above.
20a (910 mg, 3.3 mmol 33%) as a solid. Mp 138 ꢁC. H
1
3-(Methylphtaloyl)-4-phtaloyl-2-butanone O-ethyloxime
1
(18b). Yield 65%. H NMR (CDC13) d 0.9 (t,3H) 1.9
(s, 3H), 3.3 (m, 1H), 3.8 (q, 2H), 3.7–4.0 (m, 4H), 7.6–
7.9 (m, 8H).
NMR (CD3OD) d 1.9 (s, 3H), 2.9 (m, 1H), 3.4–3.7 (m,
4H), 3.85 (s, 3H), 6.25 (s, 2H), 8.0 (s, 1H). 13C NMR
(MeOD, 50 MHz) d 170.8, 154.5, 152.6, 136.6, 62.1,
41.6, 35.6, 13.4. Exact mass calcd for [M+H]+
156.1137, found 156.1112.
3-(Methylphtaloyl)-4-phtaloyl-2-butanone 0-2-propynyl-
1
oxime (18c). Yield 70%. H NMR (CDC13) d 1.8 (m,
1H), 1.95 (s, 3H), 3.3 (m, 1H), 3.7–4.0 (m, 4H), 4.4 (m,
2H), 7.6–7.9 (m, 8H).
The following compounds were prepared in a similar
manner as described above.

R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
1151
(E)-1-(1,4,5,6-Tetrahydro-5-pyrimidine)ethanone O-ethy-
loxime (Z)-2-butenedioate (20b). Mp 137 ꢁC. H NMR
concentrations and IC50 values were obtained using a
four parameter fitting procedure. Ki values were
obtained from the IC50 values by using the Chang–
Prusoff equation Ki=IC50/(1+C/Kd) in which C equals
the radiolabelled ligand concentration and Kd equals the
dissociation constant for the radiolabelled ligand. Kd
values used for these calculations were as follows: [3H]-
OXO-M binding: Kd=0.7 nM; [3H]-PZ binding:
Kd=8.3 nM.
1
(CD3OD) d 1.2(t, 3H), 1.9 (s, 3H), 2.85 (m, 1H), 3.4–3.7
(m, 4H), 4.1 (q, 2H), 6.25 (s, 2H), 8.0 (s, 1H). 13C NMR
(MeOD, 50 MHz) d 170.9, 152.6, 136.8, 70.4, 41.6, 35.7,
14.9, 13.5. Exact mass calcd for [M+H]+ 170.1293,
found 170.1294.
(E)-1-(1,4,5,6- Tetrahydro-5-pyrimidine)ethanone O-(2-
propynyl) oxime (Z)-2-butenedioate (20c). Mp 131 ꢁC.
1H NMR (CD3OD) d 1.95 (s, 3H), 2.8 (m, 1H), 2.9 (m,
1H), 3.4–3.7 (m, 4H), 4.6 (d, 2H), 6.25 (s, 2H), 8.0 (s,
1H). Exact mass calcd for [M+H]+ 180.1137, found
180.1104.
Interactions with muscarinic subtype mediated responses
in isolated organs
M1-mediated activity in the hippocampal slice. Musca-
rinic cholinergic agonists effectively suppress the elec-
trically evoked field excitatory postsynaptic potential
(fEPSP) in the rat hippocampal slice.25 This effect is due
to a decrease in release of excitatory amino acids from
the Schaffer collateral/commissural fibers, probably
mediated by a presynaptic M1 muscarinic receptor.22
(E)-1-(1,4,5,6-Tetrahydro-5-pyrimidine)ethanone O-(E)-
3-methyl-2-penten-4-ynyloxime (Z)-2-butenedioate (20d).
Mp 125 ꢁC. H NMR (CD3OD) d 1.85 (s, 3H), 1.95 (s,
1
3H), 2.9 (m, 1H), 3.4–3.8 (m, 4H), 4.6 (d, 2H), 6.0 (m,
1H), 6.25 (s, 2H), 8.0 (s, 1H). 13C NMR (MeOD,
50 MHz) d 170.8, 155.3, 152.6, 136.7, 134.9, 77.0, 70.6,
41.6, 35.7, 17.8, 13.5. Exact mass calcd for [M+H]+
220.1450, found 220.1440.
Carbachol (3E-5 mol/L) causes
a 100% decrease
(intrinsic activity=1.0) in the amplitude of the elec-
trically evoked fEPSP, an effect that can be fully antag-
onized by the M1 selective antagonist pirenzepine.26
(E)-1-(1,4,5,6-Tetrahydro-5-pyrimidine)ethanone oxime
monohydrochloride (20e). Mp 225 ꢁC. 1H NMR
(CD3OD) d 1.9 (s, 3H), 2.85 (m, 1H), 3.4–3.7 (m, 4H),
8.0 (s, 1H). Exact mass calcd for [M+H]+ 142.0980,
found 142.0989.
M2-mediated effects. Interactions with M2-muscarinic
cholinergic receptors were studied in the isolated left
atrium of the rat. An automated assay was used similar
to the b1-adrenoreceptor described previously27 but with
the following adaptation. The inhibition by cholinergic
agonists of the electrical stimulation evoked switch of
the atrium was measured using carbachol as a reference.
Cholinergic agonistic activity was compared to carba-
chol. Potential M2-activity was verified via antagonism
in the presence of the M2-selective antagonist AF-DX
116. Antagonistic activity was measured as a shift to the
right of the dose–response curve to carbachol in the
presence of the compound as described above.
In Vitro Studies
Agonist and antagonist binding studies
Binding of [methyl-3H]-OXOtremorine-M acetate (3H-
OXO-M) in homogenates of frontal cortex. The rapid
filtration method of Freedman et al.24 was used to
measure the agonist character of muscarinic cholinergic
drugs in rat cerebral cortex homogenates. For routine
measurements the concentration of [3H]-OXO-M was
0.5 nM, tissue concentration was about 1 mg/mL origi-
nal tissue and incubation was for 40 min at 30 ꢁC. Non-
specific binding was defined as the amount of binding of
[3H]-OXO-M in the presence of 2mM atropine sulfate
and represented about 10% of total binding.
M3-mediated effects. Interactions with M3-muscarinic
cholinergic receptors were measured in the isolated gui-
nea pig ileum. A fully automated method was used as
described previously.23 Contractions induced with
acetylcholine as an agonist (pD2 values between 6 and
7) were used to evaluate the potency of cholinergic
antagonist. Antagonistic activity was measured as
described above.
Binding of [N-methyl-3H]-pirenzepine [3H-PZ] in homo-
genates of rat forebrain. The rapid filtration method of
Freedman et al.21 was used to characterize M1-musca-
rinic cholinergic properties of drugs in rat forebrain
membranes. For routine measurements the concentra-
tion of [3H]-PZ was 1 nM, tissue concentration was
about 10 mg/mL original tissue and incubation was for
60 min at 25 ꢁC. Non-specific binding was defined as the
amount of binding of [3H]-PZ in the presence of 1 mM
atropine sulfate and represented about 20% of total
binding.
References and Notes
1. Mash, D. C.; Flynn, D. D.; Potter, L. T. Science 1985, 228,
1115.
2. Araujo, D. M.; Lapchak, P. A.; Robitaille, Y.; Gauthier,
S.; Quirion, R. Naunyn-Schmiedeberg’s Arch. Pharmacol.
1988, 342, 625.
3. Maltby, N.; Broe, G. A.; Creasey, H.; Jorm, A. F.; Chris-
tensen, H.; Brooks, W. S. Br. Med. J. 1994, 308, 879.
4. Eagger, S. A.; Levy, R.; Sahakian, B. J. Lancet 1991, 337,
989.
5. Lamy, P. P. C N S Drugs 1994, 1, 146.
6. Asthana, S.; Raffaele, K. C.; Berardi, A.; Greig, N. H.;
Haxby, J. V.; Schapiro, M. B.; Soncrant, T. T. Alzheimer Dis.
Assoc. Disord 1995, 9, 223.
Evaluation of the data. Displacement curves were
obtained for the various compounds by measuring the
specific binding in the present of at least four different

1152
R. Plate et al. / Bioorg. Med. Chem. 10 (2002) 1143–1152
7. Davis, K. L.; Hollander, E.; Davidson, M.; Davis, B. M.;
Mohs, R. C.; Hovath, T. B. Am. J. Psychiatry 1987, 144, 468.
8. Hollander, E.; Davidson, M.; Mohs, R. C.; Horvath, T. B.;
Davis, B. M.; Zemishlany, Z.; Davis, K. L. Biol. Psychiatry
1987, 22, 1067.
9. Soncrant, T. T.; Raffaele, K. C.; Asthana, S.; Beradi, A.;
Morris, P. P.; Haxby, J. V. Psychopharmacology 1993, 112, 421.
10. Asthana, S.; Raffaele, K. C.; Berardi, A.; Greig, N. H.;
Morris, P. P.; Schapiro, M. B.; Rapoport, S. I.; Blackman,
M. R.; Soncrant, T. T. Psychoneuroendocrinology 1995, 20,
623.
M. J. M.; Boer, Th. de; Andrews, J. S.; Rae, D. R.; Gibson, S.
Bioorg. Med. Chem. 1996, 4, 227. (c) Plate, R.; Plaum, M. J. M.;
Boer, Th. de; Andrews, J. S. Bioorg. Med. Chem. 1996, 4, 239.
(d) Plate, R.; Plaum, M. J. M.; Pintar, P.; Jans, C. G. J. M.;
Boer, Th. de; Dijcks, F. A.; Ruigt, G.; Andrews, J. S. Bioorg.
Med. Chem. 1998, 6, 1403. (e) Plate, R.; Plaum, M. J. M.;
Hulst, R. G. A.; van der Boer, Th. de Bioorg. Med. Chem.
2000, 8, 449.
18. Plate, R. WO 9412480, 1994.
19. Elvidge, J. A.; Judson, P. N.; Percival, A.; Shah, R. J.
Chem. Soc., Perkin Trans. 1 1983, 8, 1741.
11. Trends Pharmacol. Sci. (Receptor
Nomenclature Suppl.) 1997, 4.
12. Levey, A. I. Life Sci. 1993, 52, 441.
13. Flynn, D. D.; Ferrari-DiLeo, G.; Levey, A. I.; Mash, D. C.
Life Sci. 1995, 56, 869.
&
Ion Channel
20. The catalyst can be re-used at least three times.
21. Strack, E.; Schwaneberg, H.. Chem. Ber. 1934, 67, 1006.
22. The enamine A was isolated as a 1:1 Z/E mixture. This
enamine derivative A was found inert towards all attempts to
convert it into the diamino derivative 3.
14. Freedman, S. B.; Dawson, G. R.; Iversen, L. L.; Baker,
R.; Hargreaves, R. J. Life Sci. 1993, 52, 489.
15. Dawson, G. R.; Bayley, P.; Channell, S.; Iversen, S. D.
Psychopharmacology 1994, 113, 361.
23. Hellmann, H.; Aichinger, G.; Wiedemann, H. P. Justus
Liebigs Ann. Chem. 1959, 626, 35.
24. Freedman, S. B.; Beer, M. S.; Harley, E. A. Eur. J. Phar-
macol. 1988, 156, 133.
16. Sedman, A. J.; Bockbrader, H.; Schwarz, R. D. Life Sci.
1995, 56, 877.
25. Sheridan, R. D.; Sutor, B. Neurosci. Lett. 1990, 108, 273.
26. For more details, see ref 17d.
17. (a) Plate, R.; Plaum, M. J. M.; Boer, Th. de; Andrews, J. S.
Bioorg. Med. Chem. 1996, 4, 239. (b) Plate, R.; Plaum,
27. De Graaf, J. S.; De Vos, C. J.; Steenbergen, H. J. J.
Pharmacol. Meth. 1983, 10, 113.