Novel tricyclic quinazolinimines and related tetracyclic nitrogen bridgehead compounds as cholinesterase inhibitors with selectivity towards butyrylcholinesterase

Article abstract of DOI:10.1016/j.bmc.2005.10.044

Tetracyclic nitrogen bridgehead compounds, dibenzodiazecines and tricyclic quinazolinimines, in which the size of the alicyclic ring system and the length of the alkyl chain between the quinazolinimine moiety and a phenyl ring connected to the imine nitrogen atom were changed systematically, were synthesized and their ability to inhibit acetyl- and butyrylcholinesterase (AChE/BChE), respectively, was evaluated. Moderate and strong inhibitors of BChE-compared to galanthamine and rivastigmine-were identified, which show mixed affinities or are moderately or highly selective towards BChE, respectively.

Full text of DOI:10.1016/j.bmc.2005.10.044

Bioorganic & Medicinal Chemistry 14 (2006) 1966–1977
Novel tricyclic quinazolinimines and related tetracyclic
nitrogen bridgehead compounds as cholinesterase inhibitors
with selectivity towards butyrylcholinesterase
Michael Decker,* Fabian Krauth and Jochen Lehmann
Lehrstuhl fu¨r Pharmazeutische/Medizinische Chemie, Institut fu¨r Pharmazie, Friedrich-Schiller-Universita¨t Jena,
Philosophenweg 14, D-07743 Jena, Germany
Received 1 September 2005; revised 21 October 2005; accepted 25 October 2005
Available online 11 November 2005
Abstract—Tetracyclic nitrogen bridgehead compounds, dibenzodiazecines and tricyclic quinazolinimines, in which the size of the
alicyclic ring system and the length of the alkyl chain between the quinazolinimine moiety and a phenyl ring connected to the imine
nitrogen atom were changed systematically, were synthesized and their ability to inhibit acetyl- and butyrylcholinesterase (AChE/
BChE), respectively, was evaluated. Moderate and strong inhibitors of BChE—compared to galanthamine and rivastigmine—were
identified, which show mixed affinities or are moderately or highly selective towards BChE, respectively.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
the mixed AChE/BChE inhibitor rivastigmine in AD pa-
tients, because in these patients BChE inhibition corre-
lates significantly with cognitive improvement.2
Different forms of dementia, which involve impairment
of cognitive functions, like Alzheimerꢀs disease (AD),
are characterized by reduced levels of acetylcholine
(ACh) in the cortex and hippocampus. In the CNS,
the cholinergic system regulates memory and learning
processes, in which two enzymes degrade ACh: acetyl-
cholinesterase (AChE) and butyrylcholinesterase
(BChE). BChE appears in serum, liver, heart and
CNS. Unlike AChE, its physiologic function is not yet
completely revealed. Noteworthy, AChE activity
decreases progressively in certain brain regions from
mild to severe stages of AD to reach 10–15% of normal
values, while BChE activity is unchanged or even in-
creased by 20%. Therefore, a large pool of BChE is
available in glia neurons and neuritic plaques.1 It may
not be an advantage for a cholinesterase inhibitor
(ChEI) to be selective for AChE; on the contrary, a good
balance between AChE and BChE may result in a higher
efficacy. It is likely that extracellularly diffusing ACh
may come into contact with glial BChE and be effective-
ly hydrolysed, as demonstrated by the administration of
The alkaloids deoxyvasicine 1 and dehydroevodiamine
(DHED) 2, which have been described as inhibitors of
AChE (Fig. 1), served as lead structures.3,4 The antiam-
nesic properties of dehydroevodiamine 2 can only be
partially attributed to ChE inhibition, while other effects
also seem to be prominent.5
We have recently described chemically related tri- and
tetracyclic nitrogen bridgehead compounds, which are
moderate or strong inhibitors of AChE and BChE (com-
pounds 3–5, Fig. 2), showing different selectivity pro-
files:6 3 is a non-selective inhibitor with IC50 (AChE)
of 7.7 lM and IC50 (BChE) of 4.4 lM. 4 can be regarded
as a benzodehydroevodiamine, in which the benzene
ring A substitutes the indole moiety. Compound 4 is a
O
N
N
Cl
N
N
HN
CH3
2
1
Keywords: Cholinesterase inhibition; Tricyclic[2,1-b]quinazolinimines;
Ellmanꢀs assay; Butyrylcholinesterase (BChE) selectivity.
Corresponding author. Tel.: +49 3641 949817; fax: +49 3641
949802; e-mail: m.decker@uni-jena.de
*
Figure 1. Alkaloids deoxyvasicine
(DHED) 2.
1
and dehydroevodiamine
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.044

M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
1967
lead to a higher affinity or a better selectivity at the cho-
linesterases (Fig. 3).
Like dehydroevodiamine, benzodehydroevodiamine 4
shows a pH-dependent equilibrium between the cationic
structure 4 and a non-charged dicarbonyl form, in which
the C@N bond is hydrolysed at higher pH values.6,7 Our
second aim was—in order to isolate the pharmacophoric
parts of benzodehydroevodiamine
4 and to find
correlations between the structures of ChE inhibiting
compounds of 3 and 4—to synthesize a compound
with a tertiary instead of a quaternary nitrogen (i.e.,
benzoevodiamine): a structure combining features of
compounds 3 and 4, but without the ability to hydrolyse
reversibly at higher pH values. Removal of the carbonyl
group is an additional modification step yielding a
compound with a second basic nitrogen atom like in
compound 3. And finally—by a removal of the central
C–N bond—a diazecine, that is, a less rigidized structure
with two basic nitrogen atoms, should be synthesized
and its interaction with the ChEs examined (Fig. 4).
Figure 2. Structures of lead compounds 5,8-dihydro-6H-isoquino[1,2-
b]quinazoline 3, 13-methyl-8-oxo-5,8-dihydro-6H-isoquino[1,2-b]qui-
nazolin-13-ium chloride (benzodehydroevodiamine) 4 and N-(2-phen-
ylethyl)-N-(7,8,9,10-tetrahydroazepino[2,1-b]quinazolin-12(6H)-yl-
idene)amine 5, respectively.
A Prevention of oxidation-
sensitivity
CH3
B Removal of angular fixation
for greater flexibility
N
A
B
N
The third aim of our work used the moderate to good
affinities of the quinazolinimine 5 at the ChEs and its
10-fold selectivity towards BChE as a starting point
(Fig. 5).6 Since BChE possesses a larger void at the ac-
tive site gorge,8 either changing the size of the alicyclic
ring or changing the distance between the quinazolini-
mine moiety and the phenyl group might result in an im-
proved selectivity towards BChE. By investigating these
two effects systematically, we wanted to find the
optimum of these two parameters with respect to both
affinity and selectivity.
N
6
N
3
H3C
N
N
7
Figure 3. Structural modifications of quinazoline 3 to prevent its
oxidation-sensitivity (A) and to introduce greater flexibility into the
molecule (B).
selective AChE inhibitor with an IC50 (AChE) of 12 lM.
Whereas 5 shows a 10-fold selectivity towards an BChE
with IC50 (AChE) of 23 lM and IC50 (BChE) of 2.2 lM,
respectively (Fig. 2).6
By measuring the enzyme kinetics of the quinazolini-
mines, we wanted to find out what kind of inhibition
these compounds exhibit, and therefore get information,
if these compounds bind (exclusively) at the active site.
Different aims were pursued in our work: first, concern-
ing the dihydroquinazolines deoxyvasicine 1 and com-
pound 3, a drawback of these substances lies in the
fact that deoxyvasicine oxidizes to its corresponding
quinazolinone.3 This is also possible with compound 3,
although far less pronounced.6 By introducing an alkyl
group in position 8 of compound 3, a new group of alkyl
substituted dihydroquinazolines is produced. Using a
methyl group might open a possibility to yield a com-
pound without oxidation sensitivity, but with a similar
inhibitory profile (Fig. 3).
2. Results and discussion
2.1. Synthesis
5,8-Dihydro-6H-isoquino[1,2-b]quinazoline 3 can be
prepared by reduction of the corresponding quinazoli-
none under Clemmensen conditions.6,9 In order to intro-
duce a methyl group in position 8 a different synthetic
strategy had to be applied: o-aminoacetophenone was
reduced by sodium borohydride to 1-(2-aminophe-
nyl)ethanol,10 which in turn reacted after transforma-
tion into the sulfinylamino compound with hydrogen
chloride to 2-(1-chloroethyl)benzenaminium chloride (a
A formal cleavage of the C–C bond in positions 8 and 8a
should result in a more flexible structure, which might
O
O
H
C
B
N
A
N
N
N
N
N
N
N
R
A
A
CH3
CH3
CH3
10a: R = H
10b: R = CH3
Cl
4
8
9
Figure 4. Structural analogues of benzodehydroevodiamine 4 for identification of the pharmacophoric parts of the molecule, that is, removal of the
C@N double bond (A), removal of the carbonyl group (B) and ring-opening yielding diazecines (C).

1968
M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
Compound 9 was prepared by lithium aluminium
hydride reduction of compound 8 (benzoevodiamine),
which could be synthesized by the reaction of N-methyl-
n = 0, 1, 2
isatoic
anhydride
(1-methyl-2H-3,1-benzoxazine-
N
N
N
N
2,4(1H)-dione) with 3,4-dihydroisoquinoline (Scheme
2).13 For the synthesis of 3,4-dihydroisoquinoline differ-
ent methods are described in the literature, oxidation of
1,2,3,4-tetrahydroisoquinoline by sulfur in dioxane turn-
ing out to be very facile (Scheme 2).14
N
N
n = 0, 1, 2, 3
5
12a-l
Figure 5. Structural analogues of quinazolinimine 5 concerning the
ring size of the alicyclic ring system and the length of the alkylene chain
connecting phenyl group and imine-N.
Both 14-methyl-5,6,7,8,13,14-hexahydrodibenzo[b,h][1,5]dia-
zecine 10b and 5,6,7,8,13,14-hexahydrodibenzo[b,h][1,5]diaze-
cine 10a could be prepared by reduction of compound 4
and the demethylated compound 5,6-dihydro-8H-isoqu-
ino[1,2-b]quinazoline-8-one, respectively, with borane
(Scheme 3).15 The syntheses of compound 4 and of
5,6-dihydro-8H-isoquino[1,2-b]quinazoline-8-one, respective-
ly, have been described recently.6,16 Interestingly,
compound 10a is also formed by methylation of com-
pound 10b using ethyl chloroformate and subsequent
reduction of the carbamate by lithium aluminium hy-
dride. No dimethylated compound is formed. Identical
MS and NMR data proved methylation at the aromatic
nitrogen atom.
synthetic method previously applied for the prepara-
tion of a-phenylneopentyl chlorides).11 This compound
reacted with 3,4-dihydro-1(2H)-isoquinolinone,12
which was activated with POCl3, to compound 6
(Scheme 1).
The ring-opened compound 7 (Fig. 3) was synthesized
from aniline and 2-methyl-3,4-dihydro-1(2H)-isoquino-
linethione in the presence of mercury(II) bromide under
identical conditions as described for the preparation of
quinazolinimines. The quinazolinethione could be pre-
pared from 2-methyl-3,4-dihydro-1(2H)-isoquinolinone
using Lawessonꢁs reagent, the previous compound pre-
pared through cyclisation of the corresponding carba-
mate using a mixture of P4O10 and POCl3.12
The quinazolinimines were prepared according to a
modified procedure for the synthesis of compound 5,
which has been described recently by us.6 The quinazoli-
nones were obtained by reaction of anthranilic acid with
iminium ethers of lactams of different ring sizes, which
were prepared by the reaction of lactams with Meerwein
salt (triethyloxonium tetrafluoroborate).17
Despite the interesting pharmacological properties of
this compound (see below), it proved to be fairly unsta-
ble due to amidine hydrolysis yielding the benzolactam
The quinazolinones were transformed with Lawessonꢀs
reagent into quinazolinethiones (11a–d).6,9 They in turn
reacted in the presence of mercury(II) bromide or silver
(2-methyl-3,4-dihydro-1(2H)-isoquinolinone).
fore, this class of ring-opened compounds was not fur-
ther investigated.
There-
O
OH
CH3
NH2
NaBH4
CH3
1) SO2Cl2
2) HCl (g)
Cl
NH2
CH3
CH3
N
NH2 HCl
POCl3
+
N
NH
O
6
H
N
NH2
OEt
ClCOOEt
POCl3/P4O10
O
Scheme 1. Synthesis of 8-methyl-5,8-dihydro-6H-isoquino[1,2-b]quinazoline 6.
S, dioxane
O
N
N
N
toluene/
reflux
H
LiAlH4
N
+
N
O
N
N
O
CH3
CH3
8
9
O
CH3
Scheme 2. Synthesis of 13-methyl-5,6,13,13a-tetrahydro-8H-isoquino[1,2-b]quinazolin-8-one
tetrahydro-6H-isoquino[1,2-b]quinazoline 9.
8 (benzoevodiamine) and 13-methyl-5,8,13,13a-

M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
1969
O
N
H
Concerning compound 8 (benzoevodiamine), it turned
out that it neither inhibits AChE nor BChE at
10ꢀ5 M; therefore, the quaternary nitrogen atom of
compound 4 (and the ability of this compound to form
a dicarbonyl form) seems to be essential for its AChE
inhibiting properties. Compound 9, which—in contrast
to compound 8—lacks the carbonyl group and therefore
possesses two basic tertiary nitrogen atoms in the form
of a nitrogen bridgehead compound is a moderate inhib-
itor of BChE, but shows only very weak affinity towards
AChE. An interesting observation can be made regard-
ing compounds 10a and b, respectively. Whereas the tri-
cyclic diazecine 10a shows only moderate affinity
towards both ChEs (roughly 50% inhibition at
10ꢀ4 M), the diazecine 10b with two secondary nitrogen
atoms is a moderate inhibitor at both enzymes with a
threefold higher affinity at BChE. This means that the
secondary nitrogen atom in vicinity of the benzene ring
increases affinities.
N
1.0 M BH3/THF
N
N
CH3
CH3
4
10a
Cl
O
H
N
1.0 M BH3/THF
N
N
N
H
10b
Scheme 3. Synthesis of 14-methyl-5,6,7,8,13,14-hexahydrodibenzo
[b, h][1,5]diazecine 10a and 5,6,7,8,13,14-hexahydrodibenzo [b,h][1,5]
diazecine 10b.
nitrate with aniline, benzylamine and 2-phenylethyl-
amine, respectively, to quinazolinimines 12a–l (Scheme
4). The use of the above-mentioned salts proved to be
superior to the previously described use of mercury(II)
acetate, because product purification was simplified
(the use of Hg(OAc)2 leads to the formation of aceta-
mides as by-products).6
The pharmacological profiles of the quinazolinimines
12a–l are of special interest (Table 1). All of these com-
pounds show micromolar or higher affinities to BChE
with at least a 10-fold lower affinity towards AChE.
Some of these compounds (i.e., 12c, d and h) show more
than 200-fold selectivity towards BChE. Two general
trends can be observed: on the one hand, an increasing
ring size lowers the affinity towards AChE, whereas
the BChE affinity stays the same or—especially in the
case of anilino-derivatives—is even increased. These
properties could be expected, because BChE possesses
a larger void at the active site gorge.8 An increasing dis-
tance of the phenyl ring from the quinazolinimine-het-
erocycle generally increased affinity towards AChE. In
a less pronounced manner, this also applies for BChE:
the six-membered ring quinazolinimine phenylethyl-
compound 12j possesses a more than 100-fold higher
affinity as that of the respective anilino-compound 12b,
whereas the eight-membered ring quinazolinimine phen-
ylethyl-compound 12l possesses only a threefold higher
affinity than the respective anilino-compound 12d.
Taken together, the best selectivity for BChE was
reached for compound 12h, a seven-membered quinazo-
linimine with a benzyl-group. The most active com-
2.2. Pharmacology
Comparing compounds 3 (a quinazoline) and 6 (a qui-
nazoline with a methyl group in position 8), it turned
out that the affinities did not significantly change, but
a small increase towards BChE occurred (Table 1).
The methyl group did not disturb binding to the cho-
linesterases. But in contrast to compound 3 and the
alkaloid deoxyvasicine 1,3 respectively, compound 6 is
more stable towards air-oxygen, but its stability with re-
spect to oxidation has to be investigated separately.
An interesting change in the selectivity profile occurred
with the ring-opened compound 7. Affinity towards
BChE slightly increased, but its affinity towards AChE
decreased of a factor of 15. Since amidine 7 is labile to-
wards hydrolysis at higher pH values, the IC50 values
given in Table 1 might be determined under partial
hydrolysis of 7 and are therefore to be considered for
estimation only. This property of compound 7 limits
its application.
Scheme 4. Synthesis of quinazolinimines 12a–l.

1970
M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
Table 1. IC50 values (pIC50 = ꢀlgIC50 in brackets) of test compounds for AChE and BChE inhibition, respectively, and resulting selectivities
expressed as the ratio of IC50 values
Test compound
Rivastigmine
AchE inhibition, lM
[IC50 (pIC50 SEM)]
BChE inhibition, lM
[IC50 (pIC50 SEM)]
Selectivity
[IC50 (BchE)/IC50 (AChE)]
>500a,b (lit.:1 48)
0.64 (lit.:1 0.8) (6.197 0.052)
>500
3.9a (lit.:1 54) (5.409 0.050)
8.4 (lit.:1 7.3) (5.076 0.034)
19.5 (4.709 0.206)
<0.008
Galanthamine
13.1
N
N
N
<0.039
12a
N
N
98.6 (4.006 0.823)
22.3 (4.651 0.142)
4.1 (5.392 0.160)
2.3 (5.630 0.173)
0.226
<0.008
<0.005
N
12b
12c
N
>500
>500
N
N
N
N
N
12d
N
N
13.9 (4.856 0.063)
1.87 (5.728 0.028)
0.135
0.055
N
12e
12f
N
N
25.5 (4.594 0.112)
1.4 (5.844 0.088)
N
86.6c (4.063)
1.5 (5.831 0.089)
1.1 (5.942 0.165)
0.62 (6.210 0.034)
0.017
N
N
N
12g
N
>500
<0.002
N
N
12h
7.3 (5.139 0.065)
0.085
N
N
N
12i
(continued on next page)

M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
1971
Table 1 (continued)
Test compound
AchE inhibition, lM
[IC50 (pIC50 SEM)]
BChE inhibition, lM
[IC50 (pIC50 SEM)]
Selectivity
[IC50 (BchE)/IC50 (AChE)]
14.9 (4.828 0.315)
0.144 (6.84 0.031)
0.01
N
N
N
12j
27.76 (4.558 0.107)
2.26 (5.658 0.032)
0.086
N
N
N
12k
11.5 (4.940 0.092)
0.62 (6.207 0.048)
0.05
N
N
N
12l
8.5 (5.069 0.092)
2.9 (5.53 0.039)
0.34
N
N
7.76 (5.11 0.050)
4.46 (5.35 0.044)
0.576
3
H3C
N
118.6i (3.926 0.263)
82.5 (4.084 0.066)
2.0i (5.696 0.057)
25.1 (4.600 0.058)
0.017
0.30
N
HCl
O
N
N
O
N
N
38.6 (4.414 0.213)
279d (3.554)
>500
>500
>500
>12.95
>1.79
>1.16
O
N
N
O
N
N
430e (3.366)
O
N
N
>500f
>500f
CH3
8
N
N
>500e
>500g
11.9 (4.924 0.084)
<0.024
CH3
9
H
N
>500h
N
CH3
10a
(continued on next page)

1972
M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
Table 1 (continued)
Test compound
AchE inhibition, lM
BChE inhibition, lM
[IC50 (pIC50 SEM)]
Selectivity
[IC50 (BchE)/IC50 (AChE)]
[IC50 (pIC50 SEM)]
50.1 (4.300 0.86)
H
N
15.6 (4.806 0.219)
0.311
N
H
10b
a IC50 values are time-dependent.18
b 10ꢀ3 M: 55% inhibition.
c 10ꢀ5 M: 28% inhibition.
d 10ꢀ4 M: 55% inhibition.
e 10ꢀ4 M: 50% inhibition.
f No inhibition at 10ꢀ5 M.
g 10ꢀ4.3 M: 26% inhibition.
h 10ꢀ4.3 M: 33% inhibition.
i Some hydrolysis might take place under assay conditions (values for affinity estimation only).
pound is 12j with an IC50 (BChE) = 0.14 lM and a
selectivity of 1/100 towards BChE.
Substrate-velocity curve of
BChE Inhibition
by Compound 12l
1.25
1.00
0.75
0.50
0.25
0.00
The quinazolinimines show some lability towards
hydrolysis, by which the respective quinazolinones are
formed. For this reason also the quinazolinones were
tested for their ability to inhibit the cholinesterases, in
order to prove, that the effects measured are solely due
to the quinazilinimines. The six-, seven- and eight-mem-
bered quinazolinones show no activity to BChE at a
concentration of 10ꢀ5 M, but are weak inhibitors of
AChE. The six- and five-membered compounds show
IC50 values of 39 and 83 lM, respectively. Interestingly,
the five-membered quinazolinone is also a moderate
inhibitor of BChE (IC50 = 25 lM), but by far not in
the range of the respective quinazolinimines.
[12l] = 0 µM
[12l] = 0.1 µM
[12l] = 0.2 µM
[12l] = 0.8 µM
[12l] =1.0 µM
0
100
200
300
400
500
[S], µM
Lineweaver-Burk Plot
BChE Inhibition
by Compound 12l
100
75
[12l] = 0 µM
[12l] = 0.1 µM
[12l] = 0.2 µM
[12l] = 0.8 µM
[12l] = 1.0 µM
Compound 12l was selected for kinetic measurements,
because it shows high affinities towards both cholinester-
ases and chemically represents the quinazolinimines syn-
thesized and tested. The mechanism of inhibition was
analyzed by recording substrate–velocity curves in the
absence and in the presence of different concentrations
of 12l. Substrate concentration was varied between 25
and 450 lM. For BChE 0.1, 0.2, 0.8 and 1.0 lM concen-
trations, respectively, of compound 12l were applied, for
AChE 10 and 15 lM of 12l were used. Fig. 6 shows the
substrate–velocity curves for BChE and the resulting
Lineweaver–Burk plots, that is, reciprocal rates versus
reciprocal substrate concentrations for the different
inhibitor concentrations. Km values (i.e., the negative re-
ciprocal of the X intercept) differ, but in contrast the
Vmax value, (i.e., the reciprocal of the Y intercept) is
not changed with different inhibitor concentrations.
Similar results were obtained for AChE (Fig. 7). Fewer
plots were recorded for AChE, because at higher con-
centrations 12l is not sufficiently soluble anymore. The
Lineweaver–Burk plot for BChE clearly shows revers-
ible and competitive inhibition by the quinazolinimine,
meaning that these compounds compete for the same ac-
tive site as the substrate butyrylcholine. For AChE the
results are not that unequivocal due to the limited con-
centrations of inhibitor applicable, but seem to show the
same type of inhibition.
50
25
0.01 0.02 0.03 0.04 0.05
1/[S]
Figure 6. Substrate–velocity curve of BChE activity with different
substrateconcentrations (25–450 lM)in the absenceand presenceof 0.1,
0.2, 0.8 and 1.0 lM compound 12l and resulting Lineweaver–Burk plots.
Lineweaver-Burk Plot
AChE Inhibition
by Compound 12l
25
20
[12l] = 0 µM
[12l] = 10 µM
[12l] = 15 µM
15
10
5
0.01 0.02 0.03 0.04 0.05
1/[S]
Figure 7. Lineweaver–Burk plots of AChE activity over a range of
substrate concentrations (25–450 lM) in the absence and in the
presence of 10 and 15 lM 12l, respectively.

M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
1973
3. Conclusion
tal analyses were performed on a Hereaus Vario EL
apparatus. TLC was performed on silica gel F254 plates
(Merck). For detection iodine vapour or UV light
(254 nm), respectively, was used. MS data were deter-
mined by GC/MS, using a Hewlett Packard GCD-Plus
(G1800C) apparatus (HP-5MS column; J&W Scientific).
Silica gel column chromatography utilized silica gel 60
63–200 lm (Baker). UV measurements were performed
on a Jasco V-570 UV/vis/NIR spectrophotometer.
Concerning the different aims of our project, several
conclusions can be drawn: first, by introducing a methyl
group in position 8 of the quinazoline 3 it was possible
to synthesize a compound (6) with almost the same affin-
ities as the parent compound, but with a less pro-
nounced sensitivity for oxidation to the quinazolinone.
Second, a ring-opened amidine 7 structurally related to
compound 3 was synthesized, which shows 60-fold selec-
tivity towards BChE with higher affinities at BChE than
the established drugs galanthamine and rivastigmine,
respectively. But this compound turned out to be labile
to hydrolysis, therefore this class of compounds has not
been further investigated.
4.2. Chemistry
4.2.1. General procedure for the preparation of thiolac-
tams and quinazolinethiones. A solution of 5.0 mmol of
the respective lactam or quinazolinone in 50 mL of dried
toluene was stirred under reflux with 1.0 g (2.5 mmol) of
Lawessonꢁs reagent. After 4 h, the reaction mixture was
cooled to rt, the toluene removed under reduced pres-
sure and the residue was column chromatographed
using CH2Cl2 as eluent.
Third, in order to find out pharmacophoric similarities
between the AChE inhibiting compound 4 and the unse-
lective inhibitor 3, different compounds were synthesized
and evaluated pharmacologically, that contain structur-
al features of both compounds: compound 8 containing
the amide group of compound 4 but with a tertiary
nitrogen atom does not show any activity at the ChEs,
but removal of the carbonyl function creates the moder-
ate BChE inhibitor 9. Cleavage of the central C–N bond
yields the diazecine 10a, which represents a less con-
straint compound that is an unselective and only a very
weak inhibitor of both ChEs. Interestingly, the deme-
thylated compound 10b with two hydrogen donating
nitrogen atoms again represents a moderate inhibitor
with threefold selectivity towards BChE.
4.2.1.1. 2-Methyl-3,4-dihydro-1(2H)-isoquinolinethi-
one. Yellow crystals. Mp 98 ꢁC (lit.19 mp 98 ꢁC). EI-
MS m/z 177, 162, 144, 134, 115, 103, 89, 77. Other spec-
troscopic data are in accordance with the literature
data.19
2,3-Dihydropyrrolo[2,1-b]quinazoline-9(1H)-thione (11a)
was prepared as described above; the spectroscopic data
are in accordance with literature data.20 The preparation
and spectroscopic data of 7,8,9,10-tetrahydroazepino[2,1-
b]quinazoline-12(6H)-thione (11c) have been described
recently.6
Finally, SARs concerning the quinazolinimines 12a–l
have been performed, in which the distance between
the phenyl ring and the heterocycle was shortened and
in which the size of the alicyclic ring was changed sys-
tematically, in order to increase affinity and especially
to improve selectivity towards BChE, since BChE pos-
sesses a larger void at the active site of the enzyme. This
strategy was highly successful, because compounds with
both micromolar and submicromolar affinities towards
BChE were identified with 100-fold and higher selectiv-
ities. The compounds synthesized show similar or higher
affinities than galanthamine and especially the mixed
inhibitor rivastigmine with highly improved selectivity
profiles (rivastigmine is a very slow inhibitor of ChEs,
therefore IC50 values are highly time-dependent).18 But
also a number of potent mixed inhibitors have been
found. Compound 12l representing the quinazolinimines
synthesized was chosen for kinetic measurements. A
Lineweaver–Burk plot for BChE revealed a competitive
and reversible binding mode for the quinazolinimines.
4.2.1.2. 6,7,8,9-Tetrahydro-11H-pyrido[2,1-b]quinazo-
line-11-thione (11b). Yellow crystals. 570 mg (53% yield).
Mp 132 ꢁC. H NMR (250 MHz, CDCl3): d 1.85–2.06
1
(4H, m, CH2CH2CH2CH2), 2.99–3.04 (2H, t,
J = 6.25 Hz, CH2C), 4.5–4.55 (2H, t, J = 6.25 Hz,
CH2N), 7.35–7.42 (1H, t, J = 7.5 Hz, C(3)H), 7.5–7.54
(1H, d, J = 7.5 Hz, C(4)H), 7.62–7.69 (1H, t,
J = 7.5 Hz, C(2)H), 8.69–8.73 (1H, d, J = 7.5 Hz,
C(1)H) ppm. IR (KBr): 3441, 1950, 1584, 1552, 1471,
1376, 1306, 1205, 980, 768 cmꢀ1. EI-MS m/z 216, 201,
183, 176, 169, 155, 129, 102, 77. Anal. (C12H12N2S):
C, H, N, S.
4.2.1.3. 6,7,8,9,10,11-Hexahydro-13H-azocino[2,1-
b]quinazoline-13-thione (11d). Yellow crystals. 785 mg
1
(74% yield). Mp 103 ꢁC. H NMR (250 MHz, CDCl3):
d 1.25–2.05 (8H, m, CH2(CH2)4CH2), 3.14–3.19 (2H,
m, CH2C), 4.99 (2H, br s, CH2N), 7.43–7.5 (1H, t,
J = 7.5 Hz, C(3)H), 7.6–7.64 (1H, d, J = 7.5 Hz,
C(4)H), 7.69–7.76 (1H, t, J = 7.5 Hz, C(2)H), 8.76–8.8
(1H, d, J = 7.5 Hz, C(1)H) ppm. IR (KBr): 3448, 2925,
1581, 1552, 1360, 1181, 1082, 1021, 773 cmꢀ1. EI-MS
m/z 244, 215, 203, 189, 176, 155, 129, 102, 77. Anal.
(C14H16N2S): C, H, N, S.
4. Experimental protocols
4.1. General
Melting points are uncorrected and were measured in
open capillary tubes, using a Gallenkamp melting point
apparatus. 1H NMR spectral data were obtained from a
Bruker Advance 250 spectrometer (250 MHz). Elemen-
4.2.2. General procedure for the preparation of quinazo-
linimines. To a solution of 2 mmol of the respective qui-
nazolinethione 11 in 15 mL of toluene were added
6 mmol aniline, benzylamine or 2-phenylethylamine,

1974
M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
respectively. The mixture was heated under reflux under
vigorous stirring. To the stirred solution, 1.44 g
(4 mmol) of mercury(II) bromide or—if explicitly men-
tioned in the description of the compound below—
680 mg (4 mmol) of silver nitrate was added. Using sil-
ver nitrate the suspension darkened immediately. The
mixture was heated for 4 h (2 h when using silver ni-
trate). After cooling, the precipitate was filtered off,
the toluene was removed under reduced pressure and
the residue was column chromatographed using
CH2Cl2/ethyl acetate, 2:1 as eluent.
MS m/z 274, 260, 246, 184, 172, 159, 77. Anal.
(C18H17N3): C, H, N.
4.2.2.5. N-Benzyl-N-(6,7,8,9-tetrahydro-11H-pyri-
do[2,1-b]quinazolin-11-ylidene)amine (12f). White pow-
der. 150 mg (26% yield). Mp 96 ꢁC. 1H NMR
(250 MHz,
CDCl3):
d
1.83–2.05
(4H,
m,
CH2CH2CH2CH2), 2.86–2.91 (2H, t, J = 6.25 Hz,
CH2C), 3.98–4.03 (2H, t, J = 6.25 Hz, CH2N), 5.13
(2H, s, CH2-benzyl), 7.22–7.52 (8H, m, CH-phenyl,
C(1,2,3)H), 8.03–8.06 (1H, d, J = 7.5 Hz, C(4)H)
ppm. IR (KBr): 3459, 2930, 1632, 1584, 1473, 1261,
758, 732, 698, 641 cmꢀ1. EI-MS m/z 289, 260, 212,
198, 184, 155, 91. Anal. (C19H19N3): C, H, N.
4.2.2.1.
N-(2,3-Dihydropyrrolo[2,1-b]quinazolin-
9(1H)-ylidene)aniline (12a). Silver nitrate was used for
coupling. White powder. 161 mg (31% yield). Mp
164 ꢁC. 1H NMR (250 MHz, CDCl3): d 2.09–2.21
(2H, qui, J = 7.5 Hz, CH2CH2CH2), 3.0–3.06 (2H, t,
J = 7.5 Hz, CH2C), 4.03 (2H, br s, CH2N), 6.78–
6.94 (3H, m, 3· CH-phenyl), 6.94–7.0 (1H, t,
J = 7.25 Hz, C(2)H) 7.24–7.42 (5H, m, 2· CH-phenyl,
C(1,3,4)H) ppm. IR (KBr): 3422, 2968, 1644, 1466,
1403, 1257, 1024, 770, 694, 662 cmꢀ1. EI-MS m/z
260, 245, 233, 218, 184, 159, 129, 77. Anal.
(C17H15N3): C, H, N.
4.2.2.6. N-(2-Phenylethyl)-N-(6,7,8,9-tetrahydro-11H-
pyrido[2,1-b]quinazolin-11-ylidene)amine (12j). Smeary
1
white solid. 160 mg (26% yield). Mp 52 ꢁC. H NMR
(250 MHz,
CDCl3):
d
1.85–1.98
(4H,
m,
CH2CH2CH2CH2), 2.82–2.87 (2H, t, J = 7.5 Hz,
CH2C), 3.02–3.08 (2H, t, J = 7 Hz, CH2-phenyl), 3.98–
4.03 (2H, t, J = 7 Hz, CH2CH2-phenyl), 4.07–4.13 (2H,
t, J = 7.5 Hz, CH2N), 7.15–7.32 (6H, m, CH-phenyl,
C(2)H), 7.44–7.48 (2H, m, C(1,3)H), 7.97–8.0 (1H, d,
J = 8.5 Hz, C(4)H) ppm. IR (KBr): 3442, 2945, 1629,
1579, 1483, 1402, 1246, 1176, 763, 700, 668 cmꢀ1. EI-
MS m/z 303, 281, 212, 199, 184, 155, 129, 102. Anal.
(C20H21N3): C, H, N.
4.2.2.2. N-Benzyl-N-(2,3-dihydropyrrolo[2,1-b]quinaz-
olin-9(1H)-ylidene)amine (12e). Silver nitrate was used
for coupling. White powder. 259 mg (47% yield). Mp
1
99 ꢁC. H NMR (250 MHz, CDCl3): d 2.03–2.15 (2H,
4.2.2.7. N-(7,8,9,10-Tetrahydroazepino[2,1-b]quinazo-
lin-12(6H)-ylidene)aniline (12c). White powder. 220 mg
(39% yield). Mp 152 ꢁC. 1H NMR (250 MHz,
CDCl3): d 1.85–1.9 (6H, m, CH2(CH2)3CH2), 3.0–
3.04 (2H, m, CH2C), 4.45 (2H, br s, CH2N), 6.8–
7.02 (3H, m, 3· CH-phenyl), 7.04–7.05 (1H, t,
J = 7.25 Hz, C(2)H), 7.13–7.43 (5H, m, 2· CH-phenyl,
C(1,3,4)H) ppm. IR (KBr): 3421, 2920, 1638, 1587,
1264, 1135, 980, 900, 771, 703 cmꢀ1. EI-MS m/z
289, 260, 234, 186, 172, 159, 102, 77. Anal.
(C19H19N3): C, H, N.
qui, J = 7.5 Hz, CH2CH2CH2), 2.93–2.99 (2H, t,
J = 7.5 Hz, CH2C), 4.0–4.05 (2H, t, J = 7.5 Hz,
CH2N), 5.05 (2H, s, CH2-benzyl), 7.1–7.44 (8H, m,
CH-phenyl, C(1,2,3)H), 8.05–8.08 (1H, d, J = 7.5 Hz,
C(4)H) ppm. IR (KBr): 3450, 2958, 1635, 1603, 1410,
1287, 1268, 1030, 802, 761, 736, 698, 660 cmꢀ1. EI-MS
m/z 275, 246, 198, 184, 170, 142, 129, 102, 91. Anal.
(C18H17N3): C, H, N.
4.2.2.3.
N-(2,3-Dihydropyrrolo[2,1-b]quinazolin-
9(1H)-ylidene)-N-(2-phenylethyl)amine (12i). Silver ni-
trate was used for coupling. Smeary pale yellow solid.
139 mg (24% yield). Mp 70 ꢁC (continuously melting).
1H NMR (250 MHz, CDCl3): d 2.02–2.14 (2H, qui,
J = 7.5 Hz, CH2CH2CH2), 2.92–3.01 (4H, m, CH2C,
CH2-phenyl), 3.94–4.14 (4H, m, CH2CH2-phenyl,
CH2N), 7.12–7.25 (6H, m, CH-phenyl, C(7)H), 7.43–
7.44 (2H, m, C(8,6)H), 8.0–8.03 (1H, d, J = 8 Hz,
C(5)H) ppm. IR (KBr): 3433, 2920, 1638, 1600, 1410,
1290, 1030, 761, 704, 666 cmꢀ1. EI-MS m/z 289, 259,
198, 183, 169, 142, 102, 91, 77. Anal. (C19H19N3·
1/2EtOAc): C, H, N.
4.2.2.8. N-Benzyl-N-(7,8,9,10-tetrahydroazepino[2,1-
b]quinazolin-12(6H)-ylidene)amine (12g). White powder.
316 mg (62% yield). Mp 95 ꢁC. 1H NMR (250 MHz,
CDCl3): d 1.84–1.9 (6H, m, CH2(CH2)3CH2), 2.93–
2.98 (2H, m, CH2C), 4.38 (2H, br s, CH2N), 5.1 (2H,
s, CH2-benzyl), 7.2–7.53 (8H, m, CH-phenyl,
C(1,2,3)H), 7.96–7.99 (1H, d, J = 7.5 Hz, C(4)H) ppm.
IR (KBr): 3503, 2927, 1632, 1584, 1410, 1141, 761,
729, 698, 672, 641 cmꢀ1. EI-MS m/z 303, 274, 260,
249, 212, 198, 183, 170, 155, 142, 129, 102, 91, 77. Anal.
(C20H21N3): C, H, N.
4.2.2.4. N-(6,7,8,9-Tetrahydro-11H-pyrido[2,1-b]qui-
nazolin-11-ylidene)aniline (12b). White powder. 140 mg
(25% yield). Mp 113 ꢁC. H NMR (250 MHz, CDCl3):
4.2.2.9. N-(6,7,8,9,10,11-Hexahydro-13H-azocino[2,1-
b]quinazolin-13-ylidene)aniline (12d). White powder.
320 mg (53% yield). Mp 149 ꢁC. H NMR (250 MHz,
1
1
d 1.8–1.99 (4H, m, CH2CH2CH2CH2), 2.84–2.89 (2H,
t, J = 6.25 Hz, CH2C), 3.93–3.98 (2H, t, J = 6.25 Hz,
CH2N), 6.71–6.81 (3H, m, 3· CH-phenyl), 6.91–6.98
(1H, t, J = 7.25 Hz, C(2)H) 7.12–7.36 (5H, m, 2· CH-
phenyl, C(1,3,4)H) ppm. IR (KBr): 3447, 2941, 1629,
1586, 1482, 1398, 1263, 1100, 769, 700, 666 cmꢀ1. EI-
CDCl3): d 1.2–2.2 (8H, m, CH2(CH2)4CH2), 2.96–3.01
(2H, m, CH2C), 4.42 (2H, br s, CH2N), 6.79–6.89 (3H,
m, 3· CH-phenyl), 7.04–7.05 (1H, t, J = 7.25 Hz,
C(2)H), 7.23–7.49 (5H, m, 2· CH-phenyl, C(1,3,4)H)
ppm. IR (KBr): 3448, 2915, 2850, 1649, 1582, 1471,
1408, 1246, 1150, 773, 701, 666 cmꢀ1. EI-MS m/z 303,

M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
1975
274, 260, 248, 235, 172, 159, 129, 77. Anal. (C20H21N3):
C, H, N.
Colourless oil. 120 mg (16% yield). 1H NMR (250 MHz,
CDCl3): d 1.35–1.38 (3H, d, J = 8 Hz, CH3), 2.97–3.06
(2H, m, CH2C), 3.33–3.44 (1H, m, CH2CH2), 3.56–3.71
(1H, m, CH2CH2), 4.58–4.67 (1H, q, J = 8 Hz, CH),
6.94–7.39 (7H, m, CH-arom), 8.34–8.38 (1H, m, C(1)H)
ppm. IR (KBr): 3423, 2923, 1670, 1548, 1475, 769,
739 cmꢀ1. EI-MS m/z 248 (7), 233 (100), 218 (24), 190,
124, 116 (25), 102, 89, 77. Anal. (C17H16N2): C, H, N.
4.2.2.10. N-Benzyl-N-(6,7,8,9,10,11-hexahydro-13H-
azocino[2,1-b]quinazolin-13-ylidene)amine (12h). Silver
nitrate was used for coupling. White powder. 510 mg
1
(81% yield). Mp 105 ꢁC. H NMR (250 MHz, CDCl3):
d 1.15–1.96 (8H, m, CH2(CH2)4CH2), 2.81–2.86 (2H,
m, CH2C), 4.32 (2H, br s, CH2N), 5.06 (2H, s, benzyl-
CH2), 7.1–8.0 (8H, m, CH-phenyl, C(1,2,3)H), 8.16–
8.2 (1H, d, J = 7.5 Hz, C(4)H) ppm. IR (KBr): 3450,
2917, 2855, 1632, 1576, 1408, 1356, 1238, 1137, 762,
729, 696, 669, 640 cmꢀ1. EI-MS m/z 317, 288, 276,
262, 249, 226, 183, 155, 129, 102, 91, 77. Anal.
(C21H23N3): C, H, N.
4.2.4.
N-(2-Methyl-3,4-dihydroisoquinolin-1(2H)-yli-
dene)aniline (hydrochloride) (7). This compound was pre-
pared analogously to the quinazolinimines (see Section
4.2.2). Equimolar amounts of 2-methyl-3,4-dihydro-
1(2H)-isoquinolinethione (6 mmol, for preparation, see
Section 4.2.1) and mercury(II) acetate were used. Direct-
ly after preparation of the amidine and its purification
by column chromatography (EtOAc/MeOH, 1:1) 6 M
MeOHÆHCl were added to form the corresponding
hydrochloride.
4.2.2.11. N-(2-Phenylethyl)-N-(6,7,8,9,10,11-hexahy-
dro-13H-azocino[2,1-b]quinazolin-13-ylidene)amine (12l).
1
Smeary white solid. 264 mg (40% yield). Mp 55 ꢁC. H
NMR (250 MHz, CDCl3):
d 1.24–1.98 (8H, m,
1
Colourless oil. 324 mg (15% yield). H NMR (hydro-
CH2(CH2)4CH2), 2.85–2.9 (2H, m, CH2C), 3.01–3.06
(2H, t, J = 7 Hz, CH2-phenyl), 4.14–4.17 (2H, t, J = 7
Hz, CH2CH2-phenyl), 4.27 (2H, br s, CH2N), 7.15–
7.48 (8H, m, CH-phenyl, C(1,2,3)H), 8.01–8.04 (1H,
d, J = 7.5 Hz, C(4)H) ppm. IR (KBr): 3442, 2922,
2855, 1629, 1579, 1443, 1358, 1238, 1140, 761, 700,
668, 640 cmꢀ1. EI-MS m/z 331, 302, 276, 263, 240,
227, 184, 171, 159, 129, 102, 91, 77. Anal. (C22H25N3):
C, H, N.
chloride; 250 MHz, DMSO-d6): d 3.10–3.14 (2H, t,
J = 6.25 Hz, CH2C), 3.14 (3H, s, CH3), 3.76–3.81 (2H,
t, J = 6.25 Hz, CH2N), 7.2–7.85 (9H, m, CH-arom),
11.06 (1H, br s, NH) ppm. IR (hydrochloride; KBr):
3395, 3050, 2898, 2594, 1625, 1444, 1256, 1074, 765,
692, 479 cmꢀ1. EI-MS m/z 235, 219, 192, 165, 145,
132, 116, 89, 77. Anal. (free base: C16H16N2): C, H, N.
4.2.5. 13-Methyl-5,6,13,13a-tetrahydro-8H-isoquino[1,2-
b]quinazolin-8-one (benzoevodiamine, 8). A solution of
3 g (22.9 mmol) of N-methylisatoic anhydride (1-meth-
4.2.3. 8-Methyl-5,8-dihydro-6H-isoquino[1,2-b]quinazo-
line (6). To a solution of 1 g (7.3 mmol) of 1-(2-amino-
phenyl)ethanol10 in 20 mL of toluene were added
dropwise 5 mL of thionyl chloride. A solid was formed.
The mixture was heated for 24 h under reflux, during
which time the solid dissolved again. After cooling, tol-
uene and excess thionyl chloride were removed under
reduced pressure. Without further characterization
the formed 1-[2-(sulfinylamino)phenyl]ethanol was dis-
solved in 50 mL of dried diethyl ether and dry HCl
was passed for 2 h through the solution.11 The precipi-
tated 2-(1-chloroethyl)benzenaminium chloride was
washed with dry ether and was used without further
characterization for the next step.
yl-2H-3,1-benzoxazine-2,4(1H)-dione)
and
4.45 g
(25.2 mmol) of 3,4-dihydroisoquinoline14 in 50 mL tolu-
ene was refluxed for 24 h. After cooling, the solvent was
removed under reduced pressure and the residue was
column chromatographed using n-hexane/ethyl acetate,
2:1 as eluent.
Light yellow crystals. 2.72 g (45% yield). Mp 138 ꢁC. 1H
NMR (250 MHz, CDCl3): d 2.85–2.87 (1H, m, CH2C),
2.96 (3H, s, CH3), 2.95–3.06 (1H, m, CH2C), 3.24–3.32
(1H, m, CH2N), 4.37–4.45 (1H, m, CH2N), 5.91 (1H,
s, CH), 6.85–6.88 (1H, t, J = 6.75 Hz, C(10)H), 6.97–
7.0 (1H, d, J = 8 Hz, C(12)H), 7.14–7.23 (4H, m,
C(1,2,3,4)H), 7.4–7.43 (1H, t, J = 6 Hz, C(11)H),
7.67–7.7 (1H, d, J = 6 Hz, C(9)H) ppm. IR (KBr):
3462, 2863, 1651, 1606, 1416, 1401, 1299, 1283, 1169,
1141, 929, 783 cmꢀ1. EI-MS m/z 263, 235, 219, 190,
160, 146, 133, 105, 92, 77. Anal. (C17H16N2O): C, H, N.
To a solution of 515 mg (3.5 mmol) of 3,4-dihydroiso-
quinolin-1(2H)-one in 10 mL of boiling toluene 4 mL
of phosphoric trichloride were added and the mixture
was refluxed. After 30 min, 675 mg (3.5 mmol) of 2-(1-
chloroethyl)benzenaminium chloride was added and
the mixture was refluxed for additional 4 h, during
which time a small second phase separated. To the
cooled mixture 15 mL of concentrated hydrochloric
acid and a large quantity of hot water were added.
After cooling, the solution was extracted twice with
diethyl ether. The organic layers were discarded. The
aqueous phase with alkalized with ammonia and
extracted three times with diethyl ether. The combined
organic phases of the second extraction were dried
with sodium sulfate, and the solvent removed under
reduced pressure. The residue was purified by column
chromatography using ethyl acetate/methanol, 1:1 as
eluent.
4.2.6. 13-Methyl-5,8,13,13a-tetrahydro-6H-isoquino[1,2-
b]quinazoline (9). To a suspension of 200 mg (5.3 mmol)
of lithium aluminium hydride in 20 mL of dried THF
was slowly added a solution of 1 g (3.8 mmol) of 13-
methyl-5,6,13,13a-tetrahydro-8H-isoquino[1,2-b]quin
azolin-8-one (8) in 40 mL of dried THF. After com-
pletion of addition, the mixture was refluxed for 3 h
and afterwards stirring was continued at rt overnight.
Excess hydride was destroyed by a careful addition of
diluted sodium hydroxide solution. The organic phase
was separated and the aqueous phase was extracted
twice with diethyl ether. The combined organic phas-

1976
M. Decker et al. / Bioorg. Med. Chem. 14 (2006) 1966–1977
es were dried over sodium sulfate, and the solvents
were removed under reduced pressure. The residue
was column chromatographed using methanol/ethyl
acetate, 1:1 as eluent.
4.3. Pharmacology
AChE (E.C.3.1.1.7, Type VI-S, from Electric Eel) and
BChE (E.C.3.1.1.8, from equine serum) were purchased
from Sigma–Aldrich (Steinheim, Germany). 5,50-Dithi-
obis(2-nitrobenzoic acid) (Ellmanꢁs reagent, DTNB),
acetylthiocholine (ATC) and butyrylthiocholine iodides
(BTC) were obtained from Fluka (Buchs, Switzerland).
Colourless oil. 660 mg (72% yield). 1H NMR (250 MHz,
CDCl3): d 2.64 (3H, s, CH3), 2.73–2.93 (2H, m,
CH2CH2N), 3.06–3.35 (2H, m, CH2CH2N), 3.89–3.96
(1H, d, J = 15.5 Hz, NCH2Caromat.), 4.08–4.14 (1H, d,
J = 15.5 Hz, NCH2Caromat.), 4.97 (1H, s, CH), 6.83–
7.06 (3H, m, C(9,10,12)H), 7.15–7.3 (4H, m,
C(2,3,4,11)H), 7.38–7.42 (1H, m, C(1)H) ppm. IR
(KBr): 3399, 2918, 2802, 1606, 1492, 1369, 1118, 935,
746 cmꢀ1. EI-MS m/z 250, 235, 220, 145, 132, 120,
103, 91, 77. Anal. (C17H18N2): C, H, N.
As references for ChE inhibition the established drugs
galanthamine and rivastigmine were used. Rivastigmine
hydrogentartrate was obtained from Novartis Pharma
AG, Basel, Switzerland. Galanthamine hydrobromide
was obtained from JANSSEN-CILAG GmbH, Neuss,
Germany.
4.2.7.
[1,5]diazecine (10a). To
14-Methyl-5,6,7,8,13,14-hexahydrodibenzo[b,h]-
suspension of 200 mg
The assay was performed as described in the following
procedure:21 a stock solution of the test compound
was prepared in ethanol, and the highest concentration
of the test compounds applied in the assay was
10ꢀ4 M (final concentration). In order to obtain an inhi-
bition curve, at least five different concentrations (nor-
mally in the range of 10ꢀ4 to 10ꢀ9 M) of the test
compound were measured at 25 ꢁC at 412 nm, each con-
centration in triplicate.
a
(0.67 mmol) of 13-methyl-8-oxo-5,8-dihydro-6H-isoqui-
no[1,2-b]quinazolin-13-ium chloride6 in 10 mL of dried
THF was added 6.4 mL of 1.0 M borane solution in
THF under N2-atmosphere. The solid got into solution
under evolution of hydrogen while stirring 2 h at rt. The
solution was refluxed for additional 3 h. The mixture
was cooled to rt and 5 mL of water was carefully added
drop-by-drop to the stirred mixture. After the addition
of 2 mL of 50% sodium hydroxide solution, stirring
was continued for 1 h at rt. The mixture was extracted
three times with 30 mL of dichloromethane. The com-
bined extracts were dried over sodium sulfate and were
evaporated under reduced pressure to give the crude
product, which was purified by column chromatography
using n-hexane/ethyl acetate, 1/1 as eluent.
For buffer preparation, 1.36 g of potassium dihydrogen
phosphate (10 mmol) was dissolved in 100 mL water
and adjusted with NaOH to pH 8.0 0.1. Enzyme solu-
tions were prepared to give 2.5 units/mL in 1.4 mL ali-
quots. Furthermore, 0.01 M DTNB solution, 0.075 M
ATC and BTC solutions, respectively, were used. A cuv-
ette containing 3.0 mL phosphate buffer, 100 lL of the
respective enzyme and 100 lL of the test compound solu-
tion was allowed to stand for 5 min, then 100 lL of
DTNB was added, and the reaction was started by addi-
tion of 20 lL of the substrate solution (ATC/BTC). The
solution was mixed immediately, and exactly 2.5 min after
substrate addition the absorption was measured. For the
reference value, 100 lL of water replaced the test com-
pound solution. For determining the blank value, addi-
tionally 100 lL water replaced the enzyme solution.
1
White crystals. 90 mg (54% yield). Mp 90 ꢁC. H NMR
(250 MHz, CDCl3):
d 2.7–2.74 (2H, t, J = 5 Hz,
CH2CH2N), 2.81 (3H, s, CH3), 2.86–2.91 (2H, t,
J = 5 Hz, CH2CH2N), 3.63 (2H, s, CH2NCH3), 3.69
(2H, s, CH2NH), 6.12 (1H, br s, NH), 6.63–6.72 (2H,
m, C(9, 12)H), 7.02–7.17 (5H, m, C(1,2,3,4,10)H),
7.24–7.3 (1H, t, J = 7.5 Hz, C(11)H) ppm. IR (KBr):
3465, 2914, 1606, 1508, 1448, 1274, 1081, 752 cmꢀ1
.
EI-MS m/z 252, 220, 193, 147, 132, 120, 91, 77. Anal.
(C17H20N2O): C, H, N.
The inhibition curve was obtained by plotting percent-
age enzyme activity (100% for the reference) versus log-
arithm of test compound concentration.
4.2.8. 5,6,7,8,13,14-Hexahydrodibenzo[b,h][1,5]diazecine
(10b). This compound was prepared in the same manner
as described for compound 10a. As starting material
248 mg (1 mmol) of 5,6-dihydro-8H-isoquino[1,2-b]qui-
nazolin-8-one6,16 and 10 mL of 1.0 M borane solution
in THF were used.
For the kinetic measurements the following substrate
concentrations were used: 0, 25, 50, 90, 150, 266 and
452 lM. In contrast to the above-described affinity mea-
surements, the reaction time was extended to 4 min be-
fore measurement of the absorption. Vmax and Km
values, respectively, of the Michaelis–Menten kinetics
were calculated by non-linear regression out of the sub-
strate velocity curves. Linear regression was used for
calculating the Lineweaver–Burk plots.
Ethyl acetate/n-hexane, 2:1 was used as eluent.
1
Colourless oil. 76 mg (32% yield). H NMR (250 MHz,
CDCl3): d 2.72–2.77 (2H, t, J = 6.5 Hz, CH2CH2N),
2.88–2.93 (2H, t, J = 6.5 Hz, CH2CH2N), 3.62 (2H, s,
Carom.CH2NHCarom.), 3.71 (2H, s, Carom.CH2NHCali-
phat.), 4.7 (2H, br s, 2· NH), 6.65–6.77 (2H, m,
C(9,12)H), 7.0–7.17 (6H, m, CHarom.) ppm. IR (KBr):
3428, 2803, 1615, 1495, 1460, 1289, 1085, 935,
748 cmꢀ1. EI-MS m/z 239, 233, 220, 206, 146, 132,
117, 106, 77. Anal. (C16H18N2): C, H, N.
Acknowledgments
Financial support by the ꢁFonds der Chemischen Indust-
rieꢀ (FCI) is gratefully acknowledged. Appreciation is
expressed to Petra Wiecha for technical assistance.

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