Synthesis and biological evaluation of tacrine-thiadiazolidinone hybrids as dual acetylcholinesterase inhibitors

Article abstract of DOI:10.1002/ardp.200400919

The synthesis of tacrine-thiadiazolidinone hybrids is described. These compounds are designed as dual acetylcholinesterase inhibitors binding simultaneously to the peripheral and catalytic sites of the enzyme. All tested compounds exhibit significant AChE inhibitory activity. Competition assays using propidium as reference of selective ligand for the peripheral anionic site on acetylcholinesterase indicates the influence of the designed compounds over the peripheral site. They can be considered as new leads in the optimization of Alzheimer's disease modifying agents.

Full text of DOI:10.1002/ardp.200400919

18
DOI 10.1002/ardp.200400919
Arch. Pharm. Chem. Life Sci. 2005, 338, 18−23
Synthesis and Biological Evaluation of
Tacrine-Thiadiazolidinone Hybrids as Dual Acetyl-
cholinesterase Inhibitors
Isabel Dorronsoroa, Diana Alonsoa, Ana Castrob, Maria del Montea,
a
a
´
´
Esther Garcıa-Palomero , and Ana Martınez
a Neuropharma, S.A. Avda. de la Industria, 52, 28760 Tres Cantos, Madrid, Spain
b
´
´
Instituto de Quımica Medica (CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain
The synthesis of tacrine-thiadiazolidinone hybrids is described. These compounds are designed as dual
acetylcholinesterase inhibitors binding simultaneously to the peripheral and catalytic sites of the en-
zyme. All tested compounds exhibit significant AChE inhibitory activity. Competition assays using
propidium as reference of selective ligand for the peripheral anionic site on acetylcholinesterase indica-
tes the influence of the designed compounds over the peripheral site. They can be considered as new
leads in the optimization of Alzheimer·s disease modifying agents.
Keywords: Thiadiazolidinone; Acetylcholinesterase; Dual inhibitor; Alzheimer’s disease
Received: July 12, 2004; Accepted: September 2, 2004 [FP919]
Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder which is one of the most common causes of mental
deterioration in elderly people, accounting for about
50Ϫ60% of the overall cases of dementia among persons
over 65 years of age [1]. Brain regions that are associated
with higher mental functions, particularly the neocortex and
hippocampus, are those most affected by the characteristic
pathology of AD. This includes the extracellular deposits of
β-amyloid (βA) in senile plaques [2], the intracellular forma-
tion of neurofibrillary tangles containing an abnormally
phosphorylated protein tau [3] and the loss of neuronal syn-
apsis and pyramidal neurons [4]. Nevertheless, current treat-
ment approaches in this disease continue being primarily
symptomatic, with the major therapeutic strategy based on
the cholinergic hypothesis and specifically on acetylcholin-
esterase (AChE) inhibition [5]. As result, during last decade,
due to the cholinergic hypothesis of AD various cholinergic
drugs have been launched on the market, primarily AChE
inhibitors as tacrine [6], donepezil [7] or rivastigmine [8],
and more recently galanthamine [9], modestly improving the
cognitive function of Alzheimer’s patients (Figure 1).
Figure 1. Commercial available AChE inhibitors.
transition from βA into its amyloidogenic form in vitro and
therefore to amyloid fibrils [12]. The peripheral anionic site
of the AChE is involved in the adhesion function. AChE
directly promotes the assembly of βA peptide into amyloid
fibrils forming stable βA-AChE complexes through its Trp
279 residue located at the peripheral site of the enzyme by
hydrophobic interactions [13].
Recent studies have shown that AChE has secondary non-
cholinergic functions [10]. AChE colocalizes with βA pep-
tide deposits present in the brain of Alzheimer’s patients
[11]. It is postulated that AChE binds to βA acting as a
“pathological chaperone” and inducing a conformational
Considering the non-cholinergic aspects of the cholinergic
enzyme, AChE inhibitors able to interact with both the
catalytic and the peripheral site may simultaneously allevi-
ate the cognitive deficit in Alzheimer’s patients and what it
is more important, avoid the assembly of βA which re-
presents a new way to delay the neurodegenerative process
(Figure 2) [14, 15].
´
Correspondence: Ana Martınez, NeuroPharma, S.A., Avda. de la
Industria 52., Tres Cantos, 28760, Madrid. Phone: ϩ34 91 562-29-00,
Fax: ϩ34 91 803-46-60, amartinez@neuropharma.es
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 18−23
Tacrine-Thiadiazolidinone Hybrids as Dual AChE Inhibitors
19
Figure 3. General chemical structure of tacrine-thiadiazolidi-
none hybrids.
salts. The reactivity of these oxathiadiazolium salts with
aryl amines had previously been studied in our group [19].
The synthetic steps can be summarized as follows: a) chlori-
nation of alkylisothiocyanates in an inert atmosphere, and
b) subsequent reaction with N-alkylisocyanates to yield the
3-oxathiadiazolium salts 2aϪc via the intermediate imino-
chloromethylsulfenyl chloride. It is worth-mentioning that
inert atmosphere is required in this step b) to avoid the
1,2,4-thiadiazolidin-3,5-diones formation by oxidation of
the corresponding oxathiadiazolium salts. These salts were
not isolated and solvent is removed under nitrogen atmos-
phere to continue with next reaction step.
Figure 2. An AChE cartoon showing the relative disposition
of catalytic and peripheral sites.
On the other hand, 9-alkylaminotetrahydroacridines were
synthesized following a procedure previously described [20].
Reaction of 1,3,4,10-tetrahydro-2H-acridin-9-one with an
excess of POCl3 gave the 9-chloro-1,2,3,4-tetrahydroacridine
3 in quantitative yields. Reaction of 3 with α,ω-diamino-
alkyl derivatives afforded the intermediates 9-alkylamino-
tetrahydroacridines 4aϪd in good yields (60Ϫ70%).
We previously described N-benzylpiperidine derivatives of
1,2,4-thiadiazolidinones as a new acetylcholinesterase in-
hibitors [16]. Molecular dynamic simulation using X-ray
crystal structure of AChE suggested a parallel disposition
between the indole side chain of Trp 279 and the heterocycle
˚
present in the inhibitor at an average distance of 3.84 A,
Finally, in situ reaction of the iminochloromethylsulfenyl
chloride previously synthesized with the primary alkyl am-
ines 4aϪd in a 1:1 salt:amine ratio afforded the required
dimeric compounds 5Ϫ10. The structures of these com-
pounds were confirmed in basis to their analytical and spec-
trostocopic data (1H and 13C NMR) which are collected in
the experimental part.
which allows the formation of an off-centre π-stacking inter-
action.
Following with our research in this field, and looking for
simultaneous catalytic and peripheral binding site, we report
here the synthesis, AChE inhibition, and evaluation of the
possible influence on the βA peptide aggregation process,
of novel tacrine-thiadiazolidinone compounds which pos-
sess the general structure I (Figure 3). In this first approach,
we have specially modified the length of methylenes chain
between both heterocycles units that could influence in the
required binding to the peripheral site [17]. The dual bind-
ing site was pursued in these structures using the thiadiazole
moiety that interacts with the peripheral site [16] and the
tacrine heterocycle which it is recognised as a catalytic
AChE inhibitor [18].
These heterodimeric tacrine-thiadiazole compounds 5Ϫ10
were assayed for AChE (bovine erythrocytes) inhibition by
the method of Ellman et al. [21]. As Table 1 shows, all the
new compounds exhibit significant AChE inhibitory activity
comprised in the range of the commercial available inhibi-
tors using as standard references in our assays. In a first
approach to structure-activity relationships, the inspection
of these data suggests that inhibitory potency is located
around 7-9 methylene chain in the linker between the two
specific heterocycles. Meanwhile, modifications on N2-sub-
stituent influence greater the activity (compound 6 versus
7) than differences in the substituent attached to the nitro-
gen in position 4 (compound 9 versus 19).
Results and discussion
The synthesis of the compounds was achieved following a
convergent pathway strategy summarized in Scheme 1, in
which the key synthetic step was the nucleophilic substi-
tution by primary alkyl amines on the oxathiadiazolium
With the aim to predict the potential in vivo behaviour of
the inhibitors, neuronal AChE inhibitory activity were as-
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Martınez et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 18−23
Scheme 1. Synthesis of tacrine-thiadiazolidinone hybrids. (i) Cl2, dry hexane (Ϫ15°C); (ii) RЈ-NCO, r.t., 10 h; (iii) POCl3, ᭝,
3 h; (iv) NH2-(CH2)n-NH2, pentanol, ᭝; (v) TEA 2eq, N2, THF anh, r.t., 24 h.
Table 1. Inhibition data of AChE from bovine erythrocytes
Table 3. Propidium competition assay results.
Compound
R
RЈ
n
IC50 AChE [µM]
Compound
Propidium competition
[µM]
5
Et
Et
Et
Et
Et
iPr
iPr
Pr
6
7
7
8
9
9
2.3
6
0.25
0.45
0.20
0.12
0.12
0.019
0.17
1.41
5
7
8
>100
>100
0.1
7
8
9
iPr
iPr
iPr
Donepezil
1
10
CH2CO2Et
Donepezil
Tacrine
Galantamine
Finally, a propidium competition assay was evaluated for
some compounds. Propidium has been demonstrated in pre-
vious studies to be a selective ligand for the peripheral
anionic site on acetylcholinesterase [22]. Propidium exhibits
an increase in fluorescence on binding to AChE peripheral
site, making it a useful probe for competitive ligand [23]. Its
association with this site is monitored by fluorescence. This
is an indirect method to predict the potential effect of the
inhibitors on β-A aggregation. Whilst derivatives 5 and 7
were unable to displace propidium in this assay, compound
8 displaces the peripheral inhibitor propidium at the range
of 10Ϫ7 M, find it 10 fold more potent than donepezil
(Table 3).
Table 2. Inhibition data of AChE from human neuroblastoma
cell lines.
Compound
IC50 AChE [µM]
SK-N-SY
0.41
SH-SY5Y
0.3
6
8
9
0.44
0.48
0.30
0.33
0.39
0.39
Tacrine
sayed for three of them using AChE enzyme preparations
obtained from different neuroblastoma cell line (Table 2).
These inhibition data shows that compounds present similar
inhibition potency than tacrine in human AChE.
In summary, a novel type of heterodimeric tacrine-thiadi-
azolidinone compound has been synthesized, which could
be considered as new lead compounds in the optimisation
of disease modifying agents [24]. This kind of new drugs
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 18−23
Tacrine-Thiadiazolidinone Hybrids as Dual AChE Inhibitors
21
(sept, 1H, J ϭ 6.6 Hz), 3.80 (m, 2H), 3.73 (c, 2H, J ϭ 7.0 Hz), 3.23
(t br, 2H, J ϭ 5.9 Hz), 2.98 (t, 2H, J ϭ 6.8 Hz), 2.60 (t br, 2H, J ϭ
6.8 Hz), 1.86Ϫ1.74 (m, 4H), 1.63 (t, 4H, J ϭ 6.6 Hz), 1.39 (m, 4H),
1.21 (d, 6H, J ϭ 6.6 Hz), 1.20 (t, 3H, J ϭ 7.0 Hz). 13C NMR
(CDCl3, 300 MHz, δ): 161.5, 154.4, 146.9, 148.1, 139.1, 132.0, 128.1,
124.9, 123.9, 53.3, 48.8, 46.8, 37.5, 31.3, 30.6, 29.0, 27.2, 26.7, 23.7,
22.0, 21.0, 13.0. EI-MS: m/z [Mϩ]ϩ 468.
could alleviate the cholinergic deficit occurring in different
state of Alzheimer’s disease, inhibiting both cholinesterases
and thus restoring the acetylcholine levels, and presumably
could affect on the senile plaques formation.
Experimental
2-Ethyl-4-isopropyl-5-[7-(1,2,3,4-tetrahydro-acridin-9-yl-amino)-
heptyl-imino]-[1,2,4]thiadiazolidin-3-one (6)
Chemistry
Reagents: ethylisothiocyanate (0.569 mL, 6.5 mmol), KMnO4 (500
mg, 3.16 mmol), HCl (3.1 mL, 3.4 mmol), isopropylisocyanate
(0.640 mL, 6.5 mmol), triethylamine (0.094 mL, 0.68 mmol) and 9-
(7-aminoheptylamino)-1,2,3,4-tetrahydroacridine (105 mg, 0.34
mmol). Purification: silica gel column chromatography using Ac-
OEt/MeOH (4:1). Yellow syrup (24 mg, 15%). 1H NMR (CDCl3,
300 MHz, δ): 8.36 (d, 1H, J ϭ 8.4 Hz), 8.09 (d, 1H, J ϭ 8.4 Hz),
7.63 (t, 1H, J ϭ 8.4 Hz), 7.39 (t, 1H, J ϭ 8.4 Hz), 5.25 (s br, 1H),
4.58 (sept, 1H, J ϭ 6.6 Hz), 3.80 (m, 2H), 3.73 (q, 2H, J ϭ 7.0 Hz),
3.23 (t br, 2H, J ϭ 5.9 Hz), 2.98 (t, 2H, J ϭ 6.8 Hz), 2.60 (t br, 2H,
J ϭ 6.8 Hz), 1.86Ϫ1.74 (m, 4H), 1.63 (t, 2H, J ϭ 6.6 Hz), 1.39 (m,
4H), 1.21 (d, 6H, J ϭ 6.6 Hz), 1.20 (t, 3H, J ϭ 7.0 Hz). 13C NMR
(CDCl3, 300 MHz, δ): 160.2, 154.6, 146.9, 148.1, 139.1, 131.7, 128.1,
124.9, 123.9, 53.3, 48.8, 46.8, 38.2, 31.3, 30.6, 29.0, 27.2, 26.7, 23.7,
22.0, 21.0, 12.6. EI-MS: m/z [Mϩ]ϩ 481.
Reagents and solvents were purchased from common commercial
suppliers and were used without further purification. Chromato-
graphic separations were performed on silica gel, using either flash
column chromatography (CC, using Kieselgel 60 Merck of 230Ϫ400
mesh) or preparative centrifugal thin layer chromatography (CTLC,
on a circular plate coated with a 1 mm layer of Kieselgel 60 PF254
gipshaltig, Merck, using a Chromatotron®). Compounds were de-
tected with UV light (254 nm), iodine chamber, or ninhydrin.
Nuclear magnetic resonance spectra were recorded in CDCl3 solu-
tions, using Varian Unity-500 or Varian Mercury 400 MHz spec-
trometers. Typical spectral parameters for 1H NMR were: spectral
width 10 ppm, pulse width 9 µs (57°), data size 32 K. The acqui-
sition parameters in decoupled 13C NMR spectra were: spectral
width 16 kHz, acquisition time 0.99 s, pulse width 9 µs (57°), data
size 32 K. Chemical shifts are reported in δ values (ppm) relative to
internal Me4Si and J values are reported in Hertz. Other experi-
ments such HSQC (Heteronuclear Single-Quantum Coherence) and
HMBC (Heteronuclear Multiple Bond Correlation) were obtained
in standard conditions. Mass spectra (MS) were obtained by elec-
tronic impact (EI) at 70 eV in a Hewlett-Packard 5973 spectrometer
(with direct insertion probe) or by electrospray (ES) in a Waters
ZQ2000 spectrometer.
4-Ethyl-2-propyl-5-[7-(1,2,3,4-tetrahydro-acridin-9-yl-amino)-
heptyl-imino]-[1,2,4]thiadiazolidin-3-one (7)
Reagents: ethylisothiocyanate (0.57 mL, 6.5 mmol), KMnO4 (500
mg, 3.16 mmol), HCl (3.1 mL, 3.4 mmol), propylisocyanate (0.60
mL, 6.5 mmol), triethylamine (0.12 mL, 1.26 mmol) and 9-(7-ami-
noheptylamino)-1,2,3,4-tetrahydroacridine (200 mg, 0.63 mmol).
Purification: silica gel column chromatography using CH2Cl2/
MeOH (8:1). Yellow syrup (12 mg, 4%). 1H NMR (CDCl3, 300
MHz, δ): 8.35 (d, 1H, J ϭ 8.4 Hz), 8.10 (d, 1H, J ϭ 8.4 Hz), 7.63
(t, 1H, J ϭ 8.4 Hz), 7.39 (t, 1H, J ϭ 8.4 Hz), 4.95 (s br, 1H), 3.80
(c, 2H, J ϭ 7.0 Hz), 3.66 (br, 2H), 3.40 (t , 2H, J ϭ 7.0 Hz), 3.18
(br, 2H), 2.98 (t, 2H, Jϭ 7.0 Hz), 2.60 ( br, 2H), 1.86-1.74 (m, 4H),
1.65 (q, 2H, J ϭ 7.0 Hz), 1.63 (br, 4H), 1.39 (m, 6H), 1.20 (t, 3H,
J ϭ 7.0 Hz), 0.95 (t, 3H, J ϭ 7.0 Hz). 13C NMR (CDCl3, 300 MHz,
δ): 160.4, 154.6, 147.1, 148.1, 139.0, 131.7, 128.1, 124.9, 123.9, 53.3,
48.8, 46.8, 38.2, 31.3, 30.6, 29.0, 27.2, 26.7, 23.7, 22.0, 21.0,12.8,
10.6. EI-MS: m/z [Mϩ]ϩ 481.
General method for the synthesis of compounds 5Ϫ10
Chlorine was bubbled slowly through a solution of alkylisothio-
cyanate in dry hexane (15 mL) under nitrogen atmosphere at
Ϫ15 °C to Ϫ10°C. Chlorine was generated by addition of 35% HCl
to KMnO4. The temperature of the reaction mixture was carefully
controlled during the addition step. At this point the alkyl-S-chloro-
isothiocarbamoyl chloride was formed. Afterward, alkylisocyanate
was added. The mixture was stirred at room temperature during 10
h and the solvent was evaporated to dryness. The residue was dis-
solved in anhydrous tetrahydrofurane (10 mL). After this point the
amine and triethylamine were added. The reaction mixture was
stirred for 24 h at room temperature, the white solid was filtered off,
the solvent was evaporated under reduced pressure and the residue
purified by silica gel column chromatography using as eluent mix-
tures of solvents in the proportions indicated for each case.
4-Ethyl-2-isopropyl-5-[8-(1,2,3,4-tetrahydro-acridin-9-yl-amino)-
octyl-imino]-[1,2,4]thiadiazolidin-3-one (8)
Reagents: ethylisothiocyanate (0.57 mL, 6.5 mmol), KMnO4 (500
mg, 3.16 mmol), HCl (3.1 mL, 3.4 mmol), isopropylisocyanate (0.60
mL, 6.5 mmol), triethylamine (0.12 mL, 1.26 mmol) and 9-(8-ami-
nooctylamino)-1,2,3,4-tetrahydroacridine (200 mg, 0.61 mmol).
Purification: silica gel column chromatography using CH2Cl2/
MeOH (25:1). Yellow syrup (7 mg, 2.3%). 1H NMR (CDCl3, 300
MHz, δ): 8.36 (d, 1H, J ϭ 8.4 Hz), 8.09 (d, 1H, J ϭ 8.4 Hz), 7.63
(t, 1H, J ϭ 8.4 Hz), 7.39 (t, 1H, J ϭ 8.4 Hz), 4.58 (sept, 1H, J ϭ
6.6 Hz), 3.80 (m, 2H), 3.73 (c, 2H, J ϭ 7.0 Hz), 3.23 (t br, 2H, J ϭ
5.9 Hz), 3.00 (t, 2H, J ϭ 6.8 Hz), 2.60 (t br, 2H, J ϭ 6.8 Hz),
1.86Ϫ1.74 (m, 4H), 1.63 (m, 4H), 1.39 (m, 8H), 1.21 (d, 6H, J ϭ
6.6 Hz), 1.20 (t, 3H, J ϭ 7.0 Hz). 13C NMR (CDCl3, 300 MHz, δ):
160.2, 154.6, 146.9, 148.1, 139.1, 131.7, 128.1, 124.9, 123.9, 53.3,
48.8, 46.8, 38.2, 31.3, 30.6, 29.0, 27.2, 26.7, 23.7, 22.0, 21.0, 12.6.
EI-MS: m/z [Mϩ]ϩ 496.
9-Alkylaminotetrahydroacridines has been synthesized following the
procedure previously reported in bibliography [20].
4-Ethyl-2-isopropyl-5-[6-(1,2,3,4-tetrahydro-acridin-9-yl-amino)-
hexyl-imino]-[1,2,4]thiadiazolidin-3-one (5)
Reagents: ethylisothiocyanate (0.57 mL, 6.5 mmol), KMnO4 (500
mg, 3.16 mmol), HCl (3.1 mL, 3.4 mmol), isopropylisocyanate (0.60
mL, 6.5 mmol), triethylamine (0.12 mL, 1.26 mmol) and 9-(6-ami-
nohexylamino)-1,2,3,4-tetrahydroacridine (200 mg, 0.66 mmol).
Purification: silica gel column chromatography using CH2Cl2/
MeOH (25:1). Yellow syrup (5 mg, 1.6%). 1H NMR (CDCl3, 300
MHz, δ): 8.36 (d, 1H, J ϭ 8.4 Hz), 8.09 (d, 1H, J ϭ 8.4 Hz), 7.63
(t, 1H, J ϭ 8.4 Hz), 7.39 (t, 1H, J ϭ 8.4 Hz), 5.25 (s br, 1H), 4.58
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Martınez et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 18−23
2-Ethyl-4-isopropyl-5-[9-(1,2,3,4-tetrahydro-acridin-9-yl-amino)-
nonyl-imino]-[1,2,4]thiadiazolidin-3-one (9)
in a 5% CO2 humidified incubator at 37°C. The human neuroblas-
toma cell line SK-N-SH was cultured in RPMI 1640 medium, sup-
plemented with 10% fetal bovine serum and 1% penicillin/strepto-
mycin, and grown in a 5% CO2 humidified incubator at 37°C. The
cells were plated at 250 ϫ 103 cells for each reaction, at least, 48 h
before the AChE activity measure. Cells were washed and harvested
in 0.1 M sodium phosphate buffer pH 8, at 4°C.
Reagents: ethylisothiocyanate (0.569 mL, 6.5 mmol), KMnO4 (500
mg, 3.16 mmol), HCl (3.1 mL, 3.4 mmol), isopropylisocyanate
(0.640 mL, 6.5 mmol), triethylamine (0.140 mL, 1.0 mmol) and 9-(9-
aminononylamino)-1,2,3,4-tetrahydroacridine (173 mg, 0.5 mmol).
Purification: silica gel column chromatography using AcOEt/MeOH
(15:1).Yellow syrup (36 mg, 14%). 1H NMR (CDCl3, 300 MHz, δ):
7.94 (dd, 1H, J ϭ 8.0, 0.5 Hz), 7.90 (d, 1H, J ϭ 8.8 Hz), 7.53 (ddd,
1H, J ϭ 8.2, 7.1, 1.2 Hz), 7.32 (ddd, 1H, J ϭ 8.2, 7.1, 1.1 Hz), 4.59
(sept, 1H, J ϭ 6.6 Hz), 3.74 (q, 2H, J ϭ 7.1Hz), 3.48 (t, 2H, J ϭ
7.1 Hz), 3.01 (s br, 2H), 2.98 (t, 2H, J ϭ 7.1 Hz), 2.68 (s br, 2H),
1.91-1.88 (m, 4H), 1.66Ϫ1.59 (m, 4H), 1.29 (s br, 11H), 1.21 (d, 6H,
J ϭ 6.6 Hz), 1.20 (t, 3H, J ϭ 7.1 Hz). 13C NMR (CDCl3, 300 MHz,
δ): 158.5, 154.9, 148.5, 148.3, 139.5, 131.8, 128.7, 123.9, 123.1, 53.8,
49.8, 47.1, 38.5, 34.0, 32.0, 31.0, 29.7, 29.6, 27.6, 27.2, 25.0, 23.3,
22.9, 21.2, 12.9. EI-MS: m/z [Mϩ]ϩ 509.
Propidium competition assay
Propidium exhibits an increase in fluorescence on binding to AChE
peripheral site, making it a useful probe for competitive ligand bind-
ing to the enzyme.
Fluorescence was measured in a Fluostar optima plate reader
(BMG). Measurements were carried out in 100 µL solution volume,
in 96-well plates. The buffer used was 1 mM Tris/HCl, pH 8.0. 10
µM AChE was incubated, at least 6 h, with the molecules at differ-
ent concentrations. 20 µM propidium iodide was added 10 min be-
fore fluorescence measurement. The excitation wavelength was 485
nm, and that of emission, 620 nm.
4-Isopropyl-3-oxo-5-[9-(1,2,3,4-tetrahydro-acridin-9-yl-amino)-
nonyl-iminio]-[1,2,4]thiadiazolidine-2-carboxylic acid ethyl ester
(10)
Reagents: ethoxycarbonylmethylisothiocyanate (0.8 mL, 6.5 mmol),
KMnO4 (500 mg, 3.16 mmol), HCl (3.1 mL, 3.4 mmol), isopropyl-
isocyanate (0.640 mL, 6.5 mmol), triethylamine (0.140 mL, 1.0
mmol) and 9-(9-aminononylamino)-1,2,3,4-tetrahydroacridine (173
mg, 0.5 mmol). Purification: silica gel column chromatography
References
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using AcOEt/MeOH (15:1). Yellow syrup (10 mg, 0.1%). H NMR
haup, K. Beyreuther, C. Czech, Brain Pathol. 2001, 11, 1Ϫ11.
(CDCl3, 300 MHz, δ): 7.98 (d, 2H, J ϭ 7.8 Hz), 7.57 (t, 1H, J ϭ
7.6 Hz), 7.35 (t, 1H, J ϭ 7.6 Hz), 4.59 (sept, 1H, J ϭ 6.6 Hz),4.17
(q, 2H, J ϭ 7.1 Hz), 3.74 (q, 2H, J ϭ 7.1 Hz), 3.68Ϫ3.56 (m, 2H),
3.09 (s br, 2H), 2.98 (t, 2H, J ϭ 6.8 Hz), 2.65 (s br, 2H), 1.95Ϫ1.90
(m, 4H), 1.78Ϫ1.59 (m, 4H), 1.29Ϫ1.18 (m, 11H),1.21 (d, 6H, J ϭ
6.6 Hz), 1.20 (t, 3H, J ϭ 7.1 Hz). 13C NMR (CDCl3, 300 MHz,δ):
173.4, 151.6, 143.9, 148.3, 136.7, 131.8, 128.7, 123.6, 123.1, 77.2,
49.5, 42.3, 31.8, 30.7, 29.3, 26.9, 24.7, 23.0, 21.0, 14.1, 12.6. EI-MS:
m/z [MϩHϩ]ϩ 554.
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