Homobivalent quinazolinimines as novel nanomolar inhibitors of cholinesterases with dirigible selectivity toward butyrylcholinesterase

Article abstract of DOI:10.1021/jm060682m

Homobivalent dimers of quinazolinimines, which bridge the imine nitrogen atoms via a hepta- and an octamethylene spacer, with different ring sizes of the alicycles were synthesized from the corresponding quinazolinethiones. The resulting compounds show > 100-fold increase of inhibitory activities compared to related monomeric compounds yielding low-nanomolar inhibitors. For heptamethylene dimers, mixed inhibition profiles were obtained, whereas for the octamethylene compounds selectivity toward butyrylcholinesterase (> 180) can be achieved with an eight-membered alicycle.

Full text of DOI:10.1021/jm060682m

J. Med. Chem. 2006, 49, 5411-5413
5411
Chart 1. AChE Inhibitory Drug Tacrine and BChE Inhibitory
Quinazolinimines 2 and 3
Homobivalent Quinazolinimines as Novel
Nanomolar Inhibitors of Cholinesterases with
Dirigible Selectivity toward Butyrylcholinesterase
Michael Decker*
Lehrstuhl fu¨r Pharmazeutische/Medizinische Chemie, Institut fu¨r
Pharmazie, Friedrich-Schiller-UniVersita¨t Jena, Philosophenweg 14,
D-07743 Jena, Germany
ReceiVed June 7, 2006
Abstract: Homobivalent dimers of quinazolinimines, which bridge the
imine nitrogen atoms via a hepta- and an octamethylene spacer, with
different ring sizes of the alicycles were synthesized from the
corresponding quinazolinethiones. The resulting compounds show
>100-fold increase of inhibitory activities compared to related mon-
omeric compounds yielding low-nanomolar inhibitors. For heptameth-
ylene dimers, mixed inhibition profiles were obtained, whereas for the
octamethylene compounds selectivity toward butyrylcholinesterase
(>180) can be achieved with an eight-membered alicycle.
molar activities.9,10 Recently, on the basis of a molecular
modeling approach, suitable modification of the spacer unit led
to the discovery of tacrine-based heterobivalent inhibitors with
nanomolar or subnanomolar activities and high BChE selectivi-
ties.11,12 This rational approach makes use of the presence of
additional binding sites present in BChE.
In the search for novel compounds and templates with ChE
inhibiting properties, 5,6-dihydro-8H-isoquino[1,2-b]quinazolin-
8-imine (2, Chart 1) was developed from the alkaloids dehy-
droevodiamine and deoxyvasicine and identified as a micromolar
ChE inhibitor with >10-fold selectivity toward BChE.13 SAR
studies were performed, and quinazolinimines 3 were identified
as inhibitors with micro- and submicromolar inhibitory activities.
By variation of the distance of the phenyl ring to the heterocycle
and especially by the change of the size of the alicycle, it was
possible to obtain either activities at both enzymes or highly
selective BChE inhibitors; BChE selectivity increased with
increasing size of the alicycle, through increasing activity toward
BChE and/or decreasing activity at AChE.14 Selectivity was
most probably reached by the fact that BChE possesses a larger
void at the active site gorge compared to AChE.15 This
assumption is supported by the fact that kinetic measurements
revealed competitive and reversible inhibition of these com-
pounds at the active site.14
To further improve activities of quinazolinimines toward
cholinesterases, the bivalent approach was applied to this novel
class of ChE inhibitors. The aim was to obtain more potent
inhibitors with additional strong activity at BChE or even BChE-
selective compounds. Because of the fact that the quinazolin-
imines bind at the active site like tacrine, similar spacer lengths
of the most potent tacrine dimers (a heptamethylene spacer
exhibits the highest activities) were applied.10,16
The homobivalent inhibitors could be synthesized in a
straightforward four-step synthetic pathway described in Scheme
1. Lactams with varying ring sizes (4a-d) were activated by
reaction with Meerwein salt (triethyloxonium tetrafluoroborate)
to give their iminium ethers (5a-d), which readily reacted with
anthranilic acid (or 4-chloroanthranilic acid in the case of
compound 6a) to give the respective quinazolinones (6a-e).14,17
The quinazolinones were transformed into yellow quinazo-
linethiones (7a-e) using Lawesson’s reagent (2,4-bis(4-meth-
oxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide).14,18 The
quinazolinethiones were reacted with R,ω-diaminoheptane and
-octane, respectively, in the presence of triethylamine and silver
nitrate to give homobivalent quinazolinimines 8a-j.
The pharmacotherapy of the most common form of dementia
in elderly people, Alzheimer’s disease (AD), is very limited,
represented by just five FDA-approved drugs. Four of them are
acetylcholinesterase inhibitors (donepezil, tacrine, rivastigmine,
and galanthamine). The use of acetylcholinesterase (AChE)
inhibitors (AChEIs) represents the treatment of choice for mild
cases of AD.1
AChE also possesses a second binding site, the peripheral
anionic binding site (PAS). A hydrophobic environment close
to the PAS interacts with â-amyloid peptide (Aâ) and thus
accelerates amyloid fibril formation.2 This widens the application
of AChEIs from purely symptomatic treatment to a possibly
causative one. Recently the less substrate-specific butyrylcho-
linesterase (BChE) also got into the focus. BChE appears in
serum, liver, heart, and the central nervous system, where it
seems to play a key role in neurogenesis and cell proliferation/
differentiation.3 Its corresponding gene is amplified in some
neuronal disorders, and it is involved in tumorgenesis.3 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 not affected or
even up-regulated, making BChE very available in neuritic
plaques.4 Therefore, mixed AChE/BChE inhibition profiles may
result in higher efficacy, reflected by the fact that the inhibitor
rivastigmine, which is active at both enzymes, exhibits a good
correlation of BChE inhibition and improvement of cognition.5
As a novel tool in drug development, the bivalent ligand
approach has been established in which two moieties of a known
ligand are covalently connected by a suitable spacer. Especially
for G-protein-coupled receptors and also transporter systems,
this approach proved highly successful for increasing affinities
and also selectivities.6,7
This method has also been applied to AChE inhibitors
yielding both homo- and heterobivalent inhibitors.8 In particular,
the high-activity (also toward BChE) ChE inhibitor tacrine (1,
Chart 1), although its use is limited by its strong hepatotoxicity,
served as a suitable monomer and led to the discovery of several
homobivalent compounds with nanomolar and even subnano-
Another possible method to prepare dimers by connecting
the two heterocyclic moieties by reacting unsubstituted imines
(e.g., five-membered imine 10, prepared by direct reaction of
2-pyrrolidinone with anthranilonitrile 913,19 or eight-membered
imine 11,13,19 Scheme 2) with R,ω-dicarbonic acids was not
* To whom correspondence should be addressed. Phone: +49-3641-
949817. Fax: +49-3641-949802. E-mail: m.decker@uni-jena.de.
10.1021/jm060682m CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/11/2006

5412 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Letters
Table 1. AChE and BChE Inhibition Resultsa
Scheme 1a
a Reagents: (i) (a) [(C2H5)3O]BF4, chloroform, room temp, 15 h; (b)
cold NaOH, CH2Cl2 extraction; (ii) acetone, 10 °C, 2 h f 60 °C, 4 h; (iii)
Lawesson’s reagent, toluene, reflux, 12 h; (iv) 0.5 equiv of R,ω-diamine,
AgNO3, TEA, toluene, reflux, 3 h.
Scheme 2a
a Reagents: (i) POCl3, TEA, chloroform, reflux, 24 h; (ii) 2 equiv of
acetic anhydride, room temperature, 5 h.
further developed because an acetylated imine (12) showed
strongly decreased activities probably due to a loss of basicity
of the imine-N (Scheme 2, Table 1).
n-Butylquinazolinimines with a five-membered alicycle (13a,b)
were prepared for comparison from excess n-butylamine and
7a and 7b (Scheme 3)14 and can be regarded as the exact
“monomers” of the symmetrical dimeric 8f and 8g (Table 1).
The compounds synthsized were tested for inhibition of AChE
(E.C. 3.1.1.7, type VI-S, from Electric Eel) and BChE (E.C.
3.1.1.8, from equine serum), respectively, using the Ellman
assay.13,20 This colorimetric assay is based on in situ generated
chromophores formed after enzymatic cleavage of acetyl- and
butyrylthiocholine and reaction of the resulting thiocholine with
Ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid).20 Selectiv-
ity is expressed as the ratio IC50(AChE)/IC50(BChE); higher
values therefore reflect higher BChE selectivity. Inhibitory
activities are presented in Table 1.
The imine-N-unsubstituted compounds 10 and 11 are micro-
molar ChE inhibitors; the eight-membered-ring 11 only shows
a very moderate BChE selectivity (of about a factor of 5).
This is in strong contrast to the compounds previously
described that bear a phenyl ring at the imine-N like 3, which
are highly BChE selective.14 Therefore, N-substitution with
phenyl, benzyl, and phenylethyl groups, respectively, and a
concomitant larger alicycle size lead to BChE selectivity.
Substitution with a butyl group (13b) only leads to a small
increase in inhibitory activity without any change in the
selectivity profiles. Similar to tacrine, 4-chloro substitution (13a)
further increases activity at both enzymes by a factor of 2.
Acetylation of the five-membered-ring quinazolinimine (12)
leads to a dramatic decrease in activity by a factor of 10 for
AChE and approximately 30 for BChE.
a Values are the mean of at least three independent determinations.
inhibitory activity at both enzymes compared with the mono-
meric compounds 13a,b (and also 10). The bivalent heptam-
ethylene quinazolinimines do not exhibit selectivity toward one
of the cholinesterases, a fact that might make them valuable
compounds for a potential treatment of AD because of the
importance of BChE in the later stages of AD.4,5 Interestingly,
the size of the alicycle does not significantly influence activities
or selectivities. Compounds 8a-e show more or less the same
inhibition with the highest activity for the seven-membered-
ring dimer 8d, a compound with an IC50 of 40 nM at AChE
The bivalent inhibitors connected by a heptamethylene spacer
(i.e., 8a-e) show a dramatic increase, by a factor of 100, in

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5413
Scheme 3a
(2) De Ferrari, G. V.; Canales, M. A.; Shin, I.; Weiner, L. M.; Silman,
I.; Inestrosa, N. C. A Structural Motif of Acetylcholinesterase That
Promotes Amyloid Beta-Peptide Fibril Formation. Biochemistry 2001,
40, 10447-10457.
(3) Mack, A.; Robitzki, A. The Key Role of Butyrylcholinesterase during
Neurogenesis and Neural Disorders: An Antisense-5′-butyrylcho-
linesterase-DNA Study. Prog. Neurobiol. 2000, 60, 607-628.
(4) Giacobini, E. Drugs That Target Cholinesterases. In CognitiVe
Enhancing Drugs; Buccafusco, J. J., Ed.; Birkha¨user Verlag: Basel,
Boston, Berlin, 2004; pp 11-36.
(5) Giacobini, E.; Spiegel, R.; Enz, A.; Veroff, A.; Cutler, R. E. Inhibition
of Acetyl- and Butyryl-cholinesterase in the Cerebrospinal Fluid of
Patients with Alzheimer’s Disease by Rivastigmine: Correlation with
Cognitive Benefit. J. Neural Transm. 2002, 109, 1053-1065.
(6) Messer, W. S. Bivalent Ligands for G Protein-Coupled Receptors.
Curr. Pharm. Des. 2004, 10, 2015-2020.
(7) Decker, M.; Lehmann, J. Agonistic and Antagonistic Bivalent Ligands
of Serotonin and Dopamine Receptors Including Their Transporters.
Curr. Top. Med. Chem., in press.
(8) Mun˜oz-Torrero, D.; Camps, P. Dimeric and Hybrid Anti-Alzheimer
Drug Candidates. Curr. Med. Chem. 2006, 13, 399-422 and
references therein.
(9) Carlier, P. R.; Han, Y. F.; Chow, E. S.-H.; Li, C. P.-L.; Wang, H.;
Lieu, T. X.; Wong, H. S.; Pang, Y. P. Evaluation of Short Tether
Bis-THA AChE Inhibitors. A Further Test of the Dual Binding Site
Hypothesis. Bioorg. Med. Chem. 1999, 7, 351-357.
(10) Savini, L.; Campiani, G.; Gaeta, A.; Pellerano, C.; Fattorusso, C.;
Chiasserini, L.; Fedorko, J. M.; Saxena, A. Novel and Potent Tacrine-
Related Hetero- and Homobivalent Ligands for Acetylcholinesterase
and Butyrylcholinesterase. Bioorg. Med. Chem. Lett. 2001, 11, 1779-
1782.
(11) Savini, L.; Gaeta, A.; Fattorusso, C.; Catalanotti, B.; Campiani, G.;
Chiasserini, L.; Pellerano, C.; Novellino, E.; McKissic, D.; Saxena,
A. Specific Targeting of Acetylcholinesterase and Butyrylcholinest-
erase Recognition Sites. Rational Design of Novel, Selective, and
Highly Potent Cholinesterase Inhibitors. J. Med. Chem. 2003, 46,
1-4.
(12) Campiani, G.; Fattorusso, C.; Butini, S.; Gaeta, A.; Agnusdei, M.;
Gemma, S.; Persico, M.; Catalanotti, B.; Savini, L.; Nacci, V.;
Novellino, E.; Holloway, H. W.; Greig, N. H.; Belinskaya, T.;
Fedorko, J. M.; Saxena, A. Development of Molecular Probes for
the Identification of Extra Interaction Sites in the Mid-Gorge and
Peripheral Sites of Butyrylcholinesterase (BuChE). Rational Design
of Novel, Selective, and Highly Potent BuChE Inhibitors. J. Med.
Chem. 2005, 48, 1919-1929.
a Reagents: (i) 3 equiv of n-butylamine, AgNO3, TEA, toluene, reflux,
2 h.
and 24 nM at BChE. Therefore, 8d is 16 times more potent at
AChE than the drug galantamine and 350 times more potent at
BChE. In contrast to the N-butylimines, 4-chloro substitution
only leads to a small increase (1.5-fold) in activity.
In contrast to the bis-tacrine compounds,10,16 the octameth-
ylene-bridged quinazolinimines showed even higher activities
than the heptamethylene-bridged compounds. Apart from the
chloro-substituted dimer 8f, all of the octamethylene dimers
showed BChE selectivity (from 5-fold to almost 200-fold). This
is on one hand due to a higher activity at BChE, which is highest
for the smaller ring sizes, especially the compounds with five-
and six-membered alicycles, i.e., 8g and 8h, which reach low-
nanomolar activities. On the other hand, activity at AChE
decreases with increasing size of the alicyclus; for the seven-
membered-ring dimer 8i 100-fold selectivity is reached, and for
the eight-membered-ring dimer 8j selectivity is 190-fold. But
because of the high nanomolar activities, 8i is still almost as
potent as galantamine at AChE, whereas 8i is 650 times more
potent at BChE than galantamine. The chloro-substituted 8f is
as potent as the unsubstituted 8g at AChE, whereas activity at
BChE is 10-fold lower.
The structure-activity relationships between tacrine and
quinazolinimines differ in two major points: chloro substitution
of the bivalent 8 does not (significantly) improve activity, and
optimal distance in terms of activity and selectivity toward BChE
is achieved by eight and not seven methylene groups, as for
bis-tacrines.10,16
In summary, the application of the bivalent ligand approach
to quinazolinimines as a novel class of cholinesterase inhibitors
proved highly successful. Inhibitory activities increased by a
factor of 100 for the heptamethylene-bridged compounds and
by a factor of 1000 for octamethylene-bridged inhibitors (at
BChE). Heptamethylene-bridged compounds are inhibitors
exhibiting similar activities at both enzymes with two-digit
nanomolar activities, whereas octamethylene-bridged inhibitors
exhibit increasing selectivity toward BChE with increasing size
of the alicycle, reaching a selectivity of approximately 190 for
the eight-membered-ring dimer. Small ring sizes show the lowest
selectivity but the highest activity.
(13) Decker, M. Novel Inhibitors of Acetyl- and Butyrylcholinesterase
Derived from the Alkaloids Dehydroevodiamine and Rutaecarpine.
Eur. J. Med. Chem. 2005, 40, 305-313.
(14) Decker, M.; Krauth, F.; Lehmann, J. Novel Tricyclic Quinazolin-
imines and Related Tetracyclic Nitrogen Bridgehead Compounds as
Cholinesterase Inhibitors with Selectivity towards Butyrylcholinest-
erase. Bioorg. Med. Chem. 2006, 14, 1966-1977.
(15) Saxena, A.; Redman, A. M. G.; Jiang, X.; Lockridge, O.; Doctor, B.
P. Differences in Active Site Gorge Dimensions of Cholinesterases
Revealed by Binding of Inhibitors to Human Butyrylcholinesterase.
Biochemistry 1997, 36, 14642-14651.
(16) Pang, Y. P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S. Highly
Potent, Selective, and Low Cost Bis-tetrahydroaminacrine Inhibitors
of Acetylcholinesterase. Steps toward Novel Drugs for Treating
Alzheimer’s Disease. J. Biol. Chem. 1996, 271, 23646-23649.
(17) Petersen, S.; Tietze, E. Die Reaktion cyclischer Lactima¨ther mit
Aminocarbonsa¨uren. (Reaction of Cyclic Iminium Ethers with
Aminocarbonic Acids.) Liebigs Ann. Chem. 1959, 623, 166-176.
(18) Jae´n, J. C.; Gregor, V. E.; Lee, C.; Davies, R.; Emmerling, M.
Acetylcholinesterase Inhibition by Fused Dihydroquinazoline Com-
pounds. Bioorg. Med. Chem. Lett. 1996, 6, 737-742.
All bivalent inhibitors synthesized greatly exceed the activity
of the established drug galantamine at BChE and, apart from
the most BChE-selective compounds 8i,j, also at AChE.
Acknowledgment. Financial support by the “Fonds der
Chemischen Industrie” (FCI) is gratefully acknowledged. Ap-
preciation is expressed to Petra Wiecha for technical assitance.
(19) Brown, D. J.; Ienaga, K. Dimroth Rearrangement. XVIII. Synthesis
and Rearrangement of 4-Iminoquinazolines and Related Systems. J.
Chem. Soc., Perkin Trans. 1 1975, 21, 2182-2185.
(20) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. A
New and Rapid Colorimetric Determination of Acetylcholinesterase
Activity. Biochem. Pharmacol. 1961, 7, 88-95.
Supporting Information Available: Synthetic procedures,
analytical characterization for 7a, 8a-j, 10-12, 13a,b, and
pharmacological procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Johnson, C. N.; Roland, A.; Upton, N. New Symptomatic Strategies
in Alzheimer’s Disease. Drug DiscoVery Today: Ther. Strategies
2004, 1, 13-19.
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