Effects of two-carbon bridge region methoxylation of benztropine: Discovery of novel chiral ligands for the dopamine transporter

Article abstract of DOI:10.1016/S0960-894X(01)00068-3

6-Methoxylated and 8-oxygenated benztropines were prepared and evaluated for their DAT and SERT activity (binding and uptake inhibition). Methoxylation at the two-carbon bridge of benztropine produced a novel class of potent and selective DAT ligands. An

Full text of DOI:10.1016/S0960-894X(01)00068-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 823±827
E€ects of Two-Carbon Bridge Region Methoxylation of
Benztropine: Discovery of Novel Chiral Ligands for the Dopamine
Transporter
Daniele Simoni,a,* Marinella Roberti,b Riccardo Rondanin,a Riccardo Baruchello,a
Marcello Rossi,a Francesco Paolo Invidiata,c Stefania Merighi,d Katia Varani,d
Stefania Gessi,d Pier Andrea Borea,d Silvia Marino,d Sabrina Cavallini,d
Clementina Bianchid and Anna Siniscalchid
a
Á
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17±19, 44100 Ferrara, Italy
b
Á
Dipartimento di Scienze Farmaceutiche, Universita di Bologna, Italy
Á
Á
Dipartimento di Medicina Clinica e Sperimentale: Sezione di Farmacologia, Universita di Ferrara, Italy
c
Dipartimento Farmacochimico Tossicologico e Biologico, Universita di Palermo, Italy
d
Received 24 October 2000; revised 8 January 2001; accepted 26 January 2001
AbstractÐ6-Methoxylated and 8-oxygenated benztropines were prepared and evaluated for their DAT and SERT activity (binding
and uptake inhibition). Methoxylation at the two-carbon bridge of benztropine produced a novel class of potent and selective DAT
ligands. An interesting enantioselectivity was also observed for this new class of chiral benztropines. The inactivity of the 8-oxyge-
nated analogues seems to point out that, unlike cocaine and its analogues, interactions of benztropine ligands with DAT may be
strongly governed by the nitrogen atom. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
transporter domains involved in interaction with block-
ers, as opposed to those interacting with DA, could lead
to medications able to block the e€ects of cocaine with-
out themselves blocking DAuptake, which may prove
useful in the treatment of cocaine addiction.1,2 There-
fore, one approach in the discovery of potential cocaine
antagonists is to identify appropriate drugs that bind to
a region of the cocaine binding site without inhibiting
DAuptake.
Aloss of balance in monoaminergic neurotransmission
plays a key role in a myriad of neurological and psy-
chiatric diseases (Parkinson's disease, depression, schi-
zophrenia, obsessive compulsive disorder (OCD), panic
disorder). Reuptake of released norepinephrine (NE),
dopamine (DA), serotonin (5-HT) by Na+±ClÀ-depen-
dent membrane transporters is the primary mechanism
for termination of the monoaminergic signal. Although
most antidepressants display a low anity for DA
transporter (DAT), drugs like bupropione have been
proven to be useful as atypical antidepressants for the
management of resistant patients. The DAT system
represents the obligatory target for the behavioral and
biochemical action of cocaine1,2 and there is a correla-
tion between craving and OCD; moreover, clinical trials
have suggested that a combination of DAT inhibitors
with drugs enhancing 5-HT levels may be a successful
approach in the treatment of cocaine withdrawal symp-
toms.3,4 It is now well accepted that the delineation of
In this context, we have recently demonstrated that
methoxylation of the cocaine's two-carbon bridge pro-
vides compounds of pharmacological interest:5À9
although the introduction of the methoxy function uni-
formly resulted in signi®cant reduction in binding a-
nity to DAT, at least one of these methoxylated
analogues was found capable of countering, to a minor
5
extent, the e€ects of cocaine on DAreuptake.
Among the structural classes of compounds interacting
with DAT, which have provided interesting results in
the quest of potential cocaine antagonists, the benz-
tropine group has recently received particular attention.
After considerable research in this area Newman and
co-workers have hypothesized that this class of DA
*Corresponding author. Tel.: +39-0532-291291; fax: +39-0532-
291296; e-mail: smd@dns.unife.it
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00068-3

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D. Simoni et al. / Bioorg. Med. Chem. Lett. 11 (2001) 823±827
uptake inhibitors may access a DAT binding site dis-
tinct from that of cocaine, which would explain their
discrepant behavioral pro®le.10À12
halogenated-diphenylmethoxy benztropines. Moreover,
we also describe here notable enantioselective
a
approach to 6-methoxylated benztropines. We think
that the data reported in this communication further
expands the understanding of benztropine's SARs, and
may provide useful information to aid the discovery of
cocaine antagonists or `partial agonists' potentially use-
ful in abuse treatment.
Thus, since benztropine contains a tropane ring as
cocaine, we were intrigued to verify whether the same
structural perturbation at the C6±C7 region as well as at
the nitrogen bridge of the tropane skeleton might pro-
duce novel ligands for the dopamine transporter. To
explore this possibility, we considered it to be of interest
to prepare 6-methoxylated analogues 3 of the molecule.
Moreover, since 8-oxygenated cocaines and their WIN
derivatives proved to be of particular interest for their
DAT activity,13,14 we also evaluated the biological
activity of the 8-oxa analogues 4. These modi®cations
might not only lead to compounds with altered biolog-
ical activity, but also provide further relevant informa-
tion upon the question of whether benztropine and
cocaine bind to di€erent binding sites.
Chemical Synthesis
For the preparation of the racemic 3a±c we took, in
part, advantage of known procedures.10 Commercially
available acetonedicarboxylic acid was reacted with
methylamine
hydrochloride
and
2-methox-
ysuccindialdehyde (6) at room temperature in a citrate
bu€er solution at pH 4 to give the key 6-methoxy-
tropinone (7) in 60% yield. The starting 6 was obtained
by exposure of a mixture of 2,3,5-trimethoxytetrahy-
drofurane, in turn obtained from 2,5-dimethoxy-2,5-
dihydrofurane, by reaction with methanol as reported in
the literature, to a 0.2 M aqueous sulfuric acid solution.
The stereoselective reduction of 7 by means of hydrogen
and PtO2 produced in 70% yield the 6-methoxytropine
(8) which was easily reacted with benzhydryl chlorides
9a±c to a€ord, in appreciable yields, the desired benz-
tropines 3a±c (Scheme 1).
In this communication we describe our ®ndings on a
novel class of 6-methoxylated and 8-oxygenated benz-
tropines. We found that the methoxylation of benz-
tropine yieldsÐin some casesÐcompounds possessing a
higher DAT binding activity than benztropine itself,
together with interesting discrepancies in the binding
versus DAuptake inhibition data. The compounds
possess a binding potency comparable to the parent
Scheme 1.

D. Simoni et al. / Bioorg. Med. Chem. Lett. 11 (2001) 823±827
825
Biological Results and Discussion
Since the synthesized compounds 3a±c, di€erent from
the parent benztropine and analogues, bear a chiral
center, we were intrigued to verify whether stereo-
chemistry could play a role in their binding to the
recognition site on DAT and, consequently, we needed
access to the enantiomers of our racemic methoxylated
benztropines. Indeed, it has been well documented by
Carroll and others that stereochemistry plays an
important role in the binding of cocaine and its ana-
logues to the recognition site on DAT and, for example,
the seven stereoisomers of cocaine are less potent than
the natural product.2 Moreover, a profound enantio-
selectivity has previously been demonstrated in the 2-
Enantiomers (+)-3b and (À)-3b, diastereoisomers (+)-
3c and (À)-3c, and the racemic mixtures 3a and 12a,b
were examined for their ability to displace [3H]WIN
35,428 and [3H]paroxetine from DAand 5-HT trans-
porters in the rat caudate putamen. Additionally, com-
pounds possessing an appreciable binding anity to
DAT were tested for their ability to inhibit high-anity
uptake of [3H]DAinto striatal nerve endings (synapto-
somes). In general, compounds with the tropane skele-
ton retain activity relative to their parent structures,
namely benztropine and cocaine, in both binding and
functional assays (Table 1). In the case of the enantio-
meric pairs (+)-3b and (À)-3b as well as of the diaster-
eoisomers (+)-3c and (À)-3c the levo 6R isomers were
the most potent. Interestingly, compound (À)-3c
showed a binding activity comparable to the corre-
sponding 40-chlorobenztropine whereas (À)-3b demon-
strated only a 2-fold decrease in binding anity as
compared to the parent ligand 40,400-di¯uorobenz-
tropine.10 On the contrary, the 8-oxabenztropine analo-
gues 12a,b were devoid of any activity. Additionally,
none of the newly synthesized benztropines displayed
anity to SERT.
carbomethoxy-substituted
3a-[bis(40-¯uorophenyl)-
methoxy]tropane series of DAT ligands.15 Therefore, we
have investigated methods aimed at obtaining our
methoxybenztropine analogues in non-racemic form.
We considered that methoxytropinones (+)-7 and (À)-7
possessing the known con®guration at the carbon atom
bearing the methoxy functionality, would constitute
appropriate starting materials for our purposes.
Accordingly, the stereoselective reduction of the ketones
(+)-7 and (À)-7 produced the corresponding alcohol
derivatives (À)-8 and (+)-8 in optically pure form,
which were easily transformed, as described above for
the racemic 8, into the desired enantiomeric (+)-3b and
(À)-3b and diastereomeric (+)-3c and (À)-3c which
retain the same absolute con®guration at the tropane
skeleton as the starting compounds.
Among the results obtained, perhaps the most interest-
ing observation relates to the di€erences in binding ver-
sus DAreuptake inhibition for compounds ( À)-3b and
(À)-3c; in the case of the 6R isomers the IC50 for bind-
ing is about 4 to 5 times smaller than the IC50 for inhi-
bition of DAuptake. Vice versa, a di€erent behavior
has been observed with the S isomers: the IC50 for
binding is only slightly lower or higher than the IC50 for
inhibition of DAuptake. Consequently, notwithstand-
ing that only a relatively small number of chiral com-
pounds have been described here, the current ®ndings
Finally, the synthetic route depicted in Scheme 2
allowed facile preparation of the 8-oxygenated ana-
logues 12a,b. The ketone 10 was easily reduced by
means of PtO2 in methanol solution to provide the
alcohol derivative 11 in 80% yield. Reaction with the
appropriate benzhydryl chlorides 9a,b a€ords in appre-
ciable yields the desired 12a,b.
Scheme 2.

826
D. Simoni et al. / Bioorg. Med. Chem. Lett. 11 (2001) 823±827
Table 1. IC50 values for the 6-methoxylated and 8-oxygenated benztropine analogues in DA[3H]WIN 35,428 and 5HT[3H]paroxetine binding and
dopamine uptake experiments
Compd
DA[3H]WIN 35,428
Binding, IC50 (nM)
[3H]DAUptake
IC50 (nM)
5HT[3H]Paroxetine
Binding, IC50 (mM)
Cocaine
Benztropine
89Æ8
208Æ12
118Æ9
403Æ115
(+)-3b
95Æ5
165Æ10
>100
(À)-3b
(Æ)-3a
(+)-3c
(À)-3c
25Æ3
650Æ50
750Æ70
32Æ2
104Æ11
2280Æ73
519Æ29
179Æ9
>100
>100
>100
>100
12a
12b
>10,000
2000Æ200
Ð
Ð
Ð
Ð
clearly demonstrate that also in the benztropine class of
DAT inhibitors an interesting enantioselectivity may
exist when chiral carbon atoms are present in the tro-
pane carbon skeleton. In particular, a marked enantio-
selectivity is seen for (À)-3c, which is 23 times more
active in binding anity than (+)-3c. It is also worth
noting that, although a large body of literature exists
concerning the di€erent biological activity of cocaine's
stereoisomers, very few observations have hitherto been
reported regarding chiral benztropines, probably
because of the absence of chiral carbon atoms in the
starting benztropine. However, Newman et al. have
recently demonstrated that chiral benztropines could be
of particular interest in the quest for analogues that
retain high anity for DAT but less for muscarinic
receptors.16 Therefore, the study of chiral benztropines
could be of particular interest in research dealing with
selective DAT ligands.
Since in the last decade there has been considerable
e€ort to determine the SARs of cocaine and congeneres,
it has become evident that DAT inhibitors do not all
interact with DAT in the same manner. It has been
found that neither nitrogen nor oxygen14,17,18 are

D. Simoni et al. / Bioorg. Med. Chem. Lett. 11 (2001) 823±827
827
prerequisites for binding of cocaine-based ligands to
monoamine transporters and Meltzer et al.17 had pos-
tulated that the three-dimensional topology of the
ligand may be more important for binding to the biolo-
gical macromolecule (DAT or SERT) than the speci®c
functionality present. However, the inactivity of the 8-
oxygenated benztropines 12a,b seems to point out that,
unlike cocaine and its analogues, interaction of this
class of tropane-based DAT inhibitors may be governed
to a large extent by the nitrogen atom. To this regard
it is worth noting that N-acyl- and N-sulfonyl-benz-
tropines were also reported to be inactive.19
must be given to the stereochemistry of the substituents
attached to the tropane ring system.
Acknowledgements
This work was in part supported by the Ministero
dell'Universita e della Ricerca Scienti®ca e Tecnologica
(Rome).
References
1. Carroll, F. I.; Howell, L. L.; Kuhar, M. J. J. Med. Chem.
1999, 42, 2721.
2. Carroll, F. I.; Lewin, A. H.; Boja, J. W.; Kuhar, M. J. J.
Med. Chem. 1992, 35, 969.
3. Frances, A.; Manning, D.; Marin, D.; Kocsis, J.; McKin-
ney, K.; Hall, W.; Klein, M. Psychopharmacology Suppl. 1992,
106, S82.
4. Hitzig, P. Maryland, Med. J. 1993, 42, 153.
5. Simoni, D.; Stoelwinder, J.; Kozikowski, A. P.; Johnson,
K. M.; Bergmann, J. S.; Ball, R. G. J. Med. Chem. 1993, 36,
3975.
Additionally, we wish to draw attention to the biologi-
cal results obtained from this class of methoxylated
benztropines with respect to the two-carbon bridge
methoxylated cocaines. In fact, as the introduction of a
methoxyl function at 6 and 7 positions of cocaine
strongly compromises mazindol binding at DAT, the
methoxylated benztropines still retain potent activity
and, in some cases, the compounds are more active than
the parent benztropine. Therefore, in our opinion, these
®ndings may add further support to the notion that the
domains associated with the cocaine and the benztropine
recognition sites are not identical.
6. Simoni, D.; Roberti, M.; Andrisano, V.; Manferdini, M.;
Rondanin, R.; Kozikowski, A. P. J. Org. Chem. 1998, 63,
4834 and references cited therein.
7. Kozikowski, A. P.; Simoni, D.; Baraldi, P. G.; Lampronti,
I.; Manfredini, S. Bioorg. Med. Chem. Lett. 1996, 6, 441.
8. Simoni, D.; Roberti, M.; Andrisano, V.; Manferdini, M.;
Rondanin, R.; Invidiata, F. P. IlFarmaco 1999, 54, 275.
9. Simoni, D.; Rondanin, R.; Roberti, M. Synthetic Approa-
ches to Cocaine and its Analogues. In Targets in Heterocyclic
Chemistry; Attanasi, O. A., Spinelli, D., Eds.; Societa Chimica
Italiana: Rome, 2000; pp 147±183.
10. Newman, A. H.; Izenwasser, S.; Allen, A. C.; Katz, J. L.
J. Med. Chem. 1994, 37, 2258.
11. Newman, A. H.; Izenwasser, S.; Robarge, M. J.; Kline,
R. H. J. Med. Chem. 1999, 42, 3502.
12. Katz, J. L.; Izenwasser, S.; Kline, R. H.; Allen, A. C.;
Newman, A. H. J. Pharmacol. Exp. Ther. 1999, 288, 302.
13. Meltzer, P. C.; Liang, A. Y.; Blundell, P.; Gonzales,
M. D.; Chen, Z.; George, C.; Madras, B. K. J. Med. Chem.
1997, 40, 2661.
14. Kozikowski, A. P.; Simoni, D.; Roberti, M.; Rondanin,
R.; Wang, S. M.; Du, P. F.; Johnson, K. M. Bioorg. Med.
Chem. Lett. 1999, 9, 1831.
In summary, the results obtained with the stereoisomers
(+)-3b, (À)-3b, (+)-3c and (À)-3c demonstrate that
when a chiral center is present in the benztropine mole-
cule its activity is closely related to the stereochemistry
of the compounds and the 6R isomers are the most
interesting. Considering that methoxylation of benz-
tropine produces compounds possessing high DAT
activity, whereas the 8-oxygenated benztropines are
devoid of any activity, and that these data are in sharp
contrast with those obtained in the cocaine series of
compounds, these results further substantiate the
hypothesis that benztropine may be interacting with a
di€erent binding domain at DAT as compared to
cocaine and its analogues. Moreover, since the 8-oxy-
genated benztropines are not active, it is likely that, at
least for this class of compounds, the nitrogen atom
present in the tropane system plays an important role in
modulating the DAT activity of the compounds.
15. Meltzer, P. C.; Liang, A. Y.; Madras, B. K. J. Med. Chem.
1994, 37, 2001.
16. Robarge, M. J.; Agoston, G. E.; Izenwasser, S.; Kopajtic,
T.; George, C.; Katz, J. L.; Newman, A. H. J. Med. Chem.
2000, 43, 1085.
17. Meltzer, P. C.; Blundell, P.; Yong, Y. F.; Chen, Z.;
George, C.; Gonzalez, M. D.; Madras, B. K. J. Med. Chem.
2000, 43, 2982 and references cited therein.
18. Simoni, D.; Roberti, M.; Rondanin, R.; Kozikowski, A. P.
Tetrahedron Lett. 1999, 40, 4425.
19. Agoston, G. E.; Wu, J. H.; Izenwasser, S.; George, C.;
Katz, J.; Kline, R. H.; Newman, A. H. J. Med. Chem. 1997,
40, 4329.
In conclusion, methoxylation at the benztropine's two-
carbon bridge provides important leads toward map-
ping the topology of DAT. Furthermore, these novel
molecular probes, and the structural information they
provide, will very likely contribute to the development
of novel ligands for DAT and possible novel pharma-
cotherapeutic tools for cocaine abuse. The study of
other two-carbon bridge modi®ed analogues of benz-
tropine would therefore be strongly warranted, and as
evidenced by the foregoing work, particular attention