Enantioselective synthesis of S-(+)-2β-carboalkoxy-3α-[bis(4-fluorophenyl)methoxy]tropanes as novel probes for the dopamine transporter

Article abstract of DOI:10.1016/S0960-894X(02)00155-5

Synthesis of a series of pure S-(+)-2β-carboalkoxy-3α-[bis(4-fluorophenyl)methoxy]tropanes (>99% ee) was achieved by employing a chiral amine-induced asymmetric reaction of tropinone with methyl cyanoformate as the key step. In this series, all of the S-(+)-enantiomers were 2-fold more potent than their racemic mixtures and all displayed high-affinity binding for DAT (K_i=13-40 nM). These data support previous findings of significant divergence in structural requirements for high-affinity DAT binding among tropane-based inhibitors. Furthermore, the 2-substituent in the 3α-[bis(4-fluorophenyl)methoxy]tropane series is well tolerated at the DAT but not at SERT (K_i=690-2040 nM), or muscarinic M₁ receptors (K_i=133-4380 nM) resulting in highly selective DAT ligands that may provide new leads toward a cocaine-abuse therapeutic.

Full text of DOI:10.1016/S0960-894X(02)00155-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1249–1252
Enantioselective Synthesis of S-(+)-2ꢀ-Carboalkoxy-3ꢁ-
[bis(4-fluorophenyl)methoxy]tropanes as Novel Probes for the
Dopamine Transporter
Mu-Fa Zou,a Gregory E. Agoston,a,y Yuri Belov,b Theresa Kopajtic,c Jonathan L. Katzc
and Amy Hauck Newmana,*
aMedicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, NIH, Baltimore, MD 21224, USA
bChromBA, State College, PA 16803, USA
cPsychobiology Section, National Institute on Drug Abuse-Intramural Research Program, NIH, Baltimore, MD 21224, USA
Received 17 January 2002; accepted 27 February 2002
Abstract—Synthesis of a series of pure S-(+)-2b-carboalkoxy-3a-[bis(4-fluorophenyl)methoxy]tropanes (>99% ee) was achieved
by employing a chiral amine-induced asymmetric reaction of tropinone with methyl cyanoformate as the key step. In this series, all
of the S-(+)-enantiomers were 2-fold more potent than their racemic mixtures and all displayed high-affinity binding for DAT
(Ki=13–40 nM). These data support previous findings of significant divergence in structural requirements for high-affinity DAT
binding among tropane-based inhibitors. Furthermore, the 2-substituent in the 3a-[bis(4-fluorophenyl)methoxy]tropane series is
well tolerated at the DAT but not at SERT (Ki=690–2040 nM), or muscarinic M1 receptors (Ki=133–4380 nM) resulting in highly
selective DAT ligands that may provide new leads toward a cocaine-abuse therapeutic. # 2002 Elsevier Science Ltd. All rights reserved.
Identifying molecular mechanisms that are associated
with the pharmacological and reinforcing effects of
cocaine has been deemed essential toward the discovery
of a medication to be used in the treatment of cocaine
abuse. Cocaine binds with moderate affinity to all three
monoamine transporters, dopamine (DAT), serotonin
(SERT), and norepinephrine (NET). However, the
reinforcing actions of cocaine are primarily associated
with increases in basal dopamine, as a result of blockade
at the DAT (for review, see refs 1–4). Hence the inves-
tigation of dopaminergic agents and in particular,
selective dopamine transporter ligands, has received
considerable attention toward the development of a
cocaine-abuse medication (for review, see refs 1 and 5–8).
many of the 3a-(diphenylmethoxy)tropane analogues
are potent inhibitors of dopamine uptake, and yet they
do not produce significant locomotor stimulation or
cocaine-like subjective effects, in rodents.15À17 Further-
more, several of these compounds have been evaluated
and are not appreciably self administered, in monkeys
that readily self administer cocaine.18,19 SAR for this
series of compounds revealed a striking divergence from
cocaine and related tropane analogues at the
DAT.11À13,17,20À23 Furthermore, immunologic and pep-
tide mapping studies using the photoaffinity label,
[
125I]N-[n-butyl-4-(4000-azido-3000-iodophenyl)]-bis-(4-fluo-
rophenyl)methoxytropane (GA 2–34), suggested that
these ligands bind to transmembrane domains on the
DAT that differ from those at which cocaine analogues
bind.24,25 In total, these studies suggest that the binding
domain on the DAT, that is accessed by the 3a-(diphe-
nylmethoxy)tropane analogues, is not identical with
that of cocaine, and further that these binding differ-
ences may, in part, result in different pharmacological
actions of these drugs.
The 3-phenyltropane class of dopamine uptake inhibi-
tors has been investigated extensively9,10 and has pro-
vided SAR for the ‘cocaine’ binding site on the DAT.
Another class of tropane-based dopamine uptake inhi-
bitors, the 3a-(diphenylmethoxy)tropane (Benztropine)
analogues, have been investigated in our laboratory and
others11À14 in recent years. Our studies have shown that
The R-configuration at the 2-position of (À)-cocaine
and all of the 3-phenyltropane analogues is required for
high affinity binding at the DAT.9 In contrast, when all
eight isomers of 2-carbomethoxy-3a-[bis(4-fluorophenyl)-
methoxy]tropane were synthesized, it was discovered
*Corresponding author. Fax: +1-410-550-1455; e-mail: anewman@
intra.nida.nih.gov
yCurrent address: EntreMed, Inc., 9640 Medical Center Drive, Rock-
ville, MD 20874, USA.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00155-5

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M.-F. Zou et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1249–1252
that the S-enantiomer, [S-(+)-difluoropine], was con-
siderably more potent and selective than any of the
other seven isomers.11 The S-(+)-2b-carbomethoxy-3a-
[bis(4-fluorophenyl)methoxy]tropane (IC50=10.9 nM),11
appears to be equiactive to 3a-[bis(4-fluorophenyl)-
methoxy]tropane (Ki=11.8 nM),15 although this com-
parison is across studies employing different assay con-
ditions. Nonetheless, this relative equivalence of activity
suggests that the 2-position, in this series, may provide
an excellent opportunity for maintaining or potentially
improving binding affinities at the DAT. Furthermore,
it was reported that S-(+)-difluoropine was highly
selective for DAT over SERT (IC50=3580 nM),11 which
suggests that this substituent may not be as well toler-
ated at the other monoamine transporters or, possibly
muscarinic receptors, where many of the benztropine
analogues bind with high affinity.16,20 Thus, the dis-
covery of chemical modifications to the parent com-
pound that decreases binding affinity at muscarinic
receptors while retaining high affinity at DAT was desir-
able. Herein, we report the enantioselective synthesis
and binding profiles of a series of S-(+)-2b-carboalkoxy-
3a-[bis(4-fluorophenyl)methoxy]tropane analogues. The
racemates of these analogues were also prepared for
biological and chemical comparison.
Synthesis of the racemic (ꢀ)-2b-carboalkoxy-3a-[bis(4-
fluorophenyl)methoxy]tropanes was performed as a
model for the esterifications and for biological compar-
ison using literature methods.11,26 The synthesis of the
optically pure S-(+)-2b-carboalkoxy-3a-[bis(4-fluoro-
phenyl)methoxy]tropanes [(+)-6a–6d] was achieved
using the asymmetric deprotonation strategy of
Majewski and Lanny (Scheme 1).26 Synthesis of the
chiral amine (R)-N-(2,2-dimethylpropyl)-1-phenyl-2-
piperidinoethylamine (NDPPA) was prepared from
commercially available R-phenylglycine by modifying
Shirai’s original procedure.27 3-Tropinone was asym-
metrically deprotonated by the lithium salt of chiral
amine NDPPA and then treated with methyl cyano-
formate (NCCO2ME) to give (À)-2- carbomethoxy-
tropinone [(À)-2] with ꢁ92% ee. Resolution with D-
tartaric acid of (À)-2, followed by two recrystallizations
gave the enantiomerically pure (À)-2.28 Catalytic reduc-
tion of (À)-2 gave (+)-3 as the only isomer, which was
converted to (+)-4 and esterified to (+)-5a–d upon
treatment with HCl gas in the respective alcohol. Con-
version of (+)-5a–d to (+)-6a–d was achieved using
literature methods.11 Spectral data for the enantiomers
((+)-6a–6d) were identical to their racemates.29 The
enantiomeric analysis was conducted on the chiral
HPLC column ‘Klassix Chiral-A’ (ChromBA, State
College, PA, USA).29
The binding affinities of the four enantiomerically pure
S-(+)-2-carboalkoxy-3a-[bis(4-fluorophenyl)methoxy]-
tropanes analogues [(+)-6a–d] and their respective
racemic mixtures, (ꢀ)-6a–d, were evaluated in radio-
labeled ligand displacement assays for DAT, SERT,
NET and muscarinic M1 receptors, in rat brain, using
previously described methods.23 In addition, inhibition
of dopamine uptake in rat synaptosomes was also eval-
uated and these data were compared to those for the
unsubstituted 3a-[bis(4-fluorophenyl)methoxy]tropane
(7; Table 1). Table 2 shows the selectivities of the
enantioselective compounds for the DAT over SERT,
NET, and muscarinic M1 binding sites. All of the (ꢀ)-
and enantiomerically pure S-(+)-2-carboalkoxy-3a-
[bis(4-fluorophenyl)methoxy]tropane analogues (6a–6d)
bind with high affinity and selectivity at the DAT, with
the S-(+)-enantiomers showing approximately 2-fold
Scheme 1. Reagents and conditions: (a) 1. n-BuLi, NDPPA, THF; 2.
NCCO2Me, THF, 77%; (b) d-Tartaric acid, 80%; (c) H2 (50 psi)/
PtO2, EtOH, 4 days, 86%; (d) H2O, reflux; (e) HCl gas, ROH, rt or
50 ꢂC; (f) 4,40-difluorobenzhydrol, p-toluenesulfonic acid hydrate,
benzene, reflux.
Table 1. Binding results at the monoamine transporters and muscarinic M1 receptor
Compd
[3H]WIN 35, 428 DAT
[3H]DA UPTAKE
IC50 (nM)ꢀSEMa
[3H]Citalopram SERT
[3H]Nisoxetine NET
Ki (nM)ꢀSEMa
[3H]Pirenzepine M1
Ki (nM)ꢀSEMa
Ki (nM)ꢀSEMa
Ki (nM)ꢀSEMa
(+)-6a
(ꢀ)-6a
(+)-6b
(ꢀ)-6b
(+)-6c
(ꢀ)-6c
(+)-6d
(ꢀ)-6d
7
12.9ꢀ1.8
21.3ꢀ3.6
16.8ꢀ2.0
26.2ꢀ3.4
23.3ꢀ3.3
49.1ꢀ6.4
40.2ꢀ9.3
90.3ꢀ18
11.2ꢀ1.3b
1.5ꢀ0.2
2.9ꢀ0.4
1.8ꢀ0.2
3.6ꢀ0.4
6.2ꢀ0.3
6.8ꢀ0.2
2.2ꢀ0.3
3.5ꢀ0.2
13.8ꢀ1.7
690ꢀ58
1750ꢀ240
1850ꢀ270
3740ꢀ490
12,000ꢀ1280
13,800ꢀ680
2040ꢀ300
3320ꢀ290
3260ꢀ110
269ꢀ39
474ꢀ65
133ꢀ4.2
302ꢀ43
629ꢀ31
1890ꢀ130
1860ꢀ190
2680ꢀ140
7640ꢀ1040
4380ꢀ530
5100ꢀ310
11.6ꢀ0.9
1020ꢀ120
642ꢀ13
1990ꢀ380
2230ꢀ200
4110ꢀ73
610ꢀ81
aEach Ki value represents data from at least three independent experiments, each performed in triplicate.
bData from ref 15.

M.-F. Zou et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1249–1252
1251
Table 2. DAT selectivities of S-(+)-2-carboalkoxy-3-[bis(4-fluoro-
phenyl)-methoxy]tropanes
determine whether the unique pharmacology often
observed within this class of compounds is duplicated,
and if there is the possibility of developing a potential
cocaine abuse pharmacotherapeutic from one of these
agents.
Compd
DAT/SERT
DAT/NET
DAT/M1
(+)-6a
(+)-6b
(+)-6c
(+)-6d
7
53
110
515
51
21
37
28
56
55
10
112
120
110
1
Acknowledgements
290
M. Zou was supported by an NIH visiting fellowship
and G. Agoston was supported by an NIH Intramural
Research Training Award fellowship. This research was
supported by the National Institute on Drug Abuse-
Intramural Research Program. The authors acknowl-
edge Ms. J. J. Cao for expert technical support.
higher affinity than their corresponding racemates.
These data support previously described enantio-
selectivity for S-(+)-6a. Interestingly, in rat brain, this
analogue does not demonstrate the reported11 >300-
fold selectivity for DAT over SERT, but rather ꢁ50-
fold selectivity. This difference may reflect differing
assay conditions or species differences wherein monkey
SERT is less sensitive to this class of compounds than
rat. However, increasing the methyl ester (6a) to ethyl
(6b) or 2-propyl (6c), significantly decreases binding
affinities to SERT, resulting in 110- and 515-fold selec-
tivity, respectively. Likewise, muscarinic binding affi-
nities of the larger alkyl esters (+)-6b, c, and d are
significantly reduced resulting in >100-fold selectivity
for DAT over M1 receptors, which has previously only
been achieved with a few N-substituted analogues, in
this class of compounds.23 All of the compounds were
>20-fold selective over NET, with the benzyl ester (+)-6d
demonstrating the highest selectivity.
References and Notes
1. Carroll, F. I.; Howell, L. l.; Kuhar, M. J. J. Med. Chem.
1999, 42, 2721.
2. Amara, S. G.; Sonders, M. S. Drug Alcohol Depend. 1998,
51, 87.
3. Bardo, M. T. Crit. Rev. Neurobiol. 1998, 12, 37.
4. Spanagal, R.; Weiss, F. Trends Neuosci. 1999, 22, 521.
5. Newman, A. H. Exp. Opin. Ther. Pat. 2000, 10, 1095.
6. Newman, A. H. Med. Chem. Res. 1998, 8, 1.
7. Smith, M. P.; Hoepping, A.; Johnson, K. M.; Trzcinska,
M.; Kozikowski, A. P. Drug Discov. Today 1999, 4, 322.
8. Howell, L. L.; Wilcox, K. M. J. Pharmacol. Exp. Ther.
2001, 298, 1.
The DAT binding affinities for S-(+)-6a (Ki=12.9 nM)
and S-(+)-6b (Ki=16.8 nM), for example, are equiva-
9. Carroll, F. I.; Lewin, A. H.; Kuhar, M. J. In Neuro-
transmitter Transporters: Structure, Function, and Regulation
Reith, M. E. A., Ed.; Humana; Totowa, NJ, 1997; p 263.
10. Carroll, F. I.; Lewin, A. H.; Kuhar, M. J. Med. Chem.
Res. 1998, 8, 59.
11. Meltzer, P. C.; Liang, A. Y.; Madras, B. K. J. Med. Chem.
1994, 37, 2001.
12. Meltzer, P. C.; Liang, A. Y.; Madras, B. K. J. Med. Chem.
1996, 39, 371.
13. Meltzer, P. C.; Liang, A. Y.; Blundell, P.; Gonzales,
M. D.; Chen, Z.; George, C.; Madras, B. K. J. Med. Chem.
1997, 40, 2662.
14. Simoni, D.; Roberti, M.; Rondanin, R.; Baruchello, R.;
Rossi, M.; Invidiata, P.; Merighi, S.; Varani, K.; Gessi, S.;
Borea, P. A.; Marino, S.; Cavallini, S.; Bianchi, C.; Siniscalchi,
A. Bioorg. Med. Chem. Lett. 2001, 11, 823.
lent to the unsubstituted
7
(3a-[bis(4-fluoro-
phenyl)methoxy]tropane; Ki=11.8 nM),15 suggesting, as
we speculated previously, that this substituent may not
contribute significantly to the binding interaction at
DAT. As the steric bulk of the 2-position ester is
increased, DAT binding affinities decreased only
slightly. Equipotent inhibition of dopamine uptake in
rat synaptosomes was observed for all of the S(+)-ana-
logues and the enantiomers were ꢁ2-fold more potent
than the racemates. Likewise, binding affinities at SERT
and NET were uniformly poor for the 2-carboalkoxy
analogues, with the 2-propyl ester 6c showing the lowest
binding affinity at SERT (Ki=12,000 nM). Profound
losses in binding affinities were also observed at the
muscarinic receptors resulting in significant increases in
DAT selectivity (>100 for 6b, c, d vs 1 for 7).
15. Newman, A. H.; Allen, A. C.; Izenwasser, S.; Katz, J. L.
J. Med. Chem. 1994, 37, 2258.
16. Katz, J. K.; Izenwasser, S.; Kline, R. H.; Allen, A. C.;
Newman, A. H. J. Pharmacol. Exp. Ther. 1999, 288, 302.
17. Katz, J. L.; Agoston, G. E.; Alling, K. L.; Kline, R. H.;
Forster, M. J.; Woolverton, W. L.; Izenwasser, S.; Kopajtic,
T. A.; Newman, A. H. Psychopharmacology 2001, 154, 362.
18. Woolverton, W. L.; Hecht, G. S.; Agoston, G. E.; New-
man, A. H.; Katz, J. L. Psychopharmacology 2001, 154, 375.
19. Woolverton, W. L.; Rowlett, J. K.; Wilcox, K. M.; Paul,
I. A.; Kline, R. H.; Newman, A. H.; Katz, J. L. Psycho-
pharmacology 2000, 147, 426.
20. Newman, A. H.; Kline, R. H.; Allen, A. C.; Izenwasser,
S.; George, C.; Katz, J. L. J. Med. Chem. 1995, 38, 3933.
21. Newman, A. H.; Robarge, M.; Izenwasser, S.; Kline, R. H.
J. Med. Chem. 1999, 42, 3502.
22. 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 summary, a series of S-(+)-2-carboalkoxy-3a-[bis(4-
fluorophenyl)methoxy]tropane analogues was prepared
via an enantioselective synthetic strategy giving high
yields of >99% ee products that were highly potent and
selective DAT ligands. All of the analogues potently
inhibited [3H]dopamine uptake in synaptosomes
(IC50=1.5–6.8 nM). Although all four S-(+)-2-carbo-
alkoxy-analogues showed DAT selectivity, S-(+)-6b
demonstrated overall the most potent and DAT selec-
tive binding profile. Further investigation into the
pharmacology of this series of 2-carboalkoxy-sub-
stituted-3a-(diphenylmethoxy)tropane analogues, with
an emphasis on in vivo studies, will be pursued to

1252
M.-F. Zou et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1249–1252
23. Robarge, M. J.; Agoston, G. E.; Izenwasser, S.; Kopajtic,
T.; George, C.; Newman, A. H. J. Med. Chem. 2000, 43, 1085.
24. Agoston, G. E.; Vaughan, R.; Lever, J. R.; Izenwasser, S.;
Terry, P. D.; Newman, A. H. Bioorg. Med. Chem. Lett. 1997,
7, 3027.
(6H, m), 2.18 (3H, s), 2.68 (1H, s), 3.08 (1H, m), 3.58 (1H, m),
3.98 (1H, d, J=5.2 Hz), 4.0–4.2 (2H, m), 5.34 (1H, s), 6.95–
7.05 (4H, m), 6.20–6.30 (4H, m); 13C NMR (CDCl3) d 14.2,
24.6, 25.2, 36.2, 41.8, 60.4, 61.0, 63.2, 70.2, 80.3, 115.2, 115.4,
128.4, 138.4, 160.5, 163.7, 172.5; EIMS m/z 415 (M+). Anal.
(C24H27NF2O3) for C, H, N. Compound (+)-6c: colorless oil;
[a]2D6 +20.2 (c 1.03, MeOH); IR: 1726, 1603 cmÀ1; H NMR
(CDCl3) d 1.21 (3H, d, J=5.8 Hz), 1.23 (3H, d, J=5.6 Hz),
1.75–2.17 (6H, m), 2.20 (3H, s), 2.66 (1H, m), 3.10 (1H, m),
3.57 (1H, m), 3.98 (1H, d, J=5.1 Hz), 5.06 (1H, m), 5.36 (1H,
s), 6.97–7.04 (4H, m), 7.24–7.31 (4H, m); 13C NMR (CDCl3) d
22.2, 25.0, 25.6, 36.5, 42.1, 52.3, 61.4, 63.6, 67.9, 70.6, 80.7,
115.5, 115.8, 128.8, 172.3; EIMS m/z 429 (M+). Anal.
(C25H29NF2O3) for C, H, N. Compound (+)-6d: colorless oil;
[a]2D6 +12.6ꢂ (c 1.05, MeOH). Analytic HPLC (Klassix Chiral-
25. Vaughan, R. A.; Agoston, G. E.; Lever, J. R.; Newman,
A. H. J. Neurosci. 1999, 19, 630.
ꢂ
1
26. Majewski, M.; Lanny, R. J. Org. Chem. 1995, 60, 5825.
27. Shirai, R.; Aoki, K.; Sato, D.; Kim, H.-D.; Murakata, M.;
Yasukata, T.; Koga, K. Chem. Pharm. Bull. 1994, 42, 690.
28. Note 1. Compound (À)-2: mp 107–108 ꢂC (sublimed; mp
lit. 108.5–109.5 ꢂC); [a]D27 À22.8ꢂ (c 1.0, MeOH; lit. [a]D18
À20.2ꢂ; c 1, MeOH); lit: Carroll, F. I.; Lewin, A. H.; Abra-
ham, P.; Parham, K.; Boja, J. W.; Kuhar, M. J. J. Med. Chem.
1
1991, 34, 883. >99% ee by H NMR with (S)-(+)-2,2,2-tri-
fluoro-1-(9-anthryl)ethanol [(S)-(+)-TFAE].
A
column) eluting with hexane/2-PrOH/triethylamine
29. Note 2. Compound (+)-6a: mp 126.5–127.5 ꢂC (mp lit.11
132–133 ꢂC); [a]2D6 +19.6ꢂ (c 1.0, MeOH); lit.11 [a]2D1 +21.6ꢂ (c
1, MeOH); analytic HPLC (Klassix Chiral-A column) eluting
with hexane/2-PrOH/triethylamine (97.5:2.5:0.5) tR 9.29 min,
>99% ee; Anal. (C23H25NF2O3) for C,H,N. Compound (+)-
6b: mp 60–61 ꢂC; [a]2D6 +20.5ꢂ (c 1.0, MeOH); analytic HPLC
(Klassix Chiral-A column) eluting with hexane/2-PrOH/tri-
ethylamine (100:1:1) tR 6.6 min, >99% ee. IR: 1726, 1605
(100:1.5:1) tR 7.4 min, >99% ee; IR: 1733, 1605 cmÀ1 1H
;
NMR (CDCl3) d 1.75–2.17 (6H, m), 2.17 (3H, s), 2.75 (1H, m),
3.09 (1H, m), 3.60 (1H, m), 4.02 (1H, d, J=4.8 Hz), 5.03 (1H,
d, J=12.5 Hz), 5.19 (1H, d, J=12.5 Hz), 5.34 (1H, s), 6.93–
7.04 (4H, m), 7.21–7.39 (9H, m); 13C NMR (CDCl3) d 25.0,
25.6, 36.6, 42.1, 52.3, 61.4, 63.5, 66.6, 70.5, 80.7, 115.5, 115.8,
128.4, 129.0, 136.5, 138.6, 160.8, 164.1, 172.6; EIMS m/z
477(M+). Anal. (C29H27NF2O3) for C, H, N.
1
cmÀ1; H NMR (CDCl3) d 1.22 (3H, t, J=7.0 Hz), 1.70–2.10