Synthesis and monoamine uptake inhibition of conformationally constrained 2β-carbomethoxy-3β-phenyl tropanes

Article abstract of DOI:10.1039/b902863c

A series of 2β-carbomethoxy-3β-phenyl tropanes with conformationally constrained nitrogen substituents were synthesized as potential selective dopamine transporter ligands. These novel compounds were examined for their monoamine uptake inhibition potency at the human dopamine transporter (hDAT), the human serotonin transporter (hSERT) and the human noradrenalin transporter (hNET), stably expressed in human embryonic kidney cells (HEK). A SAR-study was conducted to determine the contribution of extended, 4-fluorinated, conformationally constrained C₄ chains at the tropane nitrogen to human monoamine transporter affinity and selectivity. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b902863c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and monoamine uptake inhibition of conformationally constrained
2b-carbomethoxy-3b-phenyl tropanes†
Patrick Johannes Riss,*a Rene´ Hummerichb and Patrick Schlossb
Received 11th February 2009, Accepted 31st March 2009
First published as an Advance Article on the web 29th April 2009
DOI: 10.1039/b902863c
A series of 2b-carbomethoxy-3b-phenyl tropanes with conformationally constrained nitrogen
substituents were synthesized as potential selective dopamine transporter ligands. These novel
compounds were examined for their monoamine uptake inhibition potency at the human dopamine
transporter (hDAT), the human serotonin transporter (hSERT) and the human noradrenalin
transporter (hNET), stably expressed in human embryonic kidney cells (HEK). A SAR-study was
conducted to determine the contribution of extended, 4-fluorinated, conformationally constrained C4
chains at the tropane nitrogen to human monoamine transporter affinity and selectivity.
Nevertheless, modifications of the phenyltropane carboxy-
late lead have already generated promising examples of hDAT
The dopaminergic system remains an important molecular target
or hSERT selective radio-ligands. These successful molecular
modifications indicate that the selectivity for the DAT can be
This is due to its relevance to addiction, psychiatric and neurode-
affected by substitutions at the ester function, at the phenyl
Introduction
for basic research, drug development and diagnostic imaging.1
generative diseases.2 The dopamine transporter (DAT) mediated
dopamine reuptake, in particular, has been of significant interest.3
Quantification of the availability of neuronal DAT-binding sites
with positron emission tomography (PET) is a sensitive measure
of the function and integrity of the dopaminergic system.4
Two different classes of compounds display high in vitro affinity
and selectivity to the DAT. These are benzhydryl substituted
piperazine derivatives and ligands based on the methyl 3b-
phenyltropane-2b-carboxylate lead (Fig. 1).5 Radiolabelled high
affinity analogues of the latter proved to be the most promising
candidates for DAT imaging.6,7
ring as well as at the nitrogen.8–9,11,12 With regard to a potential
application as fluorinated DAT radio-ligands for PET, variation of
w-fluorohydrocarbon chains at the tropane nitrogen showed the
most promising results.7,11 A trend in selectivity as well as in affinity
was found in the w-fluoroalkyl series.9,11 While nortropanes are
more potent at the SERT, DAT affinity, SERT–DAT and NET–
DAT ratios correlate with increasing chain lengths.8,9,11,12 The
4-fluorobutyl moiety has rarely been investigated in this relation-
ship. Instead, (E)-configured 3-iodoallyl substituted nortropanes
showed encouraging improvements.13a Isosteric substitution of the
(E)-iodoallyl moiety by an (E)-4-fluorobut-2-ene-1-yl residue led
to the development of (E)-fluorobutenyl substituted 4¢-halophenyl
(FBCFT) and 4¢-methylphenyl (LBT999, 7q) derivatives. Most of
these displayed remarkable selectivity and affinity (Fig. 2).13,14
Fig. 1 2b-Carbomethoxy-3b-phenyl tropane, lead structure for DAT-
selective monoamine transporter ligands.
Fig. 2 FBCFT, LBT-999 and PE2I.
Considerable effort has already been spent on the modification
of the available lead structure.8 However, the development of
highly potent, selective DAT inhibitors was often complicated by
the mixed binding profile of this class of compounds. Various
highly potent cocaine-derived DAT inhibitors show a similar inhi-
bition of norepinephrine or serotonin uptake.8,9 In fact, the hDAT
is closely related to the human serotonin transporter (hSERT; 49%
amino acid homology) and the human norepinephrine transporter
(hNET; 67% amino acid homology).10
It has been proposed that this improvement might be par-
ticularly effected by an olefinic residue in the vicinity of the
nitrogen. These findings may also indicate preferred binding of
extended conformations of the N-C4 substituted derivatives to the
DAT. This is presumably due to their reduced flexibility upon
binding to the DAT in the slow isomerisation step.15 Although
the concept of conformational restriction based monoamine
transporter ligand design has been studied previously,16 no further
systematic elucidation of the contribution of conformational
restriction at the N-substituent has been reported so far.17
aInstitute of NuclearChemistry,JohannesGutenberg-UniversityMainz,Fritz
Strassmann-Weg 2, 55128, Mainz, Germany. E-mail: riss@uni-mainz.de;
Fax: +49 6131 3924692; Tel: +49 6131 3925302
Para-substitution at the phenyl ring is known to have a
significant effect on potency and selectivity of cocaine-analogue
tropanes. Bulky groups may increase SERT affinity, and even
4-iodo and 4-bromo derivatives exhibit a remarkable increase
bInstitute of Mental Health, J 5, 68159, Mannheim, Germany
† Electronic supplementary information (ESI) available: 1H NMR and 13
C
NMR spectra of compounds 7c and 7j. See DOI: 10.1039/b902863c
2688 | Org. Biomol. Chem., 2009, 7, 2688–2698
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in SERT and NET affinity. In contrast, their DAT-inhibition-
potencies and binding affinities are in a similar range as the ones
of the methyl analogues.18
Herein, the influence of conformationally constrained, extended
C4-chains in rigid tropane-based monoamine transporter ligands
to hDAT, hSERT and hNET selectivity and inhibition potency
is examined. The 4-position at the phenyl ring was varied from
hydrogen via fluoride and chloride to methyl. For compari-
son, the non-restricted 4-fluorobutyl derivatives have also been
prepared.
Scheme 2 Synthetic route to 4-fluorobut-2-yne-1-yl chloride.
Cyclopropane 6b and the opposite enantiomer 6c (Fig. 3) were
synthesised stereoselectively as reported recently.21
Nortropanes 5a–d were alkylated using electrophiles 6a–c
(Scheme 3) with acetonitrile as solvent and diisopropylethy-
lamine as base in 75–97% yield. Compounds 7a–d were ob-
tained directly from 7e–h via catalytic hydrogenation with Pd◦
on activated carbon. Compounds 7q and 7r were obtained
via reduction of alkynes 7e and 7h, respectively, with sodium
Results and discussion
The synthesis of Michael-acceptor 3 was performed as published
elsewhere (Scheme 1).19a,b Stereoselective 1,4-addition of the
corresponding Normant-cuprates furnished phenyltropanes 4a–d
in high yield (88%) and high diastereomeric excess (de >95%).20
Demethylation to nortropanes 5a–d was achieved via a modified
procedure,19c providing up to 97% of nortropane. More than 85%
of the demethylated product was isolated after flash chromatogra-
phy in a preparative scale run.
R
(bismethoxyethoxy)aluminium hydride (RedAlꢀ).
Compound 6a (Fig. 3) was obtained from but-2-yne-1,4-diol
via acetylation of both alcohol functions (to give compound 14)
followed by enzyme catalysed selective hydrolysis of one acetyl
group using porcine pancreatic lipase (PPL, E.C. 3.1.1.1.; 96
and 97% respectively). The alcohol product 15 was mesylated
in dichloromethane at 0 ◦C. Subsequent fluorination with CsF
in 2-propanol furnished fluoride 17 in 85% yield. Fluoride 17
was deprotected in methanol containing KOH (10%) at room
temperature to afford alcohol 18 in 88% yield. Appel halogenation
using hexachloroethane in 1,2-dichloroethane followed by bulb-
to-bulb distillation afforded 4-fluorobut-2-yne-1-yl chloride 6a in
88% yield, resulting in an overall yield of 60% over six steps
(Scheme 2).
Scheme 3 Synthesis of compounds 7a–r; see Table 1 for compound
numbers and structures.
The final compounds were purified by semi-preparative HPLC
and converted into water soluble hydrochlorides for cell studies.
The purity of all assayed compounds exceeded 99% (by HPLC
peak-area at UV254).
Saturation analyses of [3H]dopamine ([3H]DA), [3H]serotonin
([3H]5HT) and [3H]noradrenalin ([3H]NE) uptake into HEK293
cells stably expressing the human monoamine transporters hDAT,
hNET and hSERT, respectively, were performed. The resulting
IC50 values are summarized in Table 2. A representative experiment
Fig. 3 Electrophilic building blocks for the introduction of terminally
fluorinated C4-residues.
Scheme 1 Synthesis of nortropanes 5a–d; see Table 1 for compound numbers and structures.
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Table 1 Ligand structures of new compounds and references
Compounda
Structureb
Rc
Named
7a
7b
7c
7d
Me
Cl
F
PRD01
PRD07
PRD11
PRD15
H
7e
7f
7g
7h
Me
Cl
F
PRD04
PRD08
PRD12
PRD16
H
7i
7j
7k
7l
Me
Cl
F
PRD05
PRD09
PRD13
PRD17
Fig.
4
Inhibition of [3H]DA, [3H]NE and [3H]5-HT uptake into
HEK293 cells stably expressing human DAT, NET or SERT, respectively.
HEKhDAT, HEKhNET and HEKhSERT cells were incubated with
transporter buffer containing 250 nM [3H]DA (ꢀ), [3H]NE (ꢁ) and
[3H]5-HT (᭢), respectively, and increasing concentrations of 7l. The IC50
values for the inhibition of the single monoamines obtained in these
representative experiments were as follows: 10.5 0.2 nM for [3H]DA,
172.5 0.3 nM for [3H]NE and 1456 0.2 nM for [3H]5-HT.
H
7m
7n
7o
7p
Me
Cl
F
PRD06
PRD10
PRD14
PRD18
H
7q
7r
Me
H
LBT999
PRD19
This effect is even more pronounced at the hSERT. Inhibition po-
tency significantly increases with the size of the para-substituent:
H (4 mM) < F (2.6 mM) < Cl (1.4 mM) < Me (0.8 mM).
At the hNET, both electron withdrawing substituents show a
similar potency, comparable to the hDAT. Potency decreases from
chlorine to hydrogen (Cl ª F > Me > H). Both compounds
with an electron donating substituent (7a and 7d) show a
remarkable selectivity over the hSERT (>100). They also show
a reasonable selectivity over the hNET (4–5). However, hydrogen
derivative 7d is ~4-fold less potent at the hDAT (IC50 = 33 nM),
compared to 7a (8 nM). On the other hand, both compounds
with electron withdrawing substituents show a similar potency at
hDAT and hNET. As a result, 7b and 7c display a threefold lower
selectivity over the hNET. Selectivity over the hSERT is retained
(60–140).
8
9
F
I
b-CFT
b-CIT
10
11
Cl
I
FECNT
FE-b-CIT
To examine the contribution of both a linear C4-segment as
well as the necessity of an olefinic nitrogen-residue, alkynes
7e–h were prepared. Both compounds containing an electron
donating substituent 7e and 7h display a low nanomolar potency
of 3 nM and 6 nM at the hDAT, respectively. Compared to
the flexible analogues 7a and 7d, a 2-fold to 6-fold increase in
potency is achieved. The results at the hSERT are even more
significant. The introduction of a 4-fluorobut-2-yne-1-yl chain
strikingly increases hSERT potency for 7e–h. The size dependency
of potency and selectivity, observed for the flexible analogues 7a–d,
is absent within the alkyne series. Interestingly, methyl derivative
7e displays outstanding characteristics. This is consistent with the
known restricted methyl analogues e.g. PE2I and LBT999 (7q). Its
high potency is combined with good selectivity over the hSERT
(73-fold) and the NET (10-fold). A significant increase in hDAT
potency is observed for 7h. This is accompanied by even higher
increases at both the hSERT (16 fold) and the hNET (11-fold). The
4-fluorophenyl and 4-chlorophenyl derivatives 7f and 7g show a
moderate potency (~16 nM) at the hDAT. This is comparable
to their flexible analogues. Nonetheless, their hSERT-potency is
partially augmented. In particular 7f displays a 4-fold loss in
12
13
I
FP-b-CIT
Cocaine
a Compound number. b General structure. c Phenyl substituent. d Project
tag or common name of compound.
is shown in Fig. 4.9 The selectivity was expressed as IC50-ratios
between the hSERT and the hDAT as well as the hNET and the
hDAT, respectively.
To elucidate the effect of non-constrained C4-chains, com-
pounds 7a–d were synthesised. Among these, the degree of inhibi-
tion of DA reuptake increases in the sequence H < Cl ª F < Me.
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Table 2 b IC50 values and monoamine transporter selectivity of novel phenyl tropanes, as determined in human embryonic kidney cells (HEK 293) stably
transfected with hDAT, hSERT and hNET-RNA
c
c
Compound
hDATIC c (hDATK )/nM
hSERTIC (hSERTK )/nM
hNETIC (hNETK )/nM
hSERT/hDAT
hNET/hDAT
50
i
50
i
50
i
7a
7b
7c
7d
7e
7f
7g
7h
7i
7.8 0.3
810
5
37
29 1.2
31
136
31
1
104
64
137
121
74
17
89
44
70
50
22
134
51
18
34
16
27
50
57
4.5
210
1.5
4
5
1.5
1.5
4
10
2.5
1.5
2
22
19
33
1
1
1
1400 0.3
2600 0.4
4000 0.5
1
1
1
1
3.3 0.5
240
4
17
16
1
1
270 0.2
1400 0.2
250 0.1
950
220 0.4
690
1400
290
160
420
560
700
2650 200
2300 0.3
(29 6.4)
530 1.5
41
21 1.3
13 0.5
56 0.4
16 0.4
76
175
25 0.5
24
37
84
150
210 0.6
120
(33 13)
10.6 0.5
(130 50)
(70 15)
5.8 0.6
14 0.5
4.3 0.5
3
4
7j
7k
7l
3.5
2.5
17
4.5
3
3
2.5
6
4
3
5
4.2
1.5
2.5
0.6
31
1
5
3
4
2
2
3
2
1
1
11 0.5
5.7 0.3
8.9 0.3
12 0.6
7m
7n
7o
7p
7q
7r
1
1
1
2
34
26
1
1
53.3 0.8
40
8
1
2
9a
(6.3 1.7)
2.5 0.2
(91 5)
(28 7)
(320 130)
10a
11a
12a
13
(130 31)
(110 64)
(580 110)
(180 25)
2
a Taken from ref. 9: IC50 values were converted to Ki values using the Cheng and Prusoff equation by the authors, Ki = IC50/(1 + [L]/Km) (where [L] is the
concentration of [3H]DA, [3H]5-HT or [3H]NE; Km values from Eadie–Hofstee-plots. b Values are mean SD (nM). c Compounds 7a–7r did not exhibit
competitive inhibition of substrate transport.
hSERT selectivity. Also, 7f has a 2-fold lower potency at the hNET,
resulting in a higher hNET selectivity.
diastereoisomer 7i is more selective over the hSERT. Finally,
the introduction of the (S,S)-2-fluoromethylcyclopropylmethyl-
residue led to the discovery of a potent hDAT inhibitor 7l (11 nM)
which provides remarkable selectivity over the hSERT (134-fold)
and the hNET (17-fold). Interestingly, the correlation between
activity and the characteristics of the phenyl substituent is absent
between 7i–l.
To investigate the effect of (E)-configuration, trans-
cyclopropanes 7i–p were examined. These derivatives facilitate
the evaluation of particular effects of olefinic double bonds in
the same position. Both diastereoisomeric forms lead to clearly
distinguishable characteristics. Within the (S,S)-configurated
cyclopropanes, the hDAT activity increases in the sequence
F < Me < H < Cl. In contrast, the hDAT potency within the
(R,R)-analogues increases in the sequence H < F < Cl < Me.
The hSERT and the hNET potencies follow the order H < F <
Me < Cl, completely independent of the absolute configuration.
Compared to the 4-fluorobutyl- and 4-fluorobutynyl-residues,
profound changes can be observed for the compounds containing
an electron withdrawing phenyl substituent. Chloro derivatives
7j and 7n display a remarkably high hDAT potency of 4 and
9 nM, respectively. However, the (S,S)-analogue 7j is 2.5-fold
more selective to the hSERT (SERT/DAT ~ 50) and 1.3-fold
more selective to the hNET (NET/DAT ~3.6). In contrast, the
overall activities of fluoro-analogues 7k and 7n are significantly
lower (31 nM and 12 nM, respectively). In this case the (R,R)
analogue 7o exhibits a 1.5-fold higher hSERT selectivity and a
1.25-fold higher selectivity over the hNET. Among the compounds
containing an electron donating phenyl substituent, the hSERT
and hNET potencies of the (S,S)-derivatives are slightly (at least
2-fold) lower than the corresponding (R,R)-analogues. Conversely,
(R,R)-analogue 7p shows the lowest hDAT potency (34 nM) of all
the novel compounds. Methyl derivative 7i has a lower hDAT-
potency (14 nM) than its (R,R)-counterpart 7m (6 nM). Both
display similar selectivity over the hNET. However, the (S,S)-
Conclusion
In summary, the overall potency within the 4-fluorobutyl series is
dependent on the size of the phenyl-substituent. In comparison,
the introduction of a linear alkyne strongly increases activity at
all transporters. Among the cyclopropane containing ligands,
both diastereoisomeric isomers lead to clearly distinguishable
characteristics.
The above findings indicate a strong influence of inflexible ni-
trogen substituents. In particular, the extended (S,S)-configurated
side chain. The N-substituent might influence the overall orien-
tation of the molecule, which leads to a different arrangement of
the phenyl substituent in the proximity of the aromatic binding
site. This preset orientation might furthermore limit the beneficial
effect of aromatic interactions on binding and thereby inhibition
potency.
In all other cases, the overall effect of the phenyl-substituents
remains similar as described for the N-methyl analogues. More or
less the same order of affinity is found within the present study.
This could give rise to the conclusion that the derivatives presented
herein still bind in the same environment as the non-N-modified
tropanes.
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Most novel derivatives provide low to moderate nanomolar IC50
at the hDAT (7a,c,e–j,l–o) and selectivity over the hSERT (7a–p)
and also over the hNET (7a,d–f,h–p). Two potent compounds,
7e and 7l, emerge as outstandingly selective over the hNET (10
and 17-fold, respectively), while maintaining significant selectivity
over the hSERT (74 and 134-fold, respectively). In both cases, the
inhibition potencies remain in a range comparable to the reference
compounds (7q, 8–9, 11–12), whereas their selectivity ratios exceed
the values for these reference compounds.
to -78 ◦C and TFA (4.56 g, 40 mmol) in CH2Cl2 was added
dropwise over 30 min. The solvents were removed in vacuo to leave
a semisolid residue that was partitioned between CH2Cl2 and cold
28% ammonium hydroxide solution (25 ml). The aqueous phase
was extracted with CH2Cl2 (2 ¥ 40 ml) followed by Et2O (40 ml).
Purification by flash column chromatography (Et2O–hexanes, 1 :
9, 10% NEt3) provided 4a–d as colourless to slightly yellow solids
in 88–90% yield (4.78 g, 35 mmol). NMR-spectra and FD-mass
were in accordance with those published elsewhere.19a,b
The only exception in terms of a superior hSERT–hDAT ratio
is the clinically established imaging-agent FECNT (10) which,
on the other hand, provides only moderate selectivity over the
hNET. Furthermore, LBT-999 (7q) has recently been validated as
an appropriate radio-ligand for PET-studies of the striatal and
extra-striatal DAT. Based on these findings we conclude that two
potent DAT-inhibitors (7e and 7l) of improved in vitro selectivity
have been discovered.
Procedure A: general procedure for N-alkylation
Nortropane 5a–d (100 mg, 0.35–0.45 mmol) was added to a stirred
solution of Hu¨nig’s base (1.01 equiv.) in 10 ml of acetonitrile.
Electrophile 6a–c (1 equiv.) was added and the mixture was stirred
at 70 ◦C for 12 h. The mixture was carefully concentrated in vacuo
to leave a mobile residue that was chromatographed on silica gel
(20 g, ether–hexanes, 1 : 9, 10% NEt3) to afford products 7e–p in
75–97% yield.
Compounds 7e and 7l are currently under investigation regard-
ing their potential as 11C and 18F-labelled radio-probes for the
non-invasive quantification of DAT availability in living subjects.
Procedure B: general procedure for N-demethylation
Tropanes 4a–d (1.5 g, 5.4–6.1 mmol) were dissolved in
dichloroethane (30 ml) and refluxed with 1-chloroethyl chloro-
formate (7 equiv.) for one hour. One equiv. of N,N-diisopropyl-N-
ethylamine (700–790 mg) was added and the mixture was refluxed
for one additional hour. Subsequently, the reaction mixture was
concentrated in vacuo to leave a colourless, viscous residue that
was taken up in MeOH (30 ml) with cooling and stirring. After
refluxing for 2 additional hours, the resultant, pale yellow solution
was concentrated, and the residue was taken up in cold ammonium
hydroxide solution (28%, 15 ml) with intense cooling and extracted
with Et2O (2 ¥ 25 ml), followed by dichloromethane (2 ¥ 25 ml) and
again Et2O (25 ml). Combination, drying (anhydrous K2CO3) and
concentration of the organic layers afforded crude nortropanes
5a–d. Purification was performed on silica gel 60 (AcOEt–hexanes,
3 : 7, 10% NEt3) to obtain products 5a–d in 85–88% yield. NMR
spectra were in accordance with those published previously.13a,19a,b
Experimental section
R
Melting points were determined on an Electrothermalꢀ 9100
melting point apparatus and reported uncorrected. NMR-spectra
were recorded with a Bruker AC 300 FT-NMR-spectrometer,
J values are given in Hertz, chemical shifts are reported downfield
from TMS (d = 0 ppm), referred to the solvent residual signal
1H NMR (CHCl3 7.24 ppm) and 13C NMR (CDCl3 77.0 ppm).
Field desorption (FD) mass spectra were recorded on a Finnigan
MAT90 FD spectrometer. HRMS-spectra were measured on a
Micromass QTOF Ultima 3 spectrometer. IR-spectra were ob-
tained from a Nicolet 6700 FTIR spectrometer. Optical rotations
were determined using a Perkin-Elmer polarimeter 241 at 546
and 578 nm (Hg-lamp) and were extrapolated to the sodium D
line. [a]D-values are given in 10-1 deg cm2 g-1. Boiling points are
uncorrected. All chemicals were obtained in commercial quality
from Acros Organics, Sigma Aldrich, VWR, TCI or STREM and
used without further purification. Enzymes were obtained from
Sigma-Aldrich. TLC was conducted on self-cut Merck silica gel
60 covered aluminium plates. Detection and staining were per-
formed either using iodine on silica gel, potassium permanganate
solution, UV fluorescence, vanillin–sulfuric acid, Seebach-reagent
(phosphomolybdic acid, cerium sulfate, H2SO4) or Dragendorff-
reagent (basic bismuth nitrate, potassium iodide and tartaric acid).
Column chromatography was performed on Acros silica gel 60,
0.063–0.200 mesh, p. a. solvents for chromatography were washed
with aqueous acid and base and distilled once, prior to use.
Anhydrous solvents were used for reactions.
1,4-Diactyl but-2-yne-1,4-diol (14)
But-2-yne-1,4-diol (8.61◦ g, 0.1 mol) was dissolved in CH2Cl2
(75 ml) and cooled to 0 C. Acetic anhydride (25 ml) was added
with efficient stirring and the reaction was initiated via the addition
of 1–2 drops of 97% sulfuric acid. The reaction mixture rapidly
heated to reflux and darkened. Stirring was continued overnight
at RT. The reaction mixture was washed with 1 M potassium
carbonate solution (35 ml), followed by water (35 ml), dried
over Na2SO4 and concentrated. Distillation afforded 94% (15.9 g,
0.094 mol) of a colourless liquid that crystallized upon standing:
mp. 30–31 ◦C; C8H10O4 requires C 56.5, H 5.9, found C 56.7,
H 5.9%; dH (300 MHz, CDCl3): 4.11 (s, 2H), 2.06 (s, 3 H); dC
(100 MHz, CDCl3): 170.1, 80.7, 52.0, 20.6; nmax/cm-1 (neat): 2944,
2359, 1739, 1433, 1377, 1360, 1210, 1153, 1021, 964, 604; m/z (FD)
170.1 (100) C8H10O4 requires 170.0579.
2b-Carbomethoxy-3b-phenyltropanes (4a–d)
A solution of p-substituted phenylmagnesium bromide (40 mmol,
40 ml) 1 M in THF was added to a suspension of CuI (7.59 g,
◦
40 mmol) in THF (50 ml) under nitrogen and stirred at 0 C for
30 min. The mixture was cooled to -43 ◦C and ecgonidine methyl
ester (3.6 g, 20 mmol) in THF (6 : 4, 75 ml) was added dropwise, so
that the temperature inside the flask did not exceed -40◦ C. Stirring
was continued for 5 h after which the reaction mixture was cooled
4-Acetoxy-but-2-yne-1-ol (15)
A solution of 1,4-diactyl but-2-yne-1,4-diol (5.0 g, 0.03 mol) in
dioxane (10 ml) was added at once to 500 mg of porcine pancreatic
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lipase in 0,1 M phosphate buffer (25 ml, pH 6,9) at 25 ◦C. The
reaction mixture was stirred at 25 ◦C for 12 h while maintaining the
pH stable via the controlled addition of 1,3 M NaOH solution. To
determine the progress of the reaction, 500 ml samples were taken
from the mixture, extracted with Et2O and the organic extracts
were analyzed by TLC. After 12–14 h the reaction was interrupted
leaving less than 10% of 1,4-diactyl but-2-yne-1,4-diol unaffected.
Chromatography on silica gel (30g/g; 40% ethyl acetate in hexanes,
Rf = 0.6) afforded 3.31 g (0.029 mol) of 15 as a colourless liquid
(88%). C6H8O3 requires C 56.2, H 6.3, found C 56.4, H 6.4%;
stirred at RT for 30 min after which all acetate 17 had been
consumed. The reaction mixture was concentrated in vacuo to
leave a waxy, solid residue that was taken up in Et2O (20 ml). The
solids were filtered off and the ethereal solution was washed with
water (10 ml), followed by brine (5 ml). The organic layer was dried
and concentrated to afford 18 in 88% yield: C4H5FO requires C
54.5, H 5.7, found C 54.1, H 5.8%; nmax/cm-1 (neat): 3358, 2872,
2855, 1454, 1295, 1138, 1025, 982, 907, 731; dH (300 MHz, CDCl3):
5.00 (t, J = 1.8 Hz, JH-F = 47.4 Hz, 1 H), 4.84 (t, J = 1.8 Hz, JH-F
=
47.4 Hz, 1 H), 4.05 (t, J = 1.8 Hz, 2 H), 2.04 (s, 3 H); dC (100 MHz,
CDCl3): 89.0 (d, JC-F = 22 Hz), 85.1 (d, JC-F = 22 Hz), 70.6 (d,
JC-F = 163.9 Hz), 50.4; m/z (FD) 88.1 (100) C4H5FO requires
88.0324.
n
max/cm-1 (neat): 3437, 2940, 2860, 2359, 1736, 1436, 1378, 1359,
1219, 1136, 1015, 965, 606; dH (300 MHz, CDCl3): 4.69 (t, J =
1.8 Hz, 2 H), 4.26 (t, J = 1.8 Hz, 2 H), 2.28 (brs, 1 H, OH), 2.07
(s, 3 H); dC (100 MHz, CDCl3): 170.5, 85.1, 79.5, 52.3, 50.8, 20.7;
m/z (FD) 128.1 (100) C6H8O3 requires 128.0473.
4-Fluorobut-2-yne-1-yl chloride (6a)
4-Acetoxy-but-2-yne-1-yl mesylate (16)
4-Fluorobut-2-yne-1-ol (1.0 g, 11 mmol) was dissolved in dry
C2H4Cl2 (10 ml) containing triphenylphosphine (1.00 equiv.). The
mixture was cooled to 0 ◦C with stirring over 30 min. Then C2Cl6
(2.6 g, 11 mmol) was added in portions. The mixture was stirred for
one additional hour. Hexane was added until the mixture turned
slightly turbid. The mixture was passed through a short silica
column to remove the triphenylphosphine oxide and the solvent
was evaporated. The volatile residue was purified via bulb to bulb
distillation to obtain 6a as a colourless, mobile oil. C4H4ClF
requires C 45.10, H 3.78, found: C 45.3, H 3.7%; nmax/cm-1
(neat): 2955, 1454, 1430, 1373, 1264, 1155, 987, 787, 699, 524;
4-Acetoxy-but-2-yne-1-ol (1.28 g, 10 mmol) was dissolved in
CH2Cl2 (10 ml) and triethylamine (1.02 g, 10 mmol) was added.
After stirring at 0 ◦C for 30 min, methanesulfonyl chloride
(1.146 g, 10 mmol) was added dropwise with stirring. After all
the methanesulfonyl chloride had been added (~10 min), TLC
indicated complete conversion of 15. The reaction mixture was
filtered and the filter cake was washed with cold CH2Cl2 (2 ¥
10 ml). The filtrate was washed with 1 M K2CO3 (15 ml), followed
by water (15 ml). Subsequent drying and concentration in vacuo
afforded 16 (1.95 g, 9.5 mmol; 95%) as a slightly turbid, colourless
oil. C7H10O5S requires C 40.77, H 4.89, S 15.55, found C, 40.7 H,
4.95, S 15.6%. nmax/cm-1 (neat): 3028, 2942, 2362, 1739, 1435, 1350,
1220, 1171, 1029, 936, 803, 526; dH (300 MHz, CDCl3): 4.86 (t,
J = 2 Hz, 2 H), 4.71 (t, J = 2 Hz, 2 H), 3.10 (s, 3 H), 2.07 (s, 3 H).
dC (100 MHz, CDCl3): 170.1, 83.9, 78.7, 57.3, 51.7, 39.0, 20.6; m/z
(FD) 206.1 (100) C7H10O5S requires 206.0249.
d
H (300 MHz, CDCl3): 4.99 (dt, J = 1.8 Hz, JH-F = 47.4 Hz, 2 H),
4.19 (dt, J = 1.8 Hz, J = 7.0 Hz, 1 H), 4.17 (dt, J = 1.8 Hz,
J = 7.0 Hz, 1 H); dC (100 MHz, CDCl3): 84.6, 79.8, 70.4 (d,
JC-F = 166.2 Hz), 29.8; m/z (FD) 106.0 (100) C4H4ClF requires
106.0.
Methyl 8-(4-fluorobutyl)-3-p-tolyl-8-aza-bicyclo[3.2.1]octane-2-
carboxylate (7a, PRD01)
4-Acetoxy-but-2-yne-1-yl fluoride (17)
Caesium fluoride (1.51 g, 10 mmol) was suspended in 2-propanol
(15 ml) and heated to reflux. After the inorganic material had
dissolved, 4-acetoxy-but-2-yne-1-yl mesylate (1.35 g, 6.6 mmol)
was added dropwise with stirring. The reaction mixture was
refluxed for a further 90 min. A waxy caesium mesylate precipitate
indicated the reaction’s progress. After all the mesylate had been
consumed (TLC-monitoring) the reaction mixture was cooled to
RT, filtered and the filter cake was washed with cold Et2O and
concentrated. The oily brown residue was chromatographed on
silica gel (Et2O–hexanes) to obtain 17 (730 mg, 85%, 5.6 mmol) as
a slightly yellow liquid: C6H7FO2 requires C 55.4, H 5.4, found:
C 55.3, H 5.4%; nmax/cm-1 (neat): 2943, 2850, 1742, 1434, 1376,
1360, 1216, 1150, 1025, 988, 969; dH (300 MHz, CDCl3): 5.01
(t, J = 1.8 Hz, JH-F = 47.7 Hz, 1 H), 4.85 (t, J = 1.8 Hz,
JH-F = 47.7 Hz, 1 H), 4.05 (t, J = 1.8 Hz, 2 H), 2.04 (s,
7e (100 mg, 0.3 mmol), was dissolved in EtOH (5 ml) and Pd◦ (5%)
on activated carbon was added (10 mg). H2 was passed through
this solution until all the olefin had been consumed (monitored by
R
TLC). The reaction mixture was filtered through a pad of celiteꢀ,
the filter cake was washed with EtOH (5 ml) followed by CH2Cl2
(10 ml). The organic phases were combined and concentrated
in vacuo. The residue was purified via chromatography on silica
gel (Et2O–hexanes, 1 : 4, 10% NEt3)◦to obtain 7a as colourless
crystals (90 mg, 89%): mp. 66–77 C; [a]2D3 -39.2 (c 1.25 in
MeOH). C20H28FNO2 requires C 72.04, H 8.46, N 4.20, found:
C 72.16, H 8.47, N, 4.22%; dH (300 MHz, CDCl3): 7.13 (d, J =
8.5 Hz, 2 H, ArH), 7.05 (d, J = 8.5 Hz, 2 H, ArH), 4.42 (dt,
J = 6 Hz, JHF = 47.4 Hz, 2 H), 3.65 (brs, 1H), 3.45 (s, 3 H,
OCH3), 3.36 (brs, 1 H), 2.97 (dt, J = 4.8 Hz, J = 13.2 Hz,
1 H), 2.88 (t, J = 4 Hz, 1 H), 2.54 (J = 2.9 Hz, J = 12.5 Hz,
1 H), 2.32–2.20 (m, 2H), 2.27 (s, 3H, ArCH3), 2.10–1.90 (m, 2 H),
1.82–1.53 (m, 5 H), 1.52–1.38 (m 2 H); dC (100 MHz, CDCl3):
172.2, 138.1, 127.9, 127.3, 125.7, 84.0 (d, JC-F = 161.4 Hz), 62.8,
61.4, 56.8, 52.9, 50.9, 34.3, 33.9, 28.2, 27.9, 26.0, 25.9, 24.7,
24.6, 21.0; m/z (FD) 334.2 (100) C20H29FNO2 requires 334.2;
HRMS(ESI): exact mass calcd for C20H29FNO2: 334.2182, found:
334.2180.
3 H); dC (100 MHz, CDCl3): 170.1, 83.9, 81.3, 70.4 (d, JC-F
=
161.5 Hz), 52.1, 20.6; m/z (FD) 130.1 (100) C6H7FO2 requires
130.0430.
4-Fluorobut-2-yne-1-ol (18)
4-Acetoxy-but-2-yne-1-yl fluoride (1.3 g, 10 mmol) was dissolved
in methanol (10 ml) containing KOH (560 mg, 10 mmol) and
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Org. Biomol. Chem., 2009, 7, 2688–2698 | 2693
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Methyl 8-(4-fluorobut-2-en-1-yl)-3-p-tolyl-8-aza-bicyclo[3.2.1]
octane-2-carboxylate (7q, LBT999)
61.3, 56.5, 52.9, 50.9, 34.1, 33.8, 26.0, 25.9, 21.0, 16.6, 16.5, 16.2,
15.9, 9.5, 9.4; m/z (FD) 346.2 (100) C21H29FNO2 requires 346.2;
HRMS(ESI): exact mass calcd for C21H29FNO2: 346.2182, found:
346.2181.
7e◦ (100 mg) was dissolved in dry THF (5 ml) and cooled to
R
0
C. RedAlꢀ (0.2 mL, 0,36 mmol; 70% in toluene) was added
dropwise under nitrogen. The reaction mixture was stirred at RT
for 1 h. The reaction was terminated by the dropwise addition of
saturated ammonium chloride solution. The mixture was further
stirred and allowed to warm to RT. The reaction mixture was
Methyl 8-(((1R,2R)-2-(fluoromethyl)cyclopropyl)methyl)-3-p-
tolyl-8-aza-bicyclo[3.2.1]octane-2-carboxylate (7m, PRD06)
Synthesised according to procedure A, 86% yield. Colourless
R
filtered through a pad of celiteꢀ and the filter cake was washed
◦
crystals: mp. 77 C; [a]2D3 -27.7 (c 1.21 in MeOH). C21H28FNO2
with THF (5 ml) and acetone (2 ¥ 5 ml). The filtrate was dried
(MgSO4) and concentrated in vacuo. The residue was purified via
column chromatography (Et2O–hexanes, 1 :4, 10%NEt3) to obtain
requires C 73.01, H 8.17, N 4.05, found C 73.4, H 8.1, N 3.95%.
dH (300 MHz, CDCl3): 7.14 (d, J = 8 Hz, 2 H, ArH), 7.06 (d,
J = 8 Hz, 2 H, ArH), 4.33 (m, JH-F = 48.9 Hz, 1 H), 4.17 (m,
JH-F = 48.9 Hz, 1 H), 3.91 (brs, 1H), 3.48 (s, 1H, OCH3), 3.40
(brs, 1 H), 2.98 (dt, J = 12.9 Hz, J = 4.4 Hz, 1 H), 2.91 (t, J =
4.4 Hz, 1 H), 2.58 (td, J = 12.5 Hz, J = 2.9 Hz, 1 H), 2.47 (dd,
J = 12.5 Hz, J = 3.7 Hz, 1 H), 2.28 (s, 3 H, NCH3), 2.1 (m, 3 H),
1.83–1.53 (m, 3 H), 1.03 (m, 1 H), 0.81 (m, 1 H), 0.45 (m, 2 H); dC
◦
7q as colourless crystals (86 mg, 85%): mp. 113.5 C; [a]2D3 -16.9
(c 1.42 in MeOH). C20H26FNO2 requires C 72.48, H 7.91, N 4.23,
found: C 72.20, H 8.09, N 4.41%; dH (300 MHz, CDCl3): 7.14 (d,
J = 8 Hz, 2 H, ArH), 7.06 (d, J = 8 Hz, 2 H, ArH), 5.82–5.74 (m,
2 H), 4.89 (d, J = 5 Hz, JH-F = 47.4 Hz, 1 H), 4.73 (d, J = 3 Hz,
JH-F = 47.4 Hz, 1 H), 3.69 (brs, 1H), 3.48 (s, 1H, OCH3), 3.40 (brs,
1 H), 3.06–2.92 (m, 2 H), 2.91–2.81 (m, 2 H), 2.59 (dt, J = 12.5 Hz,
J = 2.7 Hz, 1 H), 2.28 (s, 3 H, NCH3), 2.13–1.92 (m, 2 H), 1.78–
1.57 (m, 3 H); dC (100 MHz, CDCl3): 172.0, 139.9, 135.2, 134.4 (d,
(100 MHz, CDCl3): 172.1, 140.0, 135.1, 128.6, 127.2, 87.3 (d, JC-F
=
161.5 Hz), 62.0, 61.8, 56.4, 52.7, 50.9, 34.0, 33.9, 26.0, 25.8, 21.0,
18.5, 18.2, 16.6, 16.5, 7.3, 7.2; m/z (FD) 346.2 C21H29FNO2 346.2;
HRMS(ESI): exact mass calcd for C21H29FNO2: 346.2182, found:
346.2188.
JC-F = 12 Hz), 128.6, 127.2, 126.2 (d, JC-F = 17 Hz), 83.1 (d, JC-F
=
161 Hz), 62.3, 61.3, 54.9, 52.7, 50.9, 34.1, 33.8, 26.1, 25.9, 21.0;
m/z (FD) 332.2 (100) C20H27FNO2 requires 332.2; HRMS(ESI):
exact mass calcd for C20H27FNO2: 332.2026, found: 332.2024.
Methyl 8-(4-fluorobutyl)-3-(4-chlorophenyl)-8-aza-bicyclo
[3.2.1]octane-2-carboxylate (7b, PRD07)
Methyl 8-(4-fluorobut-2-yn-1-yl)-3-p-tolyl-8-aza-bicyclo[3.2.1]
octane-2-carboxylate (7e, PRD04)
For preparative details see 7a, 75% yield. Colourless crystals: mp.
◦
70 C; [a]2D3 -44.1 (c 0.33 in MeOH). C19H25ClFNO2 requires C
64.49, H 7.12, N 3.96, found C 64.6, H 6.9, N 3.9%. dH (300 MHz,
CDCl3): 7.22 (d, J = 8.8 Hz, 2 H, ArH), 7.15 (d, J = 8.8, 2 H,
ArH), 4.50 (t, J = 6.2 Hz, JH-F = 47.4 Hz, 1 H), 4.34 (t J = 6.2 Hz,
JH-F = 47.4 Hz, 1 H), 3.66 (brs, 1 H), 3.46 (s, 3H, CH3), 3.37 (brs,
1 H), 2.95 (dt, J = 4.8 Hz, J = 12.9 Hz, 1 H), 2.86 (m, 1 H),
2.52 (dt, J = 2.9 Hz, J = 12.5 Hz, 1 H), 2.26 (m, 2 H), 2.02 (m,
2 H), 1.65 (m, 5 H), 1.46 (m, 2 H); dC (100 MHz, CDCl3): 171.8,
141.8, 131.4, 128.7, 128.0, 84.2 (d, JC-F = 161.5 Hz), 62.9, 61.3,
52.9, 50.9, 38.7, 34.0, 33.8, 28.2, 27.9, 26.0, 25.9, 24.7, 24.6; m/z
(FD) 353.2 C21H29FNO2 requires 353.2; HRMS(ESI): exact mass
calcd for C19H25ClFNO2: 353.1558, found: 353.1560.
Synthesised according to procedure A, 97% yield. Yellowish
crystals: mp. 73.5 ◦C; [a]D23 -14.7 (c 1.46 in MeOH). C20H24FNO2
requires C 72.92, H 7.34, N 4.25, found: C 72.79, H 7.03, N 4.53%.
dH (300 MHz, CDCl3): 7.14 (d, J = 8.5 Hz, 2 H, ArH), 7.06 (d,
J = 8.5 Hz, 2 H, ArH), 5.02 (t, J = 1.5 Hz, JH-F = 47.8 Hz, 1 H),
4.86 (t, J = 1.5 Hz, JH-F = 47.8 Hz, 1 H), 3.89 (brs, 1 H), 3.51 (s,
3 H, OCH3), 3.47 (brs, 1 H), 3.25 (ddt, J = 1.5 Hz, J = 7.5 Hz, J =
16.5 Hz, 1 H), 3.10 (ddt, J = 1.5 Hz, J = 7.5 Hz, J = 16.5 Hz, 1 H),
2.98 (dt, J = 5 Hz, J = 12.9 Hz, 1 H), 2.94–2.89 (m, 1 H), 2.62 (dt,
J = 2.9 Hz, J = 12.5 Hz, 1 H), 2.27 (s, 3 H, ArCH3), 2.16–1.92
(m, 2 H), 1.82–1.61 (m, 3 H); dC (100 MHz, CDCl3): 171.7, 139.6,
135.3, 128.7, 127.2, 127.1, 87.7, 87.5, 70.8 (d, JC-F = 163.5 Hz),
62.6, 61.2, 52.8, 52.6, 51.1, 42.9, 34.1, 33.7, 25.9, 25.7, 21.0; m/z
(FD) 330.2 (100) C20H25FNO2 requires 330.2; HRMS(ESI): exact
mass calcd for C20H25FNO2: 330.1869, found: 330.1885.
Methyl 8-(4-fluorobut-2-yn-1-yl)-3-(4-chlorophenyl)-8-aza-
bicyclo[3.2.1]octane-2-carboxylate (7f, PRD08)
Synthesised acc◦ording to procedure A, 95% yield. Yellowish
crystals: mp. 76 C; [a]2D3 -13.2 (c 1.58 in MeOH). C19H21ClFNO2
requires C 65.23, H 6.05, N 4.00, found C 65.5, H 6.15, N 3.8%;
dH (300 MHz, CDCl3): 7.23 (d, J = 8.5 Hz, 2 H, ArH), 7.16 (d,
J = 8.5, 2 H, ArH), 5.01 (t, J = 2.2 Hz, JH-F = 47.4 Hz, 1 H) 4.85
(t J = 2.2 Hz, JH-F = 47.4 Hz, 1 H), 3.90 (brs, 1 H), 3.51 (s, 3H,
OCH3), 3.47 (brs, 1 H), 3.25 (ddt, J = 1.5 Hz, J = 7.5 Hz, J =
16.5 Hz, 1 H), 3.10 (ddt, J = 1.5 Hz, J = 7.5 Hz, J = 16.5 Hz,
1 H) 2.97 (dt, J = 5.1 Hz, J = 12.5 Hz, 1 H), 2.90 (m, 1 H), 2.59
(dt, J = 2.9 Hz, J = 12.5 Hz, 1 H), 2.05 (m, 2 H), 1.70 (m, 4 H);
dC (100 MHz, CDCl3): 171.5, 141.3, 131.6, 128.7, 128.0, 87.4, 70.8
(d, JC-F = 163.5 Hz), 62.5, 61.0, 52.5, 51.2, 42.9, 42.8, 34.0, 33.7,
25.8, 25.6; m/z (FD) 350.1 (100) C19H22ClFNO2 350.1; HRMS-
(ESI): exact mass calcd for C19H22ClFNO2: 350.1323, found:
350.1331.
Methyl 8-(((1S,2S)-2-(fluoromethyl)cyclopropyl)methyl)-3-p-tolyl-
8-aza-bicyclo[3.2.1]octane-2-carboxylate (7i, PRD05)
Synthesised according to procedure A, 87% yield. Colourless
◦
crystals: mp. 79 C; [a]2D3 -26.0 (c 1.38 in MeOH). C21H28FNO2
requires C 73.01, H 8.17, N 4.05, found C 72.9, H 8.2, N 4.0%. dH
(300 MHz, CDCl3): 7.14 (d, J = 8 Hz, 2 H, ArH), 7.06 (d, J = 8 Hz,
2 H, ArH), 4.23 (dm, JH-F = 48.5 Hz, 2 H), 3.81 (brs, 1H), 3.48 (s,
1H, OCH3), 3.45 (brs, 1 H), 2.98 (dt, J = 12.5 Hz, J = 5 Hz, 1 H),
2.89 (t, J = 4 Hz, 1 H), 2.57 (td, J = 12.5 Hz, J = 2.6 Hz, 1 H), 2.42
(dd, J = 12.5 Hz, J = 5 Hz, 1 H), 2.28 (s, 3 H, NCH3), 2.07 (dd,
J = 12.5 Hz, J = 7 Hz, 1 H), 1.98 (m, 2 H), 1.76–1.54 (m, 3 H),
1.01 (m, 1 H), 0.82 (m, 1 H), 0.52 (m, 2 H); dC (100 MHz, CDCl3):
172.2, 140.1, 135.1, 128.6, 127.2, 87.4 (d, JC-F = 161.5 Hz), 62.9,
2694 | Org. Biomol. Chem., 2009, 7, 2688–2698
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Methyl 8-(((1S,2S)-2-(fluoromethyl)cyclopropyl)methyl)-3-
(4-chlorophenyl)-8-aza-bicyclo[3.2.1]octane-2-carboxylate (7j,
PRD09)
Methyl 8-(4-fluorobut-2-yn-1-yl)-3-(4-fluorophenyl)-8-aza-
bicyclo[3.2.1]octane-2-carboxylate (7g, PRD12)
Synthesised according to procedure A, 96% yield. Colourless
◦
Synthesised according to procedure A, 85% yield. Yellowish
crystals: mp. 72 C; [a]2D3 -60.2 (c 1.17 in MeOH). C19H21F2NO2
◦
crystals: mp. 76–77 C; [a]2D3 -90.6 (hydrochloride) (c 0.96 in
requires C 68.45, H 6.35, N 4.20, found: C 68.7, H 6.6, N 4.40%;
MeOH). dH (300 MHz, CDCl3): 7.21 (d, J = 8.8 Hz, 2 H, ArH),
dH (300 MHz, CDCl3): 7.20 (dd, J = 8.1 Hz, J = 5.5 Hz, 2 H,
7.16 (d, J = 8.5, 2 H, ArH), 4.30 (dq, J = 7.3 Hz, J = 9.56 Hz,
ArH), 6.93 (dd, J = 8.8 Hz, 2 H, ArH), 5.02 (t, J = 2.0 Hz, JH-F
=
JH-F = 48.5 Hz, 1 H), 4.14 (dq, J = 7.3 Hz, J = 9.56 Hz, JH-F
=
49.6 Hz, 1 H), 4.85 (t, J = 2.0 Hz, JH-F = 49.6 Hz, 1 H), 3.89 (brs,
1 H), 3.51 (s, 3H, CH3), 3.48 (brs, 1 H), 3.26 (ddt, J = 1.5 Hz, J =
7.4 Hz, J = 16.5 Hz, 1 H), 3.11 (ddt, J = 1.5 Hz, J = 7.4 Hz, J =
16.5 Hz, 1 H), 2.98 (dt, J = 5.1 Hz, J = 12.5 Hz, 1 H), 2.82 (m,
1 H), 2.61 (dt, J = 2.9 Hz, J = 12.5 Hz, 1 H), 2.05 (m, 2 H), 1.70
(m, 4 H); dC (100 MHz, CDCl3): 171.5, 161.4 (d, JC-F = 253 Hz),
138.3, 128.9, 128.8, 114.8, 114.5, 87.5, 70.7 (d, JC-F = 163.5 Hz),
62.5, 61.1, 52.7, 51.1, 42.9, 34.2, 33.6, 25.8, 25.7; m/z (FD) 334.2
(100) C19H22F2NO2 requires 334.2; HRMS(ESI): exact mass calcd
for C19H22F2NO2: 334.1619 found: 334.1627.
48.5 Hz, 1 H), 3.81 (brs, 1 H), 3.48 (s, 3 H, CH3) 3.45 (brs, 1H),
2.95 (dt, J = 5 Hz, J = 12.5 Hz, 1 H), 2.86 (dt, J = 4.4 Hz,
1 H), 2.53 (dt, J = 2.9 Hz, J = 12.5 Hz, 1 H), 2.40 (dd, J =
6 Hz, J = 12.5 Hz, 1 H), 2.07 (dd, J = 6 Hz, J = 12.5 Hz,
1 H), 2.05–1.85 (m, 2 H), 1.75–1.55 (m, 3 H), 0.99 (m, 1 H),
0.80 (m, 1 H), 0.50 (m, 2 H); dC (100 MHz, CDCl3): 171.9,
141.7, 131.4, 129.8, 128.7, 128.0, 87.4 (d, JC-F = 161.5 Hz), 62.9,
61.1, 56.6, 52.8, 51.0, 34.0, 33.8, 26.0, 25.9, 16.6, 16.5, 16.3, 16.0,
9.3, 9.2; m/z (FD) 366.2 (100) C20H26ClFNO2 366.2; HRMS-
(ESI): exact mass calcd for C20H26ClFNO2: 366.1636, found
366.1631.
Methyl 8-(((1R,2R)-2-(fluoromethyl)cyclopropyl)methyl)-3-
(4-fluorophenyl)-8-aza-bicyclo[3.2.1]octane-2-carboxylate (7k,
PRD13)
Methyl 8-(((1R,2R)-2-(fluoromethyl)cyclopropyl)methyl)-3-
(4-chlorophenyl)-8-aza-bicyclo[3.2.1]octane-2-carboxylate (7n,
PRD10)
Synthesised according to procedure A, 89% yield. Colourless to
◦
off white crystals: mp. 74 C; [a]2D3 -22.5 (c 0.58 in MeOH).
Synthesised acc◦ording to procedure A, 88% yield. Off white
crystals: mp. 75 C; [a]2D3 -25.5 (c 0.42 in MeOH). C20H25ClFNO2
requires C 65.66, H 6.89, N 3.83, found C 65.95, H 7.16, N 3.81%.
dH (300 MHz, CDCl3): 7.21 (d, J = 8.5 Hz, 2 H, ArH), 7.16 (d,
J = 8.5, 2 H, ArH), 4.32 (d, J = 7.4 Hz, JH-F = 48.5 Hz, 1 H), 4.17
(dq, J = 7.4 Hz, JH-F = 48.5 Hz, 1 H), 3.91 (brs, 1 H), 3.47 (s, 3 H,
CH3) 3.40 (brs, 1H), 2.96 (dt, J = 5 Hz, J = 12.5 Hz, 1 H), 2.88 (t,
J = 4.4 Hz, 1 H), 2.55 (dt, J = 12.5 Hz, J = 2.9 Hz, 1 H), 2.38 (dd,
J = 12.5 Hz, J = 5 Hz, 1 H), 2.11–1.87 (m, 3 H), 1.79–1.52 (m,
3 H), 1.01 (m, 1 H), 0.80 (m, 1 H), 0.44 (m, 2 H); dC (100 MHz,
CDCl3): 171.9, 141.8, 131.5, 128.7, 128.0, 87.2 (d, JC-F = 160.5 Hz),
62.0, 61.6, 56.4, 52.7, 51.0, 34.0, 33.8, 26.0, 25.8, 18.5, 18.2, 16.6,
16.5, 7.4, 7.3; m/z (FD) 366.2 (100) C20H26ClFNO2 366.2; HRMS-
(ESI): exact mass calcd for C20H26ClFNO2: 366.1636, found:
366.1620.
C20H25F2NO2 requires C 68.75, H 7.21, N 4.01, found: C 68.9,
H 7.1, N 3.9%. dH (300 MHz, CDCl3): 7.19 (dd, J = 8.5 Hz, J =
5.5 Hz, 2H, ArH), 6.93 (t, J = 8.8 Hz, 2 H), 4.30 (dq, J = 4.4 Hz,
J = 7.4 Hz, J = 9.6 Hz, JH-F = 48.9 Hz, 1 H), 4.14 (dq, J = 4.4 Hz,
J = 7.5 Hz, J = 9.5 Hz, JH-F = 48.9 Hz, 1 H), 3.79 (brs, 1 H), 3.49
(s, 3 H, CH3), 3.43 (brs, 1H), 2.97 (dt, J = 5 Hz, J = 12.5 Hz, 1 H),
2.86 (dt, J = 4.4 Hz, 1 H), 2.53 (dt, J = 2.9 Hz, J = 12.5 Hz, 1 H),
2.41 (dd, J = 12.8 Hz, J = 5.5 Hz, 1 H), 2.17 (dd, J = 12.1 Hz,
J = 6.6 Hz, 1 H), 2.03–1.85 (m, 2 H), 1.76–1.53 (m, 3 H), 1.00 (m,
1 H), 0.82 (m, 1 H), 0.50 (m, 2 H); dC (100 MHz, CDCl3): 172.0,
161.0 (d, JC-F = 248 Hz), 138.8, 128.8, 128.6, 114.7, 114.5, 87.5
(d, JC-F = 160.5 Hz), 86.6, 62.9, 61.2, 56.5, 53.0, 51.0, 34.2, 33.8,
26.0, 25.9, 16.6, 16.5, 16.3, 16.0, 9.4, 9.3; m/z (FD) 350.0 (100)
C20H26F2NO2 requires 350.2; HRMS(ESI): exact mass calcd for
C20H26F2NO2: 350.1932 found: 350.1940.
Methyl 8-(4-fluorobutyl)-3-(4-fluorophenyl)-8-aza-bicyclo
[3.2.1]octane-2-carboxylate (7c, PRD11)
Methyl 8-(((1S,2S)-2-(fluoromethyl)cyclopropyl)methyl)-3-
(4-fluorophenyl)-8-aza-bicyclo[3.2.1]octane-2-carboxylate (7o,
PRD14)
For experimental details see 7a, 92% yield. 7c was obtained as
colourless oil that solidified upon standing: mp. 65 ◦C; [a]D23 -90.6
(hydrochloride) (c 1.75 in MeOH). C19H25F2NO2 requires C 67.64,
H 7.47, N 4.15, found C 67.5, H 7.6, N 4.05%. dH (300 MHz,
CDCl3): 7.20 (dd, J = 5.5 Hz, J = 2.9 Hz, 2 H, ArH), 6.93 (t, J =
8.8 Hz, 2 H, ArH), 4.49 (t, J = 5.9 Hz, JH-F = 47.4 Hz, 1 H), 4.33 (t,
J = 5.9 Hz, JH-F = 47.4 Hz, 1 H), 3.67 (brs, 1 H), 3.46 (s, 3H, CH3),
3.36 (brs, 1 H), 2.96 (dt, J = 5.1 Hz, J = 12.5 Hz, 1 H), 2.85 (t, J =
3.7 Hz, 1 H), 2.53 (dt, J = 2.9 Hz, J = 12.5 Hz, 1 H), 2.26 (m, 2 H),
2.02 (m, 2 H), 1.67 (m, 6 H), 1.47 (m, 2 H); dC (100 MHz, CDCl3):
171.9, 161.2 (d, JC-F = 243 Hz), 138.8, 128.8, 128.7, 114.7, 114.4,
84.2 (d, JC-F = 162 Hz), 62.9, 61.4, 52.946, 50.893, 34.2, 33.7, 28.2,
27.9, 26.0, 25.9, 24.7, 24.6; m/z (FD) 338.3 (100) C19H26F2NO2
requires 338.2; HRMS(ESI): exact mass calcd for C19H26F2NO2:
338.1933 found: 338.1932.
Synthesised acc◦ording to procedure A, 83% yield. Colourless
crystals: mp. 72 C; [a]2D3 -75.9 (hydrochloride) (c 1.13 in MeOH).
C20H25F2NO2 requires C 68.75, H 7.21, N 4.01, found: C 68.69,
H 7.0, N 4.3%. dH (300 MHz, CDCl3): 7.21 (dd, J = 8.5 Hz, J =
5.5 Hz, 2H, ArH), 6.93 (t, J = 8.8 Hz, 2 H), 4.30 (dq, J = 7.5 Hz,
J = 9.5 Hz, JH-F = 48.5 Hz, 1 H), 4.16 (dq, J = 7.5 Hz, J = 9.5 Hz,
JH-F = 48.5 Hz, 1 H), 3.79 (brs, 1 H), 3.49 (s, 3 H, CH3), 3.43 (brs,
1H), 2.97 (dt, J = 5 Hz, J = 12.5 Hz, 1 H), 2.89 (dt, J = 4.4 Hz,
J = 1.1 Hz, 1 H), 2.53 (dt, J = 2.9 Hz, J = 12.5 Hz, 1 H), 2.41
(dd, 1 H), 2.00 (m, 3 H), 1.63 (m, 4 H), 1.00 (m, 1 H), 0.80 (m,
1 H), 0.50 (m, 2 H); dC (100 MHz, CDCl3): 172.1, 161.2 (d, JC-F
=
248 Hz), 138.6, 128.9, 128.4, 114.7, 114.5, 87.4 (d, JC-F = 160.0 Hz),
62.5, 61.5, 56.4, 52.9, 51.0, 34.2, 33.9, 26.0, 25.9, 18.5, 18.2, 16.6,
16.5, 7.4, 7.3; m/z (FD) 350.2 (100) C20H26F2NO2 requires 350.2;
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HRMS(ESI): exact mass calcd for C20H26F2NO2: 350.1932, found:
350.1926.
Methyl 8-(((1R,2R)-2-(fluoromethyl)cyclopropyl)methyl)-3-
phenyl-8-aza-bicyclo[3.2.1]octane-2-carboxylate (7p, PRD18)
Synthesised according to procedure A, 84% yield. Colourless
◦
crystals: mp. 88 C; [a]2D3 -30.0 (c 1.17 in MeOH). C20H27FNO2
Methyl 8-(4-fluorobutyl)-3-phenyl-8-aza-bicyclo[3.2.1]
octane-2-carboxylate (7d, PRD15)
requires C 72.48, H 7.91, N 4.23, found: C 72.2, H 8.1, N 4.4%.
dH (300 MHz, CDCl3): 7.25 (brs, 4H, ArH), 7.14 (m, 1H, ArH),
4.33 (dq, J = 7 Hz, J = 9.6 Hz, JH-F = 48.9 Hz, 1 H), 4.13 (dq,
J = 7 Hz, J = 9.6 Hz, JH-F = 48.9 Hz, 1 H), 3.92 (brs, 1 H), 3.46
(s, 3 H, OCH3), 3.42 (brs, 1H), 3.01 (dt, J = 5 Hz, J = 12.9 Hz,
1 H), 2.93 (t, J = 4.4 Hz, 1 H), 2.6 (dt, J = 2.9 Hz, J = 12.5 Hz,
1 H), 2.47 (dd, J = 12.5 Hz, J = 4.8 Hz, 1 H), 2.11–1.87 (m, 2 H),
1.79–1.54 (m, 3 H), 1.03 (m, 1 H), 0.82 (m, 1 H), 0.45 (m, 2 H);
dC (100 MHz, CDCl3): 172.3, 143.1, 127.9, 127.4, 125.7, 87.3 (d,
JC-F = 160.5 Hz), 62.1, 61.8, 56.4, 52.8, 50.9, 34.3, 34.0, 26.0, 25.9,
18.5, 18.2, 16.6, 16.5, 7.4, 7.3; MS (FD) 332.2 (100) C20H27FNO2
requires 323.2; HRMS(ESI): exact mass calcd for C20H27FNO2:
332.2026, found: 332.2035.
For experimental details see 7a, 8◦9% yield. Colourless oil that
solidified upon standing: mp. 61–62 C; [a]2D3 -96.5 (hydrochloride)
(c 1.75 in MeOH). C19H26FNO2 requires C 71.44, H 8.20, N 4.39,
found: C 71.7, H 8.0, N 4.5%; dH (300 MHz, CDCl3): 7.23 (m, 4 H,
ArH), 7.14 (m, 1 H, ArH), 4.51 (t, J = 5.9 Hz, JH-F = 47.4 Hz,
1 H), 4.34 (t, J = 5.9 Hz, JH-F = 47.4 Hz, 1 H), 3.66 (brs, 1 H),
3.45 (s, 3H, CH3), 3.37 (brs, 1 H), 3.01 (dt, J = 5.2 Hz, J =
12.5 Hz, 1 H), 2.92 (t, J = 4.0 Hz, 1 H), 2.57 (dt, J = 2.6 Hz,
J = 12.5 Hz, 1 H), 2.27 (m, 2 H), 2.02 (m, 2 H), 1.69 (m, 6 H),
1.47 (m, 2 H); dC (100 MHz, CDCl3): 172.0, 143.2, 127.9, 127.3,
125.8, 84.2 (d, JC-F = 161.4 Hz), 62.9, 61.4, 52.9, 50.9, 34.2, 34.0,
28.2, 26.0, 25.1, 24.6; m/z (FD) 320.2 (100) C19H27FNO2 requires
320.2; HRMS(ESI): exact mass calcd for C19H27FNO2: 320.2026,
found: 320.2021.
Methyl 8-(4-fluorobut-2-en-1-yl)-3-phenyl-8-aza-bicyclo[3.2.1]
octane-2-carboxylate (7r, PRD19)
Synthesised from 7h according to the procedure described for 7q.
Methyl 8-(4-fluorobut-2-yn-1-yl)-3-phenyl-8-aza-bicyclo
[3.2.1]octane-2-carboxylate (7h, PRD16)
◦
Colourless crystals (90 mg, 91%): mp. 107–109 C; [a]2D3 -18.6
(c 1.42 in MeOH). C19H24FNO2 requires C 71.90, H 7.62, N 4.41,
found C 72.15, H 8.0, N 4.25%. dH (400 MHz, CDCl3): 7.30–
7.26 (m, 4H, ArH), 7.21–7.14 (m, 1H, ArH), 5.81 (brs, 2H), 4.85
(dd, J = 2 Hz, J = 5 Hz, JH-F = 48.5 Hz, 2 H), 3.69 (brs, 1 H),
3.49 (s, 3 H, OCH3), 3.45 (brs, 1H), 3.06 (dt, J = 5 Hz, J =
12.5 Hz, 1 H), 2.94 (t, J = 4 Hz, 1 H), 2.94–2.87 (m, 1 H), 2.65
(dt, J = 3 Hz, J = 12.5 Hz, 1 H), 2.17–1.97 (m, 2 H), 1.82–1.63
(m, 3 H); dC (100 MHz, CDCl3): 171.9, 142.9, 134.3, 127.9, 127.4,
125.7, 83.2 (d, JC-F = 166.5 Hz), 62.3, 61.4, 54.9, 52.7, 51.0, 34.2,
34.0, 26.0, 25.9; MS (FD) 318.2 (100) C19H25FNO2 requires 318.2;
HRMS(ESI): exact mass calcd for C19H25FNO2: 318.1869, found:
318.1870.
Synthesised according to procedure A, 96% yield. Colourless
crystals, mp. 70 ◦C; [a]D23 -95.7 (hydrochloride) (c 1.75 in MeOH).
C19H22FNO2 requires C 72.4, H 7.0, N 4.4, found C 72.8, H 6.9,
N 4.2%; dH (300 MHz, CDCl3): 7.24 (m, 4 H, ArH), 7.14 (m, 1 H,
ArH), 5.02 (t, J = 2 Hz, JH-F = 47.8 Hz, 1 H), 4.86 (t, J = 2 Hz,
JH-F = 47.8 Hz, 1 H), 3.90 (brs, 1 H), 3.50 (s, 3H, CH3), 3.40 (brs,
1 H), 3.25 (ddt, J = 1.5 Hz, J = 7.5 Hz, J = 16.5 Hz, 1 H), 3.12
(ddt, J = 1.5 Hz, J = 7.5 Hz, J = 16.5 Hz, 1 H), 3.02 (dt, J =
5.2 Hz, J = 12.9 Hz, 1 H), 2.94 (t, J = 3.3 Hz, 1 H), 2.69 (dt,
J = 2.6 Hz, J = 12.9 Hz, 1 H), 2.05 (m, 2 H), 1.68 (m, 4 H); dC
(100 MHz, CDCl3): 171.6, 142.7, 127.9, 127.4, 127.3, 125.9, 87.5,
70.8 (d, JC-F = 163.5 Hz), 62.6, 61.1, 52.6, 51.1, 42.9, 42.8, 34.1,
34.0, 25.9, 25.9, 25.7; MS(FD) 316.2 (100) C19H23FNO2 requires
316.2; HRMS(ESI): exact mass calcd for C19H23FNO2: 316.1713,
found: 316.1722.
Methyl 8-(2-fluoroethyl)-3-(4-chlorophenyl)-8-aza-
bicyclo[3.2.1]octane-2-carboxylate (10, FECNT)
Synthesised according to procedure A, 83% yield. Colourless
◦
crystals: mp. C; [a]D23 -43.5 (c 1.25 in MeOH). C17H21ClFNO2
requires C 62.67, H 6.50, N 4.30, found C 62.5, H 6.9, N 4.1%.
dH (400 MHz, CDCl3): 7.24 (d, J = 8.6 Hz, 2H, ArH), 7.19 (d,
J = 8.6 Hz, 2H, ArH), 4.57–4.33 (dm, JH-F = 47.5 Hz, 2 H),
3.79 (brs, 1 H), 3.52 (s, 3 H, OCH3), 3.44 (brs, 1H), 2.98 (dt, J =
6 Hz, J = 12.5 Hz, 1 H), 2.91 (t, J = 4 Hz, 1 H), 2.66–2.53 (m,
2 H), 2.65 (dt, J = 3 Hz, J = 12.5 Hz, 1 H), 2.2–2.08 (m, 1 H),
2.06–1.96 (m, 1H), 1.77 (dt, J = 4 Hz, J = 12.5 Hz, 1 H), 1.72–
1.63 (m, 2 H); dC (100 MHz, CDCl3): 171.8, 141.6, 131.5, 128.7,
128.0, 125.7, 83.9 (d, JC-F = 166.5 Hz), 63.5, 62.3, 53.8, 53.6, 52.6,
51.1, 34.0, 33.6, 26.2, 25.7; MS (FD) 326.1 (100) C17H22ClFNO2
requires 326.1; HRMS(ESI): exact mass calcd for C17H22ClFNO2:
326.1323, found: 326.1328.
Methyl 8-(((1S,2S)-2-(fluoromethyl)cyclopropyl)methyl)-3-phenyl-
8-aza-bicyclo[3.2.1]octane-2-carboxylate (7l, PRD17)
Synthesised according to procedure A, 84% yield. Colourless to
◦
yellowish crystals: mp. 76 C; [a]2D3 -27.0 (c 1.42 in MeOH).
C20H26FNO2 requires C 72.48, H 7.91, N 4.23, found: C 72.4,
H 8.0, N 4.2%; dH (300 MHz, CDCl3): 7.25 (brs, 4H, ArH), 7.14
(m, 1H, ArH), 4.30 (dq, J = 7 Hz, J = 9.6 Hz, JH-F = 48.9 Hz,
1 H), 4.13 (dq, J = 7 Hz, J = 9.6 Hz, JH-F = 48.9 Hz, 1 H), 3.82
(brs, 1 H), 3.47 (s, 3 H, CH3), 3.45 (brs, 1H), 3.00 (dt, J = 5.2 Hz,
J = 12.9 Hz, 1 H), 2.91 (dt, J = 4.4 Hz, J = 0.7 Hz, 1 H), 2.59 (dt,
J = 2.9 Hz, J = 12.5 Hz, 1 H), 2.52 (q, J = 7 Hz, 1 H), 2.0 (m,
3 H), 1.67 (m, 4 H), 1.02 (m, 1 H), 0.82 (m, 1 H), 0.52 (m, 2 H);
dC (100 MHz, CDCl3): 172.1, 143.2, 127.9, 127.3, 125.8, 87.4 (d,
JC-F = 161.5 Hz), 63.0, 61.2, 56.5, 52.9, 50.0, 34.2, 34.0, 26.0, 25.9,
16.6, 16.5, 16.3, 16.0, 9.4, 9.3; MS (FD) 332.2 (100) C20H27FNO2
requires 323.2; HRMS(ESI): exact mass calcd for C20H27FNO2:
332.2026, found: 332.2034.
Formation of hydrochloride salts for cell assay
R
Compounds 7a–q were purified by HPLC (Phenomenex Lunaꢀ
RP-18 10 m semi-preparative HPLC-column (250 ¥ 40 mm) using
10–20% 7 mM aqueous ammonia solution in MeCN. The product
2696 | Org. Biomol. Chem., 2009, 7, 2688–2698
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fractions were collected and the eluent was evaporated in vacuo.
and
R
The residue was taken up in MilliQꢀ-water and the resultant
n
H
V
IC50
solution was lyophilised over night. The lyophilised tropanes were
re-dissolved in dry ether and 2 M ethereal HCl was added dropwise
(2 equiv.). The mother liquor was removed and the hygroscopic
hydrochlorides were dried in vacuo. 1 mg of the dried hydrochloride
was re-dissolved in 1 ml CH3CN containing 20% of 0.05 M
ammonium acetate buffer and analysed by HPLC. The HPLC-
purity of all compounds used for biological assays exceeded 99%
(by UV-area at 254 nm).
=
(2)
I nH + IC50
n
H
Vmax
(
)
V represents transport rate; Vmax, maximal transport rate; S,
substrate concentration; I, inhibitor concentration; IC50, inhibitor
concentration for half maximal transport inhibition; KM, the
Michaelis–Menten constant; and nH, the Hill-coefficient.
Acknowledgements
Generation of cell lines stably expressing the hSERT, hDAT
and hNET
The authors are grateful to the Fonds der Chemischen Industrie.
This study was financially supported by a DFG grant (Ro985/21).
The authors wish to thank Dr Alexander Hoepping for helpful
discussion and Prof. Dr F. Roesch for scientific support.
Selection of clonal lines stably expressing the human
dopamine transporter, the human norepinephrine transporter
and the human serotonin transporter (HEKhDAT, HEKhNET
and HEKhSERT), respectively, was performed as described
previously.22
Notes and references
1 (a) N. D. Volkow, J. S. Fowler, G. J. Wang, R. Baler and F. Telang,
J. Neuropharm., 2009, 56 (Supplement 1), 3–8; (b) P. H. Elsinga, K.
Hatano and K. Ishiwata, Curr. Med. Chem., 2006, 13, 2139–2153; (c) G.
Grunder and D. F. Wong, Fortschr. Neurol. Psychiatr., 2003, 71, 415–
420; (d) J. M. Swanson, The Lancet, 2000, 355, 1461–1462; (e) D. D.
Dougherty, A. A. Bonab, T. J. Spencer, S. L. Rauch, B. K. Madras and
A. J. Fischman, The Lancet, 1999, 354, 2132–2133; (f) P. K. Morrish,
Mov. Disord., 2003, 18, S63–S70.
2 (a) V. Marshall and D. Grosset, Mov. Disord., 2003, 18, 1415–1423;
(b) K. A. Bergstro¨m, E. Tupala and J. Tiihonen, Pharmacol. Toxicol.,
2001, 88, 287–239; (c) M. Laruelle, M. Slifstein and Y. Huang, Methods,
2002, 27, 287–299.
3 R. L. Clarke, S. J. Daum, A. J. Gambino, M. D. Aceto, J. Pearl, M.
Levitt, W. R. Cuminskey and E. F. Bogado, J. Med. Chem., 1973, 16,
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and C. Crouzel, Top. Curr. Chem., 2002, 222, 201; (b) L. Cai, S. Lu and
V. W. P i ke , Eur. J. Org. Chem., 2008, 2853; (c) H. J. Wester, Handbook of
Nuclear Chemistry 4, Kluwer Academic Publishers, Dordrecht, 2003,
pp. 119–202.
5 L. Mu¨ller, C. Halldin, C. Lundquist, C.-G. Swahn, C. Foged, H. Hall,
P. Karlsson, N. Ginovart, Y. Nakashima, T. Suhara and L. Farde,
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Sedvall, Nucl. Med. Biol, 1997, 24, 629–634; (b) M. M. Goodman,
C. D. Kilts, R. Keil, B. Shi, L. Martarello, D. Xing, J. Votaw, T. D. Ely,
P. Lambert, M. J. Owens, V. M. Camp, E. Malveaux and J. M. Hoffman,
Nucl. Med. Biol, 2000, 27, 1–12; (c) S. S. Zoghbi, H. U. Shetty, M. Ichise,
M. Fujita, M. Imaizumi, J.-S. Liow, J. Shah, J. L. Musachio, V. W. Pike
and R. B. Innis, J. Nucl. Med., 2006, 47, 3520–3527.
7 M. Yaqub, R. Boellaard, B. N. M. van Berckel, M. M. Ponsen, M.
Lubberink, A. D. Windhorst, H. W. Berendse and A. A. Lammertsma,
J. Cereb. Blood Flow Metab., 2007, 27, 1397–1406.
8 (a) S. Singh, Chem. Rev., 2000, 100, 925–1024; (b) F. Dolle, P. Emond,
S. Mavel, S. Demphel, F. Hinnen, Z. Mincheva, W. Saba, H. Valette, S.
Chalon, C. Halldin, J. Helfenbein, J. Legaillard, J.-C. Madelmont, J.-B.
Deloye, M. Bottlaender and D. Guilloteau, Bioorg. Med. Chem., 2006,
14, 1115–1125.
Cell culture
HEKhSERT, HEKhNET and HEKhDAT cells were maintained
in Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum, penicillin (100 U ml-1), str◦eptomycin
(100 lg ml-1), and geneticin (G418, 200 lg ml-1) at 37 C in 95%
humidified air with 5% CO2. Cells were split in a defined dilution
to reach 80% confluency at the beginning of the experiment. [3H]5-
HT, [3H]NE and [3H]DA transport measurement of monoamine
uptake was performed as described previously and modified for
IC50-determination.8a,22b Briefly, HEKhSERT, HEKhNET and
HEKhDAT cells were plated into 24-well dishes (2 cm in diameter),
which had previously been treated with poly-L-lysine (0.1 mg ml-1)
and allowed to grow to 80% confluency. Culture medium was
replaced by TB1 buffer (200 lL: containing 120 mM NaCl, 2 mM
KCl, 1 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES, pH 7.5)
containing 250 nM of [3H]5-HT, [3H]NE or [3H]DA with various
concentrations (0.1–5000 nM) of each tracer ([3H]DA: KM
=
1.37 mM, [3H]5HT: KM = 1.1 mM and [3H]NE: KM = 0.61 mM).
After 6 min at room temperature, the medium was removed
quickly and cells were washed twice with ice-cold TB1 before
lysing with 10% (w/v) sodium dodecyl sulfate. Radioactivity
was determined by scintillation counting. Specific uptake is
determined as the difference between HEKhSERT, HEKhNET
and HEKhDAT-mediated and control HEK293 uptake in parallel
culture dishes. All transport measurements were analysed by non-
linear regression analysis using the graphics program GraphPad
R
Prismꢀ.
9 (a) T. Okada, M. Fujita, S. Shimada, K. Sato, P. Schloss, Y. Watanabe,
Y. Itoh, M. Tohyama and T. Nishimura, Nucl. Med. Biol., 1998, 25,
53–58; (b) P. Frankhauser, Y. Grimmer, P. Bugert, M. Deuschle, M.
Schmidt and P. Schloss, Neurosci. Lett., 2006, 399, 197–201.
10 (a) U. Gether, P. H. Andersen, O. M. Larsson and A. Schousboe, Trends
Pharmacol. Sci., 2006, 27, 375–383; (b) N. Chen and M. E. A. Reith,
Eur. J. Pharmacol., 2000, 329–339.
11 R. M. Baldwin, Y. Zea-Ponce, M. S. Al-Tikriti, A. S. Zoghbi, J. P. Seibyl,
D. S. Charney, P. B. Hoffer, S. Wang, R. A. Milius, J. L. Neumeyer and
R. B. Innis, Nucl. Med. Biol., 1995, 22, 211–219.
Data analysis
All uptake data represent the means of quadruplicate determina-
tions; each experiment was repeated at least three times. Data were
analyzed by non-linear regression analysis program (GraphPad
R
Prismꢀ), which fitted sigmoidal uptake curves to eqn (1) and (2):
Vmax
V =
Ê
ˆnH
È
˘
˙
12 (a) J. L. Neumeyer, S. Wang, Y. Gao, R. A. Milius, N. S. Kula, A.
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2698 | Org. Biomol. Chem., 2009, 7, 2688–2698
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