Development of an (S)-1-{2-[Tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid [(S)-SNAP-5114] Carba Analogue Inhibitor for Murine γ-Aminobutyric Acid Transporter Type4

Article abstract of DOI:10.1002/cmdc.201200126

A series of GABA uptake inhibitors related to (S)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid [(S)-SNAP-5114], the most potent mGAT4 inhibitor known so far, were synthesized and biologically evaluated for their inhibitory potency at the four GABA uptake transporters mGAT1-4 stably expressed in HEK-293 cell lines. New analogues were developed with potencies that are similar to or slightly higher than those of current mGAT4 inhibitors, but with distinctly improved chemical stability. (S)-Nipecotic acid derivatives possessing a 2-[1-(4-methoxy-2-methylphenyl)-1,1-bis(4-methoxyphenyl)methoxy]ethyl (DDPM-859) or a 4,4,4-tris(4-methoxyphenyl)but-2-en-1-yl moiety (DDPM-1457) were found to exhibit pIC₅₀ values of 5.78 and 5.87, respectively. Thus, as mGAT4 inhibitors, these compounds compare well with (S)-SNAP-5114 (pIC₅₀=5.71), but are far more stable than the latter. Moreover, DDPM-859 displays a more favorable subtype selectivity for mGAT4 versus mGAT3 than does (S)-SNAP-5114.

Full text of DOI:10.1002/cmdc.201200126

MED
DOI: 10.1002/cmdc.201200126
Development of an (S)-1-{2-[Tris(4-
methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid
[(S)-SNAP-5114] Carba Analogue Inhibitor for Murine
g-Aminobutyric Acid Transporter Type 4
Jçrg Pabel,[a] Mark Faust,[b] Cornelia Prehn,[c] Babette Wçrlein,[b] Lars Allmendinger,[a]
Georg Hçfner,[a] and Klaus T. Wanner*[a]
A series of GABA uptake inhibitors related to (S)-1-{2-[tris(4-me-
thoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid [(S)-
SNAP-5114], the most potent mGAT4 inhibitor known so far,
were synthesized and biologically evaluated for their inhibitory
potency at the four GABA uptake transporters mGAT1–4 stably
expressed in HEK-293 cell lines. New analogues were devel-
oped with potencies that are similar to or slightly higher than
those of current mGAT4 inhibitors, but with distinctly im-
proved chemical stability. (S)-Nipecotic acid derivatives pos-
sessing a 2-[1-(4-methoxy-2-methylphenyl)-1,1-bis(4-methoxy-
phenyl)methoxy]ethyl (DDPM-859) or a 4,4,4-tris(4-methoxy-
phenyl)but-2-en-1-yl moiety (DDPM-1457) were found to ex-
hibit pIC50 values of 5.78 and 5.87, respectively. Thus, as
mGAT4 inhibitors, these compounds compare well with (S)-
SNAP-5114 (pIC50 =5.71), but are far more stable than the
latter. Moreover, DDPM-859 displays a more favorable subtype
selectivity for mGAT4 versus mGAT3 than does (S)-SNAP-5114.
Introduction
g-Aminobutyric acid (GABA, 1, Figure 1) is the major inhibitory
neurotransmitter in the central nervous system (CNS), with
~30–40% of synapses estimated to be GABAergic.[1] A dysregu-
lation of GABA signaling is thought to play a key role in
a number of different neurological disorders such as epilepsy,
Parkinson’s disease, Huntington’s disease, schizophrenia, and
Alzheimer’s disease.[2] Targeting the individual stages of GABA
signaling to positively modulate GABA neurotransmission has
consequently become an important strategy for the potential
therapy of these diseases. Barbiturates and benzodiazepines,
for instance, which are among the most commonly used GA-
BAergic drugs today, enhance GABA neurotransmission
through allosteric modulation of GABAA receptors. The antiepi-
leptic drug vigabatrine improves the GABA status by acting as
a suicide inhibitor of GABA transaminase, an enzyme responsi-
ble for the metabolic degradation of GABA.
synaptically released GABA from extra- to intracellular side of
glial and neuronal cells. Inhibition of these transporters leads
to an increase in extracellular GABA levels and thus to en-
hanced GABA signaling. There are four different GABA trans-
porter subtypes belonging to the solute carrier 6 (SLC6) trans-
porter that use the co-transport of sodium ions as a driving
force for the translocation of their substrates against chemical
gradients.[3] The GABA transporter subtypes cloned from
murine brain are termed mGAT1, mGAT2, mGAT3, and
mGAT4.[4] For all other species, including humans and rats, a dif-
ferent nomenclature, also adopted by the Human Genome Or-
ganization (HUGO) is used; these transporters are denoted as
GAT1, GAT2, GAT3,[5] and BGT-1, respectively (see Ref. [6] for
a detailed discussion).
mGAT1 and mGAT4 are the two most abundant GABA trans-
porters in the brain, with the former being predominant. Both
subtypes are almost exclusively expressed around the synaptic
cleft; mGAT1 is primarily located in pre-synaptic neuronal
membranes, whereas mGAT4 is mainly located on glial cells.
GABA signaling may be further controlled by manipulating
the activity of GABA transporters that mediate the transport of
[a] Dr. J. Pabel, Dr. L. Allmendinger, Dr. G. Hçfner, Prof. Dr. K. T. Wanner
Department fꢀr Pharmazie—Zentrum fꢀr Pharmaforschung
Ludwig-Maximilians-Universitꢁt Mꢀnchen
Butenandtstraße 5-13, 81377 Mꢀnchen (Germany)
E-mail: klaus.wanner@cup.uni-muenchen.de
[b] Dr. M. Faust, Dr. B. Wçrlein
3M ESPE AG, ESPE Platz, 82229 Seefeld (Germany)
[c] Dr. C. Prehn
Institute for Experimental Genetics, Genome Analysis Center
Helmholtz Zentrum Mꢀnchen
German Research Center for Environmental Health
Ingolstꢁdter Landstraße 1, 85764 Neuherberg (Germany)
Figure 1. GABA (1), Tiagabine (2) and (S)-SNAP-5114 (3).
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mGAT1 is an approved drug target for the therapy of epilepsy.
However, tiagabine (2, Figure 1), the only currently approved
antiepileptic drug that addresses mGAT1 with high affinity and
selectivity, has the disadvantage of occasionally observed side
effects such as dizziness, asthenia, nervousness, tremor, diar-
rhea, and depression.[7,8] As these side effects seem to be in-
herently coupled with the function of mGAT1, other GAT sub-
types have gained increasing research interest.[9–11] In particular,
mGAT4, which is the second most abundant subtype in the
brain and differs from mGAT1 not only with respect to its cellu-
lar localization but also to its distribution in different brain re-
gions, shows great potential as a target for the development
of antiepileptic drugs with an improved pharmacological pro-
file.[9] Although a plethora of highly potent and subtype-selec-
tive inhibitors are available for mGAT1, only few inhibitors of
mediocre potency are known for mGAT4. (S)-SNAP-5114 (3,
Figure 1) represents the most potent and selective mGAT4 in-
hibitor so far.[12] It is characterized by a pIC50 value of 5.71[13,14]
at mGAT4 (Figure 2 and Table 1 below). In in vivo studies, 3 has
been found to significantly enhance GABA levels in rat thala-
mus but not in rat hippocampus, in contrast to the mGAT1-se-
lective inhibitor tiagabine (2), which affects both brain re-
gions.[9] In addition to underscoring the different pharmacolog-
ical effects of mGAT4 versus mGAT1 inhibitors, this finding also
highlights differences in the regional localization of these
GABA transporters.
et al.[12] for the preparation of 3 and related compounds. Ac-
cordingly, to introduce the respective side chain, nipecotic acid
ethyl ester (30) was allowed to react with the appropriate alky-
lating agents, and the resulting N-alkyl derivatives were finally
subjected to hydrolysis to give the free amino acids. The alky-
lating agents required for preparation of the first set of test
compounds with a structurally modified trityl moiety were syn-
thesized as outlined in Scheme 1, again following a method
used by Dhar et al.[12] The alkylating agents 16–19 were ob-
tained by treating the carboxylic acid esters 4 and 5 or methyl
chloroformate (6) with the appropriate Grignard reagents 7–10
(2.5 equiv 7 and 8, and 4 equiv 9 and 10) and subsequent
treatment of the resulting alcohols 11–14 with 2-bromoetha-
nol (15) in the presence of sulfuric acid to yield the alkyl bro-
mides 16–19 in 15–65% yield (Scheme 1).
The single ortho-methyl group present in 16 and even more
so the two ortho-methyl groups in 17 were expected to
impede planarization of the carbenium ions that would arise
from heterolytic cleavage of the ether function in 16 and 17,
thus rendering the carbenium ions less stable, and conse-
quently the ether functions more stable.
In 18 and 19, the para-methoxy groups present in 3 were
replaced by trifluoromethyl and trifluoromethoxy moieties, re-
spectively. In this case, the electron-withdrawing nature of the
substituents was expected to warrant the integrity of the trity-
loxy moiety by hampering the formation of the carbenium ion,
responsible for the decomposition reaction.
Unfortunately, the moderate potency at and selectivity for
mGAT4 exhibited by 3 is also accompanied by poor chemical
stability. Due to the trityloxy moiety present in the side chain
capable of forming triarylcarbenium ions, compound 3 is sus-
ceptible to decomposition especially under acidic conditions.
The aim of the study presented herein was to overcome this
problem by developing new mGAT4-selective inhibitors related
to (S)-SNAP-5114 (3) with structurally modified side chains of
improved chemical stability.
To overcome the chemical instability of the N-substituent of
3, the structure of the spacer linking the trityl moiety to the ni-
pecotic acid residue was modified. The residues represented
by 23, 28, and 29, respectively, seemed best suited for this
purpose (Scheme 2). In each of these compounds the chemical
instability of the side chain was eliminated by avoiding the
critical ether function. This was reached either by insertion of
a methylene moiety in the labile CꢀO bond (compound 23) or
by switching to a saturated (in 28) or partially unsaturated all-
carbon spacer (in 29).
Results and Discussion
For residue 23 the effect on the GAT inhibitory potency of
an elongated side chain with the position of the ether function
with respect to the amino function remaining unchanged
should also be tested. The synthesis of 23 was accomplished
in two steps. Reduction of 20 with lithium aluminum hydride
Chemistry
To improve the chemical stability of the side chain of (S)-SNAP-
5114, two strategies were followed. In each case, however,
only small structural changes
were envisaged to minimize the
risk of a substantial loss in activi-
ty. One strategy was aimed at
modifications of the trityl
moiety, and the other at varia-
tion of the spacer connecting
the nipecotic acid unit with the
lipophilic moiety to decrease or
even eliminate the risk of side
chain destruction due to carbe-
nium ion formation.
For the synthesis of test com-
pounds, we adopted the syn-
thetic route used by Dhar
Scheme 1. Synthesis of lipophilic residues with ether function.
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(S)-SNAP-5114 Carba Analogue mGAT4 Inhibitors
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with N-bromosuccinimide (NBS)
and 2,2’-azobis(2-methylpropio-
nitrile) (AIBN) by following
a report for an analogous trans-
formation of 4,4,4-triphenylbu-
tene,[16] delivering the desired
compound
(Scheme 2).
in
84%
yield
In the next step of the syn-
thetic sequence, the resulting
lipophilic residues were used for
the preparation of the N-substi-
tuted nipecotic acid ester deriv-
atives 31–39. Compounds 31–
34 and 37–39 were prepared in
reasonable yields by reacting ni-
pecotic ester 30, in racemic or
in enantiopure form, with the
respective alkyl halide in the
presence of potassium iodide
and potassium carbonate (meth-
od A, Scheme 3).
N-Alkylation of (RS)-30 with
alcohol 23 was performed by
following a method reported by
Zaragoza and Stephensen[17]
based on (cyanomethyl)trime-
thylphosphonium iodide as acti-
Scheme 2. Synthesis of the lipophilic residues 23, 28, and 29.
led to primary alcohol 21 (55%), which upon deprotonation
with n-butyllithium at ꢀ788C, subsequent treatment with eth-
ylene sulfate 22, and hydrolysis of the resulting alkyl sulfate
provided alcohol 23 in 75% yield.
For the synthesis of carba analogues 28 and 29, the 4,4,4-tri-
phenylbutene derivative 26 was
vating agent (method B, Scheme 3). The yield of 35 amounted
to 75%. Reductive amination of (RS)-30 with aldehyde 28 final-
ly yielded 36 in 82% yield (sodium cyanoborohydride, meth-
od C, Scheme 3). In the final step, the nipecotic acid esters 31–
39 were subjected to alkaline hydrolysis to provide the target
required (Scheme 2). An attempt
to prepare 26 by treatment of
24 with allyltrimethylsilane and
catalytic amounts of tris(penta-
fluorophenyl)borane in analogy
to a method reported by Rubin
and
Gevorgian,[15]
however,
product
failed
to
give
(Scheme 2). The transformation
was finally successful by using
allyl(tributyl)stannane (25) as nu-
cleophile and boron trifluoride
diethyl etherate as Lewis acid to
provide 26 in 96% yield.
Hydroboration and oxidation
of 26 with borane dimethyl sul-
fide and hydrogen peroxide, re-
spectively, provided alcohol 27,
which, upon Swern oxidation,
furnished aldehyde 28 in 95%
yield (Scheme 2). Finally for the
preparation of 29, compound 26
was subjected to bromination Scheme 3. Synthesis of the esters and their hydrolysis.
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K. T. Wanner et al.
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compounds, the corresponding amino acid derivatives, 40–48
in good yields (Scheme 3).
pIC50 =4.54, the subtype selectivity of 41 for mGAT4 (pIC50 =
5.07) is less pronounced.
Low potencies at mGAT4 are also observed for compounds
42 and 43 in which the methoxy groups in 3 are replaced by
trifluoromethyl and trifluoromethoxy moieties, respectively, to
stabilize the compounds by decreasing the electron-donating
capacities of the aromatic ring systems. Thus, for 42 and 43,
pIC50 values of 4.63 and 4.95 at mGAT4 were observed. With
the pIC50 values at mGAT4 being close to those of mGAT1–
mGAT3, compounds 42 and 43 also lack any significant sub-
type selectivity (Table 1, Entries 4 and 5).
Biological evaluation
Nipecotic acid derivatives 40–48 were evaluated for their in-
hibitory potency at the four GABA transporter subtypes
mGAT1–mGAT4. The assay system used is based on [3H]GABA
uptake in HEK-293 cells stably expressing the individual GABA
transporters.[13] The results are listed in Table 1. They are given
as pIC50 values except for test compounds that are unable to
decrease specific [3H]GABA uptake to a value below 50% at
a concentration of 100 mm. For these, only the percentage of
the remaining specific [3H]GABA uptake (at 100 mm) is report-
ed. For comparison, Table 1 also includes the pIC50 values that
(S)-SNAP-5114, the reference compound, exhibits in this test
system.
Extension of the spacer between nipecotic acid and the
trityl moiety in 3 by insertion of a methylene group in the
labile CꢀO bond or direct replacement of the ether function
by carbon is even less tolerated by mGAT4. For compounds 44
and 45, the pIC50 values at mGAT4 dropped to 4.06 and 4.21,
respectively, and these compounds were devoid of any reason-
able subtype selectivity (Table 1, Entries 6 and 7).
Interestingly, compound 45, a weakly active mGAT4 inhibitor
(pIC50 =4.21), gains almost 1.5 log units in potency by replace-
ment of the carbon–carbon single bond adjacent to the trityl
moiety in 45 by a double bond, resulting in DDPM-1007 (46).
Although compound 46 is still racemic, its potency at mGAT4
is nearly equal to the potency of (S)-SNAP-5114 (3) at this
transporter. Additionally, DDPM-1007 (46) can be considered
As outlined above, compounds 40–43 (Table 1, Entry 2) had
been modified with respect to their substitution pattern of the
aromatic ring systems in order to improve their chemical sta-
bility over that of (S)-SNAP-5114 (3). Interestingly, the introduc-
tion of one ortho-methyl group in one of the three phenyl
groups in 3 is well tolerated by the GABA transporters. Thus,
the potencies of the resulting compound DDPM-859 (40) at
the various GABA transporter subtypes are similar to those of
3. Only for mGAT3 is the potency of compound 40 (pIC50 =
4.85, Table 1, Entry 2) somewhat lower than that of 3 (pIC50 =
5.29). Accordingly, 40 exhibits even a slightly improved sub-
type selectivity regarding mGAT3 and mGAT4. Moreover,
1H NMR experiments revealed compound 40 to be distinctly
less prone to decomposition reactions in acidic media than 3
(pH 4.5, 3: t1/2 ~4 h; 40: t1/2 ~270 h). Thus, DDPM-859 (40) sur-
passes 3 with respect to subtype selectivity and chemical sta-
bility.
mGAT4 cannot tolerate two phenyl rings substituted with an
ortho-methyl group, as 41 is less active; its pIC50 value (5.07) is
lower than that observed for 3 and compound 40. Although
41 was tested as a racemic mixture, this would also be true if
the biological activity were to reside in one enantiomer only.
With potencies at mGAT1–mGAT3 ranging from pIC50 =4.17 to
Figure 2. Synthesized GAT inhibitors.
Table 1. Structures of synthesized GAT inhibitors (see Figure 2) and their inhibitory potencies toward mGAT1–4.
Entry
Compd
Config.
R1
R2
R3
-YꢀZ-
pIC50 (mean ꢁSEM)[a]
mGAT1
mGAT2
mGAT3
mGAT4
1
2
3
4
5
6
7
8
9
(S)-SNAP-5114 (3)
DDPM-859 (40)
S
S
OMe
OMe
OMe
CF3
OCF3
OMe
OMe
OMe
OMe
OMe
H
Me
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
-CH2-O-
-CH2-O-
-CH2-O-
-CH2-O-
-CH2-O-
-CH2-O-CH2-
-CH2-CH2-
-CH=CH-
-CH=CH-
-CH=CH-
4.07ꢁ0.09
4.19ꢁ0.07
4.17ꢁ0.02
4.88ꢁ0.11
4.87ꢁ0.02
4.12ꢁ0.07
3.94ꢁ0.09
4.32ꢁ0.05
4.19ꢁ0.09
4.40ꢁ0.05
56%
5.29ꢁ0.04
4.85ꢁ0.04
4.54ꢁ0.10
4.82ꢁ0.07
4.75ꢁ0.07
4.08ꢁ0.14
4.34ꢁ0.11
5.19ꢁ0.06
61%
5.71ꢁ0.07
5.78ꢁ0.03
5.07ꢁ0.09
4.63ꢁ0.03
4.95ꢁ0.12
4.06ꢁ0.07
4.21ꢁ0.07
5.67ꢁ0.06
4.33ꢁ0.05
5.87ꢁ0.08
4.12ꢁ0.08
4.49ꢁ0.09
4.76ꢁ0.02
4.96ꢁ0.07
4.10ꢁ0.03
59%
4.68ꢁ0.09
79%
4.42ꢁ0.11
41
42
43
44
RS
RS
RS
RS
RS
RS
R
45
DDPM-1007 (46)[b]
47
10
DDPM-1457 (48)
S
H
5.47ꢁ0.02
[a] If not stated otherwise, the results are given as the mean ꢁSEM of three independent experiments, each performed in triplicate; percentages represent
specific binding remaining in the presence of 100 mm inhibitor. [b] Compound 46 was tested as the hydrochloride.
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(S)-SNAP-5114 Carba Analogue mGAT4 Inhibitors
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chemically stable, as it lacks the critical ether function in (S)-
SNAP-5114 (3) responsible for the chemical instability of this
compound. This was indeed confirmed by H NMR experiments
Table 2. Logarithmic distribution coefficients calculated with Marvin-
Sketch and Pipeline Pilot.
1
Compd
clogD (pH 7.4)
with an aqueous solution of DDPM-1007 (46) at pH 4.5 and
1.5, for which no signs of decomposition could be detected
(reaction time: 72 h).
MarvinSketch[20]
Pipeline Pilot[21]
2
3
2.22
1.89
2.36
2.83
5.30
7.70
2.13
2.95
2.82
2.26
2.01
2.50
2.99
4.94
8.42
1.92
2.83
2.51
As for racemic (RS)-SNAP-5114[18] and also in the case of race-
mate 46, the potency resides mainly in one of the two enan-
tiomers, the S enantiomer. Thus, the S enantiomer DDPM-1457
(48) exhibits a pIC50 value of 5.87 (Table 1, Entry 8), whereas
the potency of its enantiomer 47 is distinctly lower (pIC50 =
4.33, Table 1, Entry 9). The potency of DDPM-1457 (48) is slight-
ly higher than that of (S)-SNAP-5114 (pIC50 =5.71), but only to
an extent that cannot be considered statistically significant.
Also with respect to subtype selectivity, S enantiomer DDPM-
1457 (48) compares well with (S)-SNAP-5114 (3). For both com-
pounds, the preference for mGAT4 relative to mGAT3 is identi-
cal. With the potencies at mGAT4 being only slightly higher
than those at mGAT3, however, both compounds can be con-
sidered mixed inhibitors of mGAT4 and mGAT3 with a prefer-
ence of the former. Also with reference to mGAT1 and mGAT2,
the subtype selectivities of (S)-SNAP-5114 (3) and DDPM-1457
(48) are similar, except that the potency of 48 at these trans-
porters is slightly lower. The similarities in potencies and sub-
type selectivities displayed by compounds 48 and 3 are likely
to reflect analogous binding modes of these compounds. Ob-
viously, for a reasonably high potency at mGAT4 an ether
oxygen atom as in (S)-SNAP-5114 (3) or a double bond as in
DDPM-1457 (48) is essential, as the all-carbon spacer analogue
45 is only a weak mGAT4 inhibitor. Taking into account the
aforementioned similarities in the pharmacological profile of 3
and 48, and the fact that ether oxygen atoms commonly serve
as hydrogen bond acceptors when interacting with proteins, it
seems reasonable to assume that the double bond in DDPM-
1457 (48) serves as a hydrogen bond acceptor[19] as well. Ac-
cordingly, the double bond in 48 may be considered a bioiso-
steric substitute for the OꢀCH2 group present in 3, avoiding
the chemical lability associated with the trityloxy moiety.
For the newly synthesized compounds, logD values were
calculated (clogD: calculated logD by MarvinSketch[20] and
Pipeline Pilot[21]) to estimate the lipophilicity and the potential
of 40–46 to cross the blood–brain barrier (BBB). For tiagabine
(2) and (S)-SNAP-5114 (3) under the same methods, clogD
values of ~2 were found (Table 2). It is generally accepted that
logD values should lie within the range of 2–5,[22] ideally closer
to 2,[23] to improve the potential for BBB penetration. The
clogD calculated for tiagabine, which is as an approved drug
known to enter the CNS,[24] nicely supports this rule. With
clogD values amounting to ~5 and ~8, respectively, the
fluoro-substituted compounds 42 and 43 (see Table 2) can be
expected to possess only low to negligible potential for BBB
penetration. In contrast, the remaining compounds, 40, 41,
and 44–46 display distinctly lower clogD values ranging from
1.92–2.99, and meet the criteria defined for brain penetration
reasonably well. For DDPM-859 (40) and DDPM-1457 (48,
clogD identical to that of racemate 46), which have been iden-
tified as the most efficacious substances at mGAT4 and which
40
41
42
43
44
45
46
are also active at mGAT3 to some extent, the mean clogD
values are 2.43 and 2.67, respectively (mean of MarvinSketch
and Pipeline Pilot). Thus, these compounds possess clogD
values close to optimal (logD 2), in which respect they also
compare quite well with (S)-SNAP-5114 (3; mean clogD 1.95),
which had so far been the most potent mGAT4 inhibitor
known. With the clogD value in line with common require-
ments, compounds 40 and 48 should have reasonable poten-
tial for in vivo activity and might be a valuable lead for the de-
velopment of more potent mGAT4 inhibitors.
Conclusions
As clearly documented by the results described above, the
linker and the triaryl moiety in compound 3 may be modified
to some extent while retaining the potency at mGAT4 and si-
multaneously improving chemical stability. Compound DDPM-
859 (40), with an additional methyl group at the ortho position
of one of the aromatic rings of 3, exhibits similar efficacy at
mGAT4 (40: pIC50 =5.78; 3: pIC50 =5.71) accompanied by
a slight improvement in subtype selectivity and enhanced
chemical stability over that of 3. Thus, DDPM-859 could be
a good lead molecule for the development of even more
potent and selective mGAT4 inhibitors. A structurally different
kind of mGAT4 inhibitor was found with compound DDPM-
1457 (48), characterized by substitution of the ethyloxy spacer
in 3 with a propenyl moiety. This carba analogue shows effica-
cy equal to that of 3 at mGAT4 (pIC50 =5.87; 3: pIC50 =5.71)
combined with substantially altered chemical characteristics of
the lipophilic residue.
As the in vivo effect of (S)-SNAP-5114 in animals after intra-
peritoneal administration is unexpectedly poor,[9] the synthe-
sized GAT inhibitors DDPM-859 (40) and DDPM-1457 (48)
could serve as improved pharmacological tools to study
mGAT4. Further efforts are certainly warranted to develop
more potent and more selective GAT4 inhibitors.
Experimental Section
Solvents were p.a. quality and freshly distilled before use. Pur-
chased reagents were used without further purification. As petrole-
um ether (PE) the fraction 40–808C was used. TLC plates were
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made from silica gel 60 F254 on aluminum sheets (Merck). Flash
chromatography was carried out using Merck silica gel 60 (mesh
0.040–0.063 mm) as stationary phase. Melting points: mp (uncor-
rected) were determined with a Bꢁchi 512 Melting Point apparatus.
NMR spectroscopy: 1H NMR spectra were recorded at RT with
a JNMR-GX (JEOL, 400 or 500 MHz) instrument using TMS as inter-
nal standard and integrated with the NMR software MestReNova.
IR spectroscopy: FTIR Spectrometer 410 (Jasco); samples were
measured as KBr pellets. Mass spectrometry (MS): Mass Spectrome-
ter 5989A with 59980B particle beam LC–MS interface (Hewlett
Packard) or Applied Biosystems LC–MS/MS Mass Spectrometer API
2000; analysis was carried out using chemical ionization (CH5+) or
electron impact ionization. High-resolution mass spectrometry
(HRMS): JEOL MS-Station JMS-700, FAB (Xenon, 6 kV, MBA, refer-
ence PEG), LTQ FT (Thermo Finnigan). Elementary analysis: Elemen-
taranalysator Rapid (Heraeus) and Vario EL (Elementar).
(1.0 g, 5.0 mmol, 0.71 mL), magnesium (134 mg, 5.50 mmol) in THF
(3.8 mL), 5 (332 mg, 2.00 mmol) in THF (1 mL), 2 h at 808C and
flash chromatography (pentane/EtOAc=9:1+1% NEt3). Colorless
crystals (519 mg, 68%): mp: 50–558C; 1H NMR (400 MHz, CD2Cl2):
d=2.13 (s, 6H, CH3), 2.88 (s, 1H, OH), 3.75 (s, 6H, OCH3), 3.79 (s,
3H, OCH3), 6.50 (dd, J=8.7/2.6 Hz, 2H, CHar), 6.59 (d, J=8.7 Hz, 2H,
CHar), 6.73 (d, J=2.4 Hz, 2H, CHar), 6.83 (d, J=8.9 Hz, 2H, CHar),
7.07 ppm (d, J=8.8 Hz, 2H, CHar); 13C NMR (100 MHz, CD2Cl2): d=
22.3 (CH3), 55.1 (OCH3), 55.2 (OCH3), 83.3 (COH), 109.3 (CHar), 113.0
(CHar), 118.1 (CHar), 129.0 (CHar), 130.2 (CHar), 137.4 (Car), 139.2 (Car),
139.3 (Car), 158.6 (COCH3), 158.7 ppm (COCH3); IR (KBr): n˜ = =3491,
2931, 1606, 1498, 809 cmꢀ1 HRMS-EI+: m/z [M]+ calcd for
;
C24H26O4: 378.1831, found: 378.1831.
Tris[4-(trifluoromethyl)phenyl]methanol (13):[26] According to
GP 1: 1-Bromo-4-trifluoromethylbenzene (8.1 g, 36 mmol, 4.97 mL),
magnesium (948 mg, 39.6 mmol) in THF (22 mL),
6 (851 mg,
9.00 mmol) in THF (1 mL), 1 h at RT and flash chromatography
(pentane/EtOAc=94:6). Yellow crystals (225 mg, 52%). Analytical
data are in accordance with published data.[26]
General Procedure 1 (GP 1): Under argon atmosphere, magnesium
turnings were suspended in THF, and the corresponding aryl halide
was slowly added. After 2 h, the corresponding ester was added
and stirred for the time given at the temperature stated. The reac-
tion mixture was cooled to 08C and a saturated solution of NH4Cl
was added. The aqueous layer was extracted with EtOAc, and the
combined organic layers were washed with a saturated NaCl solu-
tion, dried over Na2SO4, and concentrated in vacuo. The crude
product was purified by flash chromatography.
Tris[4-(trifluoromethoxy)phenyl]methanol (14): According to
GP 1: 1-Bromo-4-trifluoromethoxybenzene (3.9 g, 16 mmol, 2.6 mL),
magnesium (430 mg, 17.6 mmol) in THF (30 mL),
6 (378 mg,
4.00 mmol), 3 h at RT and flash chromatography (PE/EtOAc=95:5).
Colorless oil (702 mg, 34%): 1H NMR (500 MHz, CDCl3): d=7.16–
7.21 (m, 6H), 7.28–7.32 ppm (m, 6H); IR (KBr): n˜ =3474, 1509,
851 cmꢀ1; MS (EI, 70 eV): m/z (%): 512 (5) [M]+, 351 (96), 189 (100);
Anal. calcd for C22H13O4F9: C 51.58, H 2.56, found: C 51.75, H 2.41.
General Procedure 2 (GP 2): To a solution of the alcohol, conc.
H2SO4 was added, and the resulting mixture was stirred for 5 min
at 658C. After adding 15 at RT and stirring for the time given, H2O
and Et2O were added. Extraction with Et2O, drying of the combined
organic layers over MgSO4, and removing of the solvent in vacuo
resulted in the crude product, which was purified by flash chroma-
tography.
1-[1-(2-Bromoethoxy)-1,1-bis(4-methoxyphenyl)methyl]-4-meth-
oxy-2-methylbenzene (16): According to GP 2: 11 (729 mg,
2.00 mmol) in toluene (3.3 mL), conc. H2SO4 (34 mL), 15 (3.75 g,
30.0 mmol, 2.13 mL), 3 h and flash chromatography (pentane/
EtOAc=92:8). Colorless oil (224 mg, 24%): 1H NMR (400 MHz,
CD2Cl2): d=1.93 (s, 3H, CH3), 3.39 (t, J=6.0 Hz, 2H, CH2O), 3.51 (t,
J=6.0 Hz, 2H, CH2Br), 3.75 (s, 6H, OCH3), 3.78 (s, 3H, OCH3), 6.65
(dd, J=8.6/2.7 Hz, 1H, CHCHCCHC), 6.71 (d, J=2.6 Hz, 1H,
CHCHCCHC), 6.75–6.85 (m, 4H, OCCHCH), 7.25–7.33 (m, 4H,
OCCHCH), 7.35 ppm (d, J=8.6 Hz, 1H, CHCHCCHC); 13C NMR
(100 MHz, CD2Cl2): d=22.1 (CH3), 31.7 (CH2Br), 55.1 (OCH3), 55.2
(OCH3), 64.3 (CH2O), 86.8 (CO), 109.3 (CHCHCCHC), 113.2 (OCCH),
118.2 (CHCHCCHC), 128.5 (CCH), 131.8 (CHCHCCHC), 132.6 (Car),
137.6 (Car), 140.8 (Car), 158.1 (COCH3), 159.1 ppm (COCH3); IR (KBr):
n˜ =2931, 1606, 1507, 824 cmꢀ1; HRMS-FAB: m/z [M]+ calcd for
C25H27BrO4: 470.1093, found: 470.1106.
General Procedure 3 (GP 3): A solution of the alkyl bromide was
added to a solution of the appropriate nipecotic acid. K2CO3 and KI
were added, and the reaction mixture was stirred for the time
given under N2 atmosphere and under exclusion of light. The sol-
vent was removed in vacuo. The crude product was resolved in
H2O and CH2Cl2, and the aqueous layer was extracted with CH2Cl2.
The combined organic layers were dried (MgSO4) and concentrated
in vacuo.
General Procedure 4 (GP 4): The carboxylic acid ester was dis-
solved in NaOH and stirred for the given time at RT before adjust-
ing the mixture to pH 6 with HCl.
1-[1-(2-Bromoethoxy)-1-(4-methoxy-2-methylphenyl)-1-(4-meth-
oxyphenyl)methyl]-4-methoxy-2-methylbenzene (17): According
to GP 2: 12 (253 mg, 0.669 mmol) in toluene (2 mL), conc. H2SO4
(12 mL), 15 (142 mg, 1.34 mmol, 95 mL), 2 h and flash chromatogra-
phy (PE/EtOAc=97:3+1% NEtMe2). Colorless oil (49 mg, 15%):
1H NMR (500 MHz, CD2Cl2): d=2.13 (s, 6H, CH3), 3.36 (t, J=5.9 Hz,
2H, CH2O), 3.57 (t, J=5.9 Hz, 2H, CH2Br), 3.75 (s, 6H, OCH3), 3.77 (s,
3H, OCH3), 6.58 (dd, J=8.7/2.8 Hz, 2H, CHCHCCHC), 6.67 (d, J=
2.8 Hz, 2H, CHCHCCHC), 6.82 (d, J=9.0 Hz, 2H, OCCHar), 7.05 (d,
J=8.8 Hz, 2H, CHCHCCHC), 7.26 ppm (d, J=9.0 Hz, 2H, CCHar);
13C NMR (126 MHz, CD2Cl2): d=20.3 (CH3), 29.6 (CH2Br), 53.0 (OCH3),
53.2 (OCH3), 62.9 (CH2O), 86.9 (COCH2), 107.5 (CHCHCCHC), 110.7
(OCCHar), 115.9 (CHCHCCHC), 127.4 (CCHar), 128.1 (CHCHCCHC),
132.7 (Car), 132.8 (Car), 137.6 (Car), 156.0 (COCH3), 156.6 ppm
(COCH3); IR (KBr): n˜ =2928, 1605, 1497, 806 cmꢀ1; HRMS-EI+: m/z
[M]+ calcd for C26H29BrO4: 484.1249, found: 484.1231.
1-(4-Methoxy-2-methylphenyl)-1,1-bis(4-methoxyphenyl)metha-
nol (11): According to GP 1: 1-Bromo-4-methoxybenzene (935 mg,
5.00 mmol, 0.63 mL), magnesium (134 mg, 5.50 mmol) in THF
(3.8 mL), 4[25] (360 mg, 2.00 mmol) in THF (1 mL), 96 h at RT and
flash chromatography (cyclohexane/EtOAc=9:1). Colorless oil
1
(566 mg, 78%): H NMR (500 MHz, CDCl3): d=2.12 (s, 3H, CH3), 2.81
(s, 1H, OH), 3.77 (s, 3H, OCH3), 3.80 (s, 6H, OCH3), 6.53 (dd, J=8.6/
2.7 Hz, 1H, CCHCH), 6.64 (d, J=8.6 Hz, 1H, CCHCH), 6.73 (d, J=
2.6 Hz, 1H, CCH), 6.78–6.88 (m, 4H), 7.08–7.17 ppm (m, 4H);
13C NMR (126 MHz, CD2Cl2): d=20.0 (CH3), 53.1 (OCH3), 53.2 (OCH3),
80.0 (COCH2), 107.0 (CHCHCCHC), 111.0 (OCCH), 116.1 (CHCHCCHC),
126.7 (CCH), 128.6 (CHCHCCHC), 135.5 (Car), 137.6 (Car), 156.5
(COCH3), 156.8 ppm (COCH3); IR (KBr): n˜ =3506, 2933, 1606, 1508,
825 cmꢀ1; HRMS-EI+: m/z [M]+ calcd for C23H24O4: 364.1675,
found: 364.1682.
1,1-Bis(4-methoxy-2-methylphenyl)-1-(4-methoxyphenyl)metha-
1-{1-(2-Bromoethoxy)-1,1-bis[4-(trifluoromethyl)phenyl]methyl}-
nol (12): According to GP 1: 1-Bromo-4-methoxy-2-methylbenzene
4-(trifluoromethyl)benzene (18): According to GP 2: 13 (219 mg,
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(S)-SNAP-5114 Carba Analogue mGAT4 Inhibitors
MED
0.471 mmol) in toluene (1.2 mL), conc. H2SO4 (8 mL), 15 (118 mg,
0.942 mmol, 67 mL), 19 h and flash chromatography (pentane/
CH2Cl2 =98:2). Colorless crystals (171 mg, 64%): mp: 139–1438C;
1H NMR (500 MHz, CD2Cl2): d=3.41 (t, J=5.7 Hz, 2H), 3.52 (t, J=
5.7 Hz, 2H), 7.61 ppm (q, J=8.7 Hz, 12H); 13C NMR (101 MHz,
CD2Cl2): d=30.9 (CH2Br), 64.1 (OCH2), 86.0 (COCH2), 124.1 (q, J=
272 Hz, CF3), 125.4 (q, J=4 Hz, F3CCCH), 129.0, 129.8 (q, J=32 Hz,
chromatography (PE/EtOAc=9:1). Colorless crystals (171.6 mg,
95.6%): mp: 1018C; H NMR (500 MHz, CDCl3): d=3.36 (dt, J=6.6/
1
1.4 Hz, 2H, CHCH2C), 3.79 (s, 9H, OCH3), 4.95 (dq, J=10.3/1.8 Hz,
1H, CH2CH), 5.03 (dq, J=17.3/1.8 Hz, 1H, CH2CH), 5.65–5.72 (m,
1H, CH2CH), 6.78–6.81 (m, 6H, CHar), 7.09–7.12 ppm (m, 6H, CHar);
13C NMR (126 MHz, CDCl3): d=46.0 (CHCH2C), 54.3 (OCH3), 55.2
(C(C6H5)3), 113.0 (CHar), 117.0 (CH2CH), 130.3 (CHar), 136.3 (CH2CH),
140.1 (Cq), 157.5 ppm (Cq); IR (KBr): n˜ =3057, 3035, 3005, 2655,
2931, 2899, 2835, 1606 cmꢀ1; MS (CI, CH5+): m/z (%): 375 (71) [M+
H]+, 333 (37), 267 (100); Anal. calcd for C25H26O3 (374.48): C 80.18,
H 7.00, found, C 79.92, H 7.03.
F3CC), 146.4 ppm (CCH); IR (KBr): n˜ =2910, 2866, 1616, 829 cmꢀ1
;
HRMS-EI+: m/z [M]+ calcd for C24H16BrF9O: 570.0241, found:
570.0257.
1-{1-(2-Bromoethoxy)-1,1-bis[4-(trifluoromethoxy)phenyl]meth-
yl}-4-(trifluoromethoxy)benzene (19): According to GP 2: 14
(4.1 g, 8.0 mmol) in toluene (15 mL), conc. H2SO4 (0.15 mL), 15
(1.5 g, 12 mmol, 0.85 mL), 4 h and flash chromatography (PE/
EtOAc=95:5). Yellow oil (3.2 g, 65%): 1H NMR (500 MHz, CD2Cl2):
d=3.38–3.42 (m, 2H, CH2Br), 3.44–3.48 (m, 2H, CH2O), 7.15–7.21
(m, 6H, OCCHar), 7.43–7.48 ppm (m, 6H, CCHar); IR (KBr): n˜ =2868,
1594, 1504, 808 cmꢀ1; Anal. calcd for C24H16BrO4F9: C 46.55, H 2.60,
found: C 47.09; H 2.10.
4,4,4-[Tris(4-methoxyphenyl)]butan-1-ol (27): Under N2 atmos-
phere and ice cooling, 26 (1.87 g, 5.00 mmol) was dissolved in abs.
THF (20 mL) and abs. pentane (20 mL) and treated with 2m
BH3·S(CH3)2 solution in THF (5.0 mmol, 2.5 mL). The reaction mix-
ture was stirred at RT for 5 h. To oxidize the boron compound, first
ethanol (30 mL) and 3n NaOH (30 mL) and then, under ice cooling,
a 30% solution of H2O2 (20 mL) was added dropwise. The resulting
mixture was held at reflux for 2 h and stirred at RT until the boron
compound was completely oxidized. The mixture was poured in
ice water, the aqueous layer saturated with NaCl, and extracted
with Et2O. The combined organic layers were washed with saturat-
ed Na2HSO3 solution, dried over Na2SO4, and the solvent was re-
moved in vacuo. The crude product was purified by flash chroma-
tography (PE/EtOAc=3:1). Colorless oil (1.1 g, 56%): 1H NMR
(400 MHz, CDCl3): d=1.37 (m, 2H, CH2CH2OH), 2.55 (m, 2H, CCH2),
3.61 (t, J=6.4 Hz, 2H, CH2OH), 3.77 (s, 9H, OCH3), 6.77–6.80 (m, 6H,
Har), 7.15–7.18 ppm (m, 6H, Har); 13C NMR (100 MHz, CDCl3): d=
29.28 (CH2CHOH), 36.98 (CCH2), 54.30 (CCH2), 55.18 (OCH3), 63.39
(CH2OH), 113.08 (CHar), 130.04 (CHar), 140.03 (Car), 157.41 ppm
2,2,2-Tris(4-methoxyphenyl)ethanol (21): Under argon atmos-
phere, LiAlH4 (0.56 g, 15 mmol) was suspended in THF (20 mL) and
cooled to 08C. Then, a solution of 20[27] (2.3 g, 6.2 mmol) in THF
(20 mL) was added dropwise. The mixture was warmed up to RT,
stirred for 14 h, again cooled to 08C, and quenched with 1n HCl.
After adding 2n NaOH (pH>12), the mixture was extracted with
Et2O. The combined organic layers were washed with a saturated
NaCl solution (20 mL), dried over MgSO4, and concentrated in va-
cuo. The product was obtained by flash chromatography (PE/
EtOAc=90:10) as colorless crystals (1.23 g, 55%): mp: 1508C;
1H NMR (500 MHz, CD3OD): d=3.77 (s, 9H, OCH3), 4.86 (s, 2H,
CH2OH), 6.78–6.82 (m, 6H, CHar), 7.02–7.07 ppm (m, 6H, CHar);
13C NMR (125 MHz, CD3OD): d=55.1 (OCH3), 57.2 (CCH2OH), 70.2
(CH2OH), 113.6 (CHar), 131.1 (CHar), 139.4 (Car), 158.8 ppm (COCH3);
IR (KBr): n˜ =3521, 3034, 2917, 2873, 2838, 2362, 2042, 1897, 1605,
1577, 835 cmꢀ1; MS (CI, CH5+): m/z (%): 365 [M+H]+; Anal. calcd
for C23H24O4: C 75.80, H 6,64, found: C 76.03, H 6.82.
(COCH ); IR (KBr): n=3380, 2951, 2835, 1607, 1508 cmꢀ1; HRMS-EI+
˜
3
: m/z [M]+ calcd for C25H28O4: 392.1988, found: 392.1976.
4,4,4-[Tris(4-methoxyphenyl)]butanal (28): Oxalyl chloride (57 mg,
0.45 mmol) in CH2Cl2 (3.5 mL) was cooled to ꢀ788C and during
5 min DMSO (76 mg, 0.98 mmol, 69 mL) was added dropwise. After
stirring at ꢀ788C for 15 min, 27 (136 mg, 0.346 mmol) was added
over a period of 45 min. The mixture was stirred for further 15 min
before NEt3 (210 mg, 2.08 mmol, 288 mL) was added over a period
of 5 min. After stirring for 5 min at ꢀ788C and 1 h at RT, the mix-
tures was poured in NaHCO3 solution. The aqueous layer was ex-
tracted with CH2Cl2, the combined organic layers were dried over
Na2SO4 and the solvent was removed in vacuo. Yellow oil (130 mg,
95%): 1H NMR (400 MHz, CDCl3): d=2.32 (t, J=7.9 Hz, 2H,
CH2CHO), 2.83 (t, J=7.9 Hz, 2H, CCH2), 3.78 (s, 9H, OCH3), 6.78–6.81
(m, 6H, Har), 7.14–7.17 (m, 6H, Har), 9.63 ppm (s, 1H, CHO); 13C NMR
(100 MHz, CDCl3): d=32.24 (CCH2), 41.17 (CH2CHO), 53.83 (CCH2),
55.16 (OCH3), 113.25 (CHar), 129.85 (CHar), 139.17 (Car), 157.55
(COCH3), 201.91 ppm (CHO); IR (KBr): n˜ =2952, 2830, 1728, 1607,
1509 cmꢀ1; HRMS-EI+: m/z [M]+ calcd for C25H26O4: 390.1831,
found: 390.1848.
2-[2,2,2-Tris(4-methoxyphenyl)ethoxy]ethanol (23): 21 (1.12 g,
3.06 mmol) in THF (20 mL) was cooled to ꢀ788C. After 1.6m nBuLi
in hexane (3.06 mmol, 1.92 mL) was added dropwise, the mixture
was stirred at ꢀ788C for 15 min. A solution of ethylene sulfate (22;
385 mg, 3.10 mmol) in THF (5 mL) was added. The mixture was
stirred for 1 h at this temperature, additional 2 h at 08C and finally
for 16 h at RT. After adding Et2O (20 mL) and H2SO4 (20 mL), the
mixture was stirred at RT for a further 24 h. The separated aqueous
layer was extracted two times with Et2O. The combined organic
layers were dried over Na2SO4, and concentrated in vacuo. Com-
pound 23 was obtained by flash chromatography (PE/EtOAc=
70:30) as colorless solid (945 mg, 75%): mp: 1198C; 1H NMR
(400 MHz, CDCl3): d=3.58–3.60 (m, 2H, OCH2CH2O), 3.61–3.63 (m,
2H, OCH2CH2O), 3.80 (s, 9H, OCH3), 4.38 (s, 2H, CCH2O), 6.79–6.84
(m, 2H, OCCHar), 7.09–7.14 ppm (OCCHarCHar); IR (KBr): n˜ =3549,
2932, 2904, 2835, 1607, 1579, 825 cmꢀ1; MS (CI, CH5+): m/z (%): 365
(9), 347 (72), 333 (100); Anal. calcd for C25H28O5: C 73.51, H 6.91,
found: C 73.71, H 7.02.
1-Bromo-4,4,4-tris(4-methoxyphenyl)but-2-ene (29): 26 (374 mg,
1.00 mmol) dissolved in CCl4 was treated with NBS (178 mg,
1.00 mmol) and 2-[(1-cyano-1-methylethyl)azo]-2-methylpropaneni-
trile (10 mg, 0.06 mmol) and heated at reflux for 16 h. The precipi-
tate was separated and the solvent was removed in vacuo. The res-
idue was purified by crystallization from EtOH. Colorless crystals
4,4,4-Tris(4-methoxyphenyl)butene (26): 24 (168 mg, 0.479 mmol)
and 25 (205 mg, 1.00 mmol, 0.310 mL) dissolved in CH2Cl2 (1 mL)
were slowly treated with BF3·OEt2 (142 mg, 1.00 mmol) and stirred
at RT for 4 h. The reaction was stopped by adding saturated
NaHCO3 solution. The aqueous layer was separated and extracted
with CH2Cl2. The combined organic layers were dried over MgSO4
and concentrated in vacuo. The raw product was purified by flash
1
(265.9 mg, 58.6%): mp: 1048C; H NMR (500 MHz, CDCl3): d=3.81
(s, 9H, OCH3), 4.10 (d, J=7.7 Hz, 2H, CHCH2), 5.54 (dt, J=15.4/
7.7 Hz, 1H, CHCH2), 6.73 (d, J=15.4 Hz, 1H, CHCHCH2), 6.81–6.83
(m, 6H, CHar), 6.96–6.98 ppm (m, 6H, CHar); 13C NMR (100 MHz,
CDCl3): d=33.4 (CH2), 55.2 (OCH3), 58.4 (C(C6H4OCH3)3)), 113.0
(CHar), 127.3 (CHCH2), 131.0 (CHar), 138.0 (Cq), 143.1 (CHCHCH2),
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157.9 ppm (Cq); MS (CI, CH5+): m/z (%): 339 (64) [M+H]+, 297 (70),
243 (38), 123 (100); Anal. calcd for C25H25O3Br: C 66.23, H 5.56.
found: C 65.56, H 5.41.
1H, NCH2CH2CH2), 2.08 (t, J=9.6 Hz, 1H, NCH2CH2CH2), 2.24 (t, J=
10.3 Hz, 1H, NCH2CH), 2.48–2.75 (m, 4H, NCH2CH2CH2, NCH2CH2O,
NCH2CH), 2.95 (d, J=9.8 Hz, 1H, NCH2CH), 3.02–3.21 (m, 2H,
NCH2CH2O), 3.97–4.14 (m, 2H, CH2CH3), 7.61 ppm (s, 12H, CHar);
13C NMR (100 MHz, CD2Cl2): d=14.1 (CH3), 24.8 (NCH2CH2CH2), 26.8
(NCH2CH2CH2), 42.1 (NCH2CH), 54.4 (NCH2CH2CH2), 56.4 (NCH2CH),
58.3 (NCH2CH2O), 60.2 (CH2CH3), 62.6 (NCH2CH2O), 85.8 (COCH2),
124.2 (q, J=271 Hz, CF3), 125.2 (F3CCCH), 129.0 (CCH), 129.6 (q, J=
32 Hz, CCF3), 147.1 (CCH), 173.9 ppm (CO); IR (KBr): n˜ =2941, 1731,
Ethyl
(3S)-1-{2-[1-(4-methoxy-2-methylphenyl)-1,1-bis(4-meth-
oxyphenyl)methoxy]ethyl}piperidine-3-carboxylate (31): Accord-
ing to GP 3: 16 (19.5 mg, 0.414 mmol) in MeCN (1.0 mL), (S)-30·HCl
(12 mg, 0.062 mmol) in MeCN (0.2 mL), KI (0.7 mg, 0.04 mmol) and
K2CO3 (30 mg, 0.22 mmol), 13 days and flash chromatography (pen-
tane/EtOAc=85:15+1% NEtMe2). Colorless oil (19 mg, 84%):
1615, 831 cmꢀ1 HRMS-EI+: m/z [M]+ calcd for C32H30F9NO3:
;
½aꢂ2D0 +11.5 (c=0.8, CH2Cl2); H NMR (500 MHz, CD2Cl2): d=1.19 (t,
1
647.2082, found: 647.2082.
J=7.1 Hz, CH2CH3), 1.40 (ddd, J=24.2/11.8/4.0 Hz, 1H,
NCH2CH2CH2), 1.47–1.61 (m, 1H, NCH2CH2CH2), 1.63–1.75 (m, 1H,
NCH2CH2CH2), 1.83–1.91 (m, 1H, NCH2CH2CH2), 1.93 (s, 3H, CH3),
2.05 (td, J=10.9/2.2 Hz, 1H, NCH2CH2CH2), 2.19 (t, J=10.7 Hz, 1H,
NCH2CH), 2.51 (tt, J=10.4/3.7, 1H, NCH2CH), 2.62 (t, J=5.9 Hz, 2H,
NCH2CH2O), 2.73 (d, J=11.0 Hz, 1H, NCH2CH2CH2), 2.98 (dbr, J=
10.5 Hz, 1H, NCH2CH), 3.05–3.16 (m, 2H, NCH2CH2O), 3.74 (s, 6H,
OCH3), 3.77 (s, 3H, OCH3), 4.05 (q, J=7.1 Hz, CH2CH3), 6.64 (dd, J=
8.7/2.8 Hz, 1H, CHCHCCHC), 6.70 (d, J=2.7 Hz, 1H, CHCHCCHC),
6.73–6.84 (m, 4H, OCCHar), 7.24–7.36 ppm (m, 5H, CCHar,
CHCHCCHC); 13C NMR (126 MHz, CD2Cl2): d=14.1 (CH2CH3) 21.9
(CH3), 24.8 (NCH2CH2CH2), 26.9 (NCH2CH2CH2), 42.2 (NCH2CH), 54.4
(NCH2CH2CH2), 55.1 (OCH3), 55.2 (OCH3), 56.5 (NCH2CH), 58.7
(NCH2CH2O), 60.2 (CH2CH3) 62.6 (NCH2CH2O), 86.4 (COCH2), 109.1
(CHCHCCHC), 113.0 (CHCOCH3), 118.0 (CHCHCCHC), 128.5 (CCHar),
128.6 (CCHar), 131.8 (CHCHCCHC), 133.0 (Car), 138.3 (Car), 140.9 (Car),
138.8 (Car), 158.0 (COCH3), 158.9 (COCH3), 174.1 ppm (COOCH3); IR
(KBr): n˜ =2934, 1730, 1606, 1506, 824 cmꢀ1; HRMS-ESI+: m/z [M+
H]+ calcd for C33H42NO6: 548.3007, found: 548.3012.
Ethyl 1-(2-{1,1,1-tris[4-(trifluoromethoxy)phenyl]methoxy}ethyl)-
piperidine-3-carboxylate (34): According to GP 3: 19 (620 mg,
1.00 mmol) in acetone (1 mL), (RS)-30 (157 mg, 1.00 mmol) in ace-
tone (1.5 mL), KI (15 mg, 0.09 mmol), and K2CO3 (277 mg,
2.00 mmol), 65 h and flash chromatography (PE/EtOAc=85:15).
1
Colorless oil (482 mg, 69%): H NMR (500 MHz, CDCl3): d=1.21 (t,
J=7.2 Hz, 3H, CH2CH3), 1.44 (ddd, J=15.4/11.6/4.3 Hz, 1H,
NCH2CH2CH2), 1.50–1.62 (m, 1H, NCH2CH2CH2), 1.68–1.78 (m, 1H,
NCH2CH2CH2), 1.93 (dd, J=8.9/4.0 Hz, 1H, NCH2CH2CH2), 2.02–2.12
(m, 1H, NCH2CH2CH2), 2.23 (t, J=10.7 Hz, 1H„ NCH2CH), 2.54 (tt, J=
10.5/3.8 Hz, 1H, NCH2CH), 2.58–2.66 (m, 2H, NCH2CH2O), 2.70 (d,
J=11.0 Hz, 1H, NCH2CH2CH2), 2.97 (d, J=11.3 Hz, 1H, NCH2CH),
3.09–3.19 (m, 2H, NCH2CH2O), 4.03–4.18 (m, 2H, CH2CH3), 7.09–7.23
(m, 6H, OCCHar), 7.38–7.52 ppm (m, 6H, CCHar); IR (KBr): n˜ =2945,
1732, 1505, 847 cmꢀ1; MS (EI, 70 eV): m/z (%): 685 (1) [M]+, 495
(100), 190 (23); Anal. calcd for C32H30NO6F9: C 55.26, H 4.35, N 2.01,
found: C 55.24, H 4.56, N 2.02.
Ethyl 1-{2-[2,2,2-tris(4-methoxyphenyl)ethoxy]ethyl}piperidine-3-
carboxylate (35): To a mixture of 23 (481 mg, 1.18 mmol), (RS)-30
(176 mg, 1.12 mmol, 172 mL), N,N-diisopropylethylamine (190 mg,
1.47 mmol, 251 mL) and propionitrile (2.4 mL), (cyanomethyl)trime-
thylphosphonium iodide[17] (329 mg, 1.35 mmol) was added and
the mixture was heated at 908C for 2 h. After cooling to RT, addi-
tion of 1.28m solution K2CO3 (12 mL) and extraction with CH2Cl2
the resulting organic layer was washed with saturated NaCl solu-
tion, dried over MgSO4, and concentrated in vacuo. Compound 35
was obtained by flash chromatography (PE/EtOAc=60:40). Color-
Ethyl
1-{2-[1,1-Bis(4-methoxy-2-methylphenyl)-1-(4-methoxy-
phenyl)methoxy]ethyl}piperidine-3-carboxylate (32): According
to GP 3: 17 (19 mg, 0.038 mmol) in acetone (0.5 mL), (RS)-30
(9.0 mg, 0.057 mmol) in acetone (0.5 mL), KI (0.6 mg, 0.004 mmol)
and K2CO3 (19.7 mg, 0.143 mmol), 96 h and flash chromatography
(pentane/EtOAc=9:1+1% NEtMe2). Colorless oil (14 mg, 66%):
1H NMR (500 MHz, CD2Cl2): d=1.19 (t, J=7.1 Hz, 3H, CH2CH3), 1.40
(ddd, J=15.0/12.1/3.6 Hz, 1H, NCH2CH2CH2), 1.47–1.74 (m, 2H,
NCH2CH2CH2), 1.83–1.95 (m, 1H, NCH2CH2CH2), 2.05 (td, J=10.7/
2.4 Hz, 1H, NCH2CH2CH2), 2.11 (s, 3H, CH3), 2.12 (s, 3H, CH3), 2.18 (t,
J=10.5 Hz, 1H, NCH2CH), 2.52 (tt, J=10.5/3.8 Hz, 1H, NCH2CH),
2.65 (t, J=5.9 Hz, 2H, NCH2CH2O), 2.72 (d, J=11.0 Hz, 1H,
NCH2CH2CH2), 2.97 (dbr, J=10.5 Hz, 1H, NCH2CH), 3.01–3.12 (m, 2H,
NCH2CH2O), 3.75 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), 3.96–4.18 (m,
2H, CH2CH3), 6.50–6.61 (m, 2H, CHCHCCHC), 6.66 (s, 2H,
CHCHCCHC), 6.81 (d, J=8.9 Hz, 2H, OCCH), 7.01–7.12 (m, 2H,
CHCHCCHC), 7.32 ppm (d, J=8.8 Hz, 2H, CCH); 13C NMR (126 MHz,
CD2Cl2): d=12.0 (CH2CH3), 20.1 (CH3), 22.8 (NCH2CH2CH2), 24.8
(NCH2CH2CH2), 40.1 (NCH2CH), 52.4 (NCH2CH2CH2), 53.0 (OCH3), 53.1
(OCH3), 54.4 (NCH2CH), 56.6 (NCH2CH2O), 58.1 (CH2CH3), 61.0
(NCH2CH2O), 86.3 (COCH2), 107.3 (CHCHCCHC), 110.5 (OCCH), 115.7
(CHCHCCHC), 127.7 (CCH), 128.0 (CHCHCCHC), 133.1 (Car), 137.6
(Car), 137.7 (Car), 155.8 (COCH3), 156.3 ppm (COCH3), 172.0 (CO); IR
(KBr): n˜ =2937, 1730, 1606, 806 cmꢀ1; HRMS-ESI+: m/z [M+H]+
calcd for C34H44NO6: 562.3163, found: 562.3164.
1
less oil (482 mg, 75% based on 23): H NMR (400 MHz, C6D6): d=
0.97 (t, J=7.1 Hz, 3H, CH2CH3), 1.35–1.55 (m, 3H, NCH2CH2CH2,
NCH2CH2CH2), 1.74–1.89 (m, 2H, NCH2CH2CH2, NCH2CH2CH2), 2.26–
2.36 (m, 1H, NCH2CH), 2.40 (dt, J=5.7/1.2 Hz, 2H, NCH2CH2O),
2.36–2.48 (m, 1H, NCH2CH2CH2), 2.50–2.59 (m, 1H, NCH2CH), 2.87–
2.96 (m, 1H, NCH2CH), 3.37 (s, 9H, OCH3), 3.43 (t, J=5.7 Hz, 2H,
NCH2CH2O), 3.97 (q, J=7.1 Hz, 2H, CH2CH3), 4.27–4.33 (m, 2H,
CCH2O), 6.78–6.88 (m, 6H, OCCHar), 7.26–7.33 ppm (m, 6H, OC-
CHarCHar); 13C NMR (100 MHz, C6D6): d=11.2 (CH2CH3), 22.0
(NCH2CH2CH2), 24.1 (NCH2CH2CH2), 39.4 (NCH2CH), 51.0
(NCH2CH2CH2), 51.7 (OCH3), 53.3 (NCH2CH), 55.1 (NCH2CH2O), 56.9
(CH2CH3), 66.9 (NCH2CH2O), 76.3 (OCH2C), 110.4 (OCCHar), 127.9 (OC-
CHarCHar), 136.3 (OCar), 155.4 (CCar), 170.5 ppm (CO); IR (KBr): n˜ =
2936, 2834, 1729, 1607, 1509, 824 cmꢀ1; MS (CI, CH5+): m/z (%): 548
(42) [M+H]+, 333 (94), 214 (100), 170 (75); Anal. calcd for
C33H41NO6: C 72.37, H 7.55, N 2.56, found: C 72.13, H 7.49, N 2.50.
Ethyl 1-(2-{1,1,1-tris[4-(trifluoromethyl)phenyl]methoxy}ethyl)pi-
peridine-3-carboxylate (33): According to GP 3: 18 (171 mg,
0.300 mmol) in acetone (1.2 mL), (RS)-30 (70 mg, 0.45 mmol) in ace-
tone (0.5 mL), KI (5 mg, 0.03 mmol) and K2CO3 (156 mg,
1.13 mmol), 5 days and flash chromatography (pentane/EtOAc=
95:5+0.5% NEtMe2). Colorless oil (165 mg, 85%): 1H NMR
(400 MHz, CD2Cl2): d=1.18 (t, J=7.1 Hz, 3H, CH3), 1.33–1.62 (m,
2H, NCH2CH2CH2), 1.64–1.78 (m, 1H, NCH2CH2CH2), 1.81–1.94 (m,
Ethyl 1-[4,4,4-Tris(4-methoxyphenyl)butyl]piperidine-3-carboxyl-
ate (36): Under N2 atmosphere,
a solution of 28 (130 mg
0.333 mmol) in abs. MeOH (4 mL) and (RS)-30 (57.6 mg,
0.366 mmol) was stirred at RT for 30 min before NaCNBH3 (105 mg,
1.67 mmol) was added. The mixture was stirred over night at RT,
then cooled to 08C, and quenched with 2n HCl. The aqueous layer
was extracted with CH2Cl2, the combined organic layers were dried
over Na2SO4, and the solvent was removed in vacuo. The raw prod-
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(S)-SNAP-5114 Carba Analogue mGAT4 Inhibitors
MED
uct was purified by flash chromatography (PE/EtOAc=9:1). Color-
less oil (147 mg, 82%): H NMR (400 MHz, CDCl3): d=0.85–0.95 (m,
(101 MHz, CD2Cl2): d=22.0 (CH3), 22.2 (NCH2CH2CH2), 26.5
(NCH2CH2CH2), 40.6 (NCH2CH), 52.9 (NCH2CH2CH2), 55.1 (OCH3), 55.2
(OCH3), 56.0 (NCH2CH), 57.4 (NCH2CH2O), 60.2 (NCH2CH2O), 86.9
(CH2OC), 109.3 (CHCHCCHC), 113.2 (OCCHar), 118.2 (CHCHCCHC),
128.4 (CCHar), 128.5 (CCHar), 131.8 (CHCHCCHC), 132.5 (Car), 137.6
(Car), 137.7 (Car), 140.6 (Car), 158.1 (H3COC), 159.0 (H3COC),
176.5 ppm (COO); IR (KBr): n˜ =2926, 1606, 1508, 823; HRMS-ESI-:
m/z [MꢀH]ꢀ calcd for C31H36NO6: 518.2548, found: 518.2550.
1
3H, CH2CH3), 1.15–1.40 (m, 12H), 2.47–2.51 (m, 3H, NCH2CH, CCH2),
3.78 (s, 9H, OCH3), 4.12–4.15 (m, 2H, OCH2CH3), 6.78–6.80 (m, 6H,
Har), 7.13–7.16 ppm (m, 6H, Har); IR (KBr): n˜ =2927, 2360, 1731,
1608, 1509 cmꢀ1
;
HRMS-EI+: m/z [M]+ calcd for C33H41NO5:
531.2985, found: 531.2995.
Ethyl 1-[4,4,4-tris(4-methoxyphenyl)but-2-en-1-yl]piperidine-3-
carboxylate (37): According to GP 3: 29 (190 mg, 0.420 mmol) in
acetone (1.6 mL), (RS)-30 (99 mg, 0.63 mmol, 98 mL) in acetone
(0.6 mL), KI (7.0 mg, 0.042 mmol), and K2CO3 (218 mg, 1.58 mmol),
48 h. The crude product was purified by flash chromatography
(pentane/EtOAc=9:1+1% NEtMe2). Colorless oil (160 mg, 72%):
1H NMR (400 MHz, CD2Cl2): d=1.18 (t, J=7.1 Hz, 3H), 1.36–1.55 (m,
2H, NCH2CH2CH2), 1.63–1.72 (m, 1H, NCH2CH2CH2), 1.85 (dbr, J=
12.4 Hz, 1H, NCH2CH2CH2), 1.98 (tbr, J=10.5 Hz, 1H, NCH2CH2CH2),
2.09–2.17 (m, 1H, NCH2CH), 2.47 (tt, J=10.0/3.8 Hz, 1H, NCH2CH),
2.68 (dbr, J=11.0 Hz, 1H, NCH2CH2CH2), 2.89 (dbr, J=10.4 Hz, 1H,
NCH2CH), 2.96–3.20 (m, 2H, NCH2CHCH), 3.76 (s, 9H, OCH3), 3.99–
4.12 (m, 2H, OCH2), 5.23 (dt, J=15.5/6.6 Hz, 1H, NCH2CHCH), 6.54
(d, J=15.5 Hz, 1H, NCH2CHCH), 6.72–6.81 (m, 6H, Har), 6.90–
7.00 ppm (m, 6H, Har); 13C NMR (100 MHz, CD2Cl2): d=14.3 (CH3),
24.9 (NCH2CH2CH2), 27.1 (NCH2CH2CH2), 42.2 (NCH2CH), 53.9
(NCH2CH2CH2), 55.4 (OCH3), 55.8 (NCH2CH), 60.4 (OCH2), 61.5
(NCH2CHCH), 113.1 (CHar), 128.8 (NCH2CHCH), 131.3 (CHar), 139.1
(Car), 140.6 (NCH2CHCH), 158.2 (COCH3), 174.1 ppm (CO); IR (KBr):
n˜ =2932, 1729, 1606, 1507, 827 cmꢀ1; HRMS-EI+: m/z [M]+ calcd
for C33H39NO5: 529.2828, found: 529.2795.
1-{2-[1,1-Bis(4-methoxy-2-methylphenyl)-1-(4-methoxyphenyl)-
methoxy]ethyl}piperidine-3-carboxylic acid (41): According to
GP 4: 32 (56 mg, 0.10 mmol) in EtOH (0.4 mL), 12
n NaOH
(0.2 mmol, 17 mL), 27 h and 0.25n HCl. The resulting solution was
extracted with CH2Cl2. The combined organic layers were dried
over MgSO4 and the solvent was removed in vacuo. The crude
product was purified by flash chromatography (CH2Cl2/EtOH=
8:2+1% NEtMe2). Colorless crystals (27 mg, 51%): mp: 100–1208C
dec.; 1H NMR (500 MHz, CD2Cl2): d=1.53–1.63 (m, 1H,
NCH2CH2CH2), 1.66 (d, J=14.2 Hz, 1H, NCH2CH2CH2), 1.72–1.83 (m,
1H, NCH2CH2CH2), 1.94 (d, J=10.8 Hz, 1H, NCH2CH2CH2), 2.08 (s,
3H, CH3), 2.09 (s, 3H, CH3), 2.12–2.22 (m, 1H, NCH2CH2CH2), 2.38 (d,
J=10.9 Hz, 1H, NCH2CH), 2.65 (s, 1H, NCH2CH), 2.74 (dt, J=12.2/
6.0 Hz, 1H, NCH2CH2O), 2.85 (dt, J=12.3/6.0 Hz, 1H, NCH2CH2O),
3.02 (d, J=10.0 Hz, 1H, NCH2CH2CH2), 3.12 (d, J=10.9 Hz, 1H,
NCH2CH), 3.20 (t, J=5.7 Hz, 2H, NCH2CH2O), 3.76 (s, 6H, OCH3),
3.77 (s, 3H, OCH3), 6.58–6.63 (m, 2H, CHar), 6.66–6.70 (m, 2H, CHar),
6.78–6.85 (m, 2H, CHar), 7.09–7.14 (m, 2H, CHar), 7.18–7.24 ppm (m,
2H, CHar); 13C NMR (101 MHz, CD2Cl2): d=22.1 (NCH2CH2CH2), 22.2
(CH3), 26.5 (NCH2CH2CH2), 40.4 (NCH2CH), 53.5 (NCH2CH2CH2), 55.1
(OCH3), 55.2 (OCH3), 55.9 (NCH2CH), 57.6 (NCH2CH2O), 61.2
(NCH2CH2O), 88.8 (CH2OC), 109.6 (CHar), 112.8 (CHar), 117.8 (CHar),
117.9 (CHar), 129.5 (CHar), 130.2 (CHar), 130.3 (CHar), 134.4 (Car), 134.5
(Car), 134.9 (Car), 139.5 (Car), 139.6 (Car), 158.0 (Car), 158.6 (Car),
Ethyl
(3R)-1-[4,4,4-tris(4-methoxyphenyl)but-2-en-1-yl]piperi-
dine-3-carboxylate (38): According to GP 3: 29 (213 mg,
0.47 mmol) in acetone (0.7 mL), (R)-30 (111 mg, 0.705 mmol) in ace-
tone (0.7 mL), KI (7.8 mg, 0.047 mmol), and K2CO3 (244 mg,
1.763 mmol), 72 h. The crude product was purified by flash chro-
matography (pentane/EtOAc=8:2+1% NEtMe2). Colorless oil
(114 mg, 86%). Analytical data correspond to those of 37 except
176.1 ppm (CO); IR (KBr): n=2934, 1606, 1508, 1497, 811 cmꢀ1
˜
;
HRMS-ESI-: m/z [MꢀH]ꢀ calcd for C32H38NO6: 532.2704, found:
532.2704.
20
1-(2-{1,1,1-Tris[4-(trifluoromethyl)phenyl]methoxy}ethyl)piperi-
dine-3-carboxylic acid (42): According to GP 4: 33 (100 mg,
0.155 mmol) in EtOH (0.5 mL), 12n NaOH (0.31 mmol, 26 mL), 22 h
and 0.25n HCl. The resulting solution was extracted with Et2O. Col-
orless crystals (59 mg, 62%): mp: 78–908C; 1H NMR (400 MHz,
CD2Cl2): d=1.48–1.82 (m, 3H, NCH2CH2CH2), 1.84–2.03 (m, 1H,
NCH2CH2CH2), 2.05–2.24 (m, 1H, NCH2CH2CH2), 2.27–2.47 (m, 1H,
NCH2CH), 2.51–2.88 (m, 4H, NCH2CH2CH2, NCH2CH, NCH2CH2O),
2.91–3.10 (m, 1H, NCH2CH), 3.11–3.35 (m, 2H, NCH2CH2O), 7.49–
7.69 ppm (m, 12H, CHar); 13C NMR (101 MHz, CDCl3): d=22.1
(NCH2CH2CH2), 26.5 (NCH2CH2CH2), 40.4 (NCH2CH), 54.4
(NCH2CH2CH2), 55.6 (NCH2CH), 57.1 (NCH2CH2O), 60.1 (NCH2CH2O),
86.4 (COCH2), 124.1 (q, J=237 Hz, CF3), 125.2 (m, F3CCCHar), 128.9
(CHar), 129.8 (q, J=32 Hz, CCF3), 146.5 (Car), 176.2 ppm (COO); IR
(KBr): n˜ =2938, 1616, 830 cmꢀ1; HRMS-ESI+: m/z [M+H]+ calcd for
C30H27F9NO3:620.1842, found: 620.1841.
½aꢂD ꢀ12.6 (c=0.5, CH2Cl2).
Ethyl
(3S)-1-[4,4,4-tris(4-methoxyphenyl)but-2-en-1-yl]piperi-
dine-3-carboxylate (39): According to GP 3: 29 (195 mg,
0.430 mmol) in MeCN (1.6 mL), (S)-30·HCl (125 mg, 0.645 mmol) in
MeCN (1.5 mL), KI (7.1 mg, 0.043 mmol), and K2CO3 (312 mg,
2.26 mmol), 72 h. The crude product was purified by flash chroma-
tography (n-pentane/EtOAc=86:14+1% NEtMe2). Colorless oil
(165 mg, 72%). Analytical data correspond to those of 37 except
20
½aꢂD +13.2 (c=0.5, CH2Cl2).
(3S)-1-[2-[1-(4-Methoxy-2-methylphenyl)-1,1-bis(4-methoxy-
phenyl)methoxy]ethyl]piperidine-3-carboxylic acid (40): Accord-
ing to GP 4: 31 (16 mg, 0.026 mmol) in EtOH (0.1 mL), 12 n NaOH
(0.052 mmol, 4.5 mL), 72 h and 0.25 n HCl. The resulting solution
was extracted with CH2Cl2. The combined organic layers were dried
over MgSO4 and the solvent was removed in vacuo. The crude
product was purified by flash chromatography (CH2Cl2/EtOH=1:1).
1-(2-{1,1,1-Tris[4-(trifluoromethoxy)phenyl]methoxy}ethyl)piperi-
dine-3-carboxylic acid (43): According to GP 4: 34 (312 mg,
0.450 mmol) in EtOH (0.5 mL), 2n NaOH (0.31 mmol, 10 mL), 20 h
and 0.2n HCl. The resulting precipitate was isolated. Colorless crys-
tals (253 mg, 80%): mp: 45–638C; 1H NMR (500 MHz, CDCl3): d=
1.52–1.72 (m, 3H, NCH2CH2CH2), 1.72–1.89 (m, 2H, NCH2CH2CH2),
2.04 (d, J=11.6 Hz, 1H, NCH2CH2CH2), 2.08–2.18 (m, 1H,
NCH2CH2CH2), 2.34 (d, J=10.7 Hz, 1H, NCH2CH), 2.63 (dt, J=13.3/
4.6 Hz, 1H, NCH2CH), 2.67–2.77 (m, 2H, NCH2CH2O), 2.86 (d, J=
8.1 Hz, 1H, NCH2CH2CH2), 3.05 (d, J=8.9 Hz, 1H, NCH2CH), 3.17–
3.33 (m, 2H, NCH2CH2O), 7.18 (d, J=8.1 Hz, 6H, CHar), 7.42 ppm (d,
22
Colorless crystals (12 mg, 80%): mp: 88–948C; ½aꢂD ꢀ6.7 (c=0.36);
1H NMR (400 MHz, CD2Cl2): d=1.49–1.70 (m, 2H, NCH2CH2CH2),
1.70–1.85 (m, 1H, NCH2CH2CH2), 1.90 (s, 3H, CH3), 1.92–2.05 (m, 1H,
NCH2CH2CH2), 2.08–2.20 (m, 1H, NCH2CH2CH2), 2.38 (d, J=10.8 Hz,
1H, NCH2CH), 2.65 (m, 2H, NCH2CH, NCH2CH2O), 2.74–2.85 (m, 1H,
NCH2CH2O), 2.93–3.07 (m, 1H, NCH2CH2CH2), 3.08–3.26 (m, 3H,
NCH2CH, NCH2CH2O), 3.74 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), 6.65
(dd, J=8.7/2.5 Hz, 1H, CHCHCCHC), 6.70 (d, J=2.4 Hz, 1H,
CHCHCCHC), 6.79 (d, J=8.3 Hz, 4H, OCCHar), 7.22–7.31 (m, 4H,
CCHar), 7.37 ppm (d, J=8.7 Hz, 1H, CHCHCCHC); 13C NMR
ChemMedChem 0000, 00, 1 – 12
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K. T. Wanner et al.
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J=8.6 Hz, 6H, CHar); IR (KBr): n˜ =3425, 2948, 1606, 1508, 846 cmꢀ1
;
(6.6 mL). The resulting solution was extracted with CH2Cl2. The
combined organic layers were dried over MgSO4 and the solvent
was removed in vacuo; 47 was obtained as colorless crystals
(133 mg, 86%). Analytical data correspond to those of 46 except
½aꢂ2D2 +18.7 (c=0.33).
HRMS-ESI+: m/z [M+H]+ calcd for C30H27NO6F9: 668.1689, found:
668.1690.
1-{2-[2,2,2-Tris(4-methoxyphenyl)ethoxy]ethyl}piperidine-3-car-
boxylic acid (44): According to GP 4: 35 (103 mg, 0.187 mmol) in
EtOH (2 mL), 2n NaOH (0.468 mmol, 235 mL), 16 h and 0.01n HCl.
The resulting solution was extracted with CH2Cl2. The combined or-
ganic layers were dried over MgSO4 and the solvent was removed
in vacuo. Colorless solid (89.5 mg, 92%): mp: 728C; 1H NMR
(400 MHz, [D6]DMSO): d=1.19–1.42 (m, 2H, NCH2CH2CH2,
NCH2CH2CH2), 1.45–1.57 (m, 1H, NCH2CH2CH2), 1.68–1.78 (m, 1H,
NCH2CH2CH2), 1.87–2.00 (m, 1H, NCH2CH2CH2), 2.08–2.21 (m, 1H,
NCH2CH), 2.29–2.39 (m, 1H, NCH2CH), 2.50–2.60 (m, 3H, NCH2CH2O,
NCH2CH2CH2), 2.75–2.86 (m, 1H, NCH2CH), 3.46–3.56 (m, 2H,
NCH2CH2O), 3.71 (s, 9H, OCH3), 4.27 (dd, 2H, J=16.5/9.4, OCH2Cq),
6.76–6.83 (m, 6H, CHar), 6.95–7.10 ppm (m, 6H, CHar); 13C NMR
(100 MHz, [D6]DMSO): d=22.3 (NCH2CH2CH2), 24.7 (NCH2CH2CH2),
39.5 (NCH2CH), 51.4 (NCH2CH2CH2), 53.3 (OCH3), 53.8 (NCH2CH), 55.3
(NCH2CH2O), 70.7 (NCH2CH2O), 76.2 (OCH2C), 111.3 (OCCHar), 128.4
(OCCHarCHar), 136.7 (OC), 155.6 (CC), 173.4 ppm (CO); IR (KBr): n˜ =
3399, 2951, 2834, 1717, 1607, 824 cmꢀ1; MS (CI, CH5+): m/z (%): 520
(12) [M+H]+, 333 (100), 142 (34); Anal. calcd for C31H37NO6: C
71.65, H 7.18, N 2.70, found: C 71.38, H 7.11, N 2.40.
(3S)-1-[4,4,4-Tris(4-methoxyphenyl)but-2-en-1-yl]piperidine-3-
carboxylic acid (48): According to GP 4: 39 (96 mg, 0.18 mmol) in
EtOH (1 mL), 12n NaOH (0.36 mmol, 30 mL), 72 h and 0.25n HCl
(1.2 mL). The resulting solution was extracted with CH2Cl2. The
combined organic layers were dried over MgSO4 and the solvent
was removed in vacuo; 48 was obtained as colorless crystals
(84 mg, 92%). Analytical data correspond to those of 46 except
22
½aꢂD ꢀ19.5 (c=0.33).
Pharmacological methods
[3H]GABA uptake
Cells grown in 145 cm2 plates to a confluence of 70–90% were
treated with 4 mL trypsin/EDTA for ~30 s. Afterward normal
medium was added (8 mL), and the resulting cell suspension was
centrifuged for 5 min at 500 g. The cells were washed three times
in phosphate-buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl,
8 mm Na2HPO4, 1.75 mm KH2PO4, pH 7.4) by centrifugation as de-
scribed above and finally resuspended in Krebs buffer (2.5 mm
CaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4, 4.7 mm KCl, 11 mm glucose,
25 mm Tris, 119 mm NaCl, pH 7.2). The uptake assays were per-
formed with aliquots of the resulting cell suspension (~100000
cells per well for mGAT1, mGAT3, mGAT4, and ~200000 cells per
well for mGAT2) in a total volume of 250 mL in 96-well 2.2 mL poly-
ethylene deep-well plates (Abgene, Epsom, UK). The cells were
equilibrated for 25 min in Krebs buffer in the presence of the test
compound at 378C in a gently shaking water bath. Due to the
poor solubility of the test compounds, all samples contained 1%
DMSO. (The highest concentration investigated was 100 mm. Be-
cause many of the test compounds tend to precipitate at this con-
centration, it cannot be absolutely ruled out that the inhibition
assays are affected by precipitation at a test compound concentra-
tion for 100 mm. However, the inhibition curves obtained gave no
indication of any distortion effects. After addition of 25 mL of a solu-
tion containing [3H]GABA (3 TBqmmolꢀ1, Amersham Biosciences,
Freiburg, Germany) and unlabeled GABA in Krebs (final concentra-
tion 8 nm [3H]GABA and 32 nm unlabeled GABA for mGAT1,
mGAT3, mGAT4, and 20 nm [3H]GABA and 20 nm unlabeled GABA
for mGAT2), cells were incubated for a further 4 min (mGAT1,
mGAT3, mGAT4) or 10 min (mGAT2). The incubation was stopped
by filtration through Whatman GF/C filters pre-soaked for 1 h in
0.9% NaCl by means of a Brandell MWXR-96TI cell harvester (Bran-
dell, Gaithersburg, MD, USA) under reduced pressure (not
<250 mbar). The filters were rinsed four times with cold 0.9%
NaCl and subsequently transferred to 96-well sample plates (Perki-
nElmer LAS, Boston, MA, USA). After addition of Rotiszint Eco Plus
(Roth, Karlsruhe, Germany; 200 mL per well), the radioactivity was
determined in a microbeta liquid scintillation counter (PerkinElmer
LAS, Boston, MA, USA). Nonspecific uptake was defined in parallel
experiments with 10 mm NO 711 (mGAT1) or 1 mm GABA (mGAT2,
mGAT3, mGAT4) and was subtracted from total uptake (no inhibi-
tor) to yield specific uptake. IC50 values were determined by nonlin-
ear regression using GraphPad Prism 4 (“one site competition”;
bottom and top were fixed to 0 and 100%, respectively). The re-
sults are expressed as means ꢁSEM of at least three separate ex-
periments, each carried out in triplicate. Protein concentration was
1-[4,4,4-Tris(4-methoxyphenyl)butyl]piperidine-3-carboxylic acid
(45): According to GP 4: 36 (42 mg, 0.084 mmol) in EtOH (1 mL),
2n NaOH (0.2 mmol, 0.1 mL), 5 days and 2n HCl. The resulting so-
lution was extracted with CH2Cl2. The combined organic layers
were dried over Na2SO4 and the solvent was removed in vacuo.
Colorless crystals (28.6 mg, 67%): mp: 101–1048C; 1H NMR
(400 MHz, CDCl3): d=0.85 (m, 3H), 1.21 (m, 9H), 2.19 (m, 1H), 2.50
(m, 1H, CH), 3.26 (m, 1H), 3.77 (s, 9H, OCH3), 6.80 (d, J=8.6 Hz,
6H, Har), 7.14 (d, J=8.6 Hz, 6H, Har), 11.15 ppm (s, 1H, COOH);
13C NMR (100 MHz, CDCl3): d=22.01 (CH2,piperidine), 25.32 (CH2,piperidine),
29.68 (CCH2CH2), 37.44 (CCH2), 38.62 (CH), 51.71 (NCH2), 53.40
(NCH2), 54.52 (CCH2), 55.22 (OCH3), 57.98 (NCH2), 113.36 (CHar),
129.89 (CHar), 139.06 (Car), 157.57 (COCH3), 172.83 ppm (COOH); IR
(KBr): n˜ =3436, 2931, 2734, 1726, 1606, 1508 cmꢀ1; HRMS-ESI+: m/
z [M+H]+ calcd for C31H38NO5: 504.2744, found: 504.2742.
1-[4,4,4-Tris(4-methoxyphenyl)but-2-en-1-yl]piperidine-3-carbox-
ylic acid (46): According to GP 4: 37 (93 mg, 0.17 mmol) in EtOH
(0.6 mL), 12n NaOH (0.35 mmol, 30 mL), 72 h and 0.25n HCl
(1.2 mL). The resulting solution was extracted with CH2Cl2. The
combined organic layers were dried over MgSO4 and the solvent
was removed in vacuo. Colorless crystals (83 mg, 95%): mp: 98–
1
1028C; H NMR (400 MHz, CD2Cl2): d=1.56 (tt, J=13.1/4.7 Hz, 1H,
NCH2CH2CH2), 1.64 (d, J=14.1 Hz, 1H, NCH2CH2CH2), 1.70–1.85 (m,
1H, NCH2CH2CH2), 1.93 (d, J=12.6 Hz, 1H, NCH2CH2CH2), 2.14–2.31
(m, 2H, NCH2CH2CH2, NCH2CH), 2.57–2.65 (m, 1H, NCH2CH), 2.96 (d,
J=9.7 Hz, 1H, NCH2CH2CH2), 3.08 (d, J=10.6 Hz, 1H, NCH2CH),
3.17–3.31 (m, 2H, NCH2CHCH), 3.76 (s, 10H), 5.23–5.36 (m, 1H,
NCH2CHCH), 6.64 (d, J=15.5 Hz, 1H, NCH2CHCH), 6.78 (d, J=
8.8 Hz, 6H), 6.94 ppm (d, J=8.8 Hz, 6H); 13C NMR (126 MHz,
CD2Cl2): d=20.5 (NCH2CH2CH2), 24.9 (NCH2CH2CH2), 38.7 (NCH2CH),
51.5 (NCH2CH2CH2), 52.9 (NCH2CH), 53.7 (OCH3), 57.3 (C(C6H5)3), 58.2
(NCH2CHCH), 111.5 (CHar), 123.1 (NCH2CHCH), 129.4 (CHar), 136.7
(Car), 142.5 (NCH2CHCH), 156.5 (COCH3), 174.4 ppm (CO); IR (KBr):
n˜ =3433, 2933, 1606, 1507, 828 cmꢀ1; HRMS-ESI+: m/z [M+H]+
calcd for C31H36NO5: 502.2588, found: 502.2585.
(R)-1-[4,4,4-Tris(4-methoxyphenyl)but-2-en-1-yl]piperidine-3-car-
boxylic acid (47): According to GP 4: 38 (164 mg, 0.327 mmol) in
EtOH (1.5 mL), 12n NaOH (0.62 mmol, 52 mL), 96 h and 0.25n HCl
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ChemMedChem 0000, 00, 1 – 12
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(S)-SNAP-5114 Carba Analogue mGAT4 Inhibitors
MED
[14] In Ref. [12] compound 3 was reported with a pIC50 value of 5.81ꢁ0.10;
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Received: March 8, 2012
Revised: April 5, 2012
Published online on && &&, 0000
ChemMedChem 0000, 00, 1 – 12
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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These are not the final page numbers!

FULL PAPERS
J. Pabel, M. Faust, C. Prehn, B. Wçrlein,
L. Allmendinger, G. Hçfner, K. T. Wanner*
Stability is a snap! We developed
a series of analogues of (S)-SNAP-5114,
the most potent inhibitor of murine g-
aminobutyric acid transporter type 4
(mGAT4) known thus far. These ana-
logues have potencies that are similar
to or slightly higher than that of (S)-
SNAP-5114 toward mGAT4 (DDPM-1457:
pIC50 =5.87), but with distinctly
&& – &&
Development of an (S)-1-{2-[Tris(4-
methoxyphenyl)methoxy]ethyl}piperi-
dine-3-carboxylic acid [(S)-SNAP-5114]
Carba Analogue Inhibitor for Murine
g-Aminobutyric Acid Transporter
Type 4
improved chemical stability.
&12
&
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
ÝÝ
These are not the final page numbers!