Synthesis of N-substituted acyclic β-amino acids and their investigation as GABA uptake inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.04.063

In this publication, we describe the synthesis of new inhibitors for the GABA transporter subtypes GAT1 and especially GAT3. We started with 3-aminopropanoic acid possessing a distinct preference for GAT3 in comparison to GAT1 and furthermore its homolog

Full text of DOI:10.1016/j.ejmech.2013.04.063

European Journal of Medicinal Chemistry 65 (2013) 487e499
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of N-substituted acyclic b-amino acids and their
investigation as GABA uptake inhibitors
*
Ingolf Sitka, Lars Allmendinger, Günther Fülep, Georg Höfner, Klaus T. Wanner
Department Pharmazie, Zentrum für Pharmaforschung, LMU München, Butenandtstr. 5-13, D-81377 München, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In this publication, we describe the synthesis of new inhibitors for the GABA transporter subtypes GAT1
and especially GAT3. We started with 3-aminopropanoic acid possessing a distinct preference for GAT3 in
comparison to GAT1 and furthermore its homolog 3-aminobutanoic acid. A series of respective N-
substituted amino acids was synthesized by selective N-monoalkylation of these parent structures with 6
different arylalkyl alcohols via a Mitsunobu-type reaction. The resulting compounds were investigated
for their inhibitory potency GABA transporter subtypes. Among all tested compounds the 4,4-
diphenylbut-3-enyl substituted 3-aminobutanoic acid (rac)-6b showed highest potency with a pIC50
value of 5.34 at GAT1. Unfortunately, the expected GAT3 potency for 2-[tris(4-methoxyphenyl)methoxy]
ethyl substituted derivatives was not as high as observed for the respective nipecotic acid derivatives.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 27 December 2012
Received in revised form
28 March 2013
Accepted 25 April 2013
Available online 8 May 2013
Keywords:
GABA uptake inhibitors
Antiepileptic
GAT1
BGT1
GAT2
GAT3
b
-amino acids
Mitsunobu reaction
1. Introduction
(for a survey of different nomenclatures of GATs see Dalby et al.
[10]). An additional nomenclature has been proposed by the Hu-
g
-Aminobutyric acid (GABA) is the major inhibitory neuro-
man Genome Organization (HUGO). Referring to the latter the GAT
homologs are denoted as GAT1 (encoding gene: SLC6A1), BGT1
(SLC6A12), GAT2 (SLC6A13) and GAT3 (SLC6A11). For the sake of
simplicity, the HUGO nomenclature is used in terms of a species
independent nomenclature in the following, although it is e if
taken accurately e restricted to human.
transmitter in the central nervous system (CNS). In the last four
decades, several neuro-pathological and psychiatric diseases have
been connected directly or indirectly with the GABAergic neuro-
transmission such as e.g. dyskinesia, epilepsy, anxiety states,
depression, and several other behavioral disorders [1]. Interesting
pharmacological targets in search for new drugs in this field are
therefore the receptors, transporters, and metabolic enzymes that
are involved in the GABAergic neurotransmission. Thus, a prom-
ising approach to the successful treatment of cerebral disorder is
represented by drugs affecting GABA-uptake. GABA-uptake from
the synaptic cleft into the glial cells and presynaptic neurons takes
place by transmembrane proteins, the so called GABA transporters.
Of the GABA transporters (GATs) for different subtypes are known
[2e9]. These are termed mGAT1, mGAT2, mGAT3, and mGAT4 when
originating from mice but are named GAT-1, GAT-2, GAT-3, and
BGT1, respectively, for all other species, e.g. rats and humans
whereby the species is indicated by a respective prefix (e.g. hGAT-1)
Substantial differences have been found with respect to the
distribution of these four subtypes in the organism. GAT1 and GAT3
are located almost exclusively in the central nervous system, where
GAT1 has been shown to be predominantly responsible for the
transport of GABA into neurons and GAT3 into glial cells. BGT1 and
GAT2 have repeatedly been reported to be widely distributed
throughout the brain, too, though in low concentrations and in
peripheral organs like the liver and the kidneys. However, accord-
ing to recent studies from Danbolt et al. BGT1 and GAT2 are not
detectable in brain parenchyma. In the brain, both proteins show
distinct expressions in the leptomeninges and GAT2, in addition, in
a subpopulation of brain blood vessels only [11,12]. Thus, only GAT1
and GAT3 but not BGT1 and GAT2 can be considered to play a role in
neurotransmitter inactivation [13]. Accordingly, compounds
addressing GAT1 and GAT3 with high potency and selectivity are of
special interest when it comes to the development of CNS drugs
* Corresponding author. Tel.: þ49 89 2180 77249; fax: þ49 89 2180 77247.
E-mail address: Klaus.Wanner@cup.uni-muenchen.de (K.T. Wanner).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.063

488
I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
enhancing GABA neurotransmission. This is especially true for
potent and selective GAT3 inhibitors as those are lacking so far.
Nipecotic acid (1), which can be considered as a rigidified b-
alanine, has been found to be an inhibitor of GABA uptake in
neuronal and astroglial cell cultures [14]. But due to its low lip-
ophilicity, nipecotic acid (1) lacks the capability to cross the bloode
brain barrier in sufficient amount [15]. This obstacle was overcome
by attaching a lipophilic side chain to the nipecotic acid nitrogen
leading to GABA uptake inhibitors such as SK&F-89976-A [(rac)-2]
[16,17], tiagabine [(R)-3] [18,19] and (S)-SNAP-5114 [(S)-4] [20].
SK&F-89976-A [(rac)-2] displays a 4,4-diphenylbut-3-enyl side
chain whereas in case of tiagabine [(R)-3] a 4,4-bis(3-methyl
thiophene-1-yl)but-3-enyl moiety is present, both of which not
only enhance the potency of these compounds but also substan-
tially improve the subtype selectivity in favor of GAT1 inhibition as
compared to the basic compounds (rac)-1 and (R)-1, respectively.
For (S)-SNAP-5114 [(S)-4] this subtype selectivity is reversed, this
compound possessing the highest inhibitory potency at GAT3
which has largely to be ascribed to the presence of the 2-[tris(4-
methoxyphenyl)methoxy]ethyl residue. A similar effect, the rever
sal of subtype selectivity from GAT1 to GAT3, has been observed for
several other amino acids acting as GAT inhibitors when 4,4-
diarylbutenyl moieties as in (rac)-2 or (R)-3 were replaced by the
sterically more demanding N-substituent of (S)-SNAP-5114 [(S)-4]
these residues were applied [21]. Interestingly, for GAT1 and GAT3
inhibitors like 3 and 4 and related compounds the change in sub-
type selectivity is commonly paralleled by a switch in biological
enantioselectivity, the highest potency and subtype selectivity for
GAT1 inhibitors residing in the (R)- and for GAT3 inhibitors in the
(S)-enantiomer (or the corresponding homochiral compound)
(Fig. 1).
b-Alanine (3-aminopropanoic acid, 5a) is long known to inhibit
GABA uptake into glial cells [22] and in the meantime it has been
intensively studied on the four GABA transporter subtypes [23]. In
the test system that has been established in our group and is based
on HEK293 cells stably expressing the individual murine GABA
transporters,
b-alanine (5a) was found to possess a reasonable
potency at and subtype selectivity for GAT3 (pIC50 ¼ 4.66 ꢀ 0.06) as
compared to GAT1 (GAT1: pIC50 ¼ 2.55 ꢀ 0.03). Also the methyl
substituted derivative 3-aminobutanoic acid [(rac)-6a] displays
good inhibitory potency at GAT3 (pIC50 ¼ 4.08 ꢀ 0.03) but this time
Fig. 1. Structures of GABA uptake inhibitors.
yl)ethoxy]ethyl (e) moiety were selected to be employed as N-
substituents which according to the results published by Andersen
et al. [24] improve the potency at GAT1 as well as the subtype
selectivity for this transporter. With regard to the development of
more potent GAT3 inhibitors from 5a and (rac)-6a the 2-[tris(4-
methoxyphenyl)methoxy]ethyl (f) and the (E)-2-[tris(4-methoxy
phenyl)]but-2-en-1-yl (g) residue should be used as N-sub-
stituents, which are known to enhance potency and subtype
selectivity in favor of this transporter as demonstrated by (S)-SNAP-
5114 [(S)-4] and the recently introduced DDPM-1457 [25], two of
the most potent inhibitors of this subtype of GABA transporters.
also at GAT1 (pIC50 ¼ 4.30 ꢀ 0.10) thus being, in contrast to
b-
alanine (5a), almost equally potent at both transporters. With the
inhibitory potencies at GAT1 [for (rac)-6a] and at GAT3 [for 5a and
(rac)-6a] being in the range of (rac)-nipecotic acid [(rac)-1a] we
considered these compounds suitable starting points for the
development of new GABA uptake inhibitors for these GABA
transporters, especially GAT3. Thereby it was hoped the subtype
selectivity inherent to the parent compounds 5a and (rac)-6a might
also positively contribute to that of appropriately N-substituted
compounds (Fig. 2).
In this context, it should be mentioned that attachment of
lipophilic residues e known to enhance inhibitory potency at GAT1
and GAT3 e to the
b
-amino acids 5a and (rac)-6a were expected to
2. Chemistry
provide data for the establishment of structure activity relation-
ships for the respective acyclic amino acid derivatives, yet unknown
so far.
First attempts to synthesize the desired compounds were un-
dertaken by reacting methyl 3-aminopropanoate hydrochloride (9)
with the respective alkyl halides but resulted in yields that were not
fully satisfying (Scheme 1). Thus, the exemplarily performed reac-
tion of methyl 3-aminopropanoate (7) with the bromides 7b and 7f
(K2CO3, KI, CH3CN, 45 ꢁC, 24 h) led to the corresponding N-alkylated
ester derivatives 10b and 10f in moderate yields of 52% and 49%
only.
Though 5a and (rac)-6a display also reasonable potencies at
BGT1 and GAT2 no attention should be paid to the development of
inhibitors of these GABA transporter subtypes as these transporters
due to their low abundance in the brain are of no significant rele-
vance for the development of CNS active compounds [12]. For our
study the 4,4-diphenylbut-3-enyl (b), the 2-[2-(10,11-dihydro-5H-
dibenzo[b,f]azepin-5-yl)ethoxy]ethyl (c), the 2-[2-(5H-dibenzo[b,f]
azepin-5-yl)ethoxy]ethyl (d) and the 2-[2-(10H-phenothiazin-10-
Therefore, we decided to perform the synthesis of all other N-
substituted amino acids by a variant of the Mitsunobu reaction

I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
489
Fig. 2.
b-Amino acid target structures.
following a procedure published by Fukuyama et al. [26] accord-
ing to which amines were alkylated via their sulfonamides
employing alcohols 8beg as alkylating agents. This approach ap-
pears also quite favorable as it avoids the preparation of alkyl
bromides and thus an additional synthetic step, the formation of
these alkylating agents commonly prepared from the respective
alcohols.
2.2. Mitsunobu reaction
For the N-alkylation by Mitsunobu reaction the sulfonamides of
the amino acid ester hydrochlorides 9 and (rac)-16, were required.
Their synthesis was easily accomplished by treating 9 and (rac)-16
with 2,4-dinitrophenylsulfonylchloride in the presence of pyridine
to afford the desired sulfonamides 17 and (rac)-18 in 91% and 53%
yield, respectively (Scheme 3).
The N-alkylation of sulfonamides 17 and (rac)-18 was performed
in analogy to a procedure of Fukuyama et al. However, instead of
diethyl azodicarboxylate (DEAD) diisopropyl azodicarboxylate
(DIAD) was used, together with triphenylphosphane, for the acti-
vation of the alcohols, because at the time this work was performed
DEAD was commercially unavailable in Germany due to safety
problems [27]. Fortunately, DIAD proved to be suitable for this
purpose as well. Thus, N-alkylation by Mitsunobu reactions pro-
ceeded smoothly, when mixtures of the sulfonamides 17 and (rac)-
18 (1 equiv.), the respective alcohol 8beg (2 equiv.) and Ph3P
(2 equiv.) in benzene were treated with DIAD (2 equiv., addition at
10 ꢁC, subsequent reaction at rt), providing the Mitsunobu products
19cee,g and 20beg in good to excellent yields (79e95%, see
Table 1).
2.1. Synthesis of arylalkyl alcohols
The arylalkyl alcohols 8bee required for the preparation of the
N-substituted b-amino acids 5cee and (rac)-6bee by Mitsunobu
reaction were synthesized by literature methods [24].
2-[Tris(4-methoxyphenyl)methoxy]ethyl alcohol (8f) was
accessible by a two step procedure, i.e. by treating tertiary alcohol
11 with SOCl2 followed by the reaction with ethylene glycol
(Scheme 2). Unfortunately, the desired alcohol 8f could only be
isolated in moderate yield (32%), as the chlorinated derivative 12
had formed as side product, which to separate by column chro-
matography led to marked substance losses. For the synthesis of the
triaryl allyl alcohol 8g alcohol 13 was oxidized with IBX to afford
aldehyde 14. Treatment of 14 with triethyl phosphonoacetate and
sodium hydride yielded the unsaturated ester 15 (96%). Subsequent
reduction with lithium aluminum hydride afforded the desired
alcohol 8g in 71% yield.
By aiming at the free amino acids in the last two steps of the
synthetic sequence the removal of the 2,4-dinitrophenylsulfonyl
moiety and the cleavage of the carboxylic acid ester function had
to be accomplished. For the removal of the arylsulfonyl moiety we
applied
following a protocol of Fukuyama et al. which resulted in the for-
mation of the -amino acid esters 9cee,g and 21beg in good yields
a mixture of mercaptoacetic acid and triethylamine
b
(79e96%). The monoalkylated amino acid esters 9cee,g obtained
via Mitsunobu reaction and 9b,f, by direct N-alkylation as well as
(rac)-21beg were finally hydrolyzed with 2 M NaOH in MeOH at rt
to give after workup and ion exchange chromatography e where
necessary e the free amino acids 5beg and (rac)-6beg in yields of
Scheme 1. N-Alkylation with bromides 7b and 7f under SN2 condition.

490
I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
Scheme 2. Synthesis of alcohols 8f and 8g.
42e87%. Amino acid 5b was further treated with HCl, to afford after
recrystallization 5b$HCl in 84% yield.
displayed by (rac)-2 as compared to (rac)-1 as well (see Table 2).
Curiously, while according to literature data the 3-[2-(10,11-
dihydro-5H-dibenzo[b,f]azepin-5-yl)ethoxy]ethyl side chain leads
to a slightly reduced GAT1 potency for (R)-22 (pIC50 ¼ 4.40) as
compared to the parent compound (R)-1 (pIC50 ¼ 5.07), the
opposite is true when the same residue is attached to 5a and (rac)-
6a [resulting in 5c and (rac)-6c]. Here, the GAT1 potency raises for
(rac)-6a from pIC50 ¼ 4.30 to pIC50 ¼ 4.88 ((rac)-6c) and for 5a from
pIC50 ¼ 2.55 to pIC50 ¼ 4.69 (5c), the latter equaling a difference of
two log units. Here again in accord with a common trend observed
for many other N-substituents enhancing GAT1 potency [see e.g.
(rac)-2] at the same time the GAT3 potency was lowered for both
compounds, 5c and (rac)-6c. For nipecotic acid (R)-1 the 3-[2-(5H-
dibenzo[b,f]azepin-5-yl)ethoxy]ethyl and the 3-[2-(10H-pheno-
thiazin-10-yl)ethoxy]ethyl residues when attached to the amino
nitrogen give rise to highly potent GAT1 inhibitors, (R)-23 and (R)-
24. For 5a the latter substituent leads to a clear improvement of
GAT1 inhibitory potency (5e), too. This seems to be also the case for
the former substituent (leading to 5d), though the percentage of
remaining GABA uptake determined for this compound provides
only a rough estimate of its potency. Whatsoever, though the GAT1
potency of the parent compounds has increased due to the pres-
ence of the N-substituents it is still low for both compounds. Again
the opposite effect was observed for the GAT3 potency for 5d, 5e,
(rac)-6d and (rac)-6e due to the presence of these residues, d and e,
their potency being lower than that of the parent compounds 5a
and (rac)-6a.
3. Biological studies
Like the parent structures 5a and (rac)-6a before also the target
compounds, the N-substituted b-amino acids 5beg and (rac)-6beg
were tested for their inhibitory potency at GAT1, BGT1, GAT2 and
GAT3. The uptake assays were performed using HEK293 cell lines,
each stably expressing one of the four murine GABA transporter
subtypes [28].
Most of the new compounds that had been designed to address
GAT1 showed moderate to good inhibitory activity with reasonable
selectivity for this transporter subtype (Table 2). But attempts to
improve GAT3 potency of the parent compounds 5a and (rac)-6a
met only little success. As already outlined in the introduction 3-
aminopropanoic acid (5a) and 3-aminobutanoic acid [(rac)-6a]
are distinctly different with respect to their potency at GAT1 the
former possessing a far lower (pIC50 ¼ 2.55 ꢀ 0.03) and the latter an
almost similar potency (GAT1: pIC50 ¼ 4.30 ꢀ 0.10) as nipecotic acid
[(rac)-1] (GAT1: pIC50 ¼ 4.88 ꢀ 0.07) which has been widely used as
parent compound for the development of highly potent GAT in-
hibitors. In contrast, at GAT3 the potencies of all three compounds
are similar [(rac)-1: pIC50 ¼ 4.70 ꢀ 0.07; 5a: pIC50 ¼ 4.46 ꢀ 0.06;
(rac)-6a: pIC50 ¼ 4.08 ꢀ 0.03] (see Table 2).
Introduction of the 4,4-diphenylbut-3-enyl moiety as N-sub-
stituent known from SK&F-89976-A [(rac)-2] and many other ex-
amples to considerably enhance GAT1 potency improved uptake
inhibition also for 5b and (rac)-6b, but due to the low potency of the
parent compounds no high GAT1 potencies were reached (5b:
pIC50 ¼ 3.94 ꢀ 0.07; (rac)-6b: pIC50 ¼ 5.34 ꢀ 0.06). At the same time
the GAT3 potencies of 5b and (rac)-6b became lower, a phenome-
non which is also quite common for this kind of substituents being
As the parent compounds 5a and (rac)-6a display reasonable
potencies at GAT3 they were considered promising starting points for
the development of more potent inhibitors of this GABA transporter.
It was expected that the 2-[tris(4-methoxyphenyl)methoxy]ethyl
and the (E)-2-[tris(4-methoxyphenyl)]but-2-en-1-yl side chains
which are known from (S)-SNAP-5114 [(S)-4] and DDPM-1457 [(S)-

I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
491
Scheme 3. Synthesis of N-monoalkylated b-amino acids.
25], two of the most potent GAT3 inhibitors, should enhance their
GAT3 potency and selectivity. But none of the derivatives 5f, 5g, (rac)-
6f and (rac)-6g showed the expected improvements. Their inhibitory
potencies at GAT3 were moderate, still in the range of the parent
compounds 3-aminobutanoic acid derivative (rac)-6b exhibiting a
4,4-diphenylbut-3-enyl moiety at the amino nitrogen displayed the
highest inhibitory potency at this GABA transporter (GAT1:
pIC50 ¼ 5.34 ꢀ 0.06) along with a distinct subtype selectivity (BGT1:
86%, GAT2: 82% and GAT3: 98% remaining GABA uptake at a con-
centration of 100 mM of test compound). On the other hand, a potent
and highly selective inhibitor for GAT3 could not be found. To finally
conclude, the SAR established for nipecotic acid (1) is only partly
compounds (5f pIC50 ¼ 4.37 ꢀ 0.06, 5g 59%, at 100
mM; (rac)-6f;
pIC50 ¼ 4.47 ꢀ 0.11; (rac)-6g; pIC50 ¼ 4.22 ꢀ 0.01), nor did they
display a significant activity at GAT1.
In addition, most of the newly synthesized compounds have also
been studied with respect to their potency at BGT1 and GAT2. But
no reasonable potencies were found.
reflected in the acyclic
b-amino acids examined in this study.
Nevertheless, the present study is the first systematic investigation
providing SAR data for acyclic amino acids with lipophilic N-
substituents.
4. Conclusion
A series of new N-monoalkylated derivatives of 3-aminopro
panoic acid (5a) and 3-aminobutanoic acid (rac)-6a utilizing a
variant of the Mitsunobu reaction as key step has been synthesized.
5. Experimental section
5.1. Chemistry
The thus obtained N-substituted b-amino acids have been evaluated
for their potency at GAT1, BGT1, GAT2 and GAT3. In general, the
compounds derived from the 3-aminobutanoic acid [(rac)-6a] were
more potent than those delineated from 3-aminopropanoic acid (5a).
The most potent inhibitors were found for GAT1. Of all new
5.1.1. General
Benzene, toluene, n-heptane, NEt3 were distilled from sodium
under nitrogen. CH3CN, CH2Cl2 and CHCl3 were distilled from CaH2
under nitrogen. MeOH was distilled from Mg and acetone from

492
I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
Table 1
Yields of the Mitsunobu reaction.
O
N
O
CH
R
R]
O
N
O
O
O
S
S
OCH
OCH
R
O N
NO
O N
NO
ea
94% [(rac)-20b]
85% [(rac)-20c]
O
92% (19c)
86% (19d)
83% (19e)
N
O
95% [(rac)-20d]
87% [(rac)-20e]
N
O
N
S
H CO
O
ea
89% [(rac)-20f]
l96% [(rac)-20g]
OCH
H CO
H CO
79% (19g)
OCH
H CO
a
Compound not synthesized, see also text.
CaCl2 under nitrogen. Ethylene glycol and pyridine were stored
(2 equiv.) was added drop wise. The reaction mixture was stirred at
10 ꢁC for 5 min and at rt for the time given followed by concen-
tration in vacuo. The residue was purified by CC.
ꢀ
ꢀ
over mol sieve (3 A and 4 A, respectively) after distillation. Other
common solvents for recrystallization, and column chromatog-
raphy were distilled before use. Purchased chemical reagents were
used without further purification. TLC was carried out on precoated
silica gel F254 aluminum sheets (Merck). Compounds were stained
either with 5% (NH4)6Mo7O24$4H2O, 0.2% Ce(SO4)2$4H2O and 5%
conc. H2SO4 or with ninhydrin (0.3% in n-butanol þ 3% HOAc). If
nothing else is stated, Merck silica gel (mesh 230e400) was used as
stationary phase for flash chromatography (CC). Melting points:
m.p. (uncorrected) were determined with an Electrothermal
IA9100MK1 Melting Point apparatus. Elementary analysis: Ele-
mentaranalysator Vario EL (Elementar). The results of elemental
analyses for C, H, N, S were within ꢀ0.4% of the theoretical values.
IR spectroscopy: FT-IR Spectrometer 1600 and Paragon 1000 (Per-
kin Elmer), oils were measured as film, solid samples as KBr-pellets.
Mass spectrometry: EI and CI, Mass Spectrometer 5989 A with
59980 B particle beam LC/MS interface (Hewlett Packard); ESI, API
2000 (Applied Biosystems). NMR spectroscopy: NMR spectra were
recorded on JNMR-GX (JEOL, 400 MHz and 500 MHz) with TMS as
internal standard and integrated with the NMR-software Nuts (2D
Version 5.097, Acorn NMR, 1995).
5.1.3. General procedure for the cleavage 2,4-dinitrophenylsulfonyl
group (GP2)
To a stirred solution of the respective sulfonamide (1 equiv.)
in CH2Cl2 mercaptoacetic acid (1.3 equiv.) and finally NEt3
(2 equiv.) was added. After the time given the reaction mixture
was poured into a sat. NaHCO3 solution followed by extraction of
the aqueous layer with Et2O. The combined organic layers were
dried (MgSO4) and concentration in vacuo. The residue was pu-
rified by CC.
5.1.4. General procedure for the saponification of the ester
derivatives (GP3)
The respective ester derivative (1 equiv.) in MeOH (0.4 M) was
cooled to 0 ꢁC followed by the drop wise addition of aqueous NaOH
(2 M). The reaction mixture was stirred at rt for the time given and
followed by concentrating in vacuo. Purification was accomplished
as described.
5.1.5. Methyl 3-(4,4-diphenylbut-3-en-1-ylamino)propanoate (10b)
To a stirred suspension of methyl 3-aminopropanoate hydro-
chloride (209 mg, 1.50 mmol) K2CO3 (484 mg, 3.50 mmol) and KI
(25 mg, 0.15 mmol) in CH3CN (1.5 ml) a solution of 4-bromo-1,1-
diphenylbuten (431 mg, 1.50 mmol) in CH3CN (1.5 ml) was added.
5.1.2. General procedure for the preparation of N-alkylated sulpho
namides by Mitsunobu reaction (GP1)
To a stirred solution of the respective sulphonamide (1 equiv.),
Ph3P (2 equiv.) and alcohol (2 equiv.) in benzene at 10 ꢁC DIAD

Table 2
GABA uptake inhibition of b
-amino acids and their derivatives at GAT1, BGT1, GAT2 and GAT3 compared to nipecotic acid and its derivatives.I,II,III
R]
COOH
CH
R
COOH
N
H
R
COOH
N
H
N
R
pIC50 ꢀ S.E.M.
GAT1
pIC50 ꢀ S.E.M
pIC50 ꢀ S.E.M
BGT1
GAT2
GAT3
GAT1
BGT1
GAT2
GAT3
GAT1
BGT1
GAT2
GAT3
H
H
H
(rac)-1
5a
(rac)-6a
4.88 ꢀ 0.07a 3.10 ꢀ 0.09a 4.64 ꢀ 0.07a 4.70 ꢀ 0.07a 2.55 ꢀ 0.03a
3.48 ꢀ 0.11a
4.66 ꢀ 0.06a
4.46 ꢀ 0.13a
4.30 ꢀ 0.10a
3.09 ꢀ 0.10a
4.33 ꢀ 0.03a
4.08 ꢀ 0.03a
(R)-1
5.07 ꢀ 0.02a 3.28 ꢀ 0.05a 4.71 ꢀ 0.04a 4.79 ꢀ 0.05
(S)-1
4.13 ꢀ 0.05a 3.12 ꢀ 0.12a 3.71 ꢀ 0.04a 3.51 ꢀ 0.06a
SK&F-89976-A [(rac)-2]
5b$HCl
(rac)-6b
6.16 ꢀ 0.05a 3.43 ꢀ 0.07a 3.71 ꢀ 0.04a 3.56 ꢀ 0.06a 3.94 ꢀ 0.07b
3.36 ꢀ 0.04c
5.34 ꢀ 0.06a
100
m
M: 86%a 100
m
M 82%a
100 m
M: 98%a
O
(R)-22 [24]
5c
(rac)-6c
N
4.40d
e
e
e
e
e
e
e
e
e
4.69 ꢀ 0.14a
100
m
m
m
m
M: 85%a 100
m
m
m
m
M: 55%a 100
m
m
m
M: 95%a 4.88 ꢀ 0.05a
1 mM: 69%a
3.67 ꢀ 0.07a
3.47 ꢀ 0.01a
O
(R)-23 [24]
5d
100
(rac)-6d
N
6.19d
m
M: 56%a 100
M: 85%a 100
M: 88%a 100
M: 55%a 100
M: 87%a 100
M: 78%a 4.76 ꢀ 0.10a
100
100
m
m
m
M: 82%a 100
m
M: 84%a 100
m
m
M: 90%a
M: 68%a
O
(R)-24 [24]
5e
(rac)-6e
N
S
6.51d
4.45 ꢀ 0.11a
100
M: 50%a 100
M: 91%a 5.06 ꢀ 0.11a
M: 74%a 4.39 ꢀ 0.13a
100
H CO
O
(S)-SNAP-5114 [(S)-4]
5f
(rac)-6f
4.07 ꢀ 0.09a 4.51 ꢀ 0.15a 5.29 ꢀ 0.04a 5.71 ꢀ 0.20a 100
m
M: 57%a 100
M: 40%a 4.37 ꢀ 0.06a
100
m
M: 114%a 100
M: 73%a 100
m
M: 48%a 4.47 ꢀ 0.11a
OCH
H CO
H CO
DDPM-1457 (S)-25 [25]
5g
(rac)-6g
M: 59%a 100 M: 52%a
4.40 ꢀ 0.05a 4.42 ꢀ 0.11a 5.47 ꢀ 0.02a 5.87 ꢀ 0.08a 100
m
M: 77%a 100
m
M: 47%a 100
m
M: 94%a 100
m
m
100
m
M: 47%a 4.20 ꢀ 0.05a
4.22 ꢀ .01a
OCH
H CO
I
The compounds’ inhibitory potencies are given as pIC50 values (mean ꢀ S.E.M., N ¼ 3).
II
The abbreviations a,b and c specify the employed biological material: aHEK293 stably expressing mGAT1-4 (i.e. GAT1, BGT1, GAT2 and GAT3, respectively, according to HUGO), bpfcP2B membrane preparation from porcine
frontal cortex predominantly addressing the GAT1-subtype, cpbsP2C membrane preparation from porcine brainstem predominantly addressing the GAT3-subtype. dRefers to values from literature, for which the used material
can be considered as to consist predominantly of GAT1. For the sake of clarity, the reported IC50 values were converted into pIC50 values.
III
Percentages represent specific binding remaining in presence of inhibitor in the stated concentrations.

494
I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
The reaction mixture was heated to 45 ꢁC for 24 h. The solvent was
removed in vacuo and the resulting residue was resolved in H2O
which was subsequently extracted with Et2O (3ꢂ). The combined
organic layers were dried (MgSO4) and concentrated in vacuo. Pu-
rification was accomplished by CC (Et2O/n-pentane, 80:20 þ 2%
EtMe2N). Yield: 241 mg (52%), colorless oil. 1H NMR (400 MHz;
5.1.8. 2,2,2-Tris(4-methoxyphenyl)acetaldehyde (14)
2,2,2-Tris(4-methoxyphenyl)ethanol (5.47 g, 15.0 mmol) was
added to IBX (8.16 g, 29.1 mmol) in DMSO (26 ml) at rt. The re-
action mixture was stirred for 20 h. Sat. NaCl solution (150 ml) was
added and the aqueous layer was extracted with Et2O. The com-
bined organic layers were dried (MgSO4) and concentrated in
vacuo. Purification was accomplished by CC (CH2Cl2/n-pentane,
60:40). Yield: 5.24 g (96%); colorless solid, m.p.: 129 ꢁC. 1H NMR
CDCl3):
d
¼ 2.31 (q, J ¼ 7.3 Hz, 2H, ]CHCH2CH2), 2.49 (t, J ¼ 6.6 Hz,
2H, CH2COO), 2.73 (t, J ¼ 7.3 Hz, 2H, 2H, ]CHCH2CH2), 2.85 (t,
J ¼ 6.6 Hz, 2H, NCH2CH2COO), 3.67 (s, 3H, OCH3), 6.07 (t, J ¼ 7.3 Hz,
(400 MHz, CDCl3):
d
¼ 3.81 (s, 9H, 3ꢂ OCH3), 6.84e6.90 (m, 6H, 6ꢂ
ꢃ1
~
1H, C]CH), 7.15e7.40 (m, 10H, Harom). IR (film): v ¼ 3055 cm
,
OCCHCH), 6.94e7.00 (m, 6H, 6ꢂ OCCHCH), 10.19e10.21 (m, 1H,
3023, 2950, 2836, 1737, 1598. MS (CI, CHþ5 ); m/z (%): 310 (100)
[M þ H]þ. Anal. Calcd. for C20H23NO2; C, 77.64; H, 7.49; N, 4.53
Found: C, 77.61; H, 7.56; N, 4.49.
CHO). IR (KBr): v ¼ 2932 cmꢃ1, 2837, 1724, 1607, 1578, 1509, 1461,
~
1290, 1252, 1181. MS (CI, CHþ5 ); m/z (%): 363 [M þ H]þ (39), 255
(100). Anal. Calcd. for C23H22O4; C, 76.22; H, 6.12; Found: C, 76.06;
H, 6.24.
5.1.6. Methyl 3-({2-[tris(4-methoxyphenyl)methoxy]ethyl}amino)
butanoate (10f)
5.1.9. (E)-Ethyl 4,4,4-tris(4-methoxyphenyl)but-2-enoate (15)
Triethyl phosphonoacetate (3.8 ml, 18.4 mmol) was added to
NaH (538 mg, 21.3 mmol) in benzene (14 ml) at rt. The reaction
mixture was stirred for 30 min at rt, and then 1 h at 60 ꢁC. 2,2,2-
Tris(4-methoxyphenyl)acetaldehyde (5.09 g, 14.0 mmol) in ben-
zene (39 ml) was added at rt. The reaction mixture was stirred for
13 h. H2O (150 ml) was added and the aqueous layer was extracted
with EtOAc. The combined organic layers were dried (MgSO4) and
concentrated in vacuo. Purification was accomplished by CC
(CH2Cl2/n-pentane, 60:40) and recrystallized from EtOH Yield:
5.82 g (96%); colorless solid, m.p.: 132e133 ꢁC. 1H NMR (500 MHz,
To a stirred suspension of methyl 3-aminopropionate hydro-
chloride (279 mg, 2.00 mmol) K2CO3 (622 mg, 4.50 mmol) and KI
(33 mg, 0.20 mmol) in CH3CN (1.5 ml) a solution of 2-[tris(4-
methoxyphenyl)methoxy]ethyl bromide (914 mg, 2.00 mmol) in
CH3CN (1.5 ml) was added. The reaction mixture was heated to
45 ꢁC for 24 h. The solvent was removed in vacuo and the residue
was resolved in H2O which was subsequently extracted with Et2O
(3ꢂ). The combined organic layers were dried (MgSO4) and
concentrated in vacuo. Purification was accomplished by CC (Et2O/
n-pentane, 40:60 þ 2% EtMe2N). Yield: 459 mg (48%), colorless oil.
1H NMR (400 MHz; CDCl3):
d
¼ 2.53 (t, J ¼ 6.6 Hz, 2H, CH2COO), 2.80
CDCl3):
d
¼ 1.28 (t, J ¼ 7.2 Hz, 3H, CH2CH3), 3.80 (s, 9H, 3ꢂ OCH3),
(t, J ¼ 5.4 Hz, 2H, OCH2CH2N), 2.88 (t, J ¼ 6.6 Hz, 2H, 2H,
NCH2CH2COO), 3.21 (t, J ¼ 5.4 Hz, 2H, OCH2CH2N), 3.69 (s, 3H,
OCH3), 3.78 (s, 9H, OCH3), 6.78e6.84 (m, 6H, Harom), 7.28e7.34 (m,
4.20 (q, J ¼ 7.2 Hz, 2H, CH2CH3), 5.64 (d, J ¼ 15.8 Hz, 1H, COCHCH),
6.78e6.84 (m, 6H, 6ꢂ OCCHCH), 6.93e7.00 (m, 6H, 6ꢂ OCCHCH),
7.89 (d, J ¼ 15.8 Hz, 1H, COCHCH). IR (KBr): v ¼ 3432 cmꢃ1, 2959,
~
6H, Harom). IR (film): v ¼ 3036 cmꢃ1, 2999, 2951, 2836, 1737, 1608.
2834, 1709, 1645, 1606, 1508, 1292, 1250. MS (CI, CHþ5 ); m/z (%): 433
[M þ H]þ (30), 325 (100). Anal. Calcd. for C27H28O5: C, 74.98; H,
6.53; Found: C, 74.78; H, 6.50.
~
MS (CI, CHþ5 ); m/z (%): 355 (23) [M þ H]þ, 334 (100). Anal. Calcd. for
C
28H33NO6; C, 70.13; H, 6.94; N, 2.92 Found: C, 70.10; H, 6.94;
N, 2.95.
5.1.10. (E)-4,4,4-Tris(4-methoxyphenyl)but-2-en-1-ol (8g)
5.1.7. 2-[Tris(4-methoxyphenyl)methoxy]ethanol (8f) and 2-[(4-
chlorphenyl)bis(4-methoxyphenyl)methoxy]ethanol (12)
A solution of LiAlH4 (11 ml, 1.0 M in THF, 11 mmol) was added to
a solution of 15 (4.33 g, 10.0 mmol) in THF (12 ml). The reaction
mixture was stirred 46 h at 40 ꢁC. Potassium sodium tartrate so-
lution (1 M,165 ml) was added and the aqueous layer was extracted
with EtOAc. The combined organic layers were dried (MgSO4) and
concentrated in vacuo. Purification was accomplished by CC
(EtOAc/n-pentane, 30:70) and recrystallized from EtOAc and n-
pentane. Yield: 2.76 g (71%); colorless solid, m.p.: 137e138 ꢁC. 1H
To tris(4-methoxyphenyl)methanol (7.50 g, 21.4 mmol) in
toluene (15 ml) at 0 ꢁC thionyl chloride (2.95 ml, 40.7 mmol) was
added, followed by refluxing for 4.5 h. After cooling to rt n-hep-
tane (40 ml) was added and the resulting precipitate was filtered
off under N2 and washed with n-heptane (30 ml). The residue was
twice recrystallized from n-heptane/toluene to result in 6.0 g of
yellow crystals. 526 mg thereof were added to a mixture of
ethylene glycol (194 mg, 3.13 mmol) and pyridine (2.2 ml). After
stirring at rt for 25 h phosphate buffer (pH 7, 50 ml) was added,
followed by extraction with Et2O. The combined organic layers
were dried (MgSO4) and concentrated in vacuo. Purification was
accomplished by CC (EtOAc/n-pentane ¼ 35:65 þ 1% EtMe2N).
Yield: 235 mg (32%) of 8f and 37 mg (5%) of 12; 8f: colorless oil.
NMR (400 MHz, CDCl3):
d
¼ 1.32 (t, J ¼ 5.9 Hz, 1H, CH2OH), 3.80 (s,
9H, 3ꢂ OCH3), 4.26 (td, J ¼ 1.5/5.9 Hz, 2H, CH2OH), 5.45 (dt, J ¼ 5.7/
15.6 Hz, 1H, CH2CHCH), 6.67 (dt, J ¼ 1.5/15.6 Hz, 1H, CH2CHCH),
6.77e6.83 (m, 6H, 6ꢂ CH3OCCHCH), 6.96e7.01 (m, 6H, 6ꢂ
CH3OCCHCH). IR (KBr): v ¼ 3517 cmꢃ1, 3008, 2933, 2835, 1606,
~
1580, 1507, 1462, 1296, 1247. MS (CI, CHþ5 ); m/z (%): 390 [M þ H]þ
(9), 373 (100). Anal. Calcd. for C25H26O4: C, 76.90; H, 6.71; Found: C,
76.64; H, 6.79.
1H NMR (500 MHz, C6D6):
d
¼ 1.60 (t, J ¼ 6.1 Hz, 1H, CH2OH),
3.34e3.38 (m, 2H, CH2CH2OH), 3.42 (s, 9H, 3ꢂ CH3O), 3.67e3.72
(m, 2H, CH2CH2OH), 6.87e6.92 (m, 6H, 6ꢂ CH3OCCHCH), 7.57e
5.1.11. Methyl 3-(2,4-dinitrophenylsulfonylamino)propanoate (17)
7.63 (m, 6H, 6ꢂ CH3OCCHCH). IR (film): v ¼ 3428 cmꢃ1, 3001,
Pyridine (830 ml, 10.3 mmol) was added to 2,4-dinitrophenyl
~
2933, 2836, 1607, 1582, 1505, 1463, 1302, 1249, 1175, 1034. MS (EI,
70 eV): m/z (%): 394 (7) [M]þ, 333 (100). Anal. Calcd. for C24H26O5;
C, 73.08; H, 6.64; Found C, 73.08; H, 6.78. 12: colorless oil. 1H NMR
sulfonylchloride (313 mg, 1.15 mmol) and 9 (120 mg, 0.976 mmol)
in CH3CN (6 ml) at 0 ꢁC. The reaction mixture was stirred for 2 h,
than warmed to rt and stirred for additional 62.5 h. Phosphate
buffer (pH 3, 40 ml) was added and the aqueous layer was extracted
with Et2O. The combined organic layers were dried (MgSO4) and
concentrated in vacuo. Purification was accomplished by CC
(CH2Cl2) and recrystallized from CH2Cl2 and n-pentane. Yield:
259 mg (91%); colorless solid, m.p.: 100 ꢁC. 1H NMR (500 MHz,
(500 MHz, CD2Cl2):
d
¼ 1.84 (t, J ¼ 6.2 Hz, 1H, CH2OH), 3.18 (t,
J ¼ 4.8 Hz, 2H, CH2CH2OH), 3.67e3.74 (m, 2H, CH2CH2OH), 3.78 (s,
6H, 2ꢂ OCH3), 6.80e6.88 (m, 4H, 4ꢂ CH3OCCHCH), 7.24e7.34 (m,
6H, 4ꢂ CH3OCCHCH, 2ꢂ ClCCHCH), 7.38e7.44 (m, 2H, 2ꢂ
ClCCHCH). IR (film): v ¼ 3426 cmꢃ1, 3000, 2932, 2836, 1608, 1509,
~
1488, 1301, 1251. MS (EI, 70 eV): m/z (%): 398 (7) [M]þ, 337 (100).
Anal. Calcd. for C23H23O4Cl; C, 69.26; H, 5.81; Found: C, 68.99;
H, 5.61.
CDCl3):
d
¼ 2.63 (t, J ¼ 6.0 Hz, 2H, COCH2), 3.42 (q, J ¼ 6.0 Hz, 2H,
NHCH2), 3.70 (s, 3H, OCH3), 6.10 (t, J ¼ 6.0 Hz, 1H, NH), 8.37 (d,
J ¼ 8.6 Hz, 1H, SO2CCH), 8.56 (dd, J ¼ 8.6/2.2 Hz, 1H, SO2CCHCH),

I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
495
ꢃ1
~
8.69 (d, J ¼ 2.2 Hz, 1H, C(NO2)CHCNO2). IR (KBr): v ¼ 3301 cm
,
3103, 1732, 1605, 1558, 1538, 1367, 1349, 1165. MS (CI, CHþ5 ); m/z (%):
334 (36) [M þ H]þ, 302 (100). Anal. Calcd. for C10H11N3O8S; C,
36.04; H, 3.33; N,12.61; S, 9.62; Found: C, 35.79; H, 3.25; N,12.61; S,
9.33.
5.1.15. Methyl (rac)-3-({2-[2-(10,11-dihydro-5H-dibenzo[b,f]azepin-
5-yl)ethoxy]ethyl}(2,4-dinitrophenylsulfonyl)amino)butanoate [(rac)-
20c]
According to GP1 starting from (rac)-18 (339 mg, 0.977 mmol),
Ph3P (511 mg, 1.95 mmol), 8d (551 mg, 1.94 mmol) and DIAD (400 l,
m
1.96 mmol), in benzene (2.9 ml); reaction time: 45 min. Purification
by twice CC (CH2Cl2 then EtOAc/n-pentane, 20:80) Yield: 506 mg
(85%); red brown solid, m.p.: 44e49 ꢁC. 1H NMR (500 MHz, CDCl3):
5.1.12. Methyl (rac)-3-(2,4-dinitrophenylsulfonylamino)butanoate
[(rac)-18]
Pyridine (13.8 ml, 170.62 mmol) was added to 2,4-dinitrophenyl
sulfonylchloride (5.19 g, 19.1 mmol) and (rac)-16 (2.19 g, 14.3 mmol)
in CH3CN (100 ml) at 0 ꢁC. The reaction mixture was stirred for 2 h,
then warmed to rt and stirred for additional 65.5 h. Phosphate
buffer (pH 2, 600 ml) was added and the aqueous layer was
extracted with Et2O. The combined org. layers were dried (MgSO4)
and concentrated in vacuo. Purification was accomplished by CC
(CH2Cl2) and recrystallized from CH2Cl2 and n-heptane. Yield:
2.65 g (53%); colorless solid, m.p.: 65e66 ꢁC. 1H NMR (500 MHz,
d
¼ 1.23 (d, J ¼ 6.8 Hz, 3H, NCHCH3), 2.44 (dd, J ¼ 15.8/7.7 Hz, 1H,
COCH2), 2.68 (dd, J ¼ 15.8/6.5 Hz,1H, COCH2), 3.14 (s, 4H, CCH2CH2C),
3.36e3.53 (m, 4H, OCH2CH2NSO2), 3.50 (t, 6.1 Hz, 2H,
J
¼
(CAr)2NCH2CH2O), 3.58 (s, 3H, CH3O), 3.88e3.95 (m, 2H, (Car-
om)2NCH2CH2O), 4.25e4.34 (m, 1H, COCH2CHCH3), 6.93 (td, J ¼ 7.4/
1.3 Hz, 2H, 2ꢂ NCCCHCH), 7.07 (dd, J ¼ 8.1/1.1 Hz, 2H, 2ꢂ NCCHCH),
7.09 (dd, J ¼ 7.4/1.6 Hz, 2H, NCCCHCH), 7.11e7.15 (m, 2H, NCCHCH),
8.26 (d, J ¼ 8.7 Hz, 1H, SO2CCHCH), 8.33 (dd, J ¼ 8.7/2.2 Hz, 1H,
SO2CCHCH), 8.41 (d, J ¼ 2.2 Hz, 1H, C(NO2)CHCNO2). IR (KBr):
v ¼ 3441 cmꢃ1, 3099, 2948, 1736, 1553, 1537, 1487, 1350, 1207, 1166,
~
CDCl3):
d
¼ 1.26 (t, J ¼ 6.8 Hz, 3H, CHCH3), 2.56 (dd, J ¼ 5.5/0.8 Hz,
2H, COCH2), 3.63 (s, 3H, OCH3), 3.99 (dqt, J ¼ 8.2/6.8/5.5 Hz, 1H,
CH2CH), 6.00 (d, J ¼ 8.2 Hz,1H, NH), 8.40 (d, J ¼ 8.7 Hz,1H, SO2CCH),
8.56 (dd, J ¼ 8.7/2.2 Hz, 1H, SO2CCHCH), 8.69 (d, J ¼ 2.2 Hz, 1H,
1108. MS (ESIþ) m/z: 613 [M þ H]þ. Anal. Calcd. for C29H32N4O9S; C,
56.85; H, 5.27;N, 9.15; S, 5.23; Found: C, 56.63; H, 5.25; N, 9.08; S, 5.72.
C(NO2)CHCNO2). IR (KBr): v ¼ 3333 cmꢃ1, 3107, 1725, 1605, 1535,
5.1.16. Methyl 3-({2-[2-(5H-dibenzo[b,f]azepin-5-yl)ethoxy]ethyl}(2,4-
dinitrophenylsulfonyl)amino)propanoate (19d)
~
1350, 1169. MS (CI, CHþ5 ): m/z (%): 348 (4) [M þ H]þ, 118 (100). Anal.
Calcd. for C11H13N3O8S; C, 38.04; H, 3.77; N, 12.10; S 9.23; Found: C,
37.89; H, 3.79; N, 12.15; S, 9.47.
According to GP1 starting from 17 (261 mg, 0.784 mmol), Ph3P
(409 mg, 1.56 mmol), 8d (436 mg, 1.55 mmol) and DIAD (315 ml,
1.55 mmol) in benzene (2.4 ml); reaction time: 45 min. Purification
by CC (CH2Cl2 then Et2O/n-pentane, 60:40). Yield: 402 mg (86%);
5.1.13. Methyl (rac)-3-[(2,4-dinitrophenylsulfonyl)(4,4-diphenylbut-
3-en-1-yl)amino]butanoate [(rac)-20b]
According to GP1 starting from (rac)-18 (425 mg, 1.22 mmol),
Ph3P (645 mg, 2.46 mmol), 8b (549 mg, 2.45 mmol) and DIAD
orange solid, m.p.: 44e56 ꢁC. 1H NMR (500 MHz, CDCl3):
d
¼ 2.66 (t,
J ¼ 7.3 Hz, 2H, COCH2), 3.48e3.54 (m, 6H, CH2OCH2CH2NSO2), 3.63
(s, 3H, CH3O), 3.66 (t, J ¼ 7.3 Hz, 2H, COCH2CH2N), 3.78 (t, J ¼ 5.8 Hz,
2H, (Carom)2NCH2CH2O), 6.70 (s, 2H, CCHCHC), 6.91 (dd, J ¼ 8.1/
0.9 Hz, 2H, 2ꢂ NCCHCH), 6.99e7.03 (m, 2H, 2ꢂ NCCCHCH), 7.06 (dd,
J ¼ 7.6/1.8 Hz, 2H, 2ꢂ NCCCHCH), 7.27 (ddd, J ¼ 8.1/7.2/1.8 Hz, 2H,
NCCHCH), 8.11 (dd, J ¼ 8.7/2.2 Hz, 1H, SO2CCHCH), 8.21 (dd, J ¼ 8.7/
0.3 Hz, 1H, SO2CCHCH), 8.37 (dd, J ¼ 2.2/0.3 Hz, 1H, C(NO2)
(500 ml, 2.45 mmol) in benzene (3.7 ml); reaction time: 45 min.
Purification was accomplished by CC (first CH2Cl2 then Et2O/n-
pentane, 30:70). Yield: 635 mg (94%); yellow solid, m.p.: 46e49 ꢁC.
1H NMR (500 MHz, CDCl3):
d
¼ 1.13 (d, J ¼ 6.8 Hz, 3H, NCHCH3),
2.28 (dd,
J
¼
15.7/7.9 Hz, 1H, COCH2), 2.37e2.44 (m, 2H,
~
NCH2CH2CH), 2.48 (dd, J ¼ 15.7/6.6 Hz, 1H, COCH2), 3.28e3.36 (m,
1H, NCH2), 3.38e3.47 (m, 1H, NCH2), 3.58 (s, 3H, CH3O), 4.22e4.30
(m, 1H, COCH2CH), 6.05 (t, J ¼ 7.6 Hz, (C6H5)2CCH), 7.15e7.18 (m,
2H, Harom), 7.20e7.23 (m, 2H, Harom), 7.23e7.29 (m, 3H, Harom),
7.32e7.36 (m, 1H, Harom), 7.37e7.42 (m, 2H, Harom), 8.19 (d,
J ¼ 8.7 Hz, 1H, SO2CCH), 8.38 (dd, J ¼ 8.7/2.2 Hz, 1H, SO2CCHCH),
CHCNO2). IR (KBr): v ¼ 3097 cmꢃ1, 2948, 2868, 1735, 1604, 1553,
1537, 1484, 1458, 1437, 1351, 1202, 1163, 1130, 1116. MS (ESIþ) m/z:
597 [M þ H]þ. Anal. Calcd. for C28H28N4O9S; C, 56.37; H, 4.73; N,
9.39; S, 5.38; Found: C, 56.36; H, 4.78; N, 9.14; S, 5.65.
5.1.17. Methyl (rac)-3-({2-[2-(5H-dibenzo[b,f]azepin-5-yl)ethoxy]
ethyl}(2,4-dinitrophenylsulfonyl)amino)butanoate [(rac)-20d]
According to GP1 starting from (rac)-18 (412 mg, 1.19 mmol),
Ph3P (626 mg, 2.39 mmol), 8d (668 mg, 2.37 mmol) and DIAD
ꢃ1
~
8.41 (d, J ¼ 2.2 Hz, 1H, C(NO2)CHCNO2). IR (KBr): v ¼ 3101 cm
,
2952, 1736, 1604, 1555, 1538, 1495, 1442, 1351, 1302, 1205, 1166. MS
(ESIþ) m/z: 592 [M þ K]þ, 576 [M þ Na]þ. Anal. Calcd. for
C27H27N3O8S; C, 58.58; H, 4.92; N, 7.59; S, 5.79; Found: C, 58.69; H,
4.87; N, 7.59; S, 6.08.
(485
Purification by CC (CH2Cl2).Yield: 686 mg (95%); orange solid, m.p.:
45e52 ꢁC. 1H NMR (500 MHz, CDCl3):
¼ 1.24 (d, J ¼ 6.8 Hz, 3H,
ml, 2.38 mmol) in benzene (3.6 ml); reaction time 45 min.
d
5.1.14. Methyl 3-({2-[2-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)
ethoxy]ethyl}(2,4-dinitrophenylsulfonyl)amino)propanoate (19c)
According to GP1 starting from 17 (345 mg, 1.04 mmol), Ph3P
COCH2CHCH3), 2.46 (dd, J ¼ 15.9/7.9 Hz, 1H, COCH2CH), 2.72 (dd,
J ¼ 15.9/6.4 Hz, 1H, COCH2CH), 3.42e3.46 (m, 2H, OCH2CH2NSO2),
3.52e3.57 (m, 2H, OCH2CH2NSO2), 3.55 (t, J ¼ 6.1 Hz, 2H, (Car-
om)2NCH2CH2O), 3.58 (s, 3H, CH3O), 3.88 (t, J ¼ 6.1 Hz, 2H, (Car-
om)2NCH2CH2O), 4.25e4.33 (m, 1H, COCH2CH), 6.70 (s, 2H,
CCHCHC), 6.98e7.03 (m, 4H, 2ꢂ NCCCHCH, 2ꢂ NCCHCH), 7.06 (dd,
J ¼ 7.9/1.7 Hz, 2H, NCCCHCH), 7.24e7.29 (m, 2H, NCCHCH), 8.28 (d,
J ¼ 8.7 Hz, 1H, SO2CCHCH), 8.31 (dd, J ¼ 8.7/2.2 Hz, 1H, SO2CCHCH),
(545 mg, 2.08 mmol), 8c (586 mg, 2.07 mmol) and DIAD (430 ml,
2.11 mmol) in benzene (3.1 ml), reaction time: 20 min. Purification
was accomplished by twice CC (CH2Cl2 then EtOAc/n-pentane,
25:75). Yield: 570 mg (92%), brown oil. 1H NMR (400 MHz, CDCl3):
d
¼ 2.65 (t, J ¼ 7.2 Hz, 2H, COCH2), 3.11 (s, 4H, CCH2CH2C), 3.43e
ꢃ1
~
3.50 (m, 4H, CH2OCH2CH2NSO2), 3.53 (t, 5.1 Hz, 2H,
J
¼
8.41 (d, J ¼ 2.2 Hz, 1H, C(NO2)CHCNO2). IR (KBr): v ¼ 3099 cm
,
OCH2CH2NSO2), 3.60e3.66 (m, 2H, COCH2CH2N), 3.63 (s, 3H,
CH3O), 3.82 (t, J ¼ 5.9 Hz, 2H, (Carom)2NCH2CH2O), 6.92 (td, J ¼ 7.4/
1.1 Hz, 2H, 2ꢂ NCCCHCH), 7.01 (dd, J ¼ 8.1/1.1 Hz, 2H, 2ꢂ NCCHCH),
7.05e7.15 (m, 4H, 2ꢂ NCCHCH, 2ꢂ NCCCHCH), 8.19e8.22 (m, 2H,
SO2CCHCH), 8.36e8.38 (m, 1H, C(NO2)CHCNO2). IR (film):
2951, 2869, 1735, 1604, 1553, 1538, 1484, 1458, 1436, 1350, 1207,
1165, 1130, 1116. MS (ESIþ) m/z: 611 [M þ H]þ. Anal. Calcd. for
C
29H30N4O9S; C, 57.04; H, 4.95; N, 9.18; S, 5.25; Found: C, 56.76; H,
4.92; N, 9.19; S, 5.58.
v ¼ 3443 cmꢃ1, 3101, 2920, 1735, 1602, 1553, 1537, 1486, 1351, 1163.
5.1.18. Methyl 3-[(2,4-dinitrophenylsulfonyl){2-[2-(10H-phenothiazin-
10-yl)-ethoxy]ethyl}amino]propanoate (19e)
~
MS (CI, CHþ5 ); m/z (%): 599 (14) [M þ H]þ, 123 (100). Anal. Calcd. for
C
28H30N4O9S; C, 56.18; H, 5.05; N, 9.36; S, 5.36; Found: C, 56.20; H,
According to GP1 starting from 17 (258 mg, 0.775 mmol), Ph3P
5.38; N, 9.06; S, 5.58.
(408 mg, 1.56 mmol), 8e (442 mg, 1.54 mmol) and DIAD (315 ml,

496
I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
1.55 mmol) in benzene (2.3 ml), reaction time: 20 min. Purification
by CC (Et2O/n-pentane 60:40). Yield: 386 mg (83%) red brown, solid
CH3O), 6.07 (t, J ¼ 7.4 Hz, 1H, NCH2CH2CH), 7.16e7.39 (m, 10H,
arom), 7.34e7.39 (m, 2H, Harom). IR (film): v ¼ 2951 cmꢃ1, 1733,
~
H
melting range 43e50 ꢁC. 1H NMR (500 MHz, CDCl3):
d
¼ 2.61 (t,
1493, 1442, 1360, 1294. MS (CI, CHþ5 ); m/z (%): 324 (100) [M þ H]þ.
Anal. Calcd. for C21H25N1O2; C, 77.99; H, 7.79; N, 4.33; Found: C,
77.83; H, 7.92; N, 4.34.
J ¼ 7.1 Hz, 2H, COCH2), 3.53e3.57 (m, 2H, OCH2CH2NSO2), 3.57e
3.60 (m, 2H, OCH2CH2NSO2), 3.62 (s, 3H, CH3O), 3.64 (t, J ¼ 7.1 Hz,
2H, COCH2CH2), 3.74 (t, J ¼ 5.7 Hz, 2H, (Carom)2NCH2CH2O), 4.02 (t,
J ¼ 5.7 Hz, 2H, (Carom)2NCH2CH2O), 6.84 (d, J ¼ 8.1 Hz, 2H, 2ꢂ
NCCHCH), 6.94 (t, J ¼ 7.5 Hz, 2H, 2ꢂ NCCCHCH), 7.14 (dd, J ¼ 7.7/
1.4 Hz, 2H, 2ꢂ NCCCHCH), 7.12e7.19 (m, 2H, 2ꢂ NCCHCH), 8.23e
8.25 (m, 2H, SO2CCHCH), 8.40e8.42 (m, 1H, C(NO2)CHCNO2). IR
5.1.22. Methyl 3-({2-[2-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)
ethoxy]ethyl}amino)propanoate (9c)
According to GP2 starting from 19c (386 mg, 0.645 mmol),
mercaptoacetic acid (58
ml, 0.83 mmol) and NEt3 (180 ml,
(KBr): v ¼ 3448 cmꢃ1, 3097, 2871, 1735, 1594, 1552, 1537, 1463, 1350,
1.29 mmol) in CH2Cl2 (6.5 ml); reaction time: 3 h; work-up: sat.
NaHCO3 solution (50 ml) and. Purification by CC (EtOAc/n-pentane
40:60 þ 2% EtMe2N). Yield: 220 mg (92%), yellow oil. 1H NMR
~
1162. MS (ESIþ) m/z: 603 [M þ H]þ. Anal. Calcd. for C26H26N4O9S2;
C, 51.82; H, 4.35, N, 9.30; Found: C, 51.63; H, 4.23; N 9.05.
(400 MHz, CDCl3):
d
¼ 2.49 (t, J ¼ 6.8 Hz, 2H, COCH2), 2.69e2.74 (m,
5.1.19. Methyl (rac)-3-[(2,4-dinitrophenylsulfonyl){2-[2-(10H-
phenothiazin-10-yl)ethoxy]ethyl}amino]butanoate [(rac)-20e]
According to GP1 starting from (rac)-18 (389 mg, 1.12 mmol),
Ph3P (587 mg, 2.24 mmol), 8e (642 mg, 2.23 mmol) and DIAD
2H, NHCH2CH2OCH2), 2.86 (t, J ¼ 6.8 Hz, 2H, COCH2CH2NH), 3.15 (s,
4H, CCH2CH2C), 3.42e3.47 (m, 2H, NHCH2CH2OCH2), 3.56 (t,
J ¼ 6.3 Hz, 2H, (Carom)2NCH2CH2O), 3.67 (s, 3H, CH3O), 3.97 (t,
J
¼ 6.3 Hz, 2H, (Carom)2NCH2CH2O), 6.89e6.94 (m, 2H, 2ꢂ
(455
Purification by CC (Et2O/n-pentane, 60:40). Yield: 386 mg (87%);
brown solid, m.p.: 43e49 ꢁC. 1H NMR (500 MHz, CDCl3):
¼ 1.21 (d,
ml, 2.23 mmol) in benzene (3.4 ml); reaction time: 45 min.
NCCCHCH), 7.06e7.15 (m, 6H, 2ꢂ NCCCHCH, 2ꢂ NCCHCH). IR (film):
v ¼ 2889 cmꢃ1, 2846, 1735, 1486, 1457, 1337, 1236, 1175, 1127, 1109.
~
d
MS (CI, CHþ5 ); m/z (%): 369 (98) [M þ H]þ, 221 (100). Anal. Calcd.
for C22H28N2O3; C, 71.71; H, 7.66; N, 7.60; Found: C, 71.27; H, 7.51;
N, 7.53.
J ¼ 6.8 Hz, 3H, COCH2CHCH3), 2.43 (dd, J ¼ 15.8/7.7 Hz, 1H,
COCH2CH), 2.70 (dd, J ¼ 15.8/6.6 Hz, 1H, COCH2CH), 3.45e3.51 (m,
2H, OCH2CH2NSO2), 3.58 (s, 3H, CH3O), 3.59e3.64 (m, 2H,
OCH2CH2NSO2), 3.76e3.81 (m, 2H, (CAr)2NCH2CH2O), 4.08 (t,
J ¼ 5.8 Hz, 2H, (CAr)2NCH2CH2O), 4.23e4.31 (m, 1H, COCH2CHCH3),
6.87 (dd, J ¼ 8.1/0.9 Hz, 2H, 2ꢂ NCCHCH), 6.93 (td, J ¼ 7.5/1.1 Hz, 2H,
2ꢂ NCCCHCH), 7.13 (dd, J ¼ 7.6/1.5 Hz, 2H, 2ꢂ NCCCHCH), 7.15 (ddd,
J ¼ 8.1/7.5/1.5 Hz, 2H, 2ꢂ NCCHCH), 8.28 (d, J ¼ 8.7 Hz, 1H,
SO2CCHCH), 8.37 (dd, J ¼ 8.7/2.2 Hz, 1H, SO2CCHCH), 8.42 (d,
5.1.23. Methyl (rac)-3-({2-[2-(10,11-dihydro-5H-dibenzo[b,f]azepin-
5-yl)ethoxy]ethyl}amino)butanoate [(rac)-21c]
According to GP2 starting from (rac)-20c (374 mg, 0.611 mmol),
mercaptoacetic acid (56 ml, 0.81 mmol) and NEt3 (170 ml,1.22 mmol)
in CH2Cl2 (6.1 ml); reaction time: 6 h; work-up: sat. NaHCO3 solu-
tion (50 ml) and Et2O. Purification by CC (EtOAc/n-pentane
20:80 þ 2% EtMe2N). Yield: 206 mg (88%), yellow oil. 1H NMR
J ¼ 2.2 Hz,1H, C(NO2)CHCNO2). IR (KBr): v ¼ 3097 cmꢃ1, 2950, 2872,
~
1734, 1552, 1537, 1463, 1350, 1255, 1208, 1165, 1130, 1105. MS (ESIþ)
m/z: 617 [M þ H]þ. Anal. Calcd. for C27H28N4O9S2; C, 52.59; H, 4.58;
N, 9.09; S, 10.40; Found: C, 52.47; H, 4.55; N, 8.89; S, 10.72.
(500 MHz, CDCl3):
d
¼ 1.09 (d, J ¼ 6.4 Hz, 3H, NHCHCH3), 2.28 (dd,
J ¼ 15.1/6.9 Hz, 1H, COCH2), 2.46 (dd, J ¼ 15.1/6.1 Hz, 1H, COCH2),
2.67 (dt, J ¼ 12.0/5.3 Hz, 1H, NHCH2CH2OCH2), 2.73 (dt, J ¼ 12.0/
5.3 Hz, 1H, NHCH2CH2OCH2), 3.02e3.10 (m, 1H, NHCHCH3), 3.15 (s,
4H, CCH2CH2C), 3.44 (t, J ¼ 5.3 Hz, 2H, NHCH2CH2OCH2), 3.56 (t,
J ¼ 6.3 Hz, 2H, (Carom)2NCH2CH2O), 3.65 (s, 3H, CH3O), 3.97 (t,
5.1.20. Methyl (rac)-3-[(2,4-dinitrophenylsulfonyl){2-[tris(4-methoxy
phenyl)methoxy]ethyl}amino]butanoate [(rac)-20f]
According to GP1 starting from (rac)-18 (108 mg, 0.311 mmol),
Ph3P (159 mg, 0.606 mmol), 8f (238 mg, 0.604 mmol) and DIAD
J
¼ 6.3 Hz, 2H, (Carom)2NCH2CH2O), 6.89e6.94 (m, 2H, 2ꢂ
NCCCHCH), 7.07e7.14 (m, 6H, 2ꢂ NCCCHCH, 2ꢂ NCCHCH). IR (film):
v ¼ 2859 cmꢃ1, 1734, 1595, 1572, 1487, 1457, 1336, 1294, 1236. MS
~
(125
ml, 0.61 mmol) in benzene (0.9 ml); reaction time: 45 min.
Purification by CC (Et2O/n-pentane 40:60 þ 5% EtMe2N); Yield:
(CI, CHþ5 ); m/z (%): 383 (100) [M þ H]þ. Anal. Calcd. for C23H30N2O3;
200 mg (89%); yellow solid, m.p.: 67e72 ꢁC. 1H NMR (500 MHz,
C, 72.22; H, 7.91; N, 7.33; Found: C, 72.10; H, 7.77; N, 7.31.
CD2Cl2):
d
¼ 1.14 (d, J ¼ 6.8 Hz, 3H, COCH2CHCH3), 2.36 (dd, J ¼ 15.7/
8.2 Hz, 1H, COCH2CH), 2.54 (dd, J ¼ 15.7/6.1 Hz, 1H, COCH2CH), 3.27
(t, J ¼ 6.4 Hz, 2H, OCH2CH2NSO2), 3.42e3.51 (m, 2H, OCH2CH2NSO2),
3.55 (s, 3H, CH3O), 3.78 (s, 9H, 3ꢂ CH3OC6H4), 4.22e4.30 (m, 1H,
COCH2CH), 6.80e6.85 (m, 6H, 6ꢂ CH3OCCHCH), 7.27e7.31 (m, 6H,
6ꢂ CH3OCCHCH), 8.19 (d, J ¼ 8.7 Hz, 1H, SO2CCHCH), 8.35 (dd,
J ¼ 8.7/2.2 Hz, 1H, SO2CCHCH), 8.43 (d, J ¼ 2.2 Hz, 1H, C(NO2)
5.1.24. Methyl 3-({2-[2-(5H-dibenzo[b,f]azepin-5-yl)ethoxy]ethyl}
amino)propanoate (9d)
According GP2 starting from 19d (304 mg, 0.510 mmol), mer-
captoacetic acid (47 ml, 0.66 mmol) and NEt3 (145 ml, 1.04 mmol) in
CH2Cl2 (5.1 ml), reaction time: 5.5 h; work-up: sat. NaHCO3 solution
(50 ml) and Et2O. Purification by CC (Et2O/n-pentane 80:20 þ 2%
EtMe2N). Yield: 171 mg (92%), yellow oil. 1H NMR (500 MHz,
CHCNO2). IR (KBr): v ¼ 3100 cmꢃ1, 2953, 2837, 1736, 1607, 1555,
~
1540, 1508, 1463, 1440, 1351, 1302, 1250. MS (ESIþ) m/z: 762
[M þ K]þ, 746 [M þ Na]þ. Anal. Calcd. for C35H37N3O12S; C, 58.08; H,
5.15; N, 5.81; S, 4.43; Found: C, 57.99; H, 5.22; N, 5.56; S, 4.87.
CDCl3):
d
¼ 2.49 (t, J ¼ 6.8 Hz, 2H, COCH2), 2.69e2.72 (m, 2H,
NHCH2CH2OCH2), 2.86 (t, J ¼ 6.8 Hz, 2H, COCH2CH2NH), 3.47e3.50
(m, 2H, NHCH2CH2OCH2), 3.59 (t, J ¼ 6.5 Hz, 2H, (CAr)2NCH2CH2O),
3.68 (s, 3H, CH3O), 3.94 (t, J ¼ 6.5 Hz, 2H, (CAr)2NCH2CH2O), 6.72 (s,
2H, CCHCHC), 6.96e7.01 (m, 2H, 2ꢂ NCCCHCHCH), 7.03e7.07 (m,
4H, 2ꢂ NCCHCH, 2ꢂ NCCCHCHCH), 7.25 (ddd, J ¼ 8.2/7.2/1.6 Hz, 2H,
5.1.21. Methyl (rac)-3-(4,4-diphenylbut-3-en-1-ylamino)butanoate
[(rac)-21b]
2ꢂ NCCHCH). IR (film): v ¼ 3018 cmꢃ1, 2856, 1735, 1592, 1570, 1483,
~
According to GP2 starting from (rac)-20b (549 mg, 0.993 mmol),
mercaptoacetic acid (92
m
l, 1.30 mmol) and NEt3 (280
ml,
1458, 1436, 1238, 1195. MS (CI, CHþ5 ); m/z (%): 367 (100) [M þ H]þ.
Anal. Calcd. for C22H26N2O3: C, 72.11; H, 7.15; N, 7.64; Found: C,
71.69; H, 7.20; N, 7.51.
2.01 mmol) in CH2Cl2 (9.9 ml); reaction time: 5.5 h; work-up: sat.
NaHCO3 solution (100 ml) and Et2O. Purification by CC (Et2O/n-
pentane 80:20 þ 2% EtMe2N). Yield: 288 mg (89%), colorless oil. 1H
NMR (500 MHz, CDCl3):
d
¼ 1.09 (d, J ¼ 6.4 Hz, 3H, NHCHCH3), 2.30
5.1.25. Methyl (rac)-3-({2-[2(5H-dibenzo[b,f]azepin-5-yl)-ethoxy]
ethyl}amino)butanoate [(rac)-21d]
According to GP2 starting from (rac)-20d (603 mg, 0.989 mmol),
mercaptoacetic acid (92 ml,1.30 mmol) and NEt3 (275 ml, 1.98 mmol)
(q, J ¼ 7.3 Hz, 2H, NCH2CH2CH), 2.32 (dd, J ¼ 15.2/6.1 Hz, 1H,
COCH2), 2.44 (dd, J ¼ 15.2/6.7 Hz, 1H, COCH2), 2.64e2.78 (m, 2H,
NCH2CH2CH), 3.08 (sext., J ¼ 6.4 Hz, 1H, NHCHCH3), 3.65 (s, 3H,

I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
497
in CH2Cl2 (9.9 ml); reaction time 5.5 h; work-up: sat. NaHCO3 so-
lution (100 ml) and Et2O. Purification by CC (Et2O/n-pentane
80:20 þ 2% EtMe2N). Yield: 332 mg (88%), yellow oil. 1H NMR
NHCH2CH2O), 3.06 (sext., J ¼ 6.4 Hz, 1H, NHCHCH3), 3.12 (t,
J ¼ 5.5 Hz, 2H, NHCH2CH2O), 3.64 (s, 3H, CH3O), 3.77 (s, 9H, 9H, 3ꢂ
CH3OC6H4), 6.80e6.84 (m, 6H, 6ꢂ CH3OCCHCH), 7.29e7.33 (m, 6H,
6ꢂ CH3OCCHCH). IR (film): v ¼ 2952 cmꢃ1, 2836, 1733, 1607, 1555,
~
(500 MHz, CDCl3):
d
¼ 1.10 (d, J ¼ 6.4 Hz, 3H, NHCHCH3), 2.29 (dd,
J ¼ 15.1/6.9 Hz, 1H, COCH2CH), 2.47 (dd, J ¼ 15.1/6.1 Hz, 1H,
COCH2CH), 2.64e2.76 (m, 2H, NHCH2CH2OCH2), 3.07 (sext.,
J ¼ 6.4 Hz, 1H, COCH2CH), 3.49 (t, J ¼ 5.2 Hz, 2H, NHCH2CH2OCH2),
3.59 (t, J ¼ 6.6 Hz, 2H, (CAr)2NCH2CH2O), 3.66 (s, 3H, CH3O), 3.94 (t,
J ¼ 6.6 Hz, 2H, (CAr)2NCH2CH2O), 6.72 (s, 2H, CCHCHC), 6.99 (td,
J ¼ 7.4/1.1 Hz, 2H, 2ꢂ NCCCHCHCH), 7.05 (dd, J ¼ 8.1/0.8 Hz, 2H, 2ꢂ
NCCHCH), 7.06 (dd, J ¼ 7.6/1.7 Hz, 2H, 2ꢂ NCCCHCHCH), 7.25 (ddd,
1539, 1507, 1462, 1301, 1249, 1175. MS (CI, CHþ5 ); m/z (%): 333 (100)
[M þ H]þ. Anal. Calcd. for C29H35NO6; C, 70.57; H, 7.15; N, 2.84;
Found: C, 70.32; H, 6.66; N, 2.72.
5.1.29. (4,4-Diphenylbut-3en-1-yl)aminopropanoic acid 5b
NaOH solution (12 M, 98 ml, 1.17 mmol) was added drop wise at
0 ꢁC to 9b (181 mg, 0.585 mmol) in EtOH (1.2 ml). The ice bath was
removed and the reaction mixture was stirred for additional 4 h at
rt. The reaction mixture was adjusted to pH w 1 by addition of
aqueous HCl (4 M) at 0 ꢁC and subsequently extracted 5 times with
CH2Cl2. The combined organic phases were dried (MgSO4) and
concentrated in vacuo. The resulting hydrochloride was finally
recrystallized from EtOH. Yield 163 mg (84%); colorless crystals,
J ¼ 8.1/7.3/1.7 Hz, 2H, 2ꢂ NCCHCH). IR (film): v ¼ 3019 cmꢃ1, 2950,
~
2860, 1732, 1593, 1571, 1483, 1459, 1435, 1297, 1239, 1195. MS (CI,
CHþ5 ); m/z (%): 381 (100) [M þ H]þ. Anal. Calcd. for C23H28N2O3; C,
72.61; H, 7.42; N, 7.36; Found: C, 72.27; H, 7.34; N, 7.33.
5.1.26. Methyl 3-({2-[2-(10H-phenothiazin-10-y)ethoxy]ethyl}amino)
propanoate (9e)
According to GP2 starting 19e (331 mg, 0.550 mmol), mercap-
toacetic acid (50 ml, 0.71 mmol) and NEt3 (155 ml, 1.12 mmol) in
CH2Cl2 (5.5 ml); reaction time: 4.5 h; work-up: sat. NaHCO3 solu-
tion (50 ml) and Et2O. Purification was accomplished by CC (EtOAc/
n-pentane 70:30 þ 2% EtMe2N). Yield: 182 mg (87%), yellow oil. 1H
m.p.: 174 ꢁC. 1H NMR (400 MHz, CD3OD):
d
¼ 2.48 (t, J ¼ 6.6 Hz, 2H,
CH2COO), 2.52 (q, J ¼ 7.4 Hz, 2H, ]CHCH2CH2), 3.11 (t, J ¼ 7.4 Hz,1H,
]CHCH2CH2), 3.30e3.32 (m, 2H, NCH2CH2COO), 6.09 (t, J ¼ 7.4 Hz,
ꢃ1
~
1H, C]CH), 7.17e7.45 (m, 10H, Harom). IR (KBr): v ¼ 3432 cm
,
3023, 2791, 1637, 1574. MS (CI, CHþ5 ); m/z (%): 296 (100) [M þ H]þ.
Anal. Calcd. for C19H22ClN2O; C, 68.77; H, 6.68; N, 4.22; Found: C,
68.79; H, 6.67; N, 4.13.
NMR (400 MHz, CDCl3):
d
¼ 2.51 (t, J ¼ 6.8 Hz, 2H, COCH2), 2.78 (t,
J ¼ 5.2 Hz, 2H, NHCH2CH2OCH2), 2.88 (t, J ¼ 6.8 Hz, 2H, COCH2CH2),
3.54e3.59 (m, 2H, NHCH2CH2OCH2), 3.68 (s, 3H, CH3O), 3.81 (t,
J ¼ 6.2 Hz, 2H, (CAr)2NCH2CH2O), 4.10 (t, J ¼ 6.2 Hz, 2H,
(CAr)2NCH2CH2O), 6.87e6.96 (m, 4H, 2ꢂ NCCCHCH, 2ꢂ NCCHCH),
7.10e7.18 (m, 4H, 2ꢂ NCCHCH, 2ꢂ NCCCHCH). IR (film):
5.1.30. (rac)-3-[(4,4-Diphenylbut-3-enyl)amino]butanoic acid [(rac)-
6b]
According to GP3 starting from (rac)-21b (218 mg,
0.675 mmol), MeOH (1.7 ml), NaOH (2 M, 1.01 ml, 2.02 mmol);
reaction time 15 h; purification was accomplished as follows: the
residue was dissolved in H2O and Phosphate buffer pH 6 (0.45 M,
6 ml) was added followed by extraction of the aqueous layer with
CH2Cl2. The combined organic layers were washed with H2O and
concentrated in vacuo. Yield: 167 mg (80%); colorless solid, m.p.:
v ¼ 3059 cmꢃ1, 2942, 2866, 1734, 1593, 1571, 1462, 1362, 1254, 1129.
~
MS (CI, CHþ5 ); m/z (%): 373 (100) [M þ H]þ. Anal. Calcd. for
C
20H24N2O3S; C, 64.49; H, 6.50; N, 7.52; S, 8.61; Found: C, 64.21; H,
6.49; N, 7.51; S 8.52.
5.1.27. Methyl (rac)-3-({2-[2-(10H-phenothiazin-10-yl)ethoxy]
ethyl}amino)butanoate [(rac)-21e]
According to GP2 starting (rac)-20e (548 mg, 0.890 mmol),
mercaptoacetic acid (82 ml, 1.16 mmol) and NEt3 (250 ml, 1.80 mmol)
197 ꢁC. 1H NMR (500 MHz, CD3OD):
d
¼ 1.27 (d, J ¼ 6.6 Hz, 3H,
NHCHCH3), 2.31 (dd, J ¼ 16.8/8.2 Hz, 1H, COCH2), 2.48 (dd,
J ¼ 16.8/4.5 Hz, 1H, COCH2), 2.48e2.57 (m, 2H, NHCH2CH2CH),
3.06 (dt, J ¼ 12.3/7.6 Hz, 1H, NHCH2CH2CH), 3.15 (ddd, J ¼ 12.3/7.9/
6.6 Hz, 1H, NHCH2CH2CH), 3.33e3.41 (m, 1H, NHCHCH3), 6.10 (t,
J ¼ 7.3 Hz, 1H, NHCH2CH2CH), 7.17e7.29 (m, 7H, Harom), 7.32e7.37
in CH2Cl2 (8.9 ml); reaction time 5.5 h; work-up: sat. NaHCO3 so-
lution (100 ml) and Et2O. Purification by CC (Et2O/n-pentane
80:20 þ 2% EtMe2N). Yield: 317 mg (93%), yellow oil. 1H NMR
ꢃ1
~
(m, 1H, Harom), 7.39e7.44 (m, 2H, Harom). IR (KBr): v ¼ 3448 cm
,
(500 MHz, CDCl3):
d
¼ 1.11 (d, J ¼ 6.4 Hz, 3H, NHCHCH3), 2.31 (dd,
3055, 2638, 2381, 1594, 1495, 1424, 1408, 1384, 1323, 1268, 1073.
J ¼ 15.2/6.6 Hz, 1H, COCH2CH), 2.48 (dd, J ¼ 15.2/6.3 Hz, 1H,
COCH2CH), 2.74 (dt, J ¼ 12.3/5.3 Hz, 1H, NHCH2CH2OCH2), 2.79 (dt,
J ¼ 12.3/5.2 Hz, 1H, NHCH2CH2OCH2), 3.10 (sext., J ¼ 6.4 Hz, 1H,
NHCHCH3), 3.58 (t, J ¼ 5.2 Hz, 2H, NHCH2CH2OCH2), 3.67 (s, 3H,
CH3O), 3.77e3.85 (m, 2H, (CAr)2NCH2CH2O), 4.10 (t, J ¼ 6.3 Hz, 2H,
(CAr)2NCH2CH2O), 6.88e6.94 (m, 4H, 2ꢂ NCCCHCH, 2ꢂ NCCHCH),
7.10e7.17 (m, 4H, 2ꢂ NCCCHCH, 2ꢂ NCCHCH). IR (film):
MS (CI, CHþ5 ); m/z (%): 310 (100) [M þ H]þ. Anal. Calcd. for
C
20H23NO2; C, 77.64; H, 7.49; N, 4.53; Found: C, 77.13; H, 7.58;
N, 4.49.
5.1.31. 3-({2-[2-(10,11-Dihydro-5H-dibenzo[b,f]azepin-5-yl)ethoxy]
ethyl}amino)propanoic acid (5c)
According to GP3 starting from 9c (141 mg, 0.383 mmol), MeOH
(0.95 ml), NaOH (2 M, 580 ml, 1.16 mmol); reaction time 50 h; pu-
rification was accomplished by basic anion exchange chromatog-
raphy (Amberlite IRA-410 elution with 0.15 M HCl) followed by an
acidic cation exchange chromatography (DOWEX 50 WX8, elution
with sat. aqueous NH3 solution). The resulting residue was dis-
solved in CH2Cl2 and subsequently filtered. The filtrate was finally
concentrated in vacuo. Yield: 58 mg (42%); light brown solid, m.p.:
v ¼ 3331 cmꢃ1, 3059, 2955, 2867, 1733, 1594, 1570, 1463, 1362, 1286,
~
1253, 1196, 1129. MS (CI, CH5þ); m/z (%): 387 (100) [M þ H]þ. Anal.
Calcd. for C21H26N2O3S; C, 65.26; H, 6.78; N, 7.25; S, 8.30; Found: C,
65.15; H, 6.85; N, 7.25; S, 8.66.
5.1.28. Methyl (rac)-3-({2-[tris(4-methoxyphenyl)methoxy]ethyl}
amino)butanoate [(rac)-21f]
According to GP2 starting from (rac)-20f (153 mg, 0.212 mmol)
in CH2Cl2 (1.1 ml). Differing to the general procedure, the addition
160e162 ꢁC. 1H NMR (400 MHz, CD3OD):
d
¼ 2.45 (t, J ¼ 6.3 Hz, 2H,
COCH2), 3.08e3.16 (m, 8H, CH2NHCH2, CCH2CH2C), 3.57e3.61 (m,
2H, NHCH2CH2OCH2), 3.63 (t, J ¼ 5.9 Hz, 2H, (CAr)2NCH2CH2O), 4.02
(t, J ¼ 5.9 Hz, 2H, (CAr)2NCH2CH2O), 6.90 (ddd, J ¼ 7.6/6.6/1.8 Hz, 2H,
2ꢂ NCCCHCH), 7.06e7.16 (m, 6H, 2ꢂ NCCCHCH, 2ꢂ NCCHCH). IR
of mercaptoacetic acid (20
ml, 0.28 mmol) and NEt3 (60 ml,
0.43 mmol) was carried out as a solution in CH2Cl2 (1.1 ml); reaction
time 5.5 h; work-up: sat. NaHCO3 solution (50 ml) and Et2O. Puri-
fication by CC (Et2O/n-pentane, 40:60 þ 2% EtMe2N). Yield: 65 mg
(KBr): v ¼ 3447 cmꢃ1, 3013, 2912, 1616, 1485, 1464, 1394, 1341, 1253,
~
(63%), colorless oil. 1H NMR (500 MHz, CD2Cl2):
d
¼ 1.07 (d,
1235, 1211, 1134. MS (CI, CHþ5 ); m/z (%): 355 (18) [M þ H]þ, 283
(100). Anal. Calcd. for C21H26N2O3 ꢂ 0.5H2O; C, 69.40; H, 7.49; N,
7.71; Found: C, 69.81; H, 7.37; N, 7.78.
J ¼ 6.4 Hz, 3H, NHCHCH3), 2.31 (dd, J ¼ 15.1/6.1 Hz, 1H, COCH2CH),
2.43 (dd, J ¼ 15.1/6.8 Hz, 1H, COCH2CH), 2.69e2.81 (m, 2H,

498
I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
5.1.32. (rac)-3-({2-[2-(10,11-Dihydro-5H-dibenzo[b,f]azepin-5-yl)
ethoxy]ethyl}amino)butanoic acid [(rac)-6c]
According to GP3 starting from (rac)-21c (133 mg, 0.348 mmol),
MeOH (0.87 ml), NaOH (2 M, 520 ml, 1.04 mmol); reaction time:
43 h; purification was accomplished by basic anion exchange
chromatography (Amberlite IRA-410 elution with 0.15 M HCl) fol-
lowed by an acidic cation exchange chromatography (DOWEX 50
WX8, elution with sat. aqueous NH3 solution). The resulting residue
was dissolved in CHCl3 and subsequently filtered. The filtrate was
finally concentrated in vacuo. Yield: 53 mg (47%); rufous, amor-
6.0/3.5 Hz, 1H, NHCH2CH2O), 3.34e3.44 (m, 1H, NHCHCH3), 3.61e
3.73 (m, 4H, CH2OCH2), 3.95e4.04 (m, 2H, (Carom)2NCH2CH2O), 6.73
(s, 2H, CCHCHC), 6.97e7.03 (m, 2H, 2ꢂ NCCCHCHCH), 7.08 (dd,
J ¼ 7.6/1.7 Hz, 2H, 2ꢂ NCCCHCHCH), 7.11 (dd, J ¼ 8.2/1.0 Hz, 2H, 2ꢂ
NCCHCH), 7.27 (ddd, J ¼ 8.2/7.2/1.7 Hz, 2H, 2ꢂ NCCHCH). IR (KBr):
v ¼ 3423 cmꢃ1, 3018, 2870, 2729, 2414, 1593, 1483, 1458, 1406, 1127.
~
MS (CI, CHþ5 ); m/z (%): 367 (100) [M þ H]þ. Anal. Cacd. for
C
22H26N2O2 ꢂ 0.5H2O; C, 70.38; H, 7.25; N, 7.46; Found: C, 70.64; H,
7.24; N, 7.43.
phous solid, m.p.: 53e57 ꢁC. 1H NMR (400 MHz, CD3OD):
d
¼ 1.27
5.1.35. 3-({2-[2-(10H-Phenothiazin-10-yl)ethoxy]ethyl}amino)pro
panoic acid (5e)
(d, J ¼ 6.6 Hz, 3H, NHCHCH3), 2.31 (dd, J ¼ 16.9/8.7 Hz, 1H,
COCH2CH), 2.47 (dd, J ¼ 16.9/4.3 Hz, 1H, COCH2CH), 3.04e3.21 (m,
2H, NHCH2CH2O), 3.13 (s, 4H, CCH2CH2C), 3.35e3.44 (m, 1H,
NHCHCH3), 3.55e3.68 (m, 4H, NHCH2CH2OCH2), 4.02 (t, J ¼ 6.0 Hz,
2H, (Carom)2NCH2CH2O), 6.90 (ddd, J ¼ 7.6/6.9/1.6 Hz, 2H, 2ꢂ
NCCCHCH), 7.05e7.17 (m, 6H, 2ꢂ NCCCHCH, 2ꢂ NCCHCH). IR (KBr):
According to GP3 starting from 9e (126 mg, 0.339 mmol), MeOH
(0.85 ml), NaOH (2 M, 510
ml, 1.02 mmol); reaction time: 18 h;
purification was accomplished by acidic cation exchange chroma-
tography (DOWEX 50 WX 8, elution with sat. aqueous NH3 solu-
tion). The resulting residue was dissolved in MeOH and the desired
product was precipitated by addition of Et2O. This procedure was
repeated two times. Yield: 56 mg (45%); colorless solid, m.p.: 164e
v ¼ 3422 cmꢃ1, 3055, 3013, 2919, 1595, 1487, 1458, 1419, 1337, 1323,
~
1264, 1237, 1131. MS (CI, CHþ5 ); m/z (%): 369 (100) [M þ H]þ, 222
(100). Anal. Calcd. for C22H28N2O3 ꢂ 0.75H2O; C, 69.18; H, 7.78; N,
7.33; Found: C, 68.92; H, 7.88; N, 7.08.
165 ꢁC. 1H NMR (500 MHz, CD3OD):
d
¼ 2.40 (t, J ¼ 6.4 Hz, 2H,
COCH2), 3.04 (t, J ¼ 6.4 Hz, 2H, COCH2CH2NH), 3.07 (t, J ¼ 5.1 Hz, 2H,
NHCH2CH2O), 3.69 (t, J ¼ 5.1 Hz, 2H, NHCH2CH2O), 3.86 (t,
J ¼ 5.6 Hz, 2H, (Carom)2NCH2CH2O), 4.17 (t, J ¼ 5.6 Hz, 2H, (Car-
om)2NCH2CH2O), 6.93 (td, J ¼ 7.5/1.1 Hz, 2H, 2ꢂ NCCCHCH), 7.01 (dd,
J ¼ 8.2/1.0 Hz, 2H, 2ꢂ NCCHCH), 7.12 (dd, J ¼ 7.6/1.5 Hz, 2H, 2ꢂ
NCCCHCH), 7.19 (ddd, J ¼ 8.2/7.4/1.5 Hz, 2H, 2ꢂ NCCHCH). IR (KBr):
5.1.33. 3-({2-[2-(5H-Dibenzo[b,f]azepin-5-yl)ethoxy]ethyl}amino)
propanoic acid (5d)
According to GP3 starting from 9d (105 mg, 0.287 mmol), MeOH
(0.72 ml), NaOH (2 M, 430 ml, 0.86 mmol); reaction time: 64 h;
v ¼ 3442 cmꢃ1, 3057, 2877, 1590, 1458, 1355, 1288, 1256, 1222, 1109,
~
purification was accomplished as follows: the residue was dis-
solved in H2O (2 ml) and the pH was adjusted to 2 with aqueous HCl
(1 M) at 0 ꢁC. CH2Cl2 (200 ml) was added and the entire aqueous
layer was bound by addition of MgSO4. After filtration and con-
centration in vacuo the remaining viscous oil was dissolved in H2O
and sat. aqueous NH3 solution was added until pH 12 was reached.
Concentration in vacuo, subsequent dissolving in H2O and extrac-
tion with CH2Cl2 which was washed with H2O resulted after con-
centration ion vacuo in a residue which was suspended in Et2O. The
remaining yellowish crystals were finally obtained by filtration.
Yield: 73 mg (72%); yellow crystals, m.p.: 162e163 ꢁC. 1H NMR
1075, 1037. MS (CI, CHþ5 ); m/z (%): 359 (100) [M þ H]þ. Anal. Calcd.
for C19H22N2O3S 0.25H2O; C, 62.87; H, 6.25; N, 7.72, S, 8.83; Found:
C, 62.88; H, 6.13; N, 7.72; S, 9.07.
5.1.36. 3-({2-[Tris(4-methoxyphenyl)methoxy]ethyl}amino)
propanoic acid 5f
To a stirred solution of 9f (333 mg, 0.694 mmol) in EtOH (1.4 ml)
was added drop wise a NaOH solution (12 M, 116
0
ml, 1.39 mmol) at
ꢁC. The ice bath was removed and the reaction mixture was
stirred for additional 4 h at rt. The reaction mixture was adjusted to
pH w 6 by addition of aqueous HCl (0.25 M) at 0 ꢁC and subse-
quently extracted with CH2Cl2. The combined organic phases were
dried (MgSO4) and concentrated in vacuo. The resulting residue
was recrystallized from Et2O/n-pentane (1:1). Yield 261 mg (81%);
(400 MHz, CD3OD):
d
¼ 2.44 (t, J ¼ 6.3 Hz, 2H, COCH2), 3.09e3.14
(m, 4H, CH2NHCH2), 3.63e3.69 (m, 4H, CH2OCH2), 3.99 [t, J ¼ 5.8 Hz,
2H, (Carom)2NCH2CH2O], 6.74 (s, 2H, CCHCHC), 6.97e7.03 (m, 2H, 2ꢂ
NCCCHCHCH), 7.06e7.13 (m, 4H, 2ꢂ NCCCHCHCH, 2ꢂ NCCHCH),
7.28 (ddd,
~
J
¼
8.1/7.2/1.7 Hz, 2H, 2ꢂ NCCHCH). IR (KBr):
colorless crystals, m.p.: 87 ꢁC. 1H NMR (400 MHz, CDCl3):
d
¼ 2.49
v ¼ 3448 cmꢃ1, 3019, 2958, 2902, 2875, 1589, 1483, 1458, 1406,
1356, 1341, 1232, 1213, 1116. MS (CI, CHþ5 ); m/z (%): 353 (91)
[M þ H]þ, 116 (100). Anal. Calcd. for C21H24N2O3 ꢂ 0.25H2O; C,
70.67; H, 6.92; N, 7.85; Found: C, 70.47; H, 6.77; N, 7.79.
(t, J ¼ 5.8 Hz, 2H, CH2COO), 2.89 (t, J ¼ 5.0 Hz, 2H, OCH2CH2N), 2.94
(t, J ¼ 5.8 Hz, 2H, NCH2CH2COO), 3.39 (t, J ¼ 5.0 Hz, 2H, OCH2CH2N),
3.75 (s, 9H, OCH3), 6.78e6.83 (m, 6H, Harom), 7.27e7.32 (m, 6H,
~
H
arom). IR (KBr): v ¼ 3428 cmꢃ1, 2945, 2835, 1607, 1582. MS (CI,
CHþ5 ); m/z (%): 335 (100) [M-C5H10NO2þH]þ. Anal. Calcd. for
5.1.34. (rac)-3-({2-[2-(5H-Dibenzo[b,f]azepin-5-yl)ethoxy]ethyl}
amino)butanoic acid [(rac)-6d]
C27H31NO6; C, 69.66; H, 6.71; N, 3.01; Found: C, 69.43; H, 7.00;
N, 2.95.
According to GP3 starting from (rac)-21d (267 mg, 0.703 mmol),
MeOH (1.75 ml), NaOH (2 M, 1.05 ml, 2.10 mmol); reaction time.
16 h; purification was accomplished as follows: the residue was
dissolved in H2O (10 ml) and the pH was adjusted to 1 with aqueous
HCl (1 M). CH2Cl2 (100 ml) was added and the entire aqueous layer
was bound by addition of MgSO4. After filtration and concentration
in vacuo the remaining viscous oil was dissolved in H2O and sat.
aqueous NH3 solution was added until pH 12 was reached. Con-
centration in vacuo, subsequent dissolving in H2O and extraction
with CH2Cl2 which was washed with H2O resulted, after concen-
tration in vacuo, in a residue which was suspended in Et2O. The
remaining yellowish crystals were finally obtained by filtration.
Yield: 195 mg (74%); pale yellow crystals, m.p.: 125e127 ꢁC. 1H NMR
5.1.37. (rac)-3-({2-[2-(10H-Phenothiazin-10-yl)ethoxy]ethyl}amino)
butanoic acid [(rac)-6e]
According to GP3 starting from (rac)-21e (263 mg, 0.681 mmol),
MeOH (1.7 ml), NaOH (2 M, 1.02 ml, 2.04 mmol); reaction time:
15 h; purification was accomplished as follows: the residue was
dissolved in H2O (10 ml) and the pH was adjusted to 1 with aqueous
HCl (2 M). CH2Cl2 (100 ml) was added and the entire aqueous layer
was bound by addition of MgSO4. After filtration and concentration
in vacuo the remaining viscous oil was dissolved in H2O and sat.
aqueous NH3 solution was added until pH 12 was reached. Con-
centration in vacuo, subsequent dissolving in H2O, extraction with
CH2Cl2 which was washed with H2O resulted, after concentration in
vacuo, in a residue which was dissolved in MeOH. The desired
product was precipitated by addition of Et2O. Yield: 176 mg (70%);
(400 MHz, CD3OD):
J ¼ 16.9/8.6 Hz, 1H, COCH2), 2.46 (dd, J ¼ 16.9/4.3 Hz, 1H, COCH2),
d
¼ 1.26 (d, J ¼ 6.6 Hz, 3H, NHCHCH3), 2.30 (dd,
3.06 (ddd, J ¼ 13.1/7.1/3.7 Hz, 1H, NHCH2CH2O), 3.16 (ddd, J ¼ 13.1/
colorless solid, m.p.: 142 ꢁC. 1H NMR (500 MHz, CD3OD):
d
¼ 1.24 (d,

I. Sitka et al. / European Journal of Medicinal Chemistry 65 (2013) 487e499
499
J ¼ 6.6 Hz, 3H, NHCHCH3), 2.28 (dd, J ¼ 16.9/8.7 Hz,1H, COCH2), 2.44
References
(dd, J ¼ 16.9/4.2 Hz, 1H, COCH2), 3.11 (ddd, J ¼ 13.1/7.2/3.6 Hz, 1H,
NHCH2CH2O), 3.20 (ddd, J ¼ 13.1/6.0/3.4 Hz, 1H, NHCH2CH2O),
3.35e343 (m, 1H, NHCHCH3), 3.71 (ddd, J ¼ 10.9/7.2/3.4 Hz, 1H,
NHCH2CH2O), 3.76 (ddd, J ¼ 10.9/6.0/3.6 Hz, 1H, NHCH2CH2O),
3.83e3.91 (m, 2H, (Carom)2NCH2CH2O), 4.14e4.24 (m, 2H, (Car-
om)2NCH2CH2O), 6.94 (td, J ¼ 7,5/1.1 Hz, 2H, 2ꢂ NCCCHCH), 7.02 (d,
J ¼ 8.2 Hz, 2H, 2ꢂ NCCHCH), 7.12 (dd, J ¼ 7.7/1.5 Hz, 2H, 2ꢂ
NCCCHCH), 7.19 (ddd, J ¼ 8.2/7.4/1.5 Hz, 2H, 2ꢂ NCCHCH). IR (KBr):
[1] R.O. Beleboni, R.O.G. Carolino, A.B. Pizzo, L. Castellan-Baldan, J. Coutinho-
Netto, W.F. dos Santos, N.C. Coimbra, Cell. Mol. Neurobiol. 24 (2004) 707e728.
[2] J. Guastella, N.N. Nelson, H. Nelson, L. Czyzyk, S. Keynan, M.C. Miedel,
N. Davidson, H.A. Lester, B.I. Kanner, Science 249 (1990) 1303e1306.
[3] Q.-R. Liu, B. Lopez-Corcuera, S. Mandiyan, H. Nelson, N. Nelson, J. Biol. Chem.
268 (1993) 2106e2112.
[4] H. Nelson, S. Mandiyan, N. Nelson, FEBS Lett. 269 (1990) 181e184.
[5] L.A. Borden, K.E. Smith, P.R. Hartig, T.A. Branchek, R.L. Weinshank, J. Biol.
Chem. 267 (1992) 21098e21104.
[6] L.A. Borden, T.G.M. Dhar, K.E. Smith, T.A. Branchek, R.L. Weinshank,
C. Cluchowski, Recept. Channels 2 (1994) 207e213.
[7] A. Yamauchi, S. Uchida, H.M. Kwon, A.S. Preston, R.B. Robey, A. Garcia-Perez,
M.B. Burg, J.S. Handler, J. Biol. Chem. 267 (1992) 649e652.
[8] L.A. Borden, K.E. Smith, E.L. Gustafson, T.A. Branchek, R.L. Weinshank,
J. Neurochem. 64 (1995) 977e984.
v ¼ 3423 cmꢃ1, 3059, 2883, 2685, 2399,1595, 1571,1464,1407,1355,
~
1311, 1262, 1112. MS (CI, CH5þ); m/z (%): 373 (100) [M þ H]þ, Anal.
Calcd. for C20H24N2O3S; C, 64.49; H, 6.49; N, 7.52; Found: C, 64.27;
H, 6.47; N, 7.50.
[9] B. Lopez-Corcuera, Q.-R. Liu, S. Mandiyan, H. Nelson, N. Nelson, J. Biol. Chem.
267 (1992) 17491e17493.
5.1.38. (rac)-3-({2-[Tris(4-methoxyphenyl)methoxy]ethyl}amino)
butanoic acid [(rac)-6f]
According to GP3 starting from (rac)-21f (51 mg, 0.10 mmol),
MeOH (0.26 ml), NaOH (2 M, 160 ml, 0.32 mmol); reaction time 9 h;
purification was accomplished as follows: the residue was dissolved
in H2O (7 ml) followed by addition of aqueous phosphate buffer pH 6
(0.45 M, 20 ml) at 0 ꢁC. After extraction with CH2Cl2, the combined
organic layers were washed with H2O and concentrated in vacuo.
The residue was recrystallized from acetone/H2O. Yield: 44 mg
(87%); light brown crystals, m.p.: 142 ꢁC.1H NMR (500 MHz, CD2Cl2):
[10] N.O. Dalby, Eur. J. Pharmacol. 479 (2003) 127e137.
[11] A.C. Lehre, N.M. Rowley, Y. Zhou, S. Holmseth, C. Guo, T. Holen, R. Hua,
P. Laake, A.M. Olofsson, I. Poblete-Naredo, D.A. Rusakov, K.K. Madsen,
R.P. Clausen, A. Schousboe, H.S. White, N.C. Danbolt, Epilepsy Res. 98 (2011)
70e81.
[12] Y. Zhou, S. Holmseth, C. Guo, B. Hassel, G. Höfner, H.S. Huitfeldt, K.T. Wanner,
N.C. Danbolt, J. Biol. Chem. 287 (2012) 35733e35746.
[13] L.A. Borden, Neurochem. Int. 29 (1996) 335e356.
[14] P. Krogsgaard-Larsen, E. Falch, O.M. Larsson, A. Schousboe, Epilepsy Res. 1
(1987) 77e93.
[15] M.J. Croucher, B.S. Meldrum, P. Krogsgaard-Larsen, Eur. J. Pharmacol. 89
(1983) 217e228.
[16] L.M. Yunger, P.J. Fowler, P. Zarevics, P.E. Setler, J. Pharmacol. Exp. Ther. 228
(1984) 109e115.
[17] F.E. Ali, W.E. Bondinell, P.A. Dandridge, J.S. Frazee, E. Garvey, G.R. Girard,
C. Kaiser, T.W. Ku, J.J. Lafferty, G.I. Moonsammy, H.-Ja. Oh, J.A. Rush, P.E. Setler,
O.D. Stringer, J.W. Venslavsky, B.W. Volpe, L.M. Yunger, C.L. Zirkle, J. Med.
Chem. 28 (1985) 653e660.
[18] C. Braestrup, E.B. Nielsen, U. Sonnewald, L.J.S. Knutsen, K.E. Andersen,
J.A. Jansen, K. Frederiksen, P.H. Andersen, A. Mortensen, P.D. Suzdak,
J. Neurochem. 54 (1990) 639e647.
[19] E.B. Nielsen, P.D. Suzdak, K.E. Andersen, L.J.S. Knutsen, U. Sonnewald,
C. Braestrup, Eur. J. Pharmacol. 196 (1991) 257e266.
[20] T.G.M. Dhar, L.A. Borden, S. Tyagarajan, K.E. Smith, T.A. Brancheck,
R.L. Weinshank, C. Gluchowski, J. Med. Chem. 37 (1994) 2334e2342.
[21] G.H. Fülep, C.E. Hoesl, G. Höfner, K.T. Wanner, Eur. J. Med. Chem. 41 (2006)
809e824.
d
¼ 1.20 (d, J ¼ 6.5 Hz, 3H, NHCHCH3), 2.17 (dd, J ¼ 16.8/7.9 Hz, 1H,
COCH2CH), 2.46 (dd, J ¼ 16.8/3.9 Hz, 1H, COCH2CH), 2.72e2.78 (m,
1H, NHCH2CH2O), 2.85e2.91 (m, 1H, NHCH2CH2O), 2.97e3.05 (m,
1H, NHCHCH3), 3.25e3.34 (m, 2H, NHCH2CH2O), 3.78 (s, 9H, 3ꢂ
CH3O), 6.81e6.86 (m, 6H, 6ꢂ CH3OCCHCH), 7.28e7.32 (m, 6H, 6ꢂ
CH3OCCHCH). IR (KBr): v ¼ 3422 cmꢃ1, 2934, 2836, 1607, 1582, 1508,
~
1459, 1440, 1404, 1302, 1250, 1175. MS (ESIþ) m/z (%): 502
[M þ Na]þ, 480 [M þ H]þ. Anal. Calcd. for C28H33NO6 ꢂ 0.25H2O; C,
69.47; H, 6.98; N, 2.89; Found: C, 69.37; H, 6.81; N, 2.82.
5.2. Inhibition of GABA uptake
[22] F. Schon, J.S. Kelly, Brain Res. 86 (1975) 243e257.
GABA uptake assays based on porcine brain material employing
pfcP2B (GAT1) and pbsP2C membrane fraction (GAT3) were per-
formed as previously described [29]. GABA uptake assays employ-
ing HEK293 cells stably expressing mGAT1-4 (i.e. GAT1, BGT1, GAT2
and GAT3, respectively, according to HUGO) were performed as
described [25].
[23] S. Høg, J.R. Greenwood, K.B. Madsen, O.M. Larsson, B. Frølund, A. Schousboe,
P. Krogsgaard-Larsen, R.P. Clausen, Curr. Top. Med. Chem. 6 (2006) 1861e
1882.
[24] K.E. Andersen, J.L. Sørensen, J. Lau, B.F. Lundt, H. Petersen, P.O. Huusfeldt,
P.D. Suzdak, J. Med. Chem. 36 (1993) 1716e1725.
[25] J. Pabel, M. Faust, C. Prehn, B. Wörlein, L. Allmendinger, G. Höfner,
K.T. Wanner, ChemMedChem 7 (2012) 1245e1255.
[26] T. Fukuyama, M. Cheung, C.-K. Jow, Y. Hidai, T. Kan, Tetrahedron Lett. 38
(1997) 5831e5834.
[27] In the meantime DEAD is long time available again e.g. at SigmaeAldrich in
techn grade [85%] or as 40% [wt.] solution in toluene.
Appendix A. Supplementary data
[28] A. Kragler, G. Höfner, K.T. Wanner, Eur. J. Pharmacol. 519 (2005) 43e47.
[29] M.R. Faust, G. Höfner, J. Pabel, K.T. Wanner, Eur. J. Med. Chem. 45 (2010)
2453e2466.
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.04.063.