Novel Allosteric Ligands of γ-Aminobutyric Acid Transporter 1 (GAT1) by MS Based Screening of Pseudostatic Hydrazone Libraries

Article abstract of DOI:10.1021/acs.jmedchem.8b01602

This study describes the screening of dynamic combinatorial libraries based on nipecotic acid as core structure with substituents attached to the 5- instead of the common 1-position for the search of novel inhibitors of the GABA transporter GAT1. The generated pseudostatic hydrazone libraries included a total of nearly 900 compounds and were screened for their binding affinities toward GAT1 in competitive mass spectrometry (MS) based Binding Assays. Characterization of the hydrazones with the highest affinities (with cis-configured rac-16gf bearing a 5-(1-naphthyl)furan-2-yl residue and a four atom spacer being the most potent) in binding and uptake experiments revealed an allosteric interaction at GAT1, which was not reported for any other nipecotic acid derivative up to now. Therefore, the herein introduced 5-substituted nipecotic acid derivatives could serve as valuable tools for investigations of allosterically modulated GABA transport mediated by GAT1 and furthermore as starting point for a new class of GAT1 inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b01602

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Article
Novel allosteric ligands of #-aminobutyric acid transporter 1
(GAT1) by MS based screening of pseudostatic hydrazone libraries
Tobias J. Hauke, Thomas Wein, Georg Höfner, and Klaus T. Wanner
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01602 • Publication Date (Web): 30 Oct 2018
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Novel allosteric ligands of γ-aminobutyric acid
transporter 1 (GAT1) by MS based screening of
pseudostatic hydrazone libraries
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Tobias J. Hauke, Thomas Wein, Georg Höfner, Klaus T. Wanner*
Department of Pharmacy – Center of Drug Research, Ludwig-Maximilians-Universität
München, Butenandtstr. 513, 81377 Munich, Germany
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ABSTRACT
This study describes the screening of dynamic combinatorial libraries based on nipecotic
acid as core structure with substituents attached to the 5- instead of the common 1-
position for the search of novel inhibitors of the GABA transporter GAT1. The generated
pseudostatic hydrazone libraries included a total of nearly 900 compounds and were
screened for their binding affinities towards GAT1 in competitive mass spectrometry (MS)
based binding assays. Characterization of the hydrazones with the highest affinities (with
cis-configured rac-16gf bearing a 5-(1-naphthyl)furan-2-yl residue and a four atom spacer
being the most potent) in binding and uptake experiments revealed an allosteric
interaction at GAT1, which was not reported for any other nipecotic acid derivative up to
now. Therefore, the herein introduced 5-substituted nipecotic acid derivatives could serve
as valuable tools for investigations of allosterically modulated GABA transport mediated
by GAT1, and furthermore as starting point for a new class of GAT1 inhibitors.
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INTRODUCTION
γ-Aminobutyric acid (GABA; 1; Chart 1) is the most important inhibitory neurotransmitter
in the mammalian central nervous system (CNS) and pathological abnormalities of the
GABAergic neurotransmission are associated with a number of neuronal diseases such
as epilepsy,13 Parkinson’s disease,3,4 depression4,5 and neuropathic pain.6,7 For the
treatment of such diseases, GABAergic neurotransmission can be enhanced by agonists
of GABA receptors, by targeting metabolic enzymes or by inhibiting GABA transport
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proteins (GATs). With exception of one vesicular GABA transporter, GATs are
membrane bound proteins encoded by the solute carrier 6 gene family (SLC6) that
remove GABA from the synaptic cleft by utilizing a co-transport of sodium and chloride
9
through cell membranes. Amongst the four different subtypes of membrane bound
transporters designated as GAT1, BGT1, GAT2 and GAT3 (as suggested by HUGO and
corresponding to mGAT1, mGAT2, mGAT3 and mGAT4 when expressed in mice),10,11
GAT1 is mainly responsible for the neuronal reuptake of GABA in the CNS and emerged
as a drug target, while the pharmacological role and therapeutic potential of other GAT
subtypes is still less well understood.1013 Many of the known GAT inhibitors are
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derivatives of small cyclic amino acids such as nipecotic acid (2) and guvacine (3), which
already show in vitro activity as GABA uptake inhibitors by their own.14,15 By introducing
a lipophilic side chain to the cyclic amino acids a new generation of inhibitors, represented
by SK&F-89976A (4), tiagabine (5) or NO711 (6), was established that has an increased
potency and selectivity towards GAT1 compared to unsubstituted amino acids.1619
Furthermore, the increased lipophilicity of those molecules (46) enabled them to cross
the blood-brain barrier in contrast to the more hydrophilic, unsubstituted amino acids
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(23). Tiagabine (5) is well characterized with respect to its anticonvulsant activity and
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it is the only selective GAT1 inhibitor in clinical use. Recently, DDPM-2571 (7), a new
GAT1 selective compound, was found to exceed the inhibitory potency of tiagabine (5) at
GAT1 by more than one log unit and it was demonstrated to mediate anticonvulsant,
anxiolytic, antidepressant and antinociceptive effects in mouse models.22 All these
selective and potent GAT1 inhibitors possess a hydrophilic amino acid “head” and a
lipophilic aromatic moiety that is connected to the amino acid via a spacer originating from
the amino nitrogen of the amino acid. There have been extensive efforts2329 to develop
analogous GAT1 inhibitors with a different substitution pattern of the cyclic amino acid.
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For example, 4-substituted nipecotic acid derivatives including compound rac-8 and 6-
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substituted guvacine derivatives including compound 9 were synthesized and tested for
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their inhibitory potencies of the GABA transport. However, amongst these only compound
9
showed in vitro activity comparable to N-substituted derivatives, but was inactive in
anticonvulsant models in vivo, likely due to an insufficient blood/brain concentration
ratio.24
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Chart 1. Structures of GABA (1), GAT1 inhibitors (29) and hypothetical molecule 10
O
O
O
H2N
OH
OH
OH
N
N
H
H

-aminobutyric acid
nipecotic acid (2)
guvacine (3)
(
GABA) (1)
O
O
O
OH
OH
OH
N
N
N
S
O
N
S
SK&F-89976A (4)
tiagabine (5)
NO711 (6)
O
OH
O
N
OH
N
H
O
N
O
OH
N
CH3
Cl
Cl
DDPM-2571 (7)
rac-8
9
O
OH
N
H
10
In 2005 the first crystal structure of a bacterial leucine transporter (LeuT) was reported,30
which represents a homolog of the SLC6 GABA transporters. Since then, two
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investigations used this structure as base for homology modeling and analysis of the
binding of small inhibitors towards GAT1.31,32 Later, the binding of tiagabine (5) and
related compounds was evaluated using homology modeling, docking and molecular
dynamics simulations33 and different binding modes of small and large inhibitors were
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proposed. We have used our in-house hGAT1 homology model refined by molecular
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dynamics calculations and described in detail in Wein et al. to investigate the possibility
of attaching the lipophilic arylalkyl residue to the 4- or 5-position of nipecotic acid (2). For
an in silico screening we built a virtual library of 4- and 5-substituted nipecotic acid
derivatives, of which the lipophilic residues were chosen to be biphenyl or diphenyl
residues. For the linker, carbon chains with 3, 4, 5, or 6 atoms and bearing a double bond
in conjugation with the aromatic moiety were examined (example see hypothetical
molecule 10). The substitution of nipecotic acid (2) particularly in the 5-position and with
a five carbon spacer (as in molecule 10) was found to achieve the highest docking scores
amongst the hypothetical compounds. Docking calculations showed for nipecotic acid
derivative 10 and related 5-substituted derivatives that the nitrogen atom was able to point
towards the intracellular side of the binding cavity and could potentially interact with two
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different hydrogen bond acceptors in this position. Thereby, the lipophilic residue would
point to the extracellular side of the pocket (Figure 1c). So the binding pose of the
piperidine ring of 5-substituted derivatives would be more similar compared to that of the
unsubstituted nipecotic acid (2; Figure 1a) and opposing to that of tiagabine (5), of which
the piperidine nitrogen and the attached arylalkyl moiety are facing towards the
extracellular side (Figure 1b).34
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Figure 1. Side view of the hGAT1 model along the membrane plane showing the active
site of the transporter. The extracellular side is on the top and the intracellular side is at
the bottom of the picture. Docking poses of a) nipecotic acid (2; green), b) tiagabine (5;
cyan) and c) hypothetical molecule 10 (magenta) in the molecular dynamics refined
homology model of hGAT1 are shown. The transmembrane helices TM10, TM11 and
TM12 are not displayed for clarity.
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Based on these results obtained from in silico studies, we concluded that nipecotic
derivatives bearing a lipophilic moiety attached to the 5-position via a spacer might
possibly represent a new class of potent GABA uptake inhibitors. For a vast and most of
all easy to perform variation of the structure of the lipophilic residues attached to the 5-
position of the nipecotic acid moiety, we decided to analyze compound libraries generated
by dynamic combinatorial chemistry (DCC). Hence, we followed an approach that is
based on pseudostatic hydrazone libraries and uses a competitive mass spectrometry
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(
MS) based binding assay for their analysis as reported from our group, recently. MS
Binding Assays have the advantage to enable the label-free determination of binding
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affinities and can be employed analogous to radioligand binding assays but are devoid
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of the drawbacks that result from using radioactive material. The MS Binding Assay for
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the target murine GAT1 (mGAT1) that is required for this study and that uses NO711 (6)
as a native marker had already been established by us and employed in related screening
campaigns.38
For the present study we intended to synthesize nipecotic acid derivatives substituted at
the 5-position with a C1 (rac-11 and rac-12) and a C2 spacer (rac-13 and rac-14) and
with a hydrazine function at the end of the spacer. By reaction with appropriate aldehydes
1
5, these nipecotic acid derived hydrazines should allow to generate libraries with a
hydrazone function containing a total spacer length of four atoms (rac-16 and rac-17;
resulting from the conversion of hydrazines rac-11 and rac-12) and five atoms (rac-18
and rac-19; resulting from hydrazines rac-13 and rac-14) (Scheme 1), which should be
screened for their affinities towards mGAT1 and evaluated as potential GABA uptake
inhibitors. Hence, compounds with a five atom spacer, as suggested by molecular
modeling, as well as with a four atom spacer, which appeared beneficial in some
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previously reported cases when potentially new N-substituted GAT1 inhibitors were
synthesized,39,40 should be examined in this study.
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Scheme 1. Condensation of nipecotic acid derived hydrazines rac-11rac-14 with diverse
aldehydes 15 to afford hydrazones with general structures rac-16rac-19
O
O
n
n
H2N
R
N
N
OH
O
N
OH
H
H
2
H O
N
R
H
N
H
H
n = 1
n = 2
rac-11
rac-12
15
n = 1
n = 2
rac-16
rac-17
rac-13
rac-14
rac-18
rac-19
In addition to the generation and screening of the hydrazone libraries in competitive
binding assays the whole screening process would further comprise deconvolution of the
most potent libraries (testing only single hydrazones) and hit verification by resynthesis
3
and determination of pK values. Finally, the best binders should be tested in [ H]GABA
i
uptake assays for their functional activity (IC ) and subtype selectivity.
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RESULTS AND DISCUSSION
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Chemistry.
Synthesis of hydrazines. For the synthesis of the desired hydrazines (rac-11rac-14) a
carbon side chain had to be introduced in the 5-position of nipecotic acid (or derivatives
thereof) exhibiting a terminal function suitable for further derivatization, i.e. the
introduction of a hydrazine moiety. As common precursors required for all hydrazine
derivatives, the aldehydes rac-20 and rac-21 were chosen. These aldehydes were
synthesized from commercially available nicotinate 22 as shown in Scheme 2.
Compounds rac-23 and rac-24 were obtained by a hydrogenation of nicotinate 22 and a
subsequent protection of the amino function with a tert-butyloxycarbonyl (Boc) group. To
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this end, conditions as described in a patent were applied initially. However, under these
conditions the hydrogenation reaction did not lead to a conversion of the starting material
(22). Accordingly, the procedure was modified by adding sulfuric acid to the reaction
mixture in the hydrogenation step. With this modified protocol the two diastereomeres
rac-23 and rac-24 (in 3:1 ratio) were obtained in yields of 55% (rac-23) and 19% (rac-24)
after separation by flash chromatography. The major diastereomere rac-23 was oxidized
42
with Dess-Martin periodinane analogous to a patent yielding the ketone 25 (74%; the
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oxidation of the two diastereomeres rac-23 and rac-24 as a mixture afforded 25 in
approximately the same yield).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Synthesis of nipecotic acid derived hydrazines with C1 spacer (rac-11 and
rac-12) and C2 spacer (rac-13 and rac-14)a
O
O
O
OCH3
O
O
O
HO
HO
O
OCH3
OCH3
OCH3
OCH3
H
OCH3
a, b, c
d
e
f
N
N
N
N
N
Boc
for rac-23
Boc
Boc
Boc
22
rac-23 (55%)
rac-24 (19%)
25 (74%)
26 (57%)
rac-20
rac-21
(in 1:1 mixture; 78%)
H2N
N
Boc
O
OH
j
N
k
for rac-30
Boc
O
rac-32 (quant.)
Boc
NH
H2N
Boc
N
H2N
OH
O
N
O
NH
O
29
OCH3
OCH3
OH
O
h, i, c
Route for C1 spacer
l
N
N
N
H
Boc
Boc
for rac-31
g
rac-27
rac-28
rac-30 (38%)
rac-31 (43%)
rac-11 x HCl (85%)
rac-12 x HCl (87%)
(in 1:1 mixture; 76%)
rac-20
rac-21
(in 1:1 mixture)
m, c
Boc
HN
NH2
H3CO
H
O
NH
O
NH
O
O
O
OCH3
OCH3
OCH3
OH
Route for C2 spacer
n
o
l
N
N
N
N
H
Boc
Boc
Boc
rac-33 (48%)
rac-34 (33%)
rac-35 (80%)
rac-36 (88%)
rac-37 (82%)
rac-38 (77%)
rac-13 x HCl (85%)
rac-14 x HCl (83%)
a
Reagents and conditions: (a) H (10 bar), Rh/Al O , H SO , MeOH, 80 °C, 16 h; (b)
2
2
3
2
4
Boc O, NEt , dioxane, rt, 3 h; (c) separation of diastereomeres by flash chromatography;
2
3
(
d) Dess-Martin periodinane, DCM, rt, 2.5 h; (e) Ph PCH OCH Cl, t-BuOK, THF, -78
3
2
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
°
Crt, 2 h; (f) aq. HCl (2 M), THF, 0 °Crt, 2 d; (g) NaBH , EtOH, 0 °C, 1 h; (h) PPh ,
4
3
47
DIAD, 29, THF, 0 °C, 105 min; (i) CH NHNH , THF, 0 °C, 2 h; (j) aq. NaOH (1 M), MeOH,
3
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
°Crt, 15 h; (k) HCl in Et O (2 M), rt, 3 d; (l) aq. HCl (1 M), H O, 60 °C (sealed high-
2
2
pressure tube), 3 h; (m) Ph PCH OCH Cl, t-BuOK, THF, 0 °Crt, 1.5 h; (n) aq. HCl (2
3
2
3
M), THF, 0 °Crt, 79 h; (o) Boc-NHNH , NaH BCN, AcOH, MeOH, 0 °Crt, 2.53.5 h.
2
3
To introduce the side chain in the 5-position of 25 we performed a Wittig reaction
(analogously as it was described in literature for different compounds)43 with the ylide
generated from methoxymethyl triphenylphosphonium chloride by means of potassium
tert-butoxide which yielded the enol ether 26 (57%; 1:1 mixture of E- and Z-isomer;
isomers not separated). The hydrolysis of the enol ether group was accomplished by a
43
modified protocol for an analogous reaction from literature using 2 M HCl (instead of 4
and 6 M) and a higher proportion of the solvent THF (6:1 instead of 1:1 of THF/acid). That
way the undesired additional hydrolysis of the ester function could be reduced and
44
diastereomeric aldehydes rac-20 and rac-21 could be obtained in a yield of 78% as a
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1
:1 mixture, the separation of which appeared to be laborious due to their nearly identical
chromatographic retention.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For the synthesis of the nipecotic acid derived hydrazine derivatives with a C1 spacer,
rac-11 and rac-12, at first a direct reductive hydrazine formation was attempted by
converting the mixture of aldehydes rac-20 and rac-21 with tert-butyl carbazate applying
different reducing agents (e.g. sodium cyanoborohydride or sodium borohydride). This,
however, did not lead to the desired Boc-protected hydrazine derivatives. As an
alternative, the introduction of the required hydrazine function should be accomplished by
a Mitsunobu reaction following a protocol of Brosse et al.45-47 Alcohols rac-27 and rac-
44
48
28 required for this purpose were prepared by reduction of aldehydes rac-20 and rac-
21 (in 1:1 mixture) with sodium borohydride (rac-27 and rac-28; 1:1 mixture; 76%). The
thus obtained 1:1 mixture of rac-27 and rac-28 (yield 76%) was treated with hydrazine
derivative 29,47 triphenylphosphine and diisopropyl azodicarboxylate (DIAD) in a
Mitsunobu reaction. The formed product was subsequently freed from the phthalimide
protecting group with methylhydrazine yielding the hydrazine precursors rac-30 and rac-
31 in good yields (38% and 43%, respectively) after separation with flash
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
9
chromatography. Target compound rac-12 as hydrochloride was finally obtained in good
yield (87%) by simultaneous hydrolysis of the ester function and cleavage of the Boc
group of rac-31 by heating to 60 °C in aqueous HCl and in a sealed high-pressure tube.
The same procedure applied to rac-30, however, did not lead to the pure product rac-11.
Instead a mixture of rac-11 with a side product was obtained, which presumably resulted
from an intramolecular cyclization reaction of the hydrazine moiety with the carboxylic
acid ester function. Hence, the procedure was modified. To avoid the undesired
cyclization reaction, the deprotection of the functional groups was performed in two steps.
First, the ester was hydrolyzed with NaOH to give the free acid rac-32 and then the Boc
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
49
groups were cleaved in etheric HCl, giving the desired rac-11 as hydrochloride in good
yield (85% over two steps).
For the preparation of nipecotic acid derivatives with the hydrazine function attached to a
C2 spacer, we performed the Wittig reaction with aldehydes rac-20 and rac-21 analogous
as for the synthesis of the enol ether 26. The two diastereomeric enol ethers, rac-33 and
rac-34, could be isolated by flash chromatography in pure form (rac-33; 48% and rac-34;
33%). The enol ether hydrolysis of the individual diastereomeres proceeded more
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smoothly than with the analogs with shorter side chains, giving aldehydes rac-35 and rac-
36 in yields of 80% and 88%. When reacted with tert-butyl carbazate in the presence of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
sodium cyanoborohydride and acetic acid following an analogous literature method the
Boc-protected hydrazines rac-37 and rac-38 were obtained in good yields (85% and 83%,
respectively). The protective groups in rac-37 and rac-38 (Boc and ester function) could
finally be removed in one step by heating the compounds in hydrochloric acid to 60 °C in
a sealed high-pressure tube. The hydrochlorides of the desired nipecotic acid derived
49
hydrazines with C2 spacer, rac-13 and rac-14, were thus obtained in yields of 85% and
83%, respectively.
Aldehydes. Aldehydes 15a15hp (Chart 2), required for library generation, were mostly
purchased from commercial suppliers and some synthesized by literature methods.51,52
Aldehydes 15fd, 15ff, 15fh, 15fi, 15fn, 15fp, 15fs and 15gr were synthesized in a Suzuki-
Miyaura reaction51,53 (see Supporting Information). Following previous approaches,35,51,52
preferentially lipophilic, aromatic aldehydes were included in the libraries taking into
account known structure-activity relationships for benchmark GAT inhibitors typically
possessing a polar core structure (mostly an amino acid), a spacer of variable length and
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aromatic moieties (see compounds 4–9). New aldehydes were added in the order of their
availability.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of hydrazones. For the hit verification individual hydrazones, rac-16 or rac-18,
were separately synthesized by combining 1.0 equivalent of hydrazine, rac-11 x HCl or
rac-13 x HCl, with 1.0 equivalent of aldehyde 15 as shown in Scheme 1. Additionally,
stoichiometric amounts of NaOD were added to neutralize HCl, introduced with the
49
hydrazines. For practical reasons the reactions were performed in deuterated solvents
54
(DMSO-d /D O = 9:1) to be able to monitor reaction progress by NMR.
6
2
General aspects of library generation.
The generation and screening of the hydrazone libraries delineated from hydrazine
derivatives rac-11rac-14, the synthesis of which was described above, should be
accomplished analogous to a method recently published by us.35
In that case, a nipecotic acid derivative provided with a hydrazine function attached to the
N-atom via a linker was incubated with aldehyde libraries, each library containing four
constituents. For the sake of simplicity hydrazone library formation was performed in the
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presence of the target, mGAT1. The incubation time was set to four hours, which was
found sufficient for a complete conversion, and the pH adjusted to 7.1 being compatible
with the presence of the proteins. The hydrazine was applied in excess (100 µM)
compared to the aldehydes (four different aldehydes, concentration of each 10 µM) in
order to render composition of the libraries constant, pseudostatic, though they are still
dynamic. To determine the activity of the libraries, the incubation mixtures were directly
used for competitive MS binding experiments. To this end, the MS marker NO711 (6) was
directly added to the incubation mixture. After additional 40 min of incubation, the amount
of specifically bound MS marker 6, which is serving as a measure of the activity of the
library (after liberation from the target), was quantified by LC-ESI-MS/MS.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Basically, for the present study the experimental conditions were analogous to those in
the initial approach as well as in a second subsequent application.35,51 Hence, libraries
were generated in the presence of the target mGAT1 and their activities were then
analyzed by competitive MS Binding Assays as described above. However, the following
changes were made: The library size was increased from four to eight (aldehydes per
library) and the concentration of individual aldehydes was set to 1.0 µM. Finally, a library
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
containing eight constituents in a concentration of 1 µM should be considered “active”, if
it reduced the amount of bound MS marker to < 50%. Provided the activity of a library (i.e.
the reduction of MS marker binding to < 50%) was due to a single component, the affinity
of this binder (IC value) should be at least 1 µM or lower. Besides, the concentration of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
the hydrazine derivatives rac-11rac-14 was raised to 200 µM as hydrazine
concentrations of 100 µM (as in previous approaches)35,51 did not lead to a complete
hydrazone formation within the given incubation period (see Supporting Information).
Hence, the 25-fold amount of hydrazines (rac-11rac-14) as compared to total aldehyde
concentration was applied for hydrazone library generation in the present study.
Chart 2. Libraries consisting of aldehydes 15a15hp
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aldehyde library 1
aldehyde library 2
aldehyde library 3
aldehyde library 4
aldehyde library 5
O
O
O
H
O
H
O
H
F
H
S
H
O
H
O
H
O
H
H
O
OCH3
O
H
S
15a
O
F3CO
O
O
O
15n
H
O
O
15i
NO2
15ag
N
O
O
H
O
H
H
N
1
5f
15v
15ad
15al
H
H
15q
H3CO
O
15y
O
H
N
H
N
CF3
H
15ab
O
15t
H
H
O
O
O
1
5g
O
H
O
H
O
H
OH
15aj
15d
15l
O
H
S
O
O
F
15b
N
O
H
NO2
15w
N
N
15ah
Cl
H
15j
15o
15ae
15am
O
H
15r
HO
15z
H
F
O
F
O
O
O
15ac
O
H
H
O
H
S
O
O
N
H
H
F
N
F3C
H
O
O
H
O
H
H
O
15e
S
O
15m
15u
15ak
O
NO2
O
N
S
HO
OCH3
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
15c
15h
15k
15s
15af
15ai
15x
15an
H
1
5p
15aa
aldehyde library 6
aldehyde library 7
aldehyde library 8
aldehyde library 9
aldehyde library 10
Cl
CF3
H
H
O
F
H3CO
F
15be
F
O
H
OCH3
F
H
O
H
S
O
F3
C
S
F
F
H
S
Cl
O
O
O
H
O
F
15ar
O
H
1
5bb
O
F
H
15bz
15aw
H
O
O
H
1
5ao
H
15at
H
15bm
F
O
H
H
O
15bj
15br
O
H
O
Cl
H
15bp
15bu
F
S
H3CO
O
F
H
F
15bh
O
15bx
H
Cl
O
O
S
O
F
F
15bk
H
F
H
Cl
F
15bc
O
H
O
O
O
H
15az
15bf
F
Cl
S
H
H
15au
Cl
O
H
15ca
H3CO
O
15ax
F
F
15bn
F
H3CO
H
F
15ap
Cl 15bsH
O
15as
H
O
15bv
O
F
S
O
F
F
O
15bq
H3CO
O
H
H
O
H
Cl
H
15bo
H
S
H
F
H
OF
H
H
O
15bi
H
O
15bt
15by
O
Cl
O
O
F
15aq
15av
15ay
H
15ba
15bg
15bw
15cb
15bd
15bl
F3C
CF3
Cl
Cl
Cl
aldehyde library 12
aldehyde library 12
aldehyde library 13
aldehyde library 14
aldehyde library 15
H
O
H
O
O
O
O
H
O
Cl
O
H
O
O
O
15da
H
O
H
O
O
Br
O
H
H
O
H
H
Br
N
H
H
H
N
O
N
H
N
15df
O
H
N
Cl
15dn
1
5cc
H
F
15ck
15cp
15cs
N
15cx F
N
O
15di
1
5ch
15cn
F
O
O
O
15cf
O
H
Cl
O
O
15cv
H
H
O
H
H
F
O
H
O
15dg
15dl
O
H
15dd
H
N
O
N
N
O
H
O
H
Br
N
O
O
H
5cd OCH3
15ci
15cl
Br
15cq
15ct
15cy Cl
Cl
15dj
15do
15dp
1
O
O
H
O
O
H
Cl
15db
H
O
N
H
Cl
H
O
H
O
O
O
H
15co O
F
O
O15cz
H
H
O
F
H
O
O
H
H
N
H
15cw
O
O
O
N
NH
N
15dm
15cg
N
Cl
N
N
H
N
15de
15cj
Cl
Cl
O
Cl
Cl
Cl
OH 15ce
15cu
15dk
15cm
15cr
Cl
15dc
15dh
Cl
H
Cl
aldehyde library 16
aldehyde library 17
aldehyde library 18
aldehyde library 19
aldehyde library 20
O
O
H
O
O
O
H
O
O
O
S
H
H
O
H
H
H
O
H
H
H
S
CF3
CF3
F
O
N
O
H
H
O
Cl
Cl
CN
H
O
O
N
15dy
15eg
15eo
N
15ew
15ex
H
S
15dq
15dt
1
5ed
CF3
Cl
15fb
F
15dv OCF3
15eb
15el
15er
O
H
O
H
O
O
CF3
H
O
F
O
15et
H
O
H
O
Br
15ej
O
H
15ez
O
OCF3
H
S
F
H
S
H
Cl
Cl
15dr
H
O
O
Cl
O
N
Cl
H
O
15dz
O
O
15eh
H
O
F
F3
C
15dw
H
O
15em
O
H
15ee
S
H
15ep
1
5fc
H
O
H
CN
H
O
O
H
15eu
O
O
O
OCF3
H
O
O
H
Cl
H
O
CN
15ec
H3CO
HO
H
H
15es
1
5dx
Cl
H
S
15fa
15du
Cl
Cl
15ek
Cl
1
5en
S
15ea
15ei
OCH3
15eq
15ev
15ds
OCH3
15ef
15ey
Cl 15fd
aldehyde library 21
aldehyde library 22
aldehyde library 23
aldehyde library 24
aldehyde library 25
O
O
O
H
H
O
H
O
H
H
O
O
H
O
F
O
H
H
H
O
H
O
F3C
O
O
N
N
H
O
O
O
Cl
Br
O
S
H
O
I
O
O
H
15fe
CH3
N
(H3C)2N
OCH3
15gc
15gh
N
O
15fj
15fm
N
15fr
15fu
CF3
O
15gk
O
O
Cl
Cl
15fx
15fz
H
O
O
15gp
H
N
H3CO
N
O
N
NH
H
Br
15gn
H
H
N
H
O
H
O
O
1
5fp
O
O
H
H
N
O
H
S
N
O
N
O
O
Cl
O
O
15gf
H
O
H
Cl
H
N
15gi
CH3
1
5ff F
15fh
F
O
15gl
O
N
H
15fn
OCH3
O
15gd
N
S
O
H
O
F
H
O
15fs
15fy F3C
Cl
15gq
O
H3CO
N
N
O
15fk
O
15fv
Cl
CF3
Br
15ga
CF3
O
H
O
H
N
N
O
O
15go
H
H
N
H
O
O
O
O
O
H
O
H
O
NO2
O
H
H
15ge F3
C
Br
H
N
O
Cl
O
O
O
O
H
15gm
15fi
N
15fg
N
Cl
15fo
15fq
15ft
15gg
15gj
15gr
NH2
15fw
Cl
15gb
H
1
5fl
aldehyde library 26
aldehyde library 27
aldehyde library 28
O
H
O
O
CH
S
N
O
1
OCH3
5gw
15ha
3
15he
15hn
H
N
15hl
S
H3CO
H
H
N
O
H
N
H
N
Cl
15hi
N
N
O
N
O
O
O
N
H
F
OCH3
H
O
H
O
H
15ho
15gs
N
Br
Br
H
H
O
15hf
F
N
1
5gx
15hb
Cl
15hj
N
15gu
O
H
N
O
N
O
H
N
O
H
O
O
N
H
N
HO
OCH3
N
15hg
H
F
F
H
S
15gy
Cl
H
O
O
N
O
H
1
5hc
15gt
O
OCH3
O
N
N
O
N
N
F
H
H
15hk
N
H
O
N
Br
15hd
15hh
15hm
15gv
H
15hp
15gz
H
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Journal of Medicinal Chemistry
Page 22 of 117
1
2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Screening and deconvolution of hydrazone libraries.
In the present study a total of 224 aldehydes grouped in 28 libraries, each containing
eight individual aldehydes in a concentration of 1.0 µM, were used (Chart 2). Each of the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28 aldehyde libraries was converted with all four hydrazines (rac-11rac-14; applied as
hydrochlorides;49 200 µM) in separate experiments into the corresponding libraries of
hydrazones (Scheme 3). In the following hydrazone libraries are termed “cis-C1”, “trans-
C1”, “cis-C2” and “trans-C2” for hydrazones derived from rac-16, rac-17, rac-18 and rac-
19, respectively, thus indicating their relative configurations and different spacer lengths.
Scheme 3. Example for the conversion of aldehyde library 1 with hydrazine rac-11 into
cis-C1 hydrazone library 1
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Page 23 of 117
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
aldehyde library 1
cis-C1 hydrazone library 1
F3C
H
S
S
F3CO
H
Cl
H
S
O
O
S
O
rac-16e
O
15f
N
O
O
15a
N
H
OH
OH
N
rac-16a
N
OH
O
N
H
H
H
N
H
O
N
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
S
O
OCF3
1
5d
S
N
N
H
O
O
15b
Cl
H
O
N
N
H
N
OH
1
5g
rac-16b
CF3
H
O
N
H
H
O
rac-16f
N
F3C
S
O
CF3
H
S
N
1
5e
N
OH
H
N
S
O
N
N
O
15h
H
15c
rac-16c
N
N
OH
H
rac-16g
N
H
N
O
O
S
O
H2N
N
N
OH
N
OH
N
H
H
N
H
OH
N
N
H
H
rac-16d
N
rac-16h
rac-11 x HCl
H
The results of the screening experiments for the hydrazone libraries are shown in Figure
2. Control experiments with the aldehyde libraries 128 (aldehydes 15a15hp) and
hydrazines rac-11rac-14 alone were performed to ensure that in the applied
concentrations none of the building blocks affected the marker binding to a remarkable
extent (see Supporting Information).
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Journal of Medicinal Chemistry
Page 24 of 117
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a) cis-C1
b) trans-C1
120
120
100
100
80
60
40
20
0
80
60
40
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2
ry ry ry ry ry ry ry ry ry
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
rar rar rar rar rar rar rarbra rar ary ary ary ary ary ary ry ary ary ary ary ary ary ary ary ary ary ary ary
y
y
y
y
y
y
y
ry
ib
y
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
bra ra ra ra ra ra ra bra ra ary ary ary ary ary ary ry ary ary ary ary ary ary ary ary ary ary ary ary
li
l
ib ib ib ib ib ib
l
l
l
l
l
li
l
ib
ib ib ib ib ib ib
r
r
r
r
r
r
ra
ib ib ib ib ib ib ib ib ib ib ib ib
r
r
r
r
r
r
r
r
r
r
r
r
l
ib ib ib ib ib ib ib
l
l
l
l
l
l
li
l
ib ib ib ib ib ib
r
r
r
r
r
r
ra
ib ib ib ib ib ib ib ib ib ib ib ib
r
r
r
r
r
r
r
r
r
r
r
r
l
l
l
l
l
l
li
b
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
li
b
l
l
l
l
l
l
l
l
l
l
l
l
c) cis-C2
d) trans-C2
120
120
100
100
80
60
40
20
0
80
60
40
20
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2
ry ry ry ry ry ry ry ry ry
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
rar rar rar rar rar rar rarbra rar ary ary ary ary ary ary ry ary ary ary ary ary ary ary ary ary ary ary ary
y
y
y
y
y
y
y
ry
ib
y
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
bra ra ra ra ra ra ra bra ra ary ary ary ary ary ary ry ary ary ary ary ary ary ary ary ary ary ary ary
li
l
ib ib ib ib ib ib
l
l
l
l
l
li
l
ib
ib ib ib ib ib ib
r
r
r
r
r
r
ra
ib ib ib ib ib ib ib ib ib ib ib ib
r
r
r
r
r
r
r
r
r
r
r
r
l
ib ib ib ib ib ib ib
l
l
l
l
l
l
li
l
ib ib ib ib ib ib
r
r
r
r
r
r
ra
ib ib ib ib ib ib ib ib ib ib ib ib
r
r
r
r
r
r
r
r
r
r
r
r
l
l
l
l
l
l
li
b
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
li
b
l
l
l
l
l
l
l
l
l
l
l
l
Figure 2. Screening of a) cis-C1 (rac-16), b) trans-C1 (rac-17), c) cis-C2 (rac-18) and d)
trans-C2 hydrazone libraries (rac-19). The bars indicate the percentage of remaining
specific binding of NO711 (6) after an incubation time of 4 h for library generation and 40
min for marker binding to mGAT1; data represent means±SD of four replicates. The limit
for further analysis of a library was defined as 50% remaining marker binding (indicated
by the dashed line).
ACS Paragon Plus Environment
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Page 25 of 117
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
Nine cis-C1 hydrazone libraries (rac-16; libraries 1, 3, 6, 10, 23, 24, 25, 27 and 28; Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2a) and four cis-C2 hydrazone libraries (rac-18; libraries 1, 19, 23 and 24; Figure 2c) were
found to reduce the remaining MS marker binding below 50% (by mean values of four
replicates) and thus were considered active. Interestingly, all active libraries derived from
cis-configured nipecotic acid derivatives, while the trans-C1 (rac-17; Figure 2b) and trans-
C2 hydrazone libraries (rac-19; Figure 2d) did not show any striking activity towards
mGAT1. All of the 13 hydrazone libraries considered active were further examined in
deconvolution experiments in order to identify their most active components. For these
experiments single hydrazones were studied in the same way as the libraries except that
only single aldehydes were employed in the test procedure (incubation of aldehydes in a
concentration of 1.0 µM with hydrazine rac-11 and rac-13, respectively, in a concentration
of 200 µM). As summarized in Table 1, 16 hydrazones reduced MS marker binding below
the set limit of 50%, while none of the corresponding aldehydes alone showed remarkable
activity. 14 of the active compounds were represented by the shorter chained hydrazones
rac-16, showing remaining MS marker binding of 1649% (rac-16e, rac-16r, rac-16fv, rac-
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 117
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
6fw, rac-16fy, rac-16fz, rac-16ga, rac-16gb, rac-16gc, rac-16ge, rac-16gf, rac-16gg, rac-
6gk and rac-16ho; Table 1, entries 5, 10, 4243, 4549, 5153, 57 and 79). Amongst
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the longer chained derivatives only hydrazones rac-18e and rac-18fy fulfilled the criterion
for further analysis by reducing MS marker binding to 47% and 41%, respectively (Table
1, entries 5 and 45). As mentioned above, with a reduction of the marker binding to 50%
or lower when applied in a concentration of 1 µM, “active” test compounds should
correspond to a maximum IC of 1 µM. Thus, we considered it worth to subject all these
50
16 hydrazones to further analysis.
Table 1. Results of the deconvolution experiments for “active” hydrazone libraries
specific binding of NO711 [%]a
specific binding of NO711 [%]a
compd
15/rac-
6/rac-18
compd
15/rac-
aldehyd
e 15
cis-C1
cis-C2
aldehyde
cis-C1
cis-C2
entry
library
entry
library
(rac-16)
(rac-18)
15
(rac-16)
(rac-18)
1
16/rac-18
library
1
library
23
1
2
3
4
5
6
7
8
-a
-b
-c
-d
-e
-f
106±6
77±18
82±17
92±11
98±4
74±8
82±4
86±9
91±5
40±2
66±10
75±5
51±6
79±6
75±10
72±6
41
42
43
44
45
46
47
48
-fu
-fv
82±16
99±14
103±9
75±12
97±8
68±6
48±4
46±6
56±2
40±7
49±9
38±4
37±3
61±9
52±11
69±14
62±11
41±5
-fw
-fx
82±2
47±4
-fy
88±25
87±21
90±16
59±13
74±14
71±10
-fz
90±12
88±13
72±4
60±6
-g
-h
-ga
-gb
54±4
60±6
library
3
library
24
9
-q
100±14
74±15
-
49
-gc
70±5
38±2
70±4
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Page 27 of 117
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
0
1
2
3
4
5
6
-r
-s
-t
109±11
114±6
46±10
81±1
85±3
87±3
85±6
83±14
93±6
-
-
-
-
-
-
-
50
51
52
53
54
55
56
-gd
-ge
-gf
93±10
99±3
81±11
36±2
16±1
32±1
67±9
84±11
59±2
72±8
72±7
58±13
76±4
64±14
73±4
65±9
98±23
92±12
103±18
97±9
-u
-v
-w
-x
107±10
108±12
113±12
119±6
-gg
-gh
-gi
86±15
97±6
-gj
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
library
6
library
25
17
18
19
20
21
22
23
24
-ao
-ap
-aq
-ar
97±3
98±11
107±7
112±10
108±13
101±11
111±11
112±2
66±15
73±11
58±3
-
-
-
-
-
-
-
-
57
58
59
60
61
62
63
64
-gk
-gl
129±3
127±5
126±4
115±10
118±3
112±15
107±10
121±5
36±6
55±13
53±13
69±6
67±8
55±3
89±4
93±4
-
-
-
-
-
-
-
-
-gm
-gn
-go
-gp
-gq
-gr
90±11
72±11
80±9
-as
-at
-au
-av
52±2
70±15
library
10
library
27
25
26
27
28
29
30
31
32
-bu
-bv
-bw
-bx
-by
-bz
-ca
-cb
113±7
118±5
116±9
121±7
124±17
129±6
121±15
113±8
85±2
80±13
81±16
84±8
-
-
-
-
-
-
-
-
65
66
67
68
69
70
71
72
-ha
-hb
-hc
-hd
-he
-hf
98±7
85±7
52±10
75±6
-
-
-
-
-
-
-
-
108±17
107±18
104±26
101±5
61±7
80±7
87±16
50±7
88±3
122±10
118±8
84±6
-hg
-hh
64±12
55±7
80±13
113±19
library
19
library
28
33
34
35
36
37
38
39
40
-eo
-ep
-eq
-er
107±15
103±1
99±10
106±9
105±12
93±17
109±3
106±2
-
-
-
-
-
-
-
-
60±7
62±5
73
74
75
76
77
78
79
80
-hi
-hj
103±15
116±9
118±7
116±15
118±2
108±21
110±11
124±3
60±15
68±11
73±12
53±5
-
-
-
-
-
-
-
-
62±20
83±5
-hk
-hl
-es
-et
61±15
64±14
83±10
80±4
-hm
-hn
-ho
-hp
60±3
69±11
27±4
-eu
-ev
61±13
aPercentage of remaining specific binding of NO711 (6) in the presence of either pure
aldehyde 15 or cis-C1 (rac-16) or cis-C2 hydrazones (rac-18) after an incubation time of
4
h for hydrazone formation and 40 min for marker binding to mGAT1; data represent
means±SD of four replicates. The limit for further analysis of a hydrazone was defined as
5
0% remaining marker binding (hydrazones considered active are highlighted in yellow).
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Journal of Medicinal Chemistry
Page 28 of 117
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hit verification.
For hit verification the 16 hydrazones found most active in deconvolution experiments
were synthesized in pure form and their binding affinities (pK values) were established in
i
full-scale competitive MS binding experiments.
Table 2. Binding affinities (pK) determined in competitive binding assays at mGAT1 of
i
hydrazones synthesized in pure form
compda
n
R
pK
b,c
entry
compda
n
R
pK
i
b,c
entry
i
1
rac-16e
1
6.35±0.02
9
rac-16gc
1
6.15±0.01
2
3
rac-16r
1
1
6.02±0.11
5.91±0.08
10
11
rac-16ge
rac-16gf
1
1
5.90±0.04
6.67±0.03
rac-16fv
4
5
rac-16fw
rac-16fy
1
1
5.73±0.10
6.19±0.05
12
13
rac-16gg
rac-16gk
1
1
6.61±0.00
5.84±0.09
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Page 29 of 117
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
6
7
rac-16fz
rac-16ga
1
1
5.64±0.14
6.33±0.11
14
15
rac-16ho
rac-18e
1
2
6.03±0.06
6.21±0.08
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
rac-16gb
1
5.83±0.09
16
rac-18fy
2
6.32±0.05
aIndividually synthesized from appropriate aldehydes and hydrazines, see also
reference.54 bpK values are given as means±SEM of three independent experiments.
i
c
Tiagabine (5) was used as reference in all experiments and a pK of 7.56±0.06 (n = 8)
i
was found for this compound.
The pK values found for the hydrazones are in a range from 5.64 to 6.67 (Table 2). The
i
binding affinities of the best 5-substituted nipecotic acid derived hydrazones (e.g. rac-
1
6gf; Table 2, entry 11 or rac-16gg; Table 2, entry 12) are almost as good as those of yet
established potent GAT1 inhibitors such as tiagabine (5; pK = 7.56) with the difference in
i
the nominal pK values being only about one log unit.
i
Among the best binders from this study one possesses a 3-phenoxyphen-1-yl (rac-16r;
pK = 6.02; Table 2, entry 2), another a 2-(2-naphthyl)pyrimidin-5-yl (rac-16ho; pK = 6.03;
i
i
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