Focused pseudostatic hydrazone libraries screened by mass spectrometry binding assay: Optimizing affinities toward γ-aminobutyric acid transporter 1

Article

pubs.acs.org/jmc

Focused Pseudostatic Hydrazone Libraries Screened by Mass

Spectrometry Binding Assay: Optimizing Aﬃnities toward

γ‑Aminobutyric Acid Transporter 1

Miriam Sindelar, Toni A. Lutz, Marilena Petrera,^†and Klaus T. Wanner*

Center for Drug Research, Department of Pharmacy, Ludwig Maximilians University at Munich, Butenandtstrasse 9-13, D-81377

Munich, Germany

S

* Supporting Information

ABSTRACT: Mass spectrometric (MS) binding assays, a powerful

tool to determine aﬃnities of single drug candidates toward chosen

targets, were recently demonstrated to be suitable for the screening of

compound libraries generated with reactions of dynamic combinatorial

chemistry when rendering libraries pseudostatic. Screening of small

hydrazone libraries targeting γ-aminobutyric acid transporter 1

(GAT1), the most abundant γ-aminobutyric acid (GABA) transporter

in the central nervous system, revealed two nipecotic acid derived

binders with submicromolar aﬃnities. Starting from the biphenyl

carrying hit as lead structure, the objective of the present study was to

discover novel high aﬃnity GAT1 binders by screening of biphenyl

focused pseudostatic hydrazone libraries formed from hydrazine 10 and

36 biphenylcarbaldehydes 11c−al. Hydrazone 12z that carried a 2′,4′-dichlorobiphenyl residue was found to be the most potent

binder with low nanomolar aﬃnity (pK_i= 8.094 0.098). When stable carba analogues of representative hydrazones were

synthesized and evaluated, the best binder 13z was again displaying the 2′,4′-dichlorobiphenyl moiety (pK_i= 6.930 0.021).

very limited. That excludes a signiﬁcant role in termination of

GABA neurotransmission for these proteins for which high

densities have been reported in liver and kidney.¹⁶

INTRODUCTION

■

γ-Aminobutyric acid (GABA, 1, Chart 1) is the most abundant

inhibitory neurotransmitter in the central nervous system

(CNS).¹⁻³A reduced GABAergic neurotransmission, in

consequence an imbalance in the neurotransmitter equilibrium,

is associated with neurological disorders, e.g., schizophrenia,⁴

Parkinson’s disease,⁵epilepsy,⁶Alzheimer’s disease,⁷neuro-

pathic pain,⁸Huntington’s disease,⁹depression,¹⁰⁻¹²panic,^10,12

and mania.^10,12

To restore the neurotransmitter equilibrium, GABAergic

neurotransmission can be enhanced by CNS-active drugs

targeting GABA receptors, metabolic enzymes, or GABA

transport proteins (GATs). The last targets are membrane

bound proteins encoded by genes assigned to the solute carrier

6 (SLC6) family¹³and mediate the transport of GABA across

cell membranes utilizing a co-transport of sodium and chloride

ions as energy supply.

Four diﬀerent subtypes named mGAT1, mGAT2, mGAT3,

and mGAT4 when cloned from murine brain cells are known.

For other species, the nomenclature for GATs diﬀers, e.g., for

the human GATs, the declaration is given by the human

genome organization (HUGO) resulting in GAT1, BGT1,

GAT2, and GAT3 as corresponding transport proteins.¹⁴

mGAT1 and mGAT4 are the most abundant GABA trans-

porters in the CNS, from which mGAT1 is mainly responsible

for the neuronal reuptake of GABA and mGAT4 is

predominantly located in cell membranes of glial cells.¹⁵In

contrast, the expression of mGAT2 and mGAT3 in the brain is

When GATs were deﬁned as pharmacological CNS targets,¹⁷

a ﬁrst generation of inhibitors was disclosed including cyclic

analogues of GABA (1). These small cyclic amino acids like

nipecotic acid (2) and guvacine (3) (Chart 1) were in the

1970s found to be potent in vitro inhibitors.^18,19Nevertheless,

their potency was not conﬁrmed in vivo because of their

hydrophilic character preventing them from passing the blood−

brain barrier in suﬃcient amount.²⁰Consequently, a second

generation of GAT inhibitors was born by substitution of the

small amino acids with characteristic lipophilic side chains at

the amino function that enabled them to cross the blood−brain

barrier.²¹Examples of the aﬃnity enhancing side chains are

represented in SK&F-89976-A (4), tiagabine (5), and NO711

(6) that proved to have high aﬃnity at and subtype selectivity

for mGAT1 compared to the nonsubstituted amino acids

(Chart 1).²²In addition, 5 has successfully undergone clinical

trials and is now in use as add-on medication for the treatment

of epilepsy.²³

On the basis of this success, further selective and potent

inhibitors were developed in the style of these potent GAT1

inhibitors representing the third generation of mGAT1

inhibitory drugs. These were characterized by new side chains

Received: December 7, 2012

Published: January 21, 2013

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Chart 1. Structures of GABA and GAT1 Inhibitors

including the lipophilic aromatic domain attached to the amino

group of the amino acid moiety (Chart 1). To this group

belong a number of nipecotic acid based compounds in which

the amino group of the hydrophilic amino acid moiety was

connected via spacers diﬀering in length and functionalities, like

oxime and ether groups, to lipophilic aromatic residues. A

selection of these third generation inhibitors, which had been

developed by Andersen at al. in 1999 and 2001,²⁴is displayed

in Chart 1 (compounds 7−9).

For GAT1, a screening method was developed several years

ago that tested single drug candidates via a competitive MS

binding assay, employing 6 as native MS marker.²⁵This assay

allowed the indirect determination of binding aﬃnities toward

GAT1 in analogy to radioligand binding studies, circumventing

the necessity to apply radioactively labeled compounds.

Recently, we described a new method that applies MS

binding assays,^26,27which so far had been used for the

characterization of the binding aﬃnities of single test

compounds toward a target only, to the screening of compound

libraries generated by eﬃcient and the most fundamental

reactions known from dynamic combinatorial chemistry

(DCC).²⁸Both library generation and screening were

performed in the presence of the protein target to improve

assay performance. To facilitate hit detection, libraries were

rendered pseudostatic. This was achieved by reacting sets of

compounds with varying structure with a large excess of a single

compound of complementary reactivity, the thus generated

one-dimensional libraries being almost constant in composition

but still dynamic in nature. After library generation was

performed, addition of a native MS marker to the same sample

directly enabled detection of active libraries by MS binding

assays. The feasibility of this concept was demonstrated for

mGAT1, a well established target for which, as mentioned

above, a MS binding assay utilizing 6 as native marker was

already known.²⁵Screening of libraries, each of which consisted

of four hydrazone compounds 12 (Scheme 1) and had been

generated by reacting nipecotic acid derived hydrazine 10 with

four diﬀerent aldehydes 11, revealed new GAT1 inhibitors

resembling structures 8 and 9 (Chart 1).

In this study, a speciﬁc limit of maximum remaining marker

binding caused by 10 μM hydrazones was chosen to deﬁne a

library as active. As such, a 20% level (IC₂₀= 4 IC₅₀) was

selected that attributed a minimum potency (IC₅₀) of 2.5 μM

to a single hydrazone based on the assumption that all other

library members were inactive. In deconvolution experiments

testing the single aldehydes in combination with hydrazine 10,

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resulting from the reaction of hydazine 10 with biphenyl-2-

carbaldehyde was found to reduce remaining marker binding in

the deconvolution experiment to a value below the lower limit

of quantiﬁcation of the MS marker 6 (<5%), indicating an IC₅₀

equal to or below 0.53 μM (IC₀₅= 19 IC₅₀). Resynthesis of 12a

and characterization in a full range competitive MS binding

Scheme 1. Condensation of Hydrazine Derived Nipecotic

Acid Building Block 10 with Diverse Aldehydes 11

experiment ﬁnally revealed a pK_iof 6.186

0.028 for this

compound. The second hit, hydrazone 12b, was derived from

2-thiophen-2-ylbenzaldehyde and gave rise to a remaining

marker binding of 8.2 0.4% in the deconvolution experiment

and to a pK_iof 6.229 0.039 in a full range competitive MS

binding assay for the resynthesized compound.²⁸That these

mGAT1 binders are also functionally active was conﬁrmed in

[³H]GABA uptake assays with mGAT1 expressing HEK293

cells, the pIC₅₀values amounting to 5.308 0.096 (12a) and

5.542 0.107 (12b), respectively (Chart 2).

As we identiﬁed these two hits, 12a and 12b, with

submicromolar potencies in the ﬁrst library screening assays,

the aim of the present study was to develop exemplarily for one

of the two hits, compound 12a, a focused library by introducing

various substituents in the biaryl moiety to further optimize the

aﬃnity toward mGAT1 (Figure 1).

out of each of the two libraries fulﬁlling this condition, a single

hydrazone was identiﬁed to mostly represent the activity of the

individual library. Thus, comparable to structure 8 (Chart 1)

(E)-1-(2-{2-[1-(2′,4′-dichlorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic acid (12a) (Chart 2)

Chart 2. Hydrazone Based GAT1 Inhibitors

Biaryl focused pseudostatic hydrazone libraries (12c−al,

Scheme 1) each derived from libraries of four biarylaldehydes

(11c−al, Scheme 1) were therefore intended to be generated

and subsequently screened by the MS binding assay, again

employing 6 as native MS marker. As we aimed at improved

aﬃnities of desired hits compared to the hits searched in the

ﬁrst screening, the limit to deﬁne a library as active was further

tightened by lowering it from 20% used in the original study²⁸

to 10%. In theory, this equals an IC₅₀of ∼1 μM for the

compound, eﬀecting reduction of marker binding to this limit

when employed at 10 μM (IC₁₀= 9 IC₅₀). Hits contained in

active libraries (<10% marker binding) were to be identiﬁed by

applying the original deconvolution procedure and to be further

evaluated after resynthesis.²⁸As hydrazones are prone to

hydrolysis,^29,28liver-toxic,³⁰⁻³²and unstable against air,³³⁻³⁵

they are not suitable as lead structures for drug discovery. But

the results obtained from the screening of hydrazone libraries

Figure 1. Focused hydrazone library generation and screening toward GAT1. After hit identiﬁcation and validation of potent derivatives of

hydrazone 12a, corresponding carba analogues 13 were synthesized and evaluated.

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Chart 3. Libraries Consisting of Biphenylcarbaldehydes 11c−al

may nevertheless be of high value for drug discovery and

support the development of stable analogues, which to

demonstrate was a further aim of this study. Carba derivatives

in which the hydrazone linker had been replaced by an all

carbon chain with a double bond (CHN−NH−CH₂−CH₂

→ CHCH−CH₂−CH₂−CH₂) were considered a good

choice as stable substitutes of the hydrazone inhibitors. Thus,

the synthesis and biological evaluation of carba analogues of a

series of representative hydrazone inhibitors were also included

in this study.

generation were synthesized via the Suzuki−Miyaura³⁶reaction

except for 4′-ﬂuorobiphenyl-2-carbaldehyde (11e) and biphen-

yl-3-carbaldehyde (11n) that were purchased from Alfa Aesar

and Sigma-Aldrich, respectively.

Thus, 2′-ﬂuorobiphenyl-2-carbaldehyde (11c)^37,38was ob-

tained from 2-bromobenzaldehyde and 2-ﬂuorophenylboronic

acid, applying a literature method³⁹that had been successfully

employed in the synthesis of diversely substituted biphenyl-2-

carbaldehydes (reaction conditions: 2-bromobenzaldehyde/

substituted phenylboronic acid = 1:1, 5 mol % Pd(OAc)₂,

DMF/H₂O = 2:1, 25 °C). For the preparation of the biphenyl-

2-carbaldehydes 11d, 11f, 11h, 11i, 11j, 11k, 11m, 11o, 11p,

11q, 11r, 11t, 11u, 11v, 11w, 11x, 11ac, 11ad, and 11ae this

procedure had to be modiﬁed by adding common phosphine

ligands (triphenylphosphine, dibenzylideneacetone, tri-o-tolyl-

CHEMISTRY

■

Synthesis of Biarylcarbaldehydes. In general, biphenyl-

carbaldehydes 11c−11al (Chart 3) required for library

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a

Scheme 2. Synthesis of Nipecotic Acid Derived Carba Analogues 13

a

Reagents and conditions: (a) TsCl, Et₃N, CH₂Cl₂, 0 °C to rt, 20 h; (b) pinacolborane, chloridobis(cyclopentadienyl)hydridozirconium no solvent,

rt, 48 h; (c) ethyl nipecotate, no solvent, rt, 16 h; (d) Pd(dppf)Cl₂·CH₂Cl₂, 1-bromo-2-iodobenzene, K₂CO₃, 1,4-dioxane/H₂O 2:1, 72 h, rt; (e)

Pd(dppf)Cl₂·CH₂Cl₂, RPhBO₂H, K₂CO₃, 1,4-dioxane/H₂O 2:1, 3 h, 60 °C; (f) NaOH, EtOH, 4 h, rt.

phosphine, or tri-tert-butylphosphine) and increasing the

temperature from room temperature to 80 °C to improve the

reaction rates and the yields (see Supporting Information).

The latter protocol could not be successfully applied to the

synthesis of 11y starting from 2-bromobenzaldehyde and 2-

ﬂuoro-4-chlorophenylboronic. But coupling of 2-bromobenzal-

dehyde and 2-ﬂuoro-4-chlorophenylboronic acid to yield 11y

could be realized following a related literature method that had

originally been used for coupling of pentaﬂuorophenylboronic

acid with halogenated benzene derivatives by means of CsF,

Ag₂O, Pd(dba)₃·CHCl₃, and tri-o-tolylphosphine in pure DMF

at 100 °C ⁴⁰(see Supporting Information).

In the case of other sterically demanding phenylboronic acid

derivatives carrying electron withdrawing residues (2,4-

diﬂuorophenylboronic acid, 2,4-dichlorophenylboronic acid,

2,6-diﬂuorophenylboronic acid, 2,4,6-triﬂuorophenylboronic

acid, 2-chloro-4-ﬂuorophenylboronic acid), the coupling with

2-bromobenzaldehyde gave poor yields or no coupling products

at all under the applied conditions for the preparation of, for

example, 11d. However, an alternative approach starting from

2-formylphenylboronic acid, instead of 2-bromobenzaldehyde,

and an appropriate halogenated benzene derivative instead of

the corresponding phenylboronic acid gave access to the

desired aldehydes. Thus, 11g, 11z, 11aa, 11ab, and 11ah were

obtained by reacting 2-formylphenylboronic acid with the

appropriately halogen substituted benzene derivatives following

a literature method that originally had been used for coupling

of 1-bromo-2,3,4,5,6-pentaﬂuorobenzene with 2-formylphenyl-

boronic acid.⁴¹

The same reaction conditions that had been used for the

coupling reactions of 2-formylphenylboronic acid with various

aryl halides were ﬁnally also tested for the preparation of

aldehydes 11s, 11ab, 11af, 11ag, 11ai, 11aj, 11ak, and 11al by

coupling 2-bromobenzaldehyde again with the respective

phenylboronic acid derivatives, leading to the desired

compounds in yields of up to 90% (toluene/ethanol/2 M

Na₂CO₃(aq) = 1:1:1, tetrakis(triphenylphosphine)-

palladium(0)) (see Supporting Information).

Synthesis of the Hydrazones. Synthesis of individual

hydrazones required for full scale competition experiments was

carried out by combining 1 equiv of aldehyde with 1 equiv of

hydrazine 10 according to Scheme 1, again following the

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Scheme 3. Example for the Conversion of an Aldehyde Library into a Hydrazone Library (Aldehyde Library 1 → Hydrazone

Library 1)

original protocol for hydrazone synthesis described previ-

ously.²⁸

Therefore, the respective experimental conditions are given

here only in short form. To generate the hydrazone libraries,

aldehyde libraries of four aldehydes each at 10 μM were reacted

with a 2.5-fold excess of hydrazine 10 (100 μM initial

concentration) at 37 °C for 4 h, in which the time span had

turned out to be suﬃcient for complete conversion in the

former²⁸and in the present study. In addition, library

generation was again performed in the presence of the target

mGAT1 to improve assay performance. The activity of the

libaries was ﬁnally analyzed by means of competitive MS

binding experiments. To this end, following incubation of

libraries with MS marker 6 (for 40 min), the amount of

speciﬁcally protein-bound MS marker 6 was quantiﬁed by LC−

ESI-MS/MS (after isolation of protein−ligand complexes and

liberation of the bound ligands from the target), low amounts

of remaining marker binding representing high library activity

and vice versa. As for the original procedure, in addition to each

screening experiment, control experiments employing aldehyde

libraries (11) and hydrazine 10⁴⁴alone were perfomed to

ensure that the building blocks in the concentrations used do

not aﬀect marker binding to a remarkable extent.

Screening and Deconvolution of Focused Biphenyl-2-

carbaldehyde Derived Hydrazone Libraries. In our former

study²⁸that applied pseudostatic hydrazone libraries on

mGAT1, 12a, the hydrazone derived from biphenyl-2-

carbaldehyde (11a), had been uncovered to display sub-

micromolar aﬃnity to mGAT1 (pK_i= 6.186 0.028).²⁸To

further optimize the aﬃnity of 12a for mGAT1 in the present

study, 36 aldehydes mainly delineated from biphenyl-2-

carbaldehyde (11a) by additional small substituents in the

phenyl rings (biphenylcarbaldehydes 11c−11al) were selected

for hydrazone generation and screening. These were grouped

into nine libraries of four aldehydes (Chart 3), converted into

hydrazone libraries (Scheme 3), and analyzed by competitive

Synthesis of the Carba Analogues. For the synthesis of

the carba analogues of the hydrazone derivatives the reaction

sequence outlined in Scheme 2 has been developed. It features

the vinylboronic acid derivative 18 as a key compound, which

by two consecutive Suzuki−Miyaura reactions allows high

ﬂexibility with regard to construction and the substitution

pattern of the biaryl moiety present in the target compounds

13. For the synthesis of 18, ﬁrst pentynol 15 was transformed

into the tosyl ester 16 by reaction with TsCl according to a

literature method.⁴²The resulting alkyne 16 was subsequently

reacted with pinacolborane in the presence of Cp₂ZrHCl in

analogy to a literature procedure,⁴³to give the boronic acid

ester 17, which upon treatment with ethyl nipecotate provided

18 in 92% yield. Compound 18 allowed stepwise establishment

of the biaryl moiety present in the ﬁnal compounds 13. Thus, a

ﬁrst Suzuki−Miyaura cross-coupling reaction between 18 and

1-bromo-2-iodobenzene yielded the monoaryl derivative 19.

Compound 19 was then subjected to a second Suzuki−Miyaura

cross-coupling reaction with diﬀerently substituted phenyl-

boronic acid derivatives [2-ﬂuorophenylboronic acid (20c), 2,4-

diﬂuorophenylboronic acid (20g), 2-chlorophenylboronic acid

(20p), 2-chloro-4-ﬂuorophenylboronic acid (20ah), and 2,4-

dichlorophenylboronic acid (20z)] to give the desired biaryl

substituted nipecotic acid ester derivatives 21c, 21g, 21p, 21ah,

and 21z (yield 91−93%). These last compounds were ﬁnally

converted to the free nipecotic acid derivatives 13c, 13g, 13p,

13ah, and 13z by basic hydrolysis of the ethyl ester function

(yield 86−90%).

RESULTS AND DISCUSSION

■

General Aspects of Library Generation and Screening.

Libraries were generated and screened as described earlier.²⁸

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MS binding assays as described above. Clearly, a higher number

of aldehydes than four could be employed in library formation

under otherwise identical conditions. But this would require

reduction of the concentration of the individual aldehydes to

further warrant the excess of hydrazine 10 over the aldehydes

employed and thus the pseudostatic character of the libraries.

However, with lowering hydrazone concentrations, the

conditions for a library to be classiﬁed as active will also

become more stringent (IC₅₀corresponds to the initial

aldehyde concentration).

Table 1 summarizes the screening values of the aldehyde and

hydrazone libraries 1−9. All hydrazone libraries were found to

Table 1. Libary Screening Values of Aldehyde Libraries and

Hydrazone Libraries

a

library screening (%)

entry

library

aldehyde library

hydrazone library

1

2

3

4

5

6

7

8

9

library 1

library 2

library 3

library 4

library 5

library 6

library 7

library 8

library 9

82

81

88

79

2

3

<5

91 11

6

1

85

35

81

4

1

<5

a

Percentage of remaining speciﬁc binding of NO711 (6) in the

Figure 2. Deconvolution results of libraries 1−9. The limit for further

analysis of a library was deﬁned as 10% remaining marker binding.

presence of pure aldehyde libraries and corresponding hydrazone

libraries after an incubation time of 4 h for library generation and 40

min for marker binding to mGAT1, n = 4, SD, <5% = <50 pM

derived from the LLOQ of NO711 (50 pM, as 1−2 nM of 20 nM

applied NO711 is maximally bound to the membrane preparation).

Control experiment for 100 μM hydrazine 10 was obtained

comparable to published results. See ref 28.

reduction of MS marker binding. For aldehyde library 7 for

which the screening value had amounted to 35 1% (Table 1,

entry 7), a deconvolution experiment (in the absence of

hydrazine 10) revealed aldehyde 11ab to be by far the most

potent binder of this group, reducing MS marker binding to 49

1% (Figure 2). Accordingly, as a rough estimate, an IC₅₀of

∼10 μM can be attributed to this aldehyde.²⁸

Biphenyl-2-carbaldehyde Derived Hydrazone Hits.

Table 2 summarizes the pK_ivalues that had been found in

full scale competitive MS binding assays for the separately

resynthesized biphenyl-2-carbaldehyde derived hydrazones that

had turned out as the most active ligands at mGAT1 in the

deconvolution experiments (reduction of MS marker binding

to ≤10%). Hydrazones 12d, 12i, 12j, 12k, 12n, 12o, 12q, 12s,

12t, 12u, 12w, 12x, 12ai, 12aj, and 12al did not reach this limit

and were excluded from further analysis.

The compounds that achieved the desired value below 10%

are sorted according to the substitutents of the biaryl moiety

starting with hydrazones exhibiting only F-substituents (Table

2, entries 2−9), F- and CH₃O-substituents (Table 2, entries 10

and 11), followed by those with either CH₃- (Table 2, entries

12−14), CF₃- (Table 2, entry 15), Cl- (Table 2, entries 16−

18), or F- and Cl-substituents (Table 2, entries 19−22).

An F-substituent in the 2′-position increased the aﬃnity by

almost 1 order of magnitude, resulting in a pK_iof 6.914 0.061

for 12c (Table 2, entry 2) compared to the parent compound

12a (6.186 0.028, Chart 2, Table 2, entry 1), while a 4′-F-

substituent (structure 12e) caused a smaller increase, the

aﬃnity of 12e amounting to a pK_iof 6.546 0.067 (Table 2,

entry 3). With F-substituents present in both the 2′- and 4′-

position, a gain in binding energy (compared to 12a) was

observed that was roughly the sum of the increase of binding

energies eﬀected by the two F-substituents in 12c and 12e,

be highly active, reducing the remaining MS marker binding

not only below 10%, the limit set for libraries to be considered

as active, but even below the lower limit of quantitation

(LLOQ) for the MS marker 6 of 5% (Table 1). Also library 5,

which was the only exception, displayed a very low remaining

marker binding of 6 1%.

Showing very high activity in the screening step reducing MS

marker binding below 10%, all libraries were also included in

subsequent deconvolution experiments to identify the most

active compounds. These experiments were performed

analogous to the library screening except that single aldehydes

were employed instead of the libraries.

In the deconvolution study of the hydrazone libraries, 21 of

the 36 hydrazones were found to strongly inﬂuence MS marker

binding, reducing it to 10% and below (Figure 2). Taking into

account that a reduction of MS marker binding to 10%

corresponds to an IC₅₀of ∼1 μM (IC₁₀= 9 IC₅₀), we

considered it worthwhile to resynthesize most of the hydrazone

derivatives that had reached this limit in the screening and to

establish their pK_ivalues in MS competition experiments

comprising complete competition curves. The data of these

studies that had also been established to provide an improved

insight on the inﬂuence of the substituent eﬀects on the

potency of the compounds are given in the section “Biphenyl-2-

carbaldehyde Derived Hydrazone Hits”.

Interestingly, one of the pure aldehyde libraries (tested in the

absence of hydrazine 10) had also given rise to a signiﬁcant

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Table 2. Binding aﬃnities (pK_i) of Resynthesized Biphenyl Derived Hydrazones Determined from Competitive MS Binding

Assays and Inhibitory Potencies (pIC₅₀) at mGAT1 from [³H]GABA Uptake Experiments

a

compd (substitution pattern of the biphenyl

moiety of 12a)

biological testing

b

c

b

entry

compd

4

2′

3′

4′

6′

binding aﬃnity pK_i(n = 3 SEM)

inhibitory potency pIC₅₀(n = 3 SEM)

1

12a

H

F

H

F

H

F

H

F

6.186 0.028

6.914 0.061

6.546 0.067

7.449 0.023

6.690 0.036

6.204 0.054

6.883 0.035

7.514 0.030

6.578 0.057

6.354 0.062

6.119 0.037

6.666 0.044

6.325 0.065

7.135 0.095

6.769 0.081

7.184 0.063

6.762 0.001

8.094 0.098

6.981 0.060

6.630 0.040

7.607 0.043

5.308 0.096

6.277 0.061

5.746 0.078

6.891 0.078

5.824 0.033

5.479 0.095

6.001 0.014

6.594 0.046

5.763 0.037

5.648 0.103

5.134 0.094

5.806 0.064

5.468 0.118

6.078 0.020

5.549 0.047

6.320 0.070

6.012 0.121

7.213 0.085

5.909 0.081

5.708 0.078

6.899 0.147

6.535 0.082

2

12c

H

3

12e

H

F

4

12g

F

5

12h

12f

F

H

6

H

F

7

12aa

12ab

12ac

12m

12l

H

8

F

9

F

H

10

11

12

13

14

15

16

17

18

19

20

21

22

H

F

H

OCH₃

H

OCH₃

CH₃

H

F

12ae

12v

H

CH₃

CF₃

H

12af

12ag

12p

12r

CH₃

CF₃

Cl

H

Cl

H

12z

Cl

H

12ad

12ak

12y

Cl

H

F

H

Cl

F

H

F

H

12ah

Cl

H

7.835 0.081

a

b

Individually resynthesized from appropriate biphenyl-2-carbaldehydes and evaluated hydrazones. Data points for speciﬁc binding of NO711 (mean

SD from triplicate values) in the presence of diﬀerent concentrations of test compounds (M) resulted in binding curves for K_idetermination by

c

nonlinear regression. pK_iSEM obtained from three independent experiments,. pIC₅₀values from [³H]GABA uptake assays performed with

mGAT1 expressing HEK293 cells. Value SEM obtained from three independent experiments.

hydrazone 12g displaying a pK_iof 7.449

0.023 (Table 2,

outweighs the negative eﬀect of the 3′-F-substituent as the

aﬃnity of 12h is still far higher than that of the parent

compound 12a (Table 2, entry 1). In addition, 12aa (2′,6′-F₂,

Table 2, entry 7) revealed that a second F-substituent in 6′-

position is tolerated but does not improve the aﬃnity toward

mGAT1, expressed by a pK_iof 6.883 0.035 very close to the

entry 4). Comparing this eﬀect with the diﬂuoro substitution in

12h (2′,3′-F₂, Table 2, entry 5), 12f (3′,4′-F₂, Table 2, entry 6),

and 12aa (2′,6′-F₂, Table 2, entry 7), the pK_ivalues of the

compounds amounting to 6.690 0.036, 6.204 0.054, and

6.883

0.035, respectively, clearly reveals that the 2′,4′-F₂

substitution in 12g represents the most favorable substitution

pattern for the diﬂuoro substituted compounds. In contrast to

F-substituents in 2′- and 4′-position, a 3′-F-substituent

decreases aﬃnity compared to the parent compound 12a,

with hydrazone 12d reducing MS marker binding in the

deconvolution experiments far less (16 1%, 12d, Figure 2)

than the reference compound 12a (<5%). In line with that,

upon transition from the 2′-F-substituted 12c (pK_i= 6.914

0.061, Table 2, entry 2) to the 2′,3′-F₂hydrazone 12h, a

decrease in aﬃnity occurs (pK_i= 6.690 0.036, Table 2, entry

5). But the positive eﬀect of the 2′-F-substituent clearly

pK_iof 12c (2′-F₂pK_i= 6.914

0.061, Table 2, entry 2).

Accordingly, the 2′,4′,6′-triﬂuoro substituted derivative 12ab

(2′,4′,6′-F₃, Table 2, entry 8), displayed a similar aﬃnity, the

pK_ibeing 7.514 0.030, close to that of 12g (2′,4′-F₂, pK_i=

7.449 0.023, Table 2, entry 4), again indicating that a 6′-F-

substituent does not signiﬁcantly change aﬃnity. Obviously a 4-

F-substituent in the ﬁrst ring does not lead to a beneﬁcial eﬀect,

the aﬃnity of the 4,2′-diﬂuoro substituted hydrazone 12ac

(Table 2, entry 9) with a pK_iof 6.578 0.057 being lower than

that of the 2′-monoﬂuoro substituted hydrazone 12c (2′-F,

6.914 0.061, Table 2, entry 2). Like the 4-ﬂuoro substituent

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in 12ac, the 4-ﬂuoro substituent in hydrazone 12w also has a

negative eﬀect on the binding aﬃnity, which in the latter case

was even more pronounced reducing remaining MS marker

0.001, Table 2, entry 17). But this decrease was less

pronounced than that for the transition from 12p (2′-Cl,

Table 2, entry 16) to 12ad (4-F,2′-Cl, Table 2, entry 19).

Combining the 2′- and the 4′-Cl-substitution in one

compound led to the most potent biphenylhydrazone of this

study, compound 12z (2′,4′-Cl₂), with an aﬃnity in the lower

nanomolar range (pK_i= 8.094 0.098, Table 2, entry 18), as

the pK_iof this hydrazone derivative surpassed that of the parent

binding to only 13

2% in the deconvolution experiments,

thus not reaching the limit of 10%, which is the condition

hydrazones had to fulﬁll to be evaluated with regard to their pK_i

values.

A methoxy function in 6′-position of the 2′-ﬂuoro substituted

system (2′-F,6′-OCH₃, 12m, Table 2, entry 10) had an adverse

eﬀect on the aﬃnity, as well, the pK_ivalue of 12m (pK_i= 6.354

0.062) ranging distinctly below that of the 2′-ﬂuoro

monosubstituted compound 12c (pK_i= 6.914 0.061, Table

2, entry 2) and close to that of the parent compound 12a (pK_i

= 6.186 0.028, Table 2, entry 1). This was not unexpected as

the 2′-OCH₃substituted hydrazone 12o had reduced MS

marker binding in the deconvolution experiments no lower

than 19 1%. Anyway, the gain in binding aﬃnity eﬀected by

the 2′-ﬂuoro substituent in 12c (2′-F) was nearly completely

abolished by the additional 6′-OCH₃substituent (in 12m). A

similar eﬀect was observed for 12l. The additional 2′-OCH₃-

residue, by which this compound diﬀers from 12e, exhibiting a

4′-F-substituent, causes a pK_ireduction from 6.546 0.067 (of

12e, Table 2, entry 3) to 6.119 0.037 (12l, Table 2, entry

11), 12l thus being just as potent as the parent compound 12a

(pK_i= 6.186 0.028, Table 2, entry 1).

Compared to a 2′-F substituent (12c, 2′-F, pK_i= 6.914

0.061, Table 2, entry 2), the binding enhancing eﬀect of a 2′-

CH₃is less pronounced (12ae, pK_i= 6.666 0.044, Table 2,

entry 12). The 4′-CH₃substituted hydrazone 12v (4′-CH₃)

gave rise to a pK_iof 6.325 0.065 (Table 2, entry 13), the 4′-

CH₃substituent thus having slightly improved the potency of

the parent compound 12a (pK_i= 6.186 0.028, Table 2, entry

1) though the deconvolution value (percentage of remaining

MS marker binding) of 12v amounting to 10 1% had been

higher than that of 12a (<5%), suggesting the opposite eﬀect.

This discrepancy is possibly attributed to the uncertainties of

the experimental data that are commonly relatively large for

biological systems. The positive eﬀects of the 2′-methyl (as in

12ae, 2′-CH₃) and the 4′-methyl group (as in 12v, 4′-CH₃)

were combined in the 2′,4′-disubstituted hydrazone 12af (2′,4′-

CH₃, Table 2, entry 14), reaching an aﬃnity of pK_i= 7.135

0.095. Among the 2′,4′-disubstituted derivatives, 12ag (2′,4′-

(CF₃)₂) exhibiting two CF₃groups was also found to be more

potent than the parent compound 12a (pK_i= 6.186 0.028,

Table 2, entry 1) displaying a pK_iof 6.769 0.081 (Table 2,

entry 15) but less than the 2′,4′-dimethyl and 2′,4′-diﬂuoro

substituted hydrazones 12af and 12g (12af, 2′,4′-(CH₃)₂, pK_i=

7.135 0.095, Table 2, entry 14; 12g, 2′,4′-F₂, pK_i= 7.449

0.023, Table 2, entry 4).

compound 12a (pK_i= 6.186

0.028, Table 2, entry 1) by

almost 2 log units.

Further high aﬃnity ligands were obtained by mixed halogen

substitution of the 2′,4′-position of the biphenyl moiety in 12a.

Compound 12ah, the 2′-Cl,4′-F substituted compound, turned

out to be the second most potent biphenylhydrazone derivative

of this series (pK_i= 7.835 0.081, Table 2, entry 22) followed

by 12y (2′-F,4′-Cl), exhibiting the reverse halogen substitution

pattern, with a slightly diminished potency (pK_i= 7.607

0.043, Table 2, entry 21).

In conclusion of these results, substitution in 2′,4′-position of

the biphenyl moiety was most favorable to gain highly potent

derivatives of the parent compound 12a. Most of all, the

presence of halogen substitutents such as F and even more Cl

in these positions augmented binding aﬃnities remarkably. This

is best underlined by the high aﬃnity displayed by the 2′,4′-Cl₂

biphenylhydrazone 12z (2′,4′-Cl₂, pK_i= 8.094 0.098, Table

2, entry 18). Besides, as demonstrated by these results, the

screening data are in general in good accord with the results of

the MS binding assays and can thus already be considered a

good estimate for the potency of the studied compounds.

In addition to their binding aﬃnities (pK_ivalues) all

compounds were also characterized with respect to their

inhibitory potencies (IC₅₀) in functional [³H]GABA uptake

assays performed with mGAT1 expressing HEK293 cells. As

already stated in previous papers,²⁸the pIC₅₀values of the

uptake assay are generally lower than the pK_ivalues describing

the binding aﬃnity. This may be ascribed to the diﬀerent assay

conditions used for the [³H]GABA uptake and for the MS

binding assays. Most importantly MS binding assays are

performed in a buﬀer system containing 1 M NaCl whereas

for [³H]GABA uptake assays a physiological buﬀer with 120

mM NaCl is used.⁴⁵The high NaCl concentration in the MS

binding assays is needed to improve ligand aﬃnity of the MS

marker 6, which is otherwise, when NaCl is present in

physiological concentration (like in the buﬀer for the

[³H]GABA uptake assay), about 1 log unit lower^25,49under

which conditions these assays were more diﬃcult to perform

(to avoid uncontrolled loss of speciﬁcally bound MS marker 6

during the separation of the protein−ligand complex from the

rest of the incubation system).

The largest increase in aﬃnity by a single substituent was

observed for a 2′-Cl residue, the respective compound 12p (2′-

Cl, Table 2, entry 16) achieving a pK_iof 7.184 0.063 close to

that of the 2′,4′-diﬂuoro substituted derivative (12g, pK_i=

7.449 0.023, Table 2, entry 4). An additional 4-F-substituent

in the ﬁrst ring again gave rise to a reduced aﬃnity (12ad, 4-

F,2′-Cl pK_i= 6.981 0.060, Table 2, entry 19), in line with the

results described above. The aﬃnity of the 4′-Cl biphenylhy-

drazone 12r (4′-Cl) with a pK_iof 6.762 0.001 (Table 2, entry

17) was also higher than that of the 4′-F substituted analogue

12e (pK_i= 6.546 0.067, Table 2, entry 3). An additional 4-F

substituent again led to a decrease in aﬃnity, the pK_iof 12ak

(4-F,4′-Cl, pK_i= 6.630 0.040, Table 2, entry 20) being lower

than that of 12r with a single 4′-Cl substituent (pK_i= 6.762

As the high NaCl concentration (1 M) aﬀects the aﬃnity of

every test compound in a manner similar to the aﬃnity of the

MS marker 6, their pK_ivalues being a half to 1 log unit higher

than those measured in the presence of physiological NaCl

concentration, the diﬀerences observed between the pK_ivalues

from MS binding assays (in the presence of 1 M NaCl) and the

pIC₅₀values from [³H]GABA uptake assays (in the presence of

physiological NaCl concentration) can be largely ascribed to

this phenomenon. Regardless of that, the rank order of potency

that results from the pIC₅₀values of the [³H]GABA uptake

assays is in good accord with that resulting from the pK_ivalues

of the MS binding assays. Thus, in line with that, hydrazone

12z, which displays the highest binding aﬃnity (pK_i) for

mGAT1 of the compounds studied, turned out to also be the

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Table 3. Comparison of hydrazones 12c, 12p, 12g, 12z, 12ah with Carba Analogues 13c, 13p, 13g, 13z, 13ah

a

Data points for speciﬁc binding of NO711 (mean SD from triplicate values) in the presence of diﬀerent concentrations of test compounds (M)

b

resulted in binding curves for K_idetermination by nonlinear regression. pK_iSEM obtained from three independent experiments. pIC₅₀values

from [³H]GABA uptake assays performed with mGAT1 expressing HEK293 cells. Value SEM obtained from three independent experiments.

most potent inhibitor in the [³H]GABA uptake assay of

mGAT1 expressing HEK293 cells, its pIC₅₀amounting to 7.213

0.085 (Table 2, entry 18).

Carba Analogues for Biphenyl Derived Hydrazones.

The ﬁnal step of this study was aimed at the development of

stable analogues of the hydrazone derivatives that can substitute

the latter as lead structure for drug development, a purpose

hydrazones are in general not suitable for because of their

instability^28,33−35and various harmful eﬀects.³⁰⁻³²

0.037) deviated from the rank order of potency observed for

the hydrazone derivatives, this time the 2′-ﬂuoro derivative 13c

being more potent than the 2′-chloro derivative 13p. But

similar to the hydrazone derivatives 12c and 12p, the

diﬀerences in potencies were also very small for the carba

analogues 13c and 13p, rendering this change in the rank order

of potency less meaningful.

The carba analogues were ﬁnally also characterized with

respect to their potencies in [³H]GABA uptake assays (Table

3). Again the pIC₅₀values for the [³H]GABA uptake assays

were somewhat lower than the corresponding pK_ivalues

resulting from the MS binding assays, the reasons for this

phenomenon being most likely those given in the section

“Biphenyl-2-carbaldehyde Derived Hydrazone Hits”. The

sequence of potencies (pIC₅₀) was roughly in line with that

of the aﬃnities (pK_i) found for these compounds, 13c, 13p,

and 13g being nearly equally potent (13c, pIC₅₀= 5.477

A carba moiety in which the two nitrogen atoms of the

hydrazone function (CHN−NH) are replaced by carbon

atoms transforming said function into an allyl group (CH

CH−CH₂) was considered to be a suitable substitute that at

least with respect to the overall geometry should be able to

mimic the original functional group. For this transformation a

set of hydrazone derivatives was selected covering diverse

activities ranging from medium to highly potent. Thus, for the

hydrazones 12c, 12p, 12g, 12ah, and 12z the corresponding

carba analogues 13c, 13p, 13g, 13ah, and 13z were synthesized

(Table 3) and evaluated.

As shown in Table 3, the potencies of the hydrazone

derivatives 12c, 12p, 12g, 12ah, and 12z ranging from pK_iof

6.914 0.061 (12c) to 8.094 0.098 (12z) were not fully

reached by the carba analogues 13c, 13p, 13g, 13ah, and 13z,

the pK_ivalues of the latter compounds being roughly 1 log unit

lower than those of the corresponding hydrazone derivatives.

But the rank order of potency remained mostly unchanged.

Thus, also for the carba analogues, the 2′,4′-dichloro

substituted derivative 13z appeared to be the most potent

compound in this series with a pK_iof 6.930 0.021 followed

by the 2′-chloro-4′-ﬂuorocarba derivative 13ah (pK_iof 6.505

0.039) and the 2′,4′-diﬂuorocarba derivative 13g (pK_iof 6.228

0.014). The rank order of potency for the carba analogues

was in line with the respective rank order of potency for the

hydrazones. Only the 2′-ﬂuoro (13c, pK_i= 6.189 0.020) and

the 2′-chloro substituted carba analogue (13p, pK_i= 6.079

0.095; 13p, pIC₅₀= 5.448

0.032), whereas 13z showed an increased potency (13z, pIC₅₀

= 6.114 0.107). Only 13ah (pIC₅₀= 5.399 0.052) was

0.105, 13g, pIC₅₀= 5.547

slightly less potent than expected and equipotent to 13c, 13p,

and 13g.

According to the results described above, structure−activity

data obtained from screening of hydrazone libraries represent

valuable information regarding drug target binding which can

be used for the construction of stable analogues as lead

structures but should also be useful in guiding the optimization

of these compounds. The reduced potency of the carba

analogues versus the parent screening hits is attributed to the

lower polarity of the all-carbon spacer and possibly also to its

inability to participate in hydrogen bonds in contrast to the

hydrazone function.

CONCLUSION

■

Recently, competitive MS binding assays have been shown to

be applicable as a readout for the screening of compound

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1

parentheses. H NMR spectra were recorded on a JNMR-GX (JEOL,

400 or 500 MHz) at room temperature unless otherwise described.

DMSO-d₆was applied for at least 5 min to ultrasonic degassing before

libraries, generated by simple and most eﬃcient reactions

commonly used in dynamic combinatorial chemistry. A key

feature of this method is to take special means to render the

libraries pseudostatic in order to obtain well-deﬁned library

compositions with almost equal concentrations of test

compounds. Furthermore, by generation of the libraries in

the presence of the target under conditions appropriate for the

MS binding assay, library generation and screening are

performed in the same sample, thus enhancing the assay

performance. When this concept was applied to mGAT1 as

target employing pseudostatic hydrazone libraries, two hits,

hydrazones 12a and 12b, were identiﬁed as moderate

(submicromolar) binders of mGAT1. Both hits displayed a

biaryl system representing the lipophilic part that was attached

via a spacer to a more polar residue, in the present case a

nipecotic acid moiety that is characteristic of most mGAT1

inhibitors. The aim of the current study was to further improve

the aﬃnities for mGAT1 by optimizing the biphenyl system of

12a, one of the two hits that had been identiﬁed in the ﬁrst

screening round. Accordingly, focused hydrazone libraries

comprising compounds with diﬀerently substituted biphenyl

moieties were generated under pseudostatic conditions and

screened by the aforementioned method developed for

mGAT1 which is based on competitive MS binding experi-

ments utilizing NO711 (6) as native MS marker. The screening

of 9 biaryl focused pseudostatic hydrazone libraries comprising

36 hydrazones revealed 21 hydrazone hits with higher aﬃnities

than the original lead system 12a (pK_i= 6.186 0.028). The

most potent hit, 12z, displaying a 2′,4′-dichloro substituted

biphenyl moiety, exhibited an aﬃnity in the lower nanomolar

range, the pK_i= 8.094 0.098 being almost 2 log units higher

than of the original compound 12a. Actually, 2′,4′-disubstituted

biphenyl units appeared to be most favorable for increased

binding aﬃnities toward mGAT1 with Cl and F substituents

being most rewarding. A set of representative metastable

hydrazone derivatives was chosen to explore the suitability of

these compounds as models for the construction of stable

analogues as lead structures for further drug development. Of

ﬁve representative biphenylhydrazones the corresponding

stable carba analogues were synthesized and tested in

competitive MS binding experiments and [³H]GABA uptake

assays. Again, a 2′,4′-dichloro substituted compound, i.e., 13z,

was found to be the most potent mGAT1 binder (pK_i= 6.930

0.021), though the pK_ivalue of 13z was 1 log unit lower than

that of the hydrazone derivative 12z. This is possibly a result of

the reduced polarity of the carba analogue compared to the

corresponding hydrazone derivative. But pleasingly, the rank

order of potency of the ﬁve carba analogues in the binding

studies was the same as that of the corresponding hydrazone

derivatives. Thus, this study clearly demonstrated that screening

of focused hydrazone libraries under pseudostatic conditions is

a powerful tool to detect hits and that these can be successfully

used as a starting point for the development of almost equally

potent lead structures.

1

use. H chemical shifts were internally referenced to TMS or MeOH

for samples solved in D₂O or CD₃OD. The spectra were processed

with the NMR software MestReNova (version 5.1.1-3092, 2007,

Mestrelab Research S. L., Santiago de Compostela, Spain). Broadened

1

signals in H NMR spectra were supplemented by the index “br” (s_br,

d_br, t_br). IR spectroscopy was performed with an FTIR spectrometer

410 (Jasco). Samples were measured either as KBr pellets or as ﬁlms

on NaCl plates. A Hewlett-Packard 5989 A with 59.980 B particle

beam LC−MS interface was used for mass spectrometry (ionization:

+

chemical (CH₅) or electron impact (70 eV)). High resolution mass

spectrometry was carried out with a LTQ FT (ThermoFinnigan), FAB

(Xenon, 6 kV, MBA, reference PEG), or a JMS GCmate II (Jeol).

Purity testing was done by means of analytical HPLC on an Agilent

1100 instrument (G1329A ALS autosampler, G1316A column

compartment, G1315B DAD detector, G1312A binary pump,

G1322A degasser), equipped with a Lichrospher 100 RP-18 (5 μm)

in a LiChroCART 125-4 column, with elution at 1 mL/min with

phosphate buﬀer (5 mM NaH₂PO₄·H₂O, 5 mM Na₂HPO₄·2H₂O, pH

7.0) to CH₃CN 40:60. Carba analogues 13c, 13g, 13p, 13z, 13ah were

≥95% pure. Because of the tendency of hydrazones to hydrolyze,

necessitating speciﬁc means to render compound libraries pseudo-

static, HPLC analysis did not appear to be reasonable. Because of

autoxidation of hydrazones and hydrazines, starting material and

products were stored under Ar.

General Procedure for the Preparation of Hydrazones (GP1).

According to the published procedure,²⁸1 equiv of the corresponding

aldehyde in DMSO-d₆(c = 0.1 M) was added to hydrazine 10. For

practical reasons NMR spectra were directly measured from this

solution after at least ﬁve min. When all NMR measurements were

ﬁnished, DMSO-d₆was evaporated to give the desired hydrazone.⁴⁶

1

According to H NMR spectra, hydrazones existed to ≥98% as E-

isomers.

General Procedure for the Suzuki Coupling of 19 and

Phenylboronic Acid Derivatives (GP2). Under nitrogen atmos-

phere Pd(dppf)Cl₂·CH₂Cl₂(0.015 mmol, 5 mol %), the appropriate

phenylboronic acid derivative (0.33 mmol), and compound 19 (114

mg, 0.300 mmol) were dissolved in 1,4-dioxane (0.6 mL), K₂CO₃(124

mg, 0.90 mmol), and 0.3 mL of water. The resulting mixture was

heated to 60 °C and stirred for 3 h. After addition of water (5 mL) the

mixture was extracted three times with CH₂Cl₂(5 mL). The combined

organic layers were dried over Na₂SO₄, and the solvent was evaporated

in vacuo. The crude product was puriﬁed by ﬂash column

chromatography on silica gel (n-pentane/CH₂Cl₂/EtOAc = 8/1/1 +

2% Et₃N).

General Procedure for the Hydrolysis of N-Substituted

Piperidine-3-carboxylic Acid Ethyl Ester Derivatives 21 (GP3).

The chosen piperidine-3-carboxylic acid ethyl ester derivative (0.2

mmol) was dissolved in EtOH (0.8 mL) followed by addition of

NaOH (24.0 mg, 0.60 mmol, 0.050 mL, 12 M aqueous) (TLC

monitoring). Upon completion of the reaction (approximately 4 h)

water (5 mL) was added and the basic solution was washed with Et₂O

(5 mL). Afterward the pH of the aqueous phase was adjusted to pH 6

with 1 M HCl and phosphate buﬀer solution (1 M, pH 7) and the

product was extracted several times with CH₂Cl₂. The combined

organic layers were dried over Na₂SO₄, and the solvent was removed

in vacuo to give the appropriate product.

(E)-1-(2-{2-[1-(2′-Fluorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12c). According

to GP1 from hydrazine 10 (8.71 mg, 46.5 μmol) and 11c (9.31 mg,

46.5 μmol), 12c was obtained as colorless, amorphous solid (17.2 mg,

EXPERIMENTAL SECTION

■

1

yield 100%). H NMR (500 MHz, DMSO-d₆): δ 1.26−1.38 (m, 1H),

Chemistry. Solvents used were of analytical grade and freshly

destilled before use except for DMSO. Ethyl nipecotate was purchased

from Sigma-Aldrich and freshly destilled before use. Other purchased

reagents and reactants were used without further puriﬁcation. TLC

was carried out on precoated silica gel F₂₅₄glass plates (Merck). Flash

column chromatography (CC) was performed on Merck silica gel 60

(mesh 0.040−0.063 mm). Solvents used for elution are reported in

1.37−1.49 (m, 1H), 1.53−1.65 (m, 1H), 1.70−1.83 (m, 1H), 1.99 (t_br,

J = 9.5 Hz, 1H), 2.12 (t_br, J = 9.8 Hz, 1H), 2.38 (tt, J = 9.9/3.9 Hz,

1H), 2.42 (t, J = 6.8 Hz, 2H), 2.56−2.67 (m, 1H), 2.81 (d_br, J = 10.6

Hz, 1H), 3.11 (t, J = 6.6 Hz, 2H), 7.14−7.25 (s_br, 1H), 7.20 (dd, J =

7.6/1.2 Hz, 1H), 7.25−7.35 (m, 5H), 7.35−7.40 (m, 1H), 7.43−7.52

(m, 1H), 7.87 (dd, J = 7.9/1.2 Hz, 1H) ppm. ¹³C NMR (101 MHz,

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2

DMSO-d₆): δ 23.9, 26.4, 41.0, 45.4, 53.2, 55.3, 56.3, 115.5 (d, J_CF

=

Hz, 1H), 3.12 (t, J = 6.6 Hz, 2H), 7.10−7.18 (m, 1H), 7.19−7.27 (m,

2H), 7.27−7.34 (m, 3H), 7.37−7.44 (m, 1H), 7.44−7.52 (m, 1H),

7.84−7.89 (m, 1H) ppm. ¹³C NMR (126 MHz, DMSO-d₆): δ 23.9,

26.4, 40.9, 45.5, 53.2, 55.3, 56.4, 116.7 (d, ²J_CF= 17.1 Hz), 123.9, 124.8

3/4

22.1 Hz), 123.7, 124.5 (d,

Hz), 127.9, 129.7 (d, J_CF= 8.1 Hz), 130.4, 131.5, 131.8 (d,

J

= 3.6 Hz), 126.8, 127.2 (d, ²J_CF= 16.3

CF

3

3/4

J

=

CF

3.3 Hz), 132.8, 134.4, 158.9 (d, J_CF= 244.3 Hz), 175.0 ppm. ¹⁹F

NMR (471 MHz, DMSO-d₆): δ −115.00 ppm. MS (ESI+) m/z: 370

[M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for C₂₁H₂₅O₂N₃F,

370.1931; found, 370.1923.

1

3/4

2

(dd,

J

= 7.5/4.6 Hz), 126.8, 127.0 (m_CF), 128.4, 129.7 (d, J_CF

=

CF

3

12.8 Hz), 130.3, 131.0, 131.3 (s, J_CF_1/2= 2.5 Hz), 134.4, 146.8 (dd,

1/2

J

= 246.4/12.7 Hz,), 149.7 (dd, J_CF= 246.3/13.1 Hz), 175.0

CF

ppm. ¹⁹F NMR (471 MHz, DMSO-d₆): δ −140.82 (d, J = 23.21 Hz),

−138.54 (d, J = 27.07 Hz) ppm. MS (ESI+) m/z: 388.7 [M + H]⁺.

HRMS (ESI+): [M + H]⁺calcd for C₂₁H₂₄F₂N₃O₂, 388.1837; found,

388.1828.

(E)-1-(2-{2-[1-(4′-Fluorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12e). According

to GP1 from hydrazine 10 (8.55 mg, 45.6 mmol) and 11e (9.13 mg,

45.6 μmol), 12e was obtained as slightly yellow, amorphous solid

(16.9 mg, yield 100%). ¹H NMR (400 MHz, DMSO-d₆): δ 1.26−1.40

(m, 1H), 1.38−1.50 (m, 1H), 1.54−1.66 (m, 1H), 1.71−1.83 (m, 1H),

2.02 (t_br, J = 10.0 Hz, 1H), 2.15 (t_br, J = 10.2 Hz, 1H), 2.39 (tt, J = 8.9/

3.9 Hz, 1H), 2.45 (t, J = 6.7 Hz, 2H), 2.56−2.69 (m, 1H), 2.82 (d_br, J

= 10.3 Hz, 1H), 3.13 (t, J = 6.6 Hz, 2H), 7.01−7.18 (s_br, 1H), 7.17−

7.23 (m, 1H), 7.24−7.31 (m, 3H), 7.31−7.39 (m, 3H), 7.43 (s, 1H),

7.85 (dd, J = 7.7/1.3 Hz, 1H) ppm. ¹³C NMR (101 MHz, DMSO-d₆):

δ 23.9, 26.4, 41.0, 45.5, 53.2, 55.3, 56.4, 115.1 (d, ²J_CF= 21.0 Hz, 2C),

(E)-1-(2-{2-[1-(4′-Fluoro-2′-methoxybiphenyl-2-yl)-

methylidene]hydrazinyl}ethyl)piperidine-3-carboxylic Acid

(12l). According to GP1 from hydrazine 10 (13.2 mg, 70.3 μmol)

and 11l (16.2 mg, 70.3 μmol), 12l was obtained as colorless,

1

amorphous solid (28.1 mg, yield 99%). H NMR (500 MHz, DMSO-

d₆): δ 1.27−1.38 (m, 1H), 1.38−1.50 (m, 1H), 1.53−1.65 (m, 1H),

1.69−1.83 (m, 1H), 2.00 (t_br, J = 10.4 Hz, 1H), 2.14 (t_br, J = 9.8 Hz,

1H), 2.38 (tt, J = 9.7/3.9 Hz, 1H), 2.42 (t, J = 7.0 Hz, 2H), 2.56−2.67

(m, 1H), 2.81 (d_br, J = 11.3 Hz, 1H), 3.09 (t, J = 6.7 Hz, 2H), 3.71 (s,

3H), 6.84 (td, J = 8.4/2.5 Hz, 1H), 7.01 (dd, J = 11.5/2.4 Hz, 1H),

7.08 (dd, J = 7.6/1.3 Hz, 1H), 7.13 (dd, J = 8.3/7.0 Hz, 1H), 7.19 (s,

1H), 7.23 (td, J = 7.4/1.5 Hz, 1H), 7.26−7.33 (m, 1H), 7.81 (dd, J =

7.9/1.2 Hz, 1H) ppm. ¹³C NMR (126 MHz, DMSO-d₆): δ 23.9, 26.4,

3

124.2, 126.9, 127.4, 130.0, 131.3 (d, J_CF= 8.0 Hz, 2C), 132.0, 133.6,

136.2 (d, ⁴J_CF= 3.3 Hz), 138.2, 161.4 (s, ¹J_CF= 240.7 Hz), 175.0 ppm.

¹⁹F NMR (471 MHz, DMSO-d₆): δ −115.42 ppm. MS (ESI+) m/z:

370 [M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for C₂₁H₂₅O₂N₃F,

370.1931; found, 370.1923.

2

40.9, 45.5, 53.2, 55.3, 55.7, 56.4, 99.5 (d, J_CF= 25.7 Hz), 106.6 (d,

(E)-1-(2-{2-[1-(3′,4′-Diﬂuorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12f). According

to GP1 from hydrazine 10 (10.2 mg, 54.5 μmol) and 11f (11.9 mg,

54.5 μmol), 12f was obtained as a yellow, amorphous solid (21.1 mg,

4

²J_CF= 21.0 Hz), 123.3, 124.8 (d, J_CF= 2.8 Hz), 126.6, 127.2, 130.5,

3

131.8 (d, J_CF= 10.0 Hz), 132.7, 134.4, 135.3, 157.5 (d, J_CF= 10.2

Hz), 162.6 (d, J_CF= 243.5 Hz), 175.1 ppm. ¹⁹F NMR (471 MHz,

1

+

yield 100%). H NMR (500 MHz, DMSO-d₆): δ 1.26−1.39 (m, 1H),

DMSO-d₆): δ 111.80 (ddd, J = 11.4/8.4/7.2 Hz) ppm. MS (CI, CH₅)

m/z: 400 [M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for

C₂₂H₂₇N₃O₃F, 400.2036; found, 400.2029.

1.38−1.50 (m, 1H), 1.55−1.65 (m, 1H), 1.71−1.83 (m, 1H), 2.01 (t_br,

J = 9.5 Hz, 1H), 2.14 (t_br, J = 9.9 Hz, 1H), 2.39 (tt, J = 9.9/3.9 Hz,

1H), 2.45 (t, J = 6.7 Hz, 2H), 2.58−2.68 (m, 1H), 2.84 (d_br, J = 9.7

Hz, 1H), 3.15 (t, J = 6.6 Hz, 2H), 7.13−7.18 (m, 1H), 7.19−7.26 (s_br,

1H), 7.23 (dd, J = 7.6/1.3 Hz, 1H), 7.29 (td, J = 7.4/1.4 Hz, 1H), 7.35

(td, J = 7.7/1.1 Hz, 1H), 7.39−7.46 (m, 1H), 7.44 (s, 1H), 7.52 (dt, J

= 10.8/8.5 Hz, 1H), 7.85 (dd, J = 7.8/1.0 Hz, 1H) ppm. ¹³C NMR

(101 MHz, DMSO-d₆): δ 23.9, 26.5, 40.9, 45.4, 53.2, 55.3, 56.4, 117.3

(E)-1-(2-{2-[1-(2′-Fluoro-6′-methoxybiphenyl-2-yl)-

methylidene]hydrazinyl}ethyl)piperidine-3-carboxylic Acid

(12m). According to GP1 from hydrazine 10 (4.72 mg, 25.5 μmol)

and 11m (5.80 mg, 25.2 μmol), 12m was obtained as a yellow,

amorphous solid (10.8 mg, yield 100%). ¹H NMR (500 MHz, DMSO-

d₆): δ 1.25−1.36 (m, 1H), 1.35−1.48 (m, 1H), 1.52−1.64 (m, 1H),

1.70−1.81 (m, 1H), 1.96 (t_br, J = 9.9 Hz, 1H), 2.09 (t_br, J = 9.1 Hz,

1H), 2.32−2.39 (m, 1H), 2.39 (t, J = 6.7 Hz, 2H), 2.55−2.65 (m, 1H),

2.79 (d_br, J = 9.7 Hz, 1H), 3.07 (t, J = 6.6 Hz, 2H), 3.70 (s, 3H), 6.90

(t, J = 8.6 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 7.06−7.14 (s_br, 1H), 7.09

(dd, J = 7.6/1.0 Hz, 1H), 7.15 (s, 1H), 7.24 (td, J = 7.5/1.4 Hz, 1H),

7.31 (td, J = 7.7/1.0 Hz, 1H), 7.41 (td, J = 8.4/7.1 Hz, 1H), 7.83 (d, J

= 7.9 Hz, 1H) ppm. ¹³C NMR (101 MHz, DMSO-d₆): δ 23.9, 26.4,

40.9, 45.4, 53.2, 55.3, 55.9, 56.3, 107.3, 107.6 (d, 1 C, ²J_CF= 22.9 Hz),

2

(d, J_CF= 16.9 Hz), 118.4 (d, J_CF= 16.9 Hz), 124.2, 126.4 (d, J_CF

=

6.4/3.4 Hz), 126.9, 127.8, 130.0, 131.5, 133.6, 137.1 (d, J_CF= 1.4 Hz),

137.4 (dd, J_CF= 6.1/3.8 Hz), 148.9 (d, ^1/2J_CF= 245.7/12.8 Hz), 149.0

1/2

(d,

J

CF

= 245.7/12.8 Hz), 175.1 ppm. ¹⁹F NMR (471 MHz, DMSO-

d₆): δ −140.87 (m, 1 F), −138.46 (m, 1 F) ppm. MS (ESI+) m/z: 388

[M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for C₂₁H₂₄O₂N₃F₂,

388.1837; found, 388.1828.

(E)-1-(2-{2-[1-(2′,4′-Diﬂuorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12g). According

to GP1 from hydrazine 10 (10.8 mg, 57.9 μmol) and 11g (12.6 mg,

57.9 μmol), 12g was obtained as a yellow, amorphous solid (22.6 mg,

2

116.0 (d, J_CF= 19.41 Hz), 123.4, 126.5, 127.7, 128.9, 129.8 (d, 1 C,

³J_CF= 19.4 Hz), 131.0, 131.9, 134.9, 157.7 (d, ³J_CF= 7.3 Hz), 159.5 (d,

¹J_CF= 241.6 Hz), 175.0 ppm. ¹⁹F NMR (471 MHz, DMSO-d₆): δ

1

−113.70 (t, J = 7.8 Hz) ppm. MS (ESI+) m/z: 400 [M + H]⁺. HRMS

(ESI+): [M + H]⁺calcd for C₂₂H₂₇O₃N₃F, 400.2036; found, 400.2028.

(E)-1-(2-{2-[1-(2′-Chlorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12p). According

to GP1 from hydrazine 10 (12.7 mg, 68.1 μmol) and 11p (14.8 mg,

68.4 μmol), 12p was obtained as a yellow, amorphous solid (26.3 mg,

yield 100%). H NMR (400 MHz, DMSO-d₆): δ 1.25−1.51 (m, 2H),

1.53−1.66 (m, 1H), 1.70−1.83 (m, 1H), 1.99 (t_br, J = 10.4 Hz, 1H),

2.12 (t_br, J = 10.6 Hz, 1H), 2.30−2.48 (m, 3H), 2.62 (d_br, J = 11.8 Hz,

1H), 2.82 (d_br, J = 9.7 Hz, 1H), 3.12 (t, J = 6.4 Hz, 2H), 7.12−7.25

(m, 3H), 7.25−7.33 (m, 2H), 7.33−7.45 (m, 3H), 7.86 (d, J = 7.9 Hz,

1H) ppm. ¹³C NMR (126 MHz, DMSO-d₆): δ 23.9, 26.4, 40.9, 45.4,

53.2, 55.3, 56.3, 103.9 (t, ²J_CF= 26.2 Hz), 111.6 (dd, ^2/4J_CF= 21.3/3.4

1

yield 100%). H NMR (500 MHz, DMSO-d₆): δ 1.26−1.37 (m, 1H),

2/4

Hz), 123.8, (dd,

J

= 15.6/3.5 Hz) 123.8, 126.8, 128.1, 130.5,

1.37−1.48 (m, 1H), 1.54−1.63 (m, 1H), 1.69−1.82 (m, 1H), 1.97 (t_br,

J = 9.9 Hz, 1H), 2.04−2.19 (m, 1H), 2.32−2.44 (m, 3H), 2.60 (d_br, J =

10.5 Hz, 1H), 2.80 (d_br, J = 10.8 Hz, 1H), 3.08 (t, J = 6.6 Hz, 2H),

7.09−7.13 (m, 1H), 7.12 (s, 1H), 7.18 (s_br, 1H), 7.25−7.33 (m, 2H),

7.33−7.39 (m, 1H), 7.40−7.47 (m, 2H), 7.53−7.61 (m, 1H), 7.86 (dd,

J = 8.0/0.7 Hz, 1H) ppm. ¹³C NMR (126 MHz, DMSO-d₆): δ 23.9,

26.4, 40.9, 45.3, 53.2, 55.3, 56.2, 123.5, 126.6, 127.2, 127.9, 129.2,

129.3, 129.8, 131.1, 131.7, 132.4, 134.1, 136.5, 138.6, 175.0 ppm. MS

(ESI+) m/z: 386 [M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for

C₂₁H₂₅ClN₃O₂, 386.1635; found, 368.1629.

CF

3/3

131.2, 131.9, 132.8 (dd,

J

= 9.6/4.8 Hz), 134.5, 159.0 (dd, ^1/3J_CF

=

CF

1/3

246.6/12.4 Hz), 161.8 (dd,

J

= 246.6/11.9 Hz), 175.1 ppm. ¹⁹F

CF

NMR (471 MHz, DMSO-d₆): δ −110.87 (dt, J = 16.0/8.2 Hz),

−110.40 (q, J = 8.0 Hz) ppm. MS (ESI+) m/z: 388.2 [M + H]⁺.

HRMS (ESI+): [M + H]⁺calcd for C₂₁H₂₄F₂N₃O₂, 388.1837; found,

388.1829.

(E)-1-(2-{2-[1-(2′,3′-Diﬂuorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12h). According

to GP1 from hydrazine 10 (8.78 mg, 46.9 μmol) and 11h (10.2 mg,

46.9 μmol), 12h was obtained as a yellow, amorphous solid (18.0 mg,

(E)-1-(2-{2-[1-(4′-Chlorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12r). According

to GP1 from hydrazine 10 (9.65 mg, 51.6 μmol) and 11r (11.1 mg,

51.6 μmol), 12r was obtained as a yellow, amorphous solid (20.0 mg,

1

yield 99%). H NMR (500 MHz, DMSO-d₆): δ 1.25−1.38 (m, 1H),

1.38−1.50 (m, 1H), 1.54−1.65 (m, 1H), 1.71−1.83 (m, 1H), 1.99 (t_br,

J = 9.6 Hz, 1H), 2.12 (t_br, J = 9.7 Hz, 1H), 2.38 (tt, J = 9.8/3.7 Hz,

1H), 2.42 (t, J = 6.7 Hz, 2H), 2.57−2.67 (m, 1H), 2.81 (d_br, J = 10.4

1

yield 100%). H NMR (500 MHz, DMSO-d₆): δ 1.28−1.39 (m, 1H),

1334

dx.doi.org/10.1021/jm301800j | J. Med. Chem. 2013, 56, 1323−1340

Journal of Medicinal Chemistry

Article

1.39−1.50 (m, 1H), 1.55−1.66 (m, 1H), 1.69−1.84 (m, 1H), 2.02 (t_br,

J = 9.6 Hz, 1H), 2.15 (t_br, J = 10.0 Hz, 1H), 2.39 (tt, J = 9.9/3.8 Hz,

1H), 2.45 (t, J = 6.7 Hz, 2H), 2.58−2.67 (m, 1H), 2.83 (d_br, J = 11.6

Hz, 1H), 3.14 (t, J = 6.6 Hz, 2H), 7.20 (s_br, 1H), 7.21 (dd, J = 7.6/1.3

Hz, 1H), 7.29 (td, J = 7.4/1.5 Hz, 1H), 7.31−7.37 (m, 3H), 7.43 (s,

1H), 7.49−7.54 (m, 2H), 7.85 (dd, J = 7.8/1.3 Hz, 1H) ppm. ¹³C

NMR (126 MHz, DMSO-d₆): δ 23.9, 26.4, 40.9, 45.5, 53.2, 55.3, 56.4,

124.3, 126.9, 127.6, 128.2 (2C), 129.8, 131.2 (2C), 131.8, 132.0, 133.5,

137.9, 138.7, 175.0 ppm. MS (ESI+) m/z: 386.1 [M + H]⁺. HRMS

(ESI+): [M + H]⁺calcd for C₂₁H₂₅ClN₃O₂, 386.1635; found,

386.1629.

(E)-1-(2-{2-[1-(4′-Methylbiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12v). According

to GP1 from hydrazine 10 (11.1 mg, 59.5 μmol) and 11v (11.7, 59.5

μmol), 12v was obtained as colorless, amorphous solid (21.6 mg, yield

100%). ¹H NMR (500 MHz, DMSO-d₆): δ 1.26−1.39 (m, 1H), 1.39−

1.51 (m, 1H), 1.53−1.66 (m, 1H), 1.69−1.84 (m, 1H), 2.01 (t_br, J =

10.2 Hz, 1H), 2.15 (t_br, J = 10.3 Hz, 1H), 2.36 (s, 3H), 2.39 (tt, J =

9.9/2.8 Hz, 1H), 2.45 (t, J = 6.5 Hz, 2H), 2.58−2.67 (m, 1H), 2.82

(d_br, J = 9.5 Hz, 1H), 3.13 (t, J = 6.7 Hz, 2H), 7.08 (s, 1H), 7.15−7.23

(m, 3H), 7.23−7.34 (m, 4H), 7.47 (s, 1H), 7.78−7.87 (m, 1H) ppm.

¹³C NMR (126 MHz, DMSO-d₆): δ 20.6, 23.9, 26.4, 40.9, 45.5, 53.2,

(tt, J = 8.4/6.6 Hz, 1H), 7.87 (dd, J = 8.0/1.0 Hz, 1H) ppm. ¹³C NMR

(126 MHz, DMSO-d₆): δ 23.9, 26.4, 40.9, 45.4, 53.2, 55.3, 56.3, 111.7

(d, ²J_CF= 25.8 Hz, 2C), 116.3 (t, ²J_CF= 21.8 Hz), 124.1, 125.8, 126.7,

3

1

128.6, 130.3 (t, J_CF= 10.4 Hz), 130.8, 130.9, 135.1, 159.4 (d, J_CF

=

245.4 Hz, 2C), 175.0 ppm. ¹⁹F NMR (471 MHz, DMSO-d₆): δ

−112.40 (t, J = 6.5 Hz) ppm. MS (ESI+) m/z: 388.3 [M + H]⁺.

HRMS (ESI+): [M + H]⁺calcd for C₂₁H₂₄N₃O₂F₂, 388.1837; found,

388.1834.

(E)-1-(2-{2-[1-(2′,4′,6′-Triﬂuorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12ab). According

to GP1 from hydrazine 10 (10.9 mg, 58.4 μmol) and 11ab (13.8 mg,

58.4 μmol), 12ab was obtained as a yellow, amorphous solid (24.5 mg,

1

yield 100%). H NMR (500 MHz, DMSO-d₆): δ 1.27−1.39 (m, 1H),

1.38−1.50 (m, 1H), 1.53−1.66 (m, 1H), 1.68−1.85 (m, 1H), 1.99 (t_br,

J = 9.8 Hz, 1H), 2.13 (t_br, J = 10.2 Hz, 1H), 2.34−2.45 (m, 3H), 2.55−

2.69 (m, 1H), 2.82 (d_br, J = 10.7 Hz, 1H,), 3.11 (t, J = 6.4 Hz, 2H),

7.22 (dd, J = 7.6/0.9 Hz, 1H), 7.19−7.28 (s_br, 1H), 7.27 (s, 1H),

7.29−7.36 (m, 3H), 7.38−7.46 (m, 1H), 7.81−7.87 (m, 1H) ppm. ¹³C

NMR (126 MHz, DMSO-d₆): δ 23.9, 26.4, 40.9, 45.5, 53.2, 55.3, 56.3,

2

100.7 (t, J_CF= 28.0 Hz, 2C), 113.2 (td, ^2/4J_CF= 22.2/4.8 Hz), 124.4,

124.9, 126.8, 128.8, 130.9, 131.0, 135.2, 159.6 (ddd, J_CF= 246.1/15.5/

10.1 Hz, 2C), 161.7 (dt, J_CF= 246.1/15.9 Hz), 175.0 ppm. ¹⁹F NMR

(471 MHz, DMSO-d₆): δ −109.39 (t, J = 6.8 Hz), −108.58 (tt, J =

9.3/6.4 Hz) ppm. MS (ESI+) m/z: 406.0 [M + H]⁺. HRMS (ESI+):

[M + H]⁺calcd for C₂₁H₂₃F₃N₃O₂, 406.1742; found, 406.1735.

(E)-1-(2-{2-[1-(4,2′-Diﬂuorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12ac). According

to GP1 from hydrazine 10 (9.55 mg, 51.0 μmol) and 11ac (11.1 mg,

51.0 μmol), 12ac was obtained as a yellow, amorphous solid (21.0 mg,

55.3, 56.4, 124.2, 126.9, 127.1, 128.8 (2C), 129.2 (2C), 129.9, 132.5,

133.4, 136.2, 136.9, 139.3, 175.0 ppm. MS (ESI+) m/z: 366.2 [M +

H]⁺. HRMS (ESI+): [M + H]⁺calcd for C₂₂H₂₈N₃O₂, 366.2182;

found, 366.2174.

(E)-1-(2-{2-[1-(4′-Chloro-2′-ﬂuorobiphenyl-2-yl)-

methylidene]hydrazinyl}ethyl)piperidine-3-carboxylic Acid

(12y). According to GP1 from hydrazine 10 (9.40 mg, 50.2 μmol)

and 11y (11.8 mg, 50.3 μmol), 12y was obtained as a yellow,

amorphous solid (20.3 mg, yield 100%). ¹H NMR (500 MHz, DMSO-

d₆): δ 1.28−1.39 (m, 1H), 1.38−1.50 (m, 1H), 1.54−1.66 (m, 1H),

1.71−1.84 (m, 1H), 2.00 (t_br, J = 9.9 Hz, 1H), 2.14 (t_br, J = 10.1 Hz,

1H), 2.39 (tt, J = 9.8/3.8 Hz, 1H), 2.43 (t, J = 6.8 Hz, 2H), 2.57−2.68

(m, 1H), 2.82 (d_br, J = 9.2 Hz, 1H), 3.12 (t, J = 6.6 Hz, 2H), 7.14−

7.25 (s_br, 1H), 7.19 (dd, J = 7.7/1.1 Hz, 1H), 7.27−7.29 (m, 1H), 7.30

(dd, J = 7.4/1.3 Hz, 1H), 7.33−7.43 (m, 3H), 7.53 (dd, J = 9.7/1.9 Hz,

1

yield 100%). H NMR (500 MHz, DMSO-d₆): δ 1.26−1.38 (m, 1H),

1.38−1.49 (m, 1H), 1.53−1.65 (m, 1H), 1.70−1.84 (m, 1H), 1.99 (t_br,

J = 9.5 Hz, 1H), 2.12 (t_br, J = 10.0 Hz, 1H), 2.38 (tt, J = 9.9/3.8 Hz,

1H), 2.43 (t, J = 6.7 Hz, 2H), 2.58−2.66 (m, 1H), 2.81 (d, J = 9.8 Hz,

1H), 3.13 (t_br, J = 6.2 Hz, 2H), 7.11 (td, J = 8.4/2.8 Hz, 1H), 7.20 (t, J

= 2.3 Hz, 1H), 7.25 (dd, J = 8.5/5.9 Hz, 1H), 7.28−7.35 (m, 3H),

7.43−7.51 (m, 2H), 7.56 (dd, J = 10.8/2.8 Hz, 1H) ppm. ¹³C NMR

(126 MHz, DMSO-d₆): δ 23.9, 26.4, 40.9, 45.3, 53.2, 55.3, 56.2, 109.2

1H), 7.85 (dd, J = 8.0/1.0 Hz, 1H) ppm. ¹³C NMR (126 MHz,

2

(d, J_CF= 22.9 Hz), 113.6 (d, J_CF= 22.1 Hz), 115.5 (d, J_CF= 22.1

2

Hz), 124.6 (d, ⁴J_CF= 3.5 Hz), 126.3 (d, ²J_CF= 16.1 Hz), 128.8 (d, ⁴J_CF

DMSO-d₆): δ 23.9, 26.4, 40.9, 45.5, 53.2, 55.3, 56.3, 116.1 (d, J_CF

=

4

2

= 2.7 Hz), 129.4 (m_CF), 129.9 (d, ³J_CF= 8.1 Hz), 132.0 (d, J_CF= 3.2

4

26.3 Hz), 123.9, 124.8 (d, J_CF= 3.7 Hz), 126.4 (d, J_CF= 16.4 Hz),

3

Hz), 132.6 (d, ³J_CF= 8.5 Hz), 137.0 (d, ³J_CF= 9.0 Hz), 159.0 (s-d, ¹J_CF

= 244.2 Hz), 162.0 (s-d, ¹J_CF= 244.0 Hz), 175.0 ppm. ¹⁹F NMR (471

MHz, DMSO-d₆): δ −115.08 to −114.96 (td, J = 7.1/3.5 Hz),

−114.06 to −113.90 (m) ppm. MS (ESI+) m/z: 388.3 [M + H]⁺.

HRMS (ESI⁺): [M + H]⁺calcd for C₂₁H₂₄N₃O₂F₂, 388.1837; found,

388.1833.

126.8, 128.3, 130.3, 131.2, 131.6, 133.0 (d, J_CF= 4.0 Hz), 133.1 (d,

³J_CF= 10.4 Hz), 134.4, 158.9 (d, J_CF= 248.0 Hz), 175.0 ppm. ¹⁹F

1

NMR (471 MHz, DMSO-d₆): δ −111.84 (m) ppm. MS (ESI+) m/z:

404.2 [M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for C₂₁H₂₄ClFN₃O₂,

404.1541; found, 404.1534.

(E)-1-(2-{2-[1-(2′,4′-Dichlorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12z). According

to GP1 from hydrazine 10 (18.2 mg, 97.0 μmol) and 11z (24.4 mg,

97.0 μmol), 12z was obtained as a yellow, amorphous solid (40.6 mg,

yield 99%). ¹H NMR (500 MHz, DMSO-d₆, 60 °C): δ 1.28−1.50 (m,

2H), 1.56−1.66 (m, 1H), 1.72−1.83 (m, 1H), 2.02 (t, J = 9.1 Hz, 1H),

2.16 (t, J = 10.3 Hz, 1H), 2.34−2.46 (m, 3H), 2.55−2.64 (m, 1H),

2.80 (dd, J = 11.1/3.3 Hz, 1H), 3.10 (t, J = 6.7 Hz, 2H), 7.10 (dd, J =

7.6/1.0 Hz, 1H), 7.16 (s, 1H), 7.28 (td, J = 7.5/1.4 Hz, 1H), 7.32 (d, J

= 8.2 Hz, 1H), 7.34−7.39 (m, 1H), 7.48 (dd, J = 8.2/2.1 Hz, 1H), 7.68

(d, J = 2.1 Hz, 1H), 7.83 (dd, J = 8.0/1.2 Hz, 1H) ppm. ¹³C NMR

(126 MHz, DMSO-d₆, 60 °C): δ 23.9, 26.5, 41.1, 45.7, 53.2, 55.4, 56.4,

124.0, 126.6, 127.3, 128.1, 128.7, 129.7, 131.3, 132.9, 133.0, 133.6,

134.4, 135.4, 138.0, 174.9 ppm. ¹⁹F NMR (471 MHz, DMSO-d₆): δ

−111.84 (m) ppm. MS (ESI+) m/z: 420 [M + H]⁺. HRMS (ESI+):

[M + H]⁺calcd for C₂₁H₂₄N₃O₂Cl₂, 420.1246; found, 420.1210.

(E)-1-(2-{2-[1-(2′,6′-Diﬂuorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12aa). According

to GP1 from hydrazine 10 (6.51 mg, 34.7 μmol) and 11aa (7.57 mg,

34.7 μmol), 12aa was obtained as a yellow, amorphous solid (13.3 mg,

(E)-1-(2-{2-[1-(2′-Chloro-4-ﬂuorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12ad). According

to GP1 from hydrazine 10 (7.17 mg, 38.3 μmol) and 11ad (8.99 mg,

38.3 μmol), 12ad was obtained as a yellow, amorphous solid (16.2 mg,

1

yield 100%). H NMR (500 MHz, DMSO-d₆): δ 1.26−1.37 (m, 1H),

1.37−1.47 (m, 1H), 1.54−1.63 (m, 1H), 1.70−1.82 (m, 1H), 1.97 (t_br,

J = 10.2 Hz, 1H), 2.10 (t_br, J = 10.1 Hz, 1H), 2.32−2.44 (m, 3H),

2.56−2.63 (m, 1H), 2.79 (d_br, J = 9.2 Hz, 1H), 3.05−3.13 (m, 2H),

7.03 (d, J = 2.0 Hz, 1H), 7.10 (td, J = 8.4/2.8 Hz, 1H), 7.13−7.19 (m,

1H), 7.28−7.34 (m, 1H), 7.40−7.49 (m, 3H), 7.54 (dd, J = 10.8/2.7

Hz, 1H), 7.56−7.59 (m, 1H) ppm. ¹³C NMR (126 MHz, DMSO-d₆):

2

δ 23.9, 26.4, 40.9, 45.2, 53.1, 55.3, 56.1, 108.9 (d, J_CF= 23.1 Hz),

113.3 (d, ²J_CF= 22.3 Hz), 127.2, 129.2 (m_CF), 129.3 (d, ⁴J_CF= 2.4 Hz),

129.5, 131.8, 132.0 (d, ³J_CF= 8.50 Hz), 132.5 (d, ⁴J_CF= 3.0 Hz), 132.6,

136.9 (d, 1 C, ³J_CF= 8.2 Hz), 137.7, 161.8 (d, ¹J_CF= 243.3 Hz), 175.0

ppm. ¹⁹F NMR (471 MHz, DMSO-d₆): δ −114.03 ppm. MS (ESI+)

m/z: 404.3 [M + H]⁺. HRMS (ESI⁺): [M + H]⁺calcd for

C₂₁H₂₄N₃O₂F₂, 404.1541; found, 404.1538.

(E)-1-(2-{2-[1-(2′-Methylbiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12ae). According

to GP1 from hydrazine 10 (10.3 mg, 58.4 μmol) and 11ae (11.5 mg,

58.4 μmol), 12ae was obtained as a yellow, amorphous solid (21.3 mg,

1

yield 99%). H NMR (500 MHz, DMSO-d₆): δ 1.26−1.50 (m, 2H),

1.53−1.65 (m, 1H), 1.70−1.83 (m, 1H), 1.99 (t_br, J = 9.4 Hz, 1H),

2.13 (t_br, J = 10.1 Hz, 1H), 2.33−2.45 (m, 3H), 2.56−2.66 (m, 1H),

2.81 (d_br, J = 9.7 Hz, 1H), 3.10 (t, J = 6.7 Hz, 2H), 7.14−7.27 (m,

5H), 7.31 (td, J = 7.5/1.3 Hz, 1H), 7.41 (td, J = 7.7/1.0 Hz, 1H), 7.52

1

yield 100%). H NMR (500 MHz, DMSO-d₆): δ 1.26−1.37 (m, 1H),

1.36−1.47 (m, 1H), 1.53−1.63 (m, 1H), 1.71−1.81 (m, 1H), 1.93−

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2

2.02 (m, 1H), 2.00 (s, 3H), 2.06−2.18 (m, 1H), 2.33−2.40 (m, 1H),

2.40 (t, J = 6.7 Hz, 2H), 2.55−2.63 (m, 1H), 2.74−2.84 (m, 1H), 3.07

(t, J = 6.7 Hz, 2H), 7.03−7.15 (m, 3H), 7.10 (s, 1H), 7.22−7.35 (m,

5H), 7.85 (dd, J = 7.8/1.0 Hz, 1H) ppm. ¹³C NMR (126 MHz,

DMSO-d₆): δ 19.7, 23.8, 26.4, 40.9, 45.4, 53.2, 55.3, 56.3, 123.4, 125.7,

126.7, 127.2, 127.4, 129.5, 129.7, 131.7, 133.8, 135.4, 138.9, 139.6,

175.0 ppm. MS (ESI+) m/z: 366.2 [M + H]⁺. HRMS (ESI+): [M +

H]⁺calcd for C₂₂H₂₈N₃O₂, 366.2182; found, 366.2175.

(E)-1-(2-{2-[1-(2′,4′-Dimethylbiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12af). According

to GP1 from hydrazine 10 (17.6 mg, 94.1 μmol) and 11af (19.7 mg,

94.1 μmol), 12af was obtained as a yellow, amorphous solid (38.5 mg,

yield 100%). ¹H NMR (500 MHz, DMSO-d₆, 60 °C): δ 1.29−1.40 (m,

1H), 1.38−1.49 (m, 1H), 1.53−1.65 (m, 1H), 1.69−1.82 (m, 1H),

1.96 (s, 3H), 1.98−2.05 (m, 1H), 2.12−2.20 (m, 1H), 2.32 (s, 3H),

2.33−2.44 (m, 3H), 2.54−2.62 (m, 1H), 2.74−2.82 (m, 1H), 3.08 (t, J

= 6.7 Hz, 2H), 6.96 (d, J = 7.6 Hz, 1H), 7.01−7.08 (m, 2H), 7.10 (s,

1H), 7.13 (s, 1H), 7.24 (td, J = 7.4/1.4 Hz, 1H), 7.29 (td, J = 7.4/0.9

Hz, 1H), 7.83 (dd, J = 7.8/1.1 Hz, 1H) ppm. ¹³C NMR (126 MHz,

DMSO-d₆, 60 °C): δ 19.6, 20.6, 23.9, 26.5, 41.0, 45.7, 53.2, 55.3, 56.4,

123.6, 126.2, 126.7, 127.0, 129.5, 129.7, 130.4, 132.4, 134.2, 135.2,

136.4, 136.8, 139.1, 174.8 ppm. MS (ESI+) m/z: 380.5 [M + H]⁺.

HRMS (ESI+): [M + H]⁺calcd for C₂₃H₃₀N₃O₂, 379.2260; found,

380.2334.

DMSO-d₆): δ 23.9, 26.4, 41.0, 45.3, 53.2, 55.3, 56.3, 109.5 (d, J_CF

=

2

4

22.7 Hz) 113.7 (d, J_CF= 22.1 Hz), 128.3 (2C), 129.7 (d, J_CF= 2.7

3

4

Hz), 131.3 (2C), 132.1 (d, J_CF= 8.7 Hz), 132.1, 134.1 (d, J_CF= 2.5

Hz), 136.2, 137.8, 161.7 (d, J_CF= 244.3 Hz), 175.0 ppm. ¹⁹F NMR

1

(471 MHz, DMSO-d₆): δ −114.45 (m) ppm. MS (ESI+) m/z: 404.2

[M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for C₂₁H₂₄ClFN₃O₂,

404.1541; found, 404.1539.

[(E)-5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pent-1-

enyl]-4-methylbenzene Sulfonate (17). Chlorobis-

(cyclopentadienyl)hydridozirconium (258 mg, 1.00 mmol) was

added to a mixture of pent-4-ynyl-4-methylbenzenesulfonate⁴³(2.38

g, 10.0 mmol) and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.41 g,

11.0 mmol). The mixture was stirred at room temperature for 48 h and

afterward diluted with CH₂Cl₂. The solution was ﬁltered through a

short pad of silica gel (2 cm) and washed with CH₂Cl₂. The ﬁltrate

was concentrated and dried in vacuo to give 17 as a colorless, high

viscous liquid (3.48 g, 95%). ¹H NMR (400 MHz, CDCl₃): δ 1.24 (s,

12H), 1.75 (p, J = 6.9 Hz, 2H), 2.15 (q, J = 6.9 Hz, 2H), 2.44 (s, 3H),

4.01 (t, J = 6.4 Hz, 2H), 5.33 (dt, J = 17.9/1.4 Hz, 1H), 6.47 (dt, J =

18.0/6.4 Hz, 1H), 7.33 (dd, J = 8.0/0.6 Hz, 2H), 7.77 (d, J = 8.4 Hz,

2H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 21.6, 24.7 (4C), 27.3, 31.3,

69.8, 83.1 (2C), 120.0,⁴⁷127.9 (2C), 129.8 (2C), 133.0, 144.7, 151.7

ppm. MS (ESI+) m/z: 367.1 [M + H]⁺. HRMS (EI, 70 eV): M⁺calcd

for C₁₈H₂₇BO₅S, 366.1672; found, 366.1670.

Ethyl 1-[(E)-5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-

pent-1-enyl]piperidine-3-carboxylate (18). Ethyl nipecotate (1.96

g, 12.5 mmol) was added to [(E)-5-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)pent-4-enyl]-4-methylbenzene sulfonate (1.83 g,

5.00 mmol), and the mixture was stirred at room temperature for

16 h. Then water was added, and the aqueous phase was extracted with

CH₂Cl₂. The organic phase was washed with water and concentrated

under reduced pressure. The residue was dissolved in Et₂O and ﬁltered

through a short pad of silica gel (3 cm). The ﬁltrate was concentrated

and dried in high vacuum to give 18 as a pale yellow liquid (1.62 g, 4.6

(E)-1-(2-{2-[1-(2′,4′-Bistriﬂuoromethylbiphenyl-2-yl)-

methylidene]hydrazinyl}ethyl)piperidine-3-carboxylic Acid

(12ag). According to GP1 from hydrazine 10 (14.4 mg, 77.1 μmol)

and 11ag (24.5 mg, 77.1 μmol), 12ag was obtained as a yellow,

1

amorphous solid (35.1 mg, yield 93%). H NMR (500 MHz, DMSO-

d₆): δ 1.25−1.47 (m, 2H), 1.52−1.63 (m, 1H), 1.71−1.81 (m, 1H),

1.94 (t, J = 10.5 Hz, 1H), 2.02−2.15 (m, 1H), 2.25−2.41 (m, 3H),

2.53−2.60 (m, 1H), 2.71−2.84 (m, 1H), 3.03 (t, J = 6.7 Hz, 2H), 7.05

(s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.13−7.23 (s_br, 1H), 7.22−7.31 (m,

1H), 7.37−7.44 (m, 1H), 7.56−7.63 (m, 1H), 7.81 (d, J = 7.7 Hz,

1H), 8.11 (d, J = 8.1 Hz, 1H), 8.15 (s, 1H) ppm. ¹³C NMR (126 MHz,

DMSO-d₆₁): δ 23.8, 26.4, 40.9, 45.5, 53.1, 55.3, 56.4, 122.9 (m_CF),

1

mmol, 92%). The product was used without further puriﬁcation. H

NMR (400 MHz, CDCl₃): δ 1.19−1.26 (m, 15H), 1.33−1.45 (m,

1H), 1.46−1.64 (m, 3H), 1.68 (dp, J = 10.4/3.5 Hz, 1H), 1.84−1.97

(m, 2H), 2.01−2.17 (m, 3H), 2.23−2.34 (m, 2H), 2.51 (tt, J = 10.6/

3.7 Hz, 1H), 2.72 (d, J = 11.1 Hz, 1H), 2.94 (d, J = 10.1 Hz, 1H), 4.09

(q, J = 7.2 Hz, 2H), 5.40 (d, J = 18.0 Hz, 1H), 6.60 (dt, J = 17.9/6.4

Hz, 1H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 14.14, 24.53, 24.7

(4C), 25.3, 27.0, 33.6, 41.8, 53.7, 55.4, 58.2, 60.2, 82.9 (2C), 154.0,

174.2 ppm. MS (ESI+) m/z: 352 [M + H]⁺. HRMS (EI, 70 eV): M⁺

calcd for C₁₉H₃₄BNO₄, 351.2580; found, 351.2572.

1

123.1 (q, J_CF= 274.8 Hz), 123.4 (q, J_CF= 272.8 Hz), 124.0, 126.0,

2

128.5, 128.7 (q, J_CF= 32.8 Hz), 128.8 (q, J_CF= 32.2 Hz), 128.9

(m_CF), 129.3, 131.1, 133.7, 134.1, 134.5, 143.8, 175.0 ppm. ¹⁹F NMR

(471 MHz, DMSO-d₆): δ −61.12 (m), −57.61 (d, J = 2.5 Hz) ppm.

MS (ESI+) m/z: 488.3 [M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for

C₂₃H₂₄F₆N₃O₂, 487.1694; found, 488.1768.

(E)-1-(2-{2-[1-(2′-Chloro-4′-ﬂuorobiphenyl-2-yl)-

methylidene]hydrazinyl}ethyl)piperidine-3-carboxylic Acid

(12ah). According to GP1 from hydrazine 10 (9.93 mg, 53.0 μmol)

and 11ah (12.4 mg, 53.0 μmol), 12ah was obtained as a yellow,

amorphous solid (21.3 mg, yield 100%). ¹H NMR (400 MHz, DMSO-

d₆): δ 1.26−1.50 (m, 2H), 1.53−1.65 (m, 1H), 1.70−1.84 (m, 1H),

1.98 (t, J = 10.2 Hz, 1H), 2.12 (t, J = 10.0 Hz, 1H), 2.30−2.47 (m,

3H), 2.55−2.65 (m, 1H), 2.80 (d, J = 10.6 Hz, 1H), 3.09 (t, J = 6.7 Hz,

2H), 7.03−7.22 (m, 3H), 7.22−7.43 (m, 4H), 7.56 (dt, J = 8.9/2.3 Hz,

1H), 7.84 (d, J = 7.9 Hz, 1H) ppm. ¹³C NMR (126 MHz, DMSO-d₆):

Ethyl 1-[(E)-5-(2-Bromophenyl)pent-1-enyl]piperidine-3-car-

boxylate (19). Under nitrogen atmosphere Pd(dppf)Cl₂·CH₂Cl₂(41

mg, 0.025 mmol), 1-bromo-2-iodobenzene (312 mg, 1.10 mmol), and

compound 18 (351 mg, 1.00 mmol) were dissolved in a mixture of

1,4-dioxane/water 2:1 (1.5 mL) and K₂CO₃(414 mg, 3.00 mmol).

The resulting mixture was stirred at room temperature for 72 h. After

addition of water (5 mL) the mixture was extracted three times with

CH₂Cl₂(10 mL). The combined organic layers were dried over

Na₂SO₄, and the solvent was removed in vacuo. The residue was

puriﬁed by ﬂash column chromatography on silica gel (n-pentane/

CH₂Cl₂/EtOAc = 8/1/1 + 2% Et₃N, R_f= 0.3) to give the 19 as a

colorless oil (342 mg, 90%). ¹H NMR (400 MHz, CDCl₃): δ 1.25 (t, J

= 7.1 Hz, 3H), 1.44 (qd, J = 12.2/4.0 Hz, 1H), 1.52−1.65 (m, 1H),

1.65−1.77 (m, 3H), 1.90−2.03 (m, 2H), 2.15 (t, J = 10.6 Hz, 1H,),

2.27 (q, J = 6.9 Hz, 2H), 2.37−2.43 (m, 2H), 2.57 (tt, J = 10.6/3.8 Hz,

1H), 2.79 (d, J = 11.0 Hz, 1H), 3.01 (d, J = 10.4 Hz, 1H), 4.13 (q, J =

7.1 Hz, 2H), 6.16 (dt, J = 15.6/6.9 Hz, 1H), 6.71 (d, J = 15.7 Hz, 1H),

7.05 (td, J = 7.9/1.5 Hz, 1H), 7.24 (td, J = 7.5/0.7 Hz, 1H), 7.48 (dd, J

= 7.8/1.4 Hz, 1H), 7.52 (dd, J = 8.0/0.7 Hz, 1H) ppm. ¹³C NMR (101

MHz, CDCl₃): δ 14.2, 24.6, 26.3, 27.0, 30.9, 41.9, 53.8, 55.4, 58.2,

60.2, 123.1, 126.7, 127.3, 128.1, 128.9, 132.7, 133.5, 137.5, 174.2 ppm.

2

δ 24.0, 26.5, 41.0, 45.5, 53.3, 55.4, 56.4, 114.5 (d, J_CF= 21.3 Hz),

2

116.5 (d, J_CF= 24.7 Hz), 123.7, 126.7, 128.1, 130.0, 131.2, 133.0 (d,

3

4

³J_CF= 8.7 Hz), 133.4 (d, J_CF= 10.5 Hz), 134.4, 135.3 (d, J_CF= 3.6

Hz), 135.6, 161.4 (d, J_CF= 247.8 Hz), 175.1 ppm. ¹⁹F NMR (471

1

MHz, DMSO-d₆): δ −112.61 (tdd, J = 8.8/6.3/2.4 Hz) ppm. MS (ESI

+) m/z: 404.3 [M + H]⁺. HRMS (ESI+): [M + H]⁺calcd for

C₂₁H₂₄ClFN₃O₂, 404.1541; found, 404.1537.

(E)-1-(2-{2-[1-(4′-Chloro-4-ﬂuorobiphenyl-2-yl)methylidene]-

hydrazinyl}ethyl)piperidine-3-carboxylic Acid (12ak). According

to GP1 from hydrazine 10 (8.91 mg, 47.6 μmol) and 11ak (11.2 mg,

47.6 μmol), 12ak was obtained as a yellow, amorphous solid (19.3 mg,

1

yield 100%). H NMR (400 MHz, DMSO-d₆): δ 1.24−1.42 (m, 1H),

1.37−1.51 (m, 1H), 1.53−1.67 (m, 1H), 1.70−1.84 (m, 1H), 2.00 (t, J

= 10.3 Hz, 1H), 2.13 (t_br, J = 10.3 Hz, 1H), 2.38 (tt, J = 9.8/3.7 Hz,

1H), 2.44 (t, J = 6.6 Hz, 2H), 2.56−2.67 (m, 1H), 2.82 (d_br, J = 10.7

Hz, 1H), 3.15 (t, J = 6.5 Hz, 2H), 7.10 (td, J = 8.4/2.8 Hz, 1H), 7.25

(dd, J = 8.5/5.9 Hz, 1H), 7.29−7.37 (m, 3H), 7.38−7.48 (m, 1H),

7.48−7.54 (m, 2H), 7.52−7.56 (m, 1H) ppm. ¹³C NMR (101 MHz,

+

MS (CI, CH₅) m/z: 381 [M + H]⁺. HRMS (EI, 70 eV): M⁺calcd for

C₁₉H₂₆BrNO₂, 379.1147; found, 379.1128.

Ethyl 1-[(E)-5-(2′-Fluorobiphenyl-2-yl)pent-4-en-1-yl]-

piperidine-3-carboxylate (21c). According to the GP2 from 19

(114 mg, 0.300 mmol) and 20c (46 mg, 0.33 mmol), 21c was obtained

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as a slightly yellow oil (110 mg, 93%). ¹H NMR (500 MHz, CDCl₃): δ

1.24 (t, J = 7.1 Hz, 3H), 1.42 (qd, J = 11.9/3.6 Hz, 1H), 1.50−1.61 (m,

3H), 1.70 (dp, J = 13.0/3.7 Hz, 1H), 1.89−1.97 (m, 2H), 2.06−2.14

(m, 3H), 2.28−2.33 (m, 2H), 2.53 (tt, J = 10.7/3.8 Hz, 1H), 2.73 (d, J

= 11.1 Hz, 1H), 2.95 (d, J = 10.0 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H),

6.13 (dt, J = 15.7/6.7 Hz, 1H), 6.21 (d, J = 16.0 Hz, 1H), 7.13 (ddd, J

= 9.8/8.2/1.1 Hz, 1H), 7.18 (td, J = 7.4/1.1 Hz, 1H), 7.21−7.28 (m,

3H), 7.31−7.37 (m, 2H), 7.59 (d, J = 7.7 Hz, 1H) ppm. ¹³C NMR

(126 MHz, CDCl₃): δ 14.2, 24.6, 26.4, 27.0, 31.0, 41.9, 53.8, 55.4,

Hz, 1H), 2.72 (d, J = 10.8 Hz, 1H), 2.96 (d, J = 10.1 Hz, 1H), 4.12 (q,

J = 7.1 Hz, 2H), 6.03 (d, J = 15.8 Hz, 1H), 6.12 (dt, J = 15.6/6.7 Hz,

1H), 7.03 (td, J = 8.3/2.6 Hz, 1H), 7.12 (dd, J = 7.6/1.2 Hz, 1H),

7.18−7.28 (m, 3H), 7.35 (td, J = 7.7/1.1 Hz, 1H), 7.58 (d, J = 7.5 Hz,

1H) ppm. ¹³C NMR (126 MHz, CDCl₃): δ 14.2, 24.6, 26.3, 27.0, 31.0,

41.8, 53.8, 55.4, 58.1, 60.2, 113.8 (d, ²J_CF= 20.9 Hz), 116.6 (d, ²J_CF

=

24.5 Hz), 125.1, 126.5, 127.7, 128.2, 130.1, 132.0, 132.5 (d, ³J_CF= 8.6

Hz), 134.2 (d, ³J_CF= 10.3 Hz), 136.0 (d, ⁴J_CF= 3.4 Hz), 136.4, 136.6,

1

161.7 (d, J_CF= 249.3 Hz), 174.2 ppm. MS (ESI+) m/z: 430 [M +

H]⁺. HRMS (EI, 70 eV): M⁺calcd for C₂₅H₂₉ClFNO₂, 429.1871;

found, 429.1875.

2

4

58.2, 60.2, 115.5 (d, J_CF= 22.4 Hz), 123.8 (d, J_CF= 3.7 Hz), 125.3,

126.6, 128.0, 128.2, 128.5 (d, ²J_CF= 16.3 Hz), 129.1 (d, ³J_CF= 7.9 Hz),

3

1

1-[(E)-5-(2′-Fluorobiphenyl-2-yl)pent-4-en-1-yl]piperidine-3-

130.5, 131.8, 132.1 (d, J_CF= 3.5 Hz), 134.0, 136.5, 159.6 (d, J_CF

=

+

246.6 Hz), 174.3 ppm. MS (CI, CH₅) m/z: 396 [M + H]⁺. HRMS

(EI, 70 eV): M⁺calcd for C₂₅H₃₀FNO₂, 395.2261; found, 395.2265.

Ethyl 1-[(E)-5-(2′,4′-Diﬂuorobiphenyl-2-yl)pent-4-en-1-yl]-

piperidine-3-carboxylate (21g). According to GP2 from 19 (114

mg, 0.300 mmol) and 20g (52 mg, 0.33 mmol), 21g was obtained as a

carboxylic Acid (13c). According to GP3 from 21c (79 mg, 0.20

1

mmol), 13c was obtained as a slightly yellow solid (63 mg, 86%). H

NMR (400 MHz, 0.1 M NaOD/CD₃OD = 1/2, 60 °C): δ 1.37 (qd, J

= 12.7/4.1 Hz, 1H), 1.52−1.75 (m, 4H), 1.91 (td, J = 11.6/2.5 Hz,

1H), 2.02 (m, 2H), 2.11 (q, J = 7.1 Hz, 2H), 2.29−2.45 (m, 3H), 2.86

(d, J = 10.6 Hz, 1H), 3.11 (d, J = 9.8 Hz, 1H), 6.09−6.26 (m, 2H),

7.16−7.23 (m, 2H), 7.23−7.33 (m, 3H), 7.37 (t, J = 7.6 Hz, 1H),

7.40−7.48 (m, 1H), 7.61 (d, J = 7.9 Hz, 1H) ppm. ¹³C NMR (101

MHz, NaOD/CD₃OD = 1/2, 60 °C): δ 25.8 26.8, 29.4, 32.0, 46.3,

1

slightly yellow oil (113 mg, 91%). H NMR (500 MHz, CDCl₃): δ

1.25 (t, J = 7.1 Hz, 3H), 1.42 (qd, J = 11.9/3.8 Hz, 1H), 1.49−1.64 (m,

3H), 1.71 (dp, J = 13.0/3.6 Hz, 1H), 1.89−1.97 (m, 2H), 2.05−2.16

(m, 3H), 2.28−2.35 (m, 2H), 2.54 (tt, J = 10.6/3.8 Hz, 1H), 2.74 (d, J

= 10.6 Hz, 1H), 2.97 (d, J = 10.5 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H),

6.08−6.22 (m, 2H), 6.87−6.97 (m, 2H), 7.17−7.23 (m, 2H), 7.27 (td,

J = 7.6/1.3 Hz, 1H), 7.35 (td, J = 7.6/1.2 Hz, 1H), 7.59 (d, J = 7.8 Hz,

2

4

54.8, 58.2, 59.5, 116.4 (d, J_CF= 22.5 Hz), 125.1 (d, J_CF= 3.8 Hz),

2

3

126.6, 127.7, 129.1, 129.7, 130.0 (s, J_CF= 16.7 Hz), 130.5 (d, J_CF

=

7.9 Hz), 131.3, 132.8, 133.0 (d, ³J_CF= 3.4 Hz), 135.5, 138.0, 161.0 (d,

+

¹J_CF= 244.7 Hz), 182.9 ppm. MS (CI, CH₅) m/z: 368 [M + H]⁺.

1H) ppm. ¹³C NMR (126 MHz, CDCl₃): δ 14.2, 24.6, 26.4, 27.0, 31.0,

2/2

HRMS (EI, 70 eV): M⁺calcd for C₂₃H₂₆FNO₂, 367.1948; found,

367.1949. Purity: 98.5% (HPLC).

41.9, 53.8, 55.5, 58.2, 60.3, 103.9 (dd,

J

= 26.4/25.2 Hz), 111.0

CF

2/4

(dd,

J

= 21.0/3.8 Hz), 124.61 (dd,

J

= 16.6/3.8 Hz), 125.5,

CF

3/3

126.7, 128.0, 128.2, 130.6, 132.1, 132.7 (dd,

J

= 9.5/5.0 Hz),

1-[(E)-5-(2′,4′-Diﬂuorobiphenyl-2-yl)pent-4-en-1-yl]-

piperidine-3-carboxylic Acid (13g). According to the GP3, starting

from 21g (83 mg, 0.20 mmol), 13g was obtained as a slightly yellow

solid (68 mg, 88%). ¹H NMR (400 MHz, 0.1 M NaOD/CD₃OD = 1/

1, 60 °C): δ 1.25 (qd, J = 12.7/4.2 Hz, 1H), 1.35−1.52 (m, 3H), 1.58

(d, J = 13.5 Hz, 1H), 1.68 (td, J = 11.7/2.3 Hz, 1H), 1.80−1.98 (m,

4H), 2.04−2.14 (m, 1H), 2.14−2.26 (m, 1H), 2.33 (tt, J = 12.0/3.8

Hz, 1H), 2.63 (d, J = 10.1 Hz, 1H), 2.96 (d, J = 11.1 Hz, 1H), 5.90−

6.07 (m, 2H), 6.65−6.75 (m, 2H), 6.85−6.95 (m, 2H), 6.99 (t, J = 7.5

Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H) ppm. ¹³C

NMR (101 MHz, 0.1 M NaOD/CD₃OD = 1/1, 60 °C): δ 25.1, 25.9,

CF

1/3

133.0, 136.7, 159.6 (dd,

J

= 249.2/11.9 Hz), 162.4 (dd,

J

=

CF

+

248.4/11.5 Hz), 174.3 ppm. MS (CI, CH₅) m/z: 414 [M + H]⁺.

HRMS (EI, 70 eV): M⁺calcd for C₂₅H₂₉F₂NO₂, 413.2166; found,

413.2164.

Ethyl 1-[(E)-5-(2′-Chlorobiphenyl-2-yl)pent-4-en-1-yl]-

piperidine-3-carboxylate (21p). According to the GP2 from 19

(114 mg, 0.300 mmol) and 20p (52 mg, 0.33 mmol), 21p was

1

obtained as a slightly yellow oil (115 mg, 93%). H NMR (500 MHz,

CDCl₃): δ 1.24 (t, J = 7.1 Hz, 3H), 1.40 (qd, J = 11.7/3.7 Hz, 1H),

1.48−1.59 (m, 3H), 1.70 (dp, J = 13.1/3.6 Hz, 1H), 1.90 (m, 2H),

2.02−2.11 (m, 3H), 2.25−2.31 (m, 2H), 2.52 (tt, J = 10.6/3.8 Hz,

1H), 2.72 (d, J = 11.1 Hz, 1H), 2.94 (d, J = 10.4 Hz, 1H), 4.11 (q, J =

7.1 Hz, 2H), 6.02−6.15 (m, 2H), 7.15 (d, J = 7.5 Hz, 1H), 7.21−7.31

(m, 4H), 7.34 (t, J = 7.6 Hz, 1H), 7.43−7.47 (m, 1H), 7.59 (d, J = 7.8

Hz, 1H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 14.2, 24.6, 26.4, 27.0,

31.0, 41.9, 53.8, 55.4, 58.2, 60.3, 125.0, 126.4 (2C), 128.0 (2C), 128.6,

129.4, 130.0, 131.6, 131.8, 133.5, 136.2, 137.6, 139.8, 174.3 ppm. MS

2

28.7, 31.5, 45.6, 53.6, 57.4, 58.7, 104.4 (d, J_CF= 26.5 Hz), 111.8 (d,

3

2/4

²J_CF= 21.2, J_CF= 3.5 Hz), 125.4 (dd,

J

= 16.6/3.1 Hz), 126.4,

CF

3/3

127.4, 128.6, 129.1, 131.1, 133.1, 133.28 (dd,

J

= 9.5/5.0 Hz),

CF

1/3

133.6, 137.3, 160.2 (dd,

J

= 247.2/11.3 Hz), 163.0 (dd,

J

=

CF

+

247.7/11.4 Hz), 183.4 ppm. MS (CI, CH₅) m/z: 386 [M + H]⁺.

HRMS (EI, 70 eV): M⁺calcd for C₂₃H₂₅F₂NO₂, 385.1853; found,

385.1853. Purity: 100% (HPLC).

+

(CI, CH₅) m/z: 412 [M + H]⁺. HRMS (EI, 70 eV): M⁺calcd for

1-[(E)-5-(2′-Chlorobiphenyl-2-yl)pent-4-en-1-yl]piperidine-

3-carboxylic Acid (13p). According to the GP3, starting from 21p

(82 mg, 0.20 mmol), 13p was obtained as a slightly yellow solid (67

mg, 87%). ¹H NMR (400 MHz, 0.1 M NaOD/CD₃OD = 2/1, 60 °C,

MeOH): δ 1.24 (qd, J = 12.8/4.2 Hz, 1H), 1.32−1.51 (m, 3H), 1.57

(d, J = 13.2 Hz, 1H), 1.66 (td, J = 11.5/2.1 Hz, 1H), 1.74−1.98 (m,

4H), 2.00−2.11 (m, 1H), 2.11−2.23 (m, 1H), 2.32 (tt, J = 11.5/3.4

Hz, 1H), 2.61 (d, J = 10.8 Hz, 1H), 2.94 (d, J = 9.6 Hz, 1H), 5.88−

5.99 (m, 2H), 6.83 (d, J = 7.4 Hz, 1H), 6.89−6.97 (m, 2H), 6.99−7.11

(m, 3H), 7.23 (d, J = 7.1 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H) ppm. ¹³C

NMR (101 MHz, 0.1 M NaOD/CD₃OD = 2/1, 60 °C, MeOH): δ

25.1, 26.0, 28.7, 31.6, 45.5, 53.6, 57.5, 58.7, 126.1, 127.3, 127.5, 128.7,

128.9, 129.7, 130.1, 130.6, 132.3, 132.7, 133.9, 136.8, 138.3, 140.5,

C₂₅H₃₀ClNO₂, 411.1965; found, 411.1966.

Ethyl 1-[(E)-5-(2′,4′-Dichlorobiphenyl-2-yl)pent-4-en-1-yl]-

piperidine-3-carboxylate (21z). According to the GP2 from 19

(114 mg, 0.300 mmol) and 20z (63 mg, 0.33 mmol), 21z was obtained

as a slightly yellow oil (123 mg, 92%). ¹H NMR (400 MHz, CDCl₃, 21

°C, TMS): δ 1.25 (t, J = 7.1 Hz, 3H), 1.42 (qd, J = 12.0/3.7 Hz, 1H),

1.49−1.62 (m, 3H), 1.71 (dp, J = 13.2/3.6 Hz, 1H), 1.89−1.97 (m,

2H), 2.05−2.13 (m, 3H), 2.27−2.32 (m, 2H), 2.53 (td, J = 10.6/5.3

Hz, 1H), 2.73 (d, J = 11.0 Hz, 1H), 2.96 (d, J = 10.6 Hz, 1H), 4.12 (q,

J = 7.1 Hz, 2H), 6.03 (d, J = 15.8 Hz, 1H), 6.13 (dt, J = 15.7/6.7 Hz,

1H), 7.12 (dd, J = 7.5/1.0 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 7.24−

7.31 (m, 2H), 7.36 (td, J = 7.7/1.0 Hz, 1H), 7.49 (t, J = 1.7 Hz, 1H),

7.59 (d, J = 7.8 Hz, 1H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 14.2,

24.6, 26.4, 27.0, 31.0, 41.9, 53.8, 55.4, 58.2, 60.3, 125.2, 126.6, 126.8,

127.6, 128.3, 129.2, 129.9, 132.2, 132.4, 133.7, 134.4, 136.3, 136.4,

+

183.4 ppm. MS (CI, CH₅) m/z: 384 [M + H]⁺. HRMS (EI, 70 eV):

M⁺calcd for C₂₃H₂₆ClNO₂, 383.1652; found, 383.1635. Purity: 100%

(HPLC)

+

138.5, 174.3 ppm. MS (CI, CH₅) m/z: 446 [M + H]⁺. HRMS (EI, 70

1-[(E)-5-(2′,4′-Dichlorobiphenyl-2-yl)pent-4-en-1-yl]-

piperidine-3-carboxylic Acid (13z). According to GP3 from 21z

(89 mg, 0.20 mmol), 13z was obtained as a slightly yellow solid (75

mg, 90%). ¹H NMR (400 MHz, 0.1 M NaOD/CD₃OD = 2/1, 60 °C):

δ 1.28 (qd, J = 12.4/3.8 Hz, 1H), 1.40−1.54 (m, 3H), 1.60 (d, J = 12.8

Hz, 1H), 1.71 (t, J = 11.3 Hz, 1H), 1.80−2.01 (m, 4H), 2.04−2.16 (m,

1H), 2.22 (m, 1H), 2.34 (tt, J = 11.6/3.6 Hz, 1H), 2.66 (d, J = 10.4

Hz, 1H), 2.99 (d, J = 10.9 Hz, 1H), 5.88−6.03 (m, 2H), 6.81 (d, J =

7.4 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.93−7.04 (m, 2H), 7.13 (t, J =

eV): M⁺calcd for C₂₅H₂₉Cl₂NO₂, 445.1575; found, 445.1549.

Ethyl 1-[(E)-5-(2′-Chloro-4′-ﬂuorobiphenyl-2-yl)pent-4-en-1-

yl]piperidine-3-carboxylate (21ah). According to the GP2 from 19

(114 mg, 0.300 mmol) and 20ah (58 mg, 0.33 mmol), 21ah was

1

obtained as slightly yellow oil (117 mg, 91%). H NMR (500 MHz,

CDCl₃): δ 1.25 (t, J = 7.1 Hz, 3H), 1.42 (qd, J = 12.3/3.9 Hz, 1H),

1.51−1.61 (m, 3H), 1.71 (dp, J = 13.4/3.6 Hz, 1H), 1.89−1.97 (m,

2H), 2.04−2.14 (m, 3H), 2.25−2.33 (m, 2H), 2.53 (tt, J = 10.6/3.8

1337

dx.doi.org/10.1021/jm301800j | J. Med. Chem. 2013, 56, 1323−1340

Journal of Medicinal Chemistry

Article

7.4 Hz, 1H), 7.24 (s, 1H), 7.42 (d, J = 7.7 Hz, 1H). ¹³C NMR (101

MHz, 0.1 M NaOD/CD₃OD = 2/1, 60 °C): δ 25.4, 26.3, 28.9, 31.8,

45.7, 54.1, 57.9, 59.0, 126.4, 127.4, 127. 7, 128.6, 129.2, 129.8, 130.5,

133.2, 133.3, 134.4, 135.0, 137.1, 137.2, 139.5, 183.1 ppm. MS (CI,

Deconvolution Experiments. The deconvolution experiments

were carried out in the same way²⁸as described for library screening

except that the single aldehyde (10 μM per sample) was applied

instead of the mixture of four aldehydes.

Competition Experiments Applying Pure Hydrazones. These

experiments were performed as recently described,^25,49applying the

incubation buﬀer as described for “Library Screening”.

LC−ESI-MS/MS. The methanolic eluates were dried and

reconstituted with 10 mM ammonium formate buﬀer (pH 7.0)/

methanol (95:5, v/v). Quantitation by LC−ESI-MS/MS was

performed as described utilizing a API 3200 triple-quadrupole mass

spectrometer.^25,49

+

CH₅) m/z: 418 [M + H]⁺. HRMS (EI, 70 eV): M⁺calcd for

C₂₃H₂₅Cl₂NO₂, 417.1262; found, 417.1257. Purity: 99.2% (HPLC).

1-[(E)-5-(2′-Chloro-4′-ﬂuorobiphenyl-2-yl)pent-4-en-1-yl]-

piperidine-3-carboxylic Acid (13ah). According to GP3 from 21ah

(86 mg, 0.20 mmol), 13ah was obtained as a slightly yellow solid (75

mg, 90%). ¹H NMR (400 MHz, NaOD/CD₃OD = 1/2, 60 °C): δ 1.36

(qd, J = 12.6/4.1 Hz, 1H), 1.51−1.65 (m, 3H), 1.71 (dp, J = 13.5/3.3

Hz, 1H), 1.89−2.00 (m, 2H), 2.01−2.13 (m, 3H), 2.27−2.44 (m, 3H),

2.84 (d, J = 11.0 Hz, 1H), 3.06 (d, J = 11.3 Hz, 1H), 6.00−6.17 (m,

2H), 7.11 (dd, J = 7.6/1.0 Hz, 1H), 7.18 (td, J = 8.4/2.5 Hz, 1H),

7.21−7.27 (m, 1H), 7.28−7.36 (m, 2H), 7.40 (td, J = 7.4/1.0 Hz, 1H),

7.61 (d, J = 7.3 Hz, 1H) ppm. ¹³C NMR (101 MHz, NaOD/CD₃OD

= 1/2, 60 °C): δ 25.2, 26.0, 28.8, 31.7, 45.7, 54.3, 57.5, 58.9, 115.0 (d,

Analysis of Binding Experiments. Marker depletion was

negligible (<10%) in all binding experiments. Speciﬁc binding was

deﬁned as the diﬀerence between total and nonspeciﬁc binding. A

nonspeciﬁc binding less than 50 pM was not determined

experimentally but extrapolated by linear regression for nonspeciﬁcally

bound NO711 concentrations of ≥50 pM. The concentration of a

competitor that inhibits 50% of speciﬁc binding (IC₅₀) was calculated

from competition curves, plotting speciﬁcally bound NO711

concentrations vs log competitor concentration (eight diﬀerent

concentrations per competitor) with Prism 4.02 (GraphPad Software,

San Diego, CA, U.S.) using the equation for one-site competition and

nonlinear curve ﬁtting. Speciﬁc binding determined for control

samples in the absence of any competitor was set to 100%, whereas

the bottom level was set to 0% (corresponds to the nonspeciﬁc

binding of the employed NO711 concentration). K_ivalues were

calculated according to Cheng and Prussoﬀ⁵⁰and were expressed as

pK_ivalues. All results are expressed as the mean SEM (unless stated

otherwise). pK_ivalues were determined in at least three separate

experiments.

2

²J_CF= 21.3 Hz), 117.3 (d, J_CF= 25.2 Hz), 126.4, 127.7, 128.9, 129.3,

130.8, 133.1, 133.6 (d, ³J_CF= 8.8 Hz), 134.9 (s, ³J_CF= 10.2 Hz), 137.3,

1

137.5, 137.8, 163.0 (s, J_CF= 248.2 Hz), 182.9 ppm. MS (ESI+) m/z:

402 [M + H]⁺. HRMS (EI, 70 eV): M⁺calcd for C₂₃H₂₅ClFNO₂,

401.1558; found, 401.1558. Purity: 98.0% (HPLC).

Aldehydes. Detailed synthesis protocols for aldehydes are found in

the Supporting Information.

MS Binding Experiments. mGAT1 Membrane Preparation.

Membrane preparations of HEK293 cells stably expressing mGAT1⁴⁸

were prepared as described previously and stored at −80 °C.^25,49On

the day of the assay, an aliquot was rapidly thawed and diluted in a 20-

fold volume of cold aqueous 0.9% NaCl (m/v). After centrifugation at

15 000 rpm and 4 °C for 20 min (CP56GII, P70AT, Hitachi Ltd.,

Tokyo, Japan), the pellet was resuspended in ice-cold assay buﬀer (see

below) to a protein concentration of approximately 0.1 mg/mL⁴⁹as

previously described.²⁸

GABA Uptake Assays. [³H]GABA uptake assays were performed

as previously described.⁴⁸

Library Screening. Library screening experiments were performed

as reported.²⁸In short, quadruplicate samples in a total volume of 250

μL were employed. Phosphate buﬀer, pH 7.1 (12.5 mM

Na₂HPO₄·2H₂O, 12.5 mM NaH₂PO₄·H₂O, 1 M NaCl adjusted with

2 M NaOH), was used as incubation buﬀer. Each sample contained 1%

DMSO as ﬁnal concentration. All solutions were added as 10-fold

concentrated stock solutions. Aldehydes were present at 10 μM in the

sample, hydrazine 10 at 100 μM. By addition of the mGAT1

membrane preparation, directly after combining hydrazine and

aldehydes, a ﬁrst incubation period of 4 h (for library generation) at

37 °C in a shaking water bath was started. The second incubation

period of 40 min was started by addition of the MS marker 6 (20 nM

ﬁnal concentration in the sample). The binding experiment was

stopped by a vacuum ﬁltration step (96-well ﬁlter plate, Acroprep,

glass ﬁber, 1.0 μm, 350 μL, Pall, Dreieich, Germany, 12-channel pipet).

After multiple washing steps with ice-cold aqueous 1 M NaCl, the ﬁlter

was dried (60 min, 50 °C) and cooled to room temperature.

Subsequently the marker was liberated by elution of the ﬁlter plate

with MeOH into a 96-deep-well plate. Each sample was supplemented

with 200 μL of 1 nM [²H₁₀]NO711 (in MeOH) as internal standard.

For calibration, matrix blank samples (produced analogously in

absence of NO711) were supplemented with 200 μL of methanolic

calibration standards, 50 pM, 100 pM, 500 pM, or 1 nM NO711, and

200 μL of 1 nM [²H₁₀]NO711 (in MeOH). All samples were dried to

completeness (50 °C). For quantiﬁcation by LC−ESI-MS/MS,

samples were reconstituted in 200 μL of 10 mM ammonium formate

buﬀer (pH 7.0)/MeOH (95:5, v/v).

ASSOCIATED CONTENT

* Supporting Information

General procedures for the synthesis of aldehydes and

references and detailed analytic data of aldehydes. This material

is available free of charge via the Internet at http://pubs.acs.org.

■

S

AUTHOR INFORMATION

Corresponding Author

*Phone: +49 2180 77249. Fax: +49 2180 77247. E-mail: Klaus.

Wanner@cup.uni-muenchen.de.

■

Present Address

^†Clinical Trial Oncology, Ente Ospedaliero Ospedali Galliera,

Mura delle Cappuccine 14, 16128 Genova, Italy.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

Special thanks go to Silke Duensing-Kropp and Ljiljana

Galogaza for excellent technical support.

■

DEDICATION

■

Dedicated to Professor H. Zipse with warm wishes on the

occasion of his 50th birthday.

As stated before, each library screening experiment included

samples characterizing the pure aldehyde libraries and the pure

hydrazine 10, as well as matrix blanks and zero samples. Total binding

and nonspeciﬁc binding of NO711 were determined as previously

described.²⁸If the nonspeciﬁc binding of the 20 nM NO711

concentration employed was less than 50 pM, the value was

extrapolated by linear regression of the nonspeciﬁc binding of

NO711 concentrations, >20 nM.

ABBREVIATIONS USED

■

BGT, betain γ-aminobutyric acid transporter; CC, column

chromatography; DAD, diode array detector; DCC, dynamic

combinatorial chemistry; GAT, γ-aminobutyric acid trans-

porter; GP, general procedure; HUGO, human genome

organization; IC₁₀, inhibitor concentration that reduces signal

to 10%; IC₂₀, inhibitor concentration that reduces signal to

By employment of the results obtained for the calibration samples,

calibration curves for marker quantitation were generated.

1338

dx.doi.org/10.1021/jm301800j | J. Med. Chem. 2013, 56, 1323−1340

Journal of Medicinal Chemistry

Article

Rusakov, D. A.; Madsen, K. K.; Clausen, R. P.; Schousboe, A.; White,

H. S.; Danbolt, N. C. Deletion of the betaine-GABA transporter

(BGT1; slc6a12) gene does not affect seizure threshold of adult mice.

Epilepsy Res. 2011, 95, 75−81.

20%; LLOQ, lower limit of quantiﬁcation; SLC6, solute carrier

6

REFERENCES

■

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Focused pseudostatic hydrazone libraries screened by mass spectrometry binding assay: Optimizing affinities toward γ-aminobutyric acid transporter 1

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