Synthesis and biological evaluation of α- and β-hydroxy substituted amino acid derivatives as potential mGAT1–4 inhibitors

MEDICINAL

CHEMISTRY

RESEARCH

Medicinal Chemistry Research

https://doi.org/10.1007/s00044-020-02548-x

ORIGINAL RESEARCH

Synthesis and biological evaluation of α- and β-hydroxy substituted

amino acid derivatives as potential mGAT1–4 inhibitors

Janina C. Andreß¹Michael C. Böck¹Georg Höfner¹Klaus T. Wanner¹

●

Received: 23 December 2019 / Accepted: 17 April 2020

Abstract

In this study, we report the synthesis and biological evaluation of a variety of α- and β-hydroxy substituted amino acid

derivatives as potential amino acid subunits in inhibitors of GABA uptake transporters (GATs). In order to ensure that the

test compounds adopt a binding pose similar to that presumed for related larger GAT inhibitors, lipophilic residues were

introduced either at the amino nitrogen atom or at the alcohol function. Several of the synthesized compounds were found to

exhibit similar inhibitory activity at the GAT subtypes mGAT2, mGAT3, and mGAT4, respectively, as compared with the

reference N-butylnipecotic acid. Hence, these compounds might serve as starting point for future developments of more

complex GAT inhibitors.

●

Keywords Neurochemistry GABA transporters GABA uptake inhibitors mGAT4 Amino acids

Introduction

transporter gene family, these membrane-bound proteins

termed GABA transporters (GATs) use the co-transport of

sodium ions for the translocation of the substrate against the

chemical gradient (Kristensen et al. 2011). The nomenclature

of the four GAT subtypes depends on the species the trans-

porters are cloned from. When originating from mouse brain

cells, the GAT subtypes are termed mGAT1–4. For all other

species, including humans, an alternate nomenclature is used

which has also been adopted by the Human Genome Orga-

nisation, denoting the transporters GAT1 (=mGAT1), BGT1

(=mGAT2), GAT2 (=mGAT3), and GAT3 (=mGAT4)

(Madsen et al. 2009). Hereafter the nomenclature referring to

the murine transporters will be applied as the biological test

system used in our group is based on these. mGAT1 and

mGAT4 are the most abundant GATs in the mammalian

CNS, with the former being predominantly located on pre-

synaptic neuronal membranes mediating the neuronal GABA

uptake, whereas mGAT4, which is mainly expressed on glia

cells, is responsible for the glial GABA uptake (Minelli et al.

1996; Jin et al. 2011). The other two GAT subtypes, mGAT2

and mGAT3, are primarily located in the periphery, with the

highest densities being found in liver and kidneys, and hence

are thought to not play any signiﬁcant role in the termination

of GABAergic signalling in the CNS (Zhou et al. 2012).

The ﬁrst parent compound selectively targeting GATs was

4,5,6,7-tetrahydroixoxazolo[4,5-c]pyridin-3-ol (THPO, 1,

Table 1, entry 1) (White et al. 2002), which is derived from

muscimol, an alkaloid isolated from ﬂy agaric (Amanita

Gamma-aminobutyric acid (GABA) is the most abundant

inhibitory neurotransmitter in the mammalian central nervous

system (CNS) (Bowery and Smart 2006), with up to 40% of

synapses estimated to be GABAergic (Meldrum and Chap-

man 1999). Deﬁcient GABAergic neurotransmission is

assumed to play a decisive role in the pathogenesis of several

severe neurological diseases, including Alzheimer’s disease

(Lanctôt et al. 2004), depression (Kalueff and Nutt 2007),

epilepsy (Treimann 2001), and neuropathic pain (Daemen

et al. 2008). A promising therapeutical approach for the

treatment of these diseases exists in the inhibition of the

transport molecules responsible for the removal of GABA

from the synaptic cleft, resulting in the prolongation of the

effect exerted by the available GABA (Krogsgaard-Larsen

et al. 1991). Belonging to the solute carrier 6 (SLC6)

Supplementary information The online version of this article (https://

doi.org/10.1007/s00044-020-02548-x) contains supplementary

material, which is available to authorized users.

* Klaus T. Wanner

klaus.wanner@cup.uni-muenchen.de

1

Department of Pharmac - Center for Drug Research, Ludwig-

Maximilians-Universität München, Butenandtstraße 5-13, 81377

Munich, Germany

Medicinal Chemistry Research

Table 1 GAT inhibitors

Entry

Compound

GABA uptake inhibition (pIC₅₀SEM)^a

mGAT1

mGAT2

mGAT3

mGAT4

1

2

3

4

5

6

1

3.0

2.5

<2.5

2

4.87 0.06

5.19 0.03

4.24 0.05

6.88 0.12

4.07 0.09

3.31 0.03

3.39 0.05

3.13 0.14

50%^b

4.59 0.05

4.76 0.05

3.83 0.04

64%^b

4.59 0.05

4.95 0.05

3.63 0.06

73%^b

(R)-3

(S)-3

4

5

62%^b

5.29 0.04

5.71 0.20

^aResults of the [³H]GABA uptake assays are given as pIC₅₀SEM

^bPercentages represent remaining [³H]GABA uptake in presence of 100 µM test compound

muscaria) that can be considered an bioisostere of GABA

(Corvey et al. 1994). Replacement of the isoxazol-3-ol partial

structure of THPO with a carboxylic acid function led to the

more potent GAT inhibitors guvacine (2, Table 1, entry 2)

and nipecotic acid [(R)-3,(S)-3 Table 1, entry 3–4] (Krogs-

gaard-Larsen et al. 2000). However, these cyclic GABA

analogues still lacked inhibitory potency and subtype selec-

tivity (Table 1, entry 1–4). Moreover, due to being present in

the zwitterionic state under physiological conditions, guvacine

(2) and nipecotic acid [(R)-3,(S)-3] are hardly able to cross the

blood brain barrier (BBB), which strongly limits any potential

therapeutic application (Seth et al. 2018).

for being the only GAT inhibitor that has been approved for

clinical use (Nielsen et al. 1991).

In a similar way the introduction of a lipophilic residue

consisting of a triarylmethyl group, which is linked to the

nipecotic acid subunit via a spacer of three atoms, furnishes

mGAT4 selective inhibitors, with (S)-SNAP-5114 con-

stituting the prototypic representative of this group (5, Table

1, entry 6) (Dhar et al. 1994). Remarkably, the (S)-isomer 5

exhibits higher inhibitory potency than the respective (R)-

isomer, running contrary to what is observed for the

unsubstituted nipecotic acid [(R)-3, (S)-3] as well as for

mGAT1 inhibitors such as Tiagabine (4).

When bulky, lipophilic residues were introduced at the

nitrogen atom, this led to compounds with not only increased

lipophilicity and hence improved BBB penetration, but also

with signiﬁcantly higher inhibitory potency. In this context,

compounds possessing a diaryl methyl or a biaryl unit as

lipophilic domain which is connected to the nipecotic acid

partial structure via a ﬂexible linker of 3–5 atoms proved to be

highly potent and selective mGAT1 inhibitors, with the

respective pIC₅₀values rising from ~5 to almost 7 as compared

with unsubstituted (R)-nipecotic acid. Among these com-

pounds, Tiagabine (Gabatril®, 4, Table 1, entry 5) stands out

Interestingly, small amino acids including β-alanine (6,

Table 2, entry 1), isoserine (7a, Table 2, entry 2), 2,3-diami-

nopropionic acid (8, Table 2, entry 3) and (Z)-4-aminobut-2-

enoic acid (9, Table 2, entry 4) also show moderate biological

activity at mGAT3 and mGAT4 that is comparable to that

exerted by (R)-nipecotic acid [(R)-3, Table 1, entry 3], while

being distinctly less potent inhibitors of mGAT1 (Kragler et al.

2005). Thus, substitution of the nipecotic acid partial structure

in the lead compound (S)-SNAP-5114 (5) with a (S)-2-

hydroxy-2-[(R)-pyrrolidin-2-yl]acetic acid unit, which can be

understood as rigidized derivative of rac-isoserine (7a), led to

Medicinal Chemistry Research

Table 2 GAT inhibitors

Entry

GABA uptake inhibition (pIC₅₀SEM)^a

compound

mGAT1

mGAT2

mGAT3

mGAT4

1

2

3

4

5

6

2.59 0.03

2.33 0.05

3.11 0.02

2.99 0.04

80%^b

3.48 0.11

3.39 0.11

3.50 0.12

3.67 0.08

64%^b

4.66 0.06

4.87 0.05

4.66 0.08

4.95 0.04

70%^b

4.46 0.13

4.78 0.14

5.05 0.02

5.04 0.06

5.18 0.05

7a

8

9

10

^aFor consistency the values determined in our research group are listed

^bRemaining [³H]GABA uptake at 100 µM compound concentration

compound 10, which displays signiﬁcantly improved subtype

selectivity in favour of mGAT4. Unfortunately, this structure

variation is also accompanied by a moderate decrease of

inhibitory potency (pIC₅₀= 5.18 0.05, Table 2, entry 5) as

compared with the parent compound 5 (pIC₅₀= 5.71 0.20,

Table 1, entry 6) (Steffan et al. 2015).

methylene groups at various positions, and the rigidization of

the molecule by integrating the amino acid backbone into lar-

ger heterocycles. Accordingly, a set of compounds featuring

both variations and thus deviating from the original isoserine

(7a) structure should be synthesized and biologically evaluated

(Scheme 1).

For the development of mGAT4 inhibitors with increased

subtype selectivity, this study hence aims to identify further

cyclic and acyclic 2- and 3-hydroxyamino acids as possible

alternatives to the nipecotic acid partial structure present in the

scaffold of important mGAT4 inhibitors such as 5. In this

context, the hydroxyl function plays a crucial role as it may

enable additional interactions with the target, e.g., by estab-

lishing hydrogen bridges, hence increasing binding enthalpy.

Proceeding from the basic structure of isoserine 7a, it was

intended to implement several structural modiﬁcations,

including elongation of the carbon chain by insertion of

Despite the stereochemistry of amino acids such as

nipecotic acid [(R)-3, (S)-3] being known to represent an

important factor when it comes to the biological activity of

mGAT4 inhibitors, we opted for the synthesis of the target

compounds in racemic form as this provides information

about the biological activity of both enantiomers.

According to the results of molecular modelling experi-

ments previously performed by us for mGAT1 (Wein et al.

2016), small inhibitors such as nipecotic acid adapt a

binding pose at which the amino nitrogen atom is facing

towards the intracellular space. However, if bulky, lipophilic

Medicinal Chemistry Research

moieties are introduced at the nitrogen atom, as it is the case

with Tiagabine (4, Table 1, entry 5), the binding pose is

altered in a way that the nitrogen atom is orientated towards

the extracellular site, with the lipophilic side chain looming

into the extracellular vestibule, although the position of the

carboxylic acid function remains largely unchanged

(Scheme 2). Using the example of a N-butyl residue, Wein

et al. demonstrated that even the presence of small N-

substituents is sufﬁcient to cause this change of the binding

mode. Although no reliable in silico models exists so far for

the other GAT subtypes, it seems highly likely that these

ﬁndings also apply to mGAT2-4. Thus, following this

assumption, all parent compounds included in this study

were also evaluated for their inhibitory potential as N-butyl

derivatives in order to ensure that the orientation of the

parent compounds in the binding pocket corresponds to the

orientation they would have as part of larger molecules

comprising a lipophilic domain.

In addition, unsubstituted α- and β-hydroxyamino acids

exhibiting higher biological activity than their corre-

sponding N-butyl derivatives should in addition be sub-

stituted at the alcohol function. In detail, the alcohol

function should be provided with a 4,4′,4″-trimethoxy-

trityloxyethyl residue [(4-MeO-C₆H₄)₃COCH₂CH₂], which

Scheme 1 Planned structure variations of isoserine (7a)

extracellular space

vesꢀbule

a

b

intracellular space

Scheme 2 Postulated binding poses of nipecotic acid (3, a) and Tiagabine (4, b) at mGAT1

Medicinal Chemistry Research

is a characteristic structural motif of mGAT4 inhibitors

such as 5 and 10. According to the model pointed out

above, this might allow the amino acid substructure to stay

in the position with the nitrogen atom facing towards the

intracellular space, which would evidently be more

favourable than the antagonal orientation found in the N-

substituted amino acids. At the same time, the newly

introduced lipophilic domain might be accommodated in

the vestibule, which is known to contribute in general

signiﬁcantly to inhibitory potency and selectivity of GAT

inhibitors (compare Table 1, entries 3 and 4 with 6).

up to −10 °C, quenched with brine, diluted with H₂O and

extracted thrice with CH₂Cl₂. The combined CH₂Cl₂phases

were dried over sodium sulfate (Na₂SO₄) and concentrated in

vacuum to yield the crude product.

General procedure for the deprotection and N-butylation of

the amino acid amides and amino acid esters (GP2)

A mixture of the benzyl or benzhydryl-protected amino acid

derivative, palladium on charcoal (10% Pd, 0.1 eq) and

butyraldehyde (2.5 eq) in EtOH was stirred vigorously

under 15 bar hydrogen pressure for 16 h, ﬁltered over

Cealite® and reduced in vacuum.

Materials and methods

General procedure for the hydrolysis of the amides and

esters (GP3)

Chemistry

Moisture-sensitive reactions were carried out in oven-dried

glassware under inert gas atmosphere. Commercially avail-

able starting materials were used without further puriﬁcation.

Tetrahydrofuran (THF) was freshly distilled from sodium

benzophenone ketyl. All other solvents were distilled prior to

use. Microwave reactions were carried out with Biotage

Initiator™. Flash column chromatography was performed on

Merck silica gel 60 (mesh 0.040–0.063 mm) as stationary

phase; thin-layer chromatography (TLC) was carried out on

Merck silica gel 60 F₂₅₄sheets. Preparative MPLC was

performed using a Buechi instrument (C-605 binary pump

system, C-630 UV detector at 254 nm and C-660 fraction

collector) and a Sepacore B-685 (26 V, 230 mm) glass col-

umn equipped with YMC Gel Triart Prep C18-S (12 nm,

A mixture of carboxamide or ester and barium hydroxide

octahydrate (2.0–2.4 eq) was stirred under reﬂux conditions

(carboxamides) or at rt (esters) in EtOH/H₂O 1:1 for the

appropriate time. Carbon dioxide was passed through until

no further precipitate formed. The suspension was ﬁltered

over a cotton wool pad and a syringe ﬁlter (Perfect-Flow®,

WICOM Germany GmbH, PTFE, 0.2 µM) and reduced in

vacuum. The residue was solved in distilled water (2.0 ml)

and lyophilized.

General procedure for the N-butylation of the acyclic amino

acids (GP4)

The amino acid (1.0 eq) and KOH (2.0 eq) were suspended in

EtOH and H₂O was added until the reaction mixture became

homogeneous. 1-Bromobutane (0.9 eq) was added dropwise.

After stirring at rt for 16 h the reaction mixture was reduced in

vacuum. The crude compound was puriﬁed by MPLC (elu-

ent: MeOH/H₂O 1:9).

1

5–20 µm). H and ¹³C NMR spectra were, unless stated

otherwise, recorded at rt with JNMR-GX (JEOL 400 or

500 MHz) or Bruker BioSpin Avance III HD (400 or

500 MHz) and integrated with the NMR software MestRe-

Nova. Deuterated solvents for NMR, which included

CD₂Cl₂, CDCl₃, MeOD, D₂O, and 1,1,2,2-tetrachloroethane-

d2, were purchased from Eurisotop, Saint-Aubin, France.

NaOD was purchased from Sigma-Aldrich Chemie GmbH,

Munich, Germany. IR samples were measured as KBr pellets

or ﬁlm with Perkin-Elmer FT-IR 1600. HRMS data were

obtained with JMS-GCmate II (EI, Jeol) or Thermo Finnigan

LTQ FT Ultra (ESI, Thermo Finnigan).

General procedure for the protection of the acyclic amino

acids I (GP5a)

A solid, well-grounded mixture of the amino acid (1.0 eq)

and phtalic anhydride (1.0 eq) was heated to 140 °C,

resulting in a colourless melting. After 30 min, the reaction

mixture was cooled to rt and re-dissolved in EtOAc

(300 ml). The solution was washed with 1 M sodium

hydrogen sulfate solution (100 ml), water (3 × 100 ml), and

brine (100 ml). The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated in vacuum. The resulting residue

was solved in anhydrous MeOH (250 ml) and 2 M HCl in

Et₂O (250 ml) was added. The mixture was stirred at rt until

TLC indicated complete consumption of the educt and

reduced in vacuum. The crude compound was puriﬁed by

General procedure for the synthesis of the β-hydroxyamino

acid esters (GP1)

A solution of lithium-HMDS in MTBE (0.97 M) was cooled

to −78 °C and anhydrous ethyl acetate (EtOAc, 1.0 eq) was

added dropwise. After complete addition the mixture was

stirred for 25 min and a solution of ketone (1.0 eq) in dry THF

was added. The reaction mixture was allowed to slowly warm

Medicinal Chemistry Research

ﬂash column chromatography on silica (eluent: CH₂Cl₂/

EtOAc 9:1).

General procedure for the deprotection of the acyclic

amino acid derivatives I (GP9a)

General procedure for the protection of the acyclic amino

acids II (GP5b)

12 M NaOH (2.0 eq) was added to a solution of the

protected compound (1.0 eq) in MeOH (15.0 ml). After

stirring for 16 h at rt, 1,2-diaminoethane (7.0 eq) was

added and the mixture was heated in a microwave for

16 h at 140 °C. Finally, the reaction mixture was reduced

in vacuum and puriﬁed by MPLC (eluent: MeOH/

H₂O 7:3).

A solid, well-grounded mixture of the amino acid (1.0 eq)

and phtalic anhydride (1.0 eq) was heated to 140 °C,

resulting in a colourless melting. After 30 min, the reaction

mixture was cooled to rt, solved in anhydrous MeOH

(250 ml) and 2M HCl in Et₂O (250 ml) was added. The

mixture was stirred at rt until TLC indicated complete

consumption of the educt and reduced in vacuum. The

crude compound was puriﬁed by ﬂash column chromato-

graphy on silica (eluent: CH₂Cl₂/EtOAc 9:1).

General procedure for the deprotection of the acyclic

amino acid derivatives II (GP9b)

12 M NaOH (2.0 eq) was added to a solution of the pro-

tected compound (1.0 eq) in MeOH (15.0 ml). After stirring

for 16 h at rt, the mixture was freeze-dried. 1,2-Diami-

noethane (7.0 eq) was added and the mixture was heated in

a microwave for 16 h at 140 °C. Finally, the reaction mix-

ture was reduced in vacuum and puriﬁed by MPLC (eluent:

MeOH/H₂O 7:3).

General procedure for the formation of the ether function

(GP6)

tert-Butyl(2-iodoethoxy) diphenylsilane (1.4 eq) and

Ag₂CO₃(4.0 eq) were added to a suspension of the amino

acid derivative (1.0 eq) in toluene (10.0 ml). The reaction

mixture was stirred in a pressure tube at 120 °C until TLC

indicated complete consumption of the amino acid deriva-

tive, cooled to rt, ﬁltered through a paper ﬁlter and reduced

in vacuum. The residue was puriﬁed by ﬂash column

chromatography on silica (eluent: pentane/Et₂O 7:3).

3-(Butylamino)-2-hydroxypropanoic acid (7b)

GP4 was followed using 7a (90 mg, 0.86 mmol), EtOH

(1.0 ml), KOH (114 mg, 1.72 mmol), 1-bromobutane

(106 mg, 0.774 mmol). The desired compound was obtained

as amorphous white solid (110 mg, 79%). ¹H NMR

(400 MHz, MeOD) δ = 4.07 (dd, J = 8.1, 4.7 Hz, 1H,

COOHCH), 3.24 (dd, J = 12.5, 4.7 Hz, 1H, COOHCHCH₂),

3.06 (dd, J = 12.5, 8.1 Hz, COOHCHCH₂), 3.04–2.99

(m, 2H, CH₃CH₂CH₂CH₂), 1.74–1.57 (m, 2H, CH₃CH₂CH₂),

1.43 (m, 2H, CH₃CH₂CH₂), 0.99 (t, J = 7.4 Hz, 3H,

CH₃CH₂CH₂) ppm; ¹³C NMR (101 MHz, MeOD) δ = 177.2

(COOH), 68.9 (COOHCH), 52.2 (COOHCHCH₂), 48.6

(NCH₂CH₂CH₂CH₃), 29.2 (NCH₂CH₂CH₂CH₃), 20.8

(NCH₂CH₂CH₂CH₃), 13.9 (NCH₂CH₂CH₂CH₃) ppm; IR

(KBr): ṽ = 3397, 1463, 1390, 1135, 1110, 770 cm⁻¹; HRE-

SIMS m/z (pos): 162.1123 C₇H₁₆NO₃(calcd. 162.1130).

General procedure for cleavage of the TBDPS protecting

group (GP7)

The TBDPS-protected compound (1.0 eq) was solved in

THF/pyridine 9:1 (v/v) in a polypropylene tube. A 70%

solution of HF-pyridine (5.0 eq) was added dropwise at

0 °C. The suspension was stirred at rt and the progress of

the reaction was monitored by TLC. After complete con-

sumption of the educt phosphate buffer (pH = 6.0, 1.0 M,

100 ml) was added and the mixture was extracted with

ethyl acetate (100 ml). The organic phase was washed with

water (100 ml) and brine (100 ml), dried over MgSO₄and

reduced in vacuum. The crude product was puriﬁed by

ﬂash column chromatography on silica (eluent: CH₂Cl₂/

EtOAc 65:35).

Methyl 3-(1,3-dioxoisoindolin-2-yl)-2-hydroxypropanoate

(7c)

GP5b was followed using 3-amino-2-hydroxypropionic

acid 7a (1.10 g, 10.5 mmol), phthalic anhydride (1.56 g,

10.5 mmol) in MeOH (80.0 ml) and 2 M HCl in Et₂O

(80.0 ml). The desired compound was obtained as amor-

phous white solid (2.00 g, 76%). ¹H NMR (400 MHz,

CDCl₃) δ = 7.90–7.83 (m, 2H, NCOC_arCH_arCH_ar),

7.78–7.69 (m, 2H, NCOC_arCH_arCH_ar), 4.50 (td, J = 6.5,

5.1 Hz, 1H, NCH₂CH), 4.08 (dd, J = 14.1, 5.1 Hz, 1H,

NCH₂), 4.02 (dd, J = 14.1, 6.4 Hz, 1H, NCH₂), 3.82 (s, 3H,

OCH₃), 3.07 (d, J = 6.8 Hz, 1H, OH) ppm; ¹³C NMR

General procedure for the coupling of the alcohol with

4,4′,4″-trimethoxytrithyl chloride (GP8)

The alcohol (1.0 eq) was solved in pyridine. Dimethyl-

formamide (DMF, 1 drop) and 4,4′,4″-trimethoxytrithyl

chloride (1.8 eq.) were added. The mixture was stirred at

55 °C for 16 h and reduced in vacuum. The crude product

was puriﬁed by ﬂash chromatography on silica (eluent

Et₂O/pentane: 6:4).

Medicinal Chemistry Research

(101 MHz, CDCl₃) δ = 173.2 (COOCH₃), 168.4 (NCO),

134.3 (NCOC_arCH_arCH_ar), 132.0 (NCOC_arCH_arCH_ar), 123.7

(NCOC_arCH_arCH_ar), 68.7 (NCH₂CH), 53.2 (OCH₃), 41.2

(NCH₂) ppm; IR (HBr): ṽ = 3497, 1747, 1700, 1464, 1440,

1396, 1310, 1231, 1095, 983, 883, 722 cm⁻¹; HRESIMS:

m/z (pos): 272.0529 C₁₂H₁₁NO₅Na (calcd. 272.0535).

(856 mg, 2.25 mmol). The desired compound was

obtained as yellow oil (705 mg, 90%). ¹H NMR

(500 MHz, CD₂Cl₂) δ = 7.87–7.76 (m, 2H, NCOC_arCH_ar

CH_ar), 7.76–7.67 (m, 2H, NCOC_arCH_arCH_ar), 7.26–7.17

(m, 6H, C_arCH_arCH_arC_arOCH₃), 6.81–6.68 (m, 6H,

C_arCH_arCH_arC_arOCH₃), 4.38 (dd, J = 7.3, 5.5 Hz, 1H,

NCH₂CH), 4.06 (dd, J = 14.0, 7.4 Hz, 1H, NCH₂), 4.00

(dd, J = 14.1, 5.5 Hz, 1H, NCH₂), 3.77 (s, 9H, C_arOCH₃),

3.74 (s, 3H, COOCH₃), 3.73 (ddd, J = 10.5, 5.4, 3.5 Hz,

1H, OCH₂CH₂OCC_ar),3.52 (ddd, J = 10.4, 6.9,

3.5 Hz, 1H, OCH₂CH₂OCC_ar), 3.16 (ddd, J = 10.4, 6.9,

3.5 Hz, 1H, OCH₂CH₂OCC_ar), 3.06 (ddd, J = 10.3, 5.3,

3.5 Hz, 1H, OCH₂CH₂OCC_ar) ppm; ¹³C NMR (126 MHz,

CD₂Cl₂) δ = 171.3 (COOCH₃), 168.4 (NCO), 158.9

(CC_arCH_arCH_arC_arOCH₃), 137.2 (CC_arCH_ar), 134.6

(NCOC_arCH_arCH_ar), 132.5 (NCOC_ar), 130.2 (CH₃O-

Methyl 3-{2-[(tert-butyldiphenylsilyl)oxy]ethoxy}-3-{-(1,3-

dioxoisoindolin-2-yl})propanoate (7d)

GP6 was followed using 7c (4.3 mmol, 1072 mg), tert-butyl

(2-iodoethoxy)diphenylsilane (6.02 mmol, 2374 mg), Ag₂CO₃

(17.2 mmol, 4743 mg), toluene (15.0 ml), 4d. The desired

1

compound was obtained as yellow oil (1882 mg, 82%). H

NMR (500 MHz, CD₂Cl₂) δ = 7.85–7.78 (m, 2H, NCO-

C_arCH_arCH_ar), 7.76–7.68 (m, 2H, NCOC_arCH_arCH_ar),

7.66–7.57 (m, 4H_rCH_ar), 7.47–7.33 (m, 6H, CH_ar), 4.38 (dd,

J = 6.8, 6.0 Hz, 1H, NCH₂CH), 4.02 (dd, J = 14.0,

6.8 Hz,1H, NCH₂), 3.98 (dd, J = 14.0, 6.0 Hz, 1H, NCH₂),

3.78–3.68 (m, 6H, CHOCH₂CH₂Si, OCH₃), 3.58–3.49 (m,

1H, CHOCH₂CH₂Si), 0.94 (s, 9H, CCH₃) ppm; ¹³C NMR

(126 MHz, CD₂Cl₂) δ = 171.3 (COOCH₃), 168.4 (NCO),

136.1 (C_ar), 134.6 (C_ar), 134.0 (C_ar), 132.5 (C_ar), 130.2 (C_ar),

128.2 (C_ar), 123.8 (C_ar), 76.7 (NCH₂CH), 72.4 (OCH₂CH₂Si),

63.9 (OCH₂CH₂Si), 52.7 (OCH₃), 39.9 (NCH₂CH), 27.0

(SiCCH₃), 19.4 (SiC) ppm; IR (ﬁlm): ṽ = 2930, 1755, 1700,

1427, 1395, 1208, 1111, 703 cm⁻¹; HRESIMS m/z (pos):

554.1970 C₃₀H₃₃NO₆SiNa (calcd. 554.1975).

C_arCH_arCH_ar),

123.8

(NCOC_arCH_arCH_ar),

113.5

(CC_arCH_arCH_arC_arOCH₃), 86.2 (OCH₂CH₂OCC_ar), 76.8

(NCH₂CH), 70.9 (OCH₂CH₂OCC_ar), 63.5 (OCH₂-

CH₂OCC_ar), 55.7 (C_arOCH₃), 52.7 (COOCH₃), 40.0

(NCH₂CH) ppm; IR (ﬁlm): ṽ = 1776, 1607, 1506, 1464,

1395, 1249, 1176, 1034, 827 cm⁻¹; HRESIMS m/z (pos):

648.2210 C₃₆H₃₅NO₉Na (calcd. 648.2210).

3-Amino-2-{2-[tris(4-methoxyphenyl)methoxy]ethoxy}

propanoic acid (7g)

GP9a was followed using 7f (192 mg, 0.300 mmol), MeOH

(3.0 ml), 12 M NaOH (0.05 ml), 1,2-diaminoethane (126 mg,

2.10 mmol). The desired compound was obtained as amor-

phous white solid (123 mg, 83%). ¹H NMR (500 MHz, 0.1 M

NaOD/MeOD) δ = 7.46–7.14 (m, 6H, CH₃OC_arCH_arCH_ar),

7.05–6.65 (m, 6H, CH₃OC_arCH_arCH_ar), 3.85 (ddd, J = 10.6,

5.2, 4.4 Hz, 1H, OCH₂CH₂OCC_ar), 3.81 (dd, J = 6.4, 3.8 Hz,

1H, NCH₂CH), 3.77 (s, 9H, OCH₃), 3.52 (ddd, J = 10.9, 6.7,

4.1 Hz, 1H, OCH₂CH₂OCC_ar), 3.31 (m, 1H, OCH₂CH₂OC-

C_ar), 3.24 (ddd, J = 10.0, 5.4, 4.1 Hz, 1H, OCH₂CH₂OCC_ar),

2.93 (dd, J = 13.4, 3.8 Hz, 1H, NCH₂), 2.85 (dd, J = 13.4,

6.4 Hz, 1H, NCH₂) ppm; ¹³C NMR (126 MHz, 1 M NaOD/

MeOD) δ = 180.8 (COOH), 179.2 (CH₃OC_arCH_arCH_arC_ar),

159.9 (CH₃OC_arCH_arCH_arC_ar), 138.0 (CH₃OC_arCH_arCH_arC_ar),

131.0 (CH₃OC_arCH_arCH_arC_ar), 87.3 (OCH₂CH₂OCC_ar), 84.3

(NCH₂CH), 70.5 (OCH₂CH₂OCC_ar), 64.4 (OCH₂CH₂OC-

C_ar), 55.9 (OCH₃), 45.2 (NCH₂CH) ppm; IR (HBR): ṽ =

3375, 1608, 1508, 1420, 1303, 1176, 1034 cm⁻¹; HRESIMS

m/z (neg): 480.2030 C₂₇H₃₀NO₇(calcd. 480.2028).

Methyl 3-(1,3-dioxoisoindolin-2-yl)-3-(2-hydroxyethoxy)

propanoate (7e)

GP7 was followed using 7d (737 mg, 1.35 mmol), THF/

pyridine 9:1 (10.0 ml), HF-pyridine (1.93 g, 6.75 mmol).

The desired compound was obtained as a yellow oil

(350 mg, 84%). ¹H NMR (400 MHz, CD₂Cl₂) δ = 7.89–7.82

(m, 2H, NCOC_arCH_arCH_ar), 7.81–7.72 (m, 2H, NCO-

C_arCH_arCH_ar), 4.25 (dd, J = 6.5, 5.5 Hz, 1H, NCH₂CH),

4.00 (dd, J = 14.0, 6.5 Hz, 1H, NCH₂), 4.05 (dd, J = 14.0,

5.5 Hz, 1H, NCH₂), 3.75 (s, 3H, OCH₃), 3.70–3.54 (m, 4H,

OCH₂CH₂OH) ppm; ¹³C NMR (101 MHz, CD₂Cl₂) δ =

171.55 (COOCH₃), 168.6 (NCO), 134.8 (NCOC_arCH_ar

CH_ar), 132.5 (NCOC_arCH_ar), 123.9 (NCOC_arCH_ar), 76.9

(NCH₂CH), 73.4 (OCH₂CH₂OH), 62.0 (OCH₂CH₂OH),

52.9 (OCH₃), 40.0 (NCH₂) ppm; IR (ﬁlm): ṽ = 3474, 1774,

1770, 1429, 1396, 1213, 1029, 720 cm⁻¹; HRESIMS m/z

(pos): 294.0973 C₁₄H₁₆NO₆(calcd. 294.0978).

1-Butyl-3-hydroxyazetidine-3-carboxamide (11c)

Methyl 3-{1,3-dioxoisoindolin-2-yl}-2-{2-[tris(4-

methoxyphenyl)methoxy]ethoxy}propanoat (7f)

GP2 was followed using 1-benzhydryl-3-hydroxyazetidine-

3-carboxamide 11b (350 mg, 1.20 mmol), palladium on

charcoal (10% Pd, 131 mg, 0.120 mmol) and butyraldehyde

(0.28 ml, 3.1 mmol) in EtOH (2.0 ml). The crude product

GP8 was followed using 7e (367 mg, 1.25 mmol), DMF (1

drop), pyridine (4.0 ml), 4,4′,4″-trimethoxytrithyl chloride

Medicinal Chemistry Research

was puriﬁed by ﬂash column chromatography on silica

(eluent: EtOAc/MeOH 95:5 + 3% triethylamine) to afford

the desired compound as amorphous white solid (177 mg,

1451, 1304, 1231, 1080, 902, 749, 706 cm−¹; HREIMS m/z

(pos): 325.1662 C₂₀H₂₃NO₃(calcd. 325.1672).

1

83%). H NMR (400 MHz, MeOD): δ (ppm) = 3.65–3.55

Ethyl 2-(1-butyl-3-hydroxyazetidin-3-yl)acetate (11f)

(m, 2H, NCH₂COH), 3.27–3.22 (m, 2H, NCH₂COH),

2.57–2.48 (m, 2H, CH₃CH₂CH₂CH₂N), 1.41–1.28 (m, 4H,

CH₃CH₂CH₂CH₂N + CH₃CH₂CH₂CH₂N), 0.96–0.87 (m,

3H, CH₃CH₂CH₂CH₂N); ¹³C NMR (101 MHz, MeOD): δ

(ppm) = 14.4 (CH₃CH₂CH₂CH₂N), 21.5 (CH₃CH₂CH₂

CH₂N), 30.6 (CH₃CH₂CH₂CH₂N), 59.8 (CH₃CH₂CH₂

CH₂N), 65.6 (NCH₂COH), 72.2 (NCH₂COH), 178.4

GP2 was followed using 11e (263 mg, 0.810 mmol), palla-

dium on charcoal (10% Pd, 84 mg, 0.081 mmol) and butyr-

aldehyde (0.18 ml, 2.0 mmol) in EtOH (5.0 ml). The crude

product was puriﬁed by ﬂash column chromatography on

silica gel (eluent: Et₂O + 3% triethylamine) to afford the

1

desired compound as colourless oil (195 mg, 87%). H NMR

(CONH₂); IR (KBr): ν

̃

= 3467, 2930, 2361, 1694, 1383,

(500 MHz, CDCl₃): δ (ppm) = 4.19 (q, 2H, J = 7.2 Hz,

COOCH₂CH₃), 3.92 (br s, 1H, OH), 3.43–3.25 (m, 2H,

NCH₂COH), 3.14–2.96 (m, 2H, NCH₂COH), 2.86 (s, 2H,

CH₂COO), 2.52–2.40 (m, 2H, NCH₂CH₂CH₂CH₃), 1.37–

1.23 (m, 7H, NCH₂CH₂CH₂CH₃+ NCH₂CH₂CH₂CH₃+

COOCH₂CH₃), 0.98–0.71 (m, 3H, NCH₂CH₂CH₂CH₃); ¹³C

NMR (126 MHz, CDCl₃): δ (ppm) = 173.0 (COOCH₂CH₃),

68.3 (COH), 66.3 (NCH₂COH), 61.0 (COOCH₂CH₃), 59.7

(NCH₂CH₂CH₂CH₃), 42.8 (CH₂COO), 30.1 (NCH₂CH₂

CH₂CH₃), 20.6 (NCH₂CH₂CH₂CH₃), 14.3 (COOCH₂CH₃),

1252, 1164, 899, 765, 669 cm⁻¹; HRESIMS m/z (pos):

173.1288 C₈H₁₇N₂O₂(calcd. 173.1285).

1-Butyl-3-hydroxyazetidine-3-carboxylic acid (11d)

GP3 was followed using 1-butyl-3-hydroxyazetidine-3-

carboxamide 11c (88 mg, 0.51 mmol) and barium hydroxide

octahydrate (380 mg, 1.20 mmol) in EtOH/H₂O 1:1

(10.0 ml), 24 h, reﬂux. The desired compound was obtained

as amorphous white solid (84 mg, 95%). ¹H NMR

(400 MHz, D₂O + NaOD): δ (ppm) = 3.58 (d, J = 9.2 Hz,

2H, NCH₂COH), 3.21 (d, J = 9.1 Hz, 2H, NCH₂COH),

2.54–2.48 (m, 2H, CH₃CH₂CH₂CH₂N), 1.36–1.20 (m, 4H,

CH₃CH₂CH₂CH₂N + CH₃CH₂CH₂CH₂N), 0.89–0.82 (m, 3H,

CH₃CH₂CH₂CH₂N); ¹³C NMR (101 MHz, D₂O + NaOD): δ

(ppm) = 180.5 (COO), 72.6 (NCH₂COH), 64.7 (NCH₂COH),

58.8 (CH₃CH₂CH₂CH₂N), 29.4 (CH₃CH₂CH₂CH₂N), 20.5

(CH₃CH₂CH₂CH₂N), 13.4 (CH₃CH₂CH₂CH₂N); IR (KBr):

14.2 (NCH₂CH₂CH₂CH₃); IR (ﬁlm): ν = 3456, 2958, 2933,

̃

1737, 1465, 1370, 1190, 1031, 948, 879 cm⁻¹; HRESIMS m/z

(pos): 216.1589 C₁₁H₂₂NO₃(calcd. 216.1594).

2-(1-Butyl-3-hydroxyazetidin-3-yl)acetic acid (11g)

GP3 was followed using 11f (34 mg, 0.16 mmol) and barium

hydroxide octahydrate (201 mg, 0.630 mmol) in EtOH/H₂O

1:1 (6.0 ml), 19 h, rt. The desired compound was obtained as

1

ν

̃

= 3467, 2930, 2361, 1694, 1383, 1252, 1164, 899, 765,

amorphous white solid (26 mg, 87%). H NMR (400 MHz,

669 cm⁻¹; HRESIMS m/z (pos): 173.1129 C₈H₁₆NO₃

D₂O + NaOD): δ (ppm) = 3.39–3.33 (m, 2H, NCH₂COH),

3.09–3.02 (m, 2H, NCH₂COH), 2.61 (s, 2H, CCH₂COO),

2.54–2.45 (m, 2H, CH₃CH₂CH₂CH₂N), 1.40–1.27 (m, 4H,

CH₃CH₂CH₂CH₂N + CH₃CH₂CH₂CH₂N), 0.96–0.87 (m,

3H, CH₃CH₂CH₂CH₂N); ¹³C NMR (101 MHz, D₂O +

NaOD): δ (ppm) = 180.4 (COO), 69.1 (NCH₂COH), 66.1

(NCH₂COH), 59.6 (CH₃CH₂CH₂CH₂N), 46.1 (CH₂COO),

29.8 (CH₃CH₂CH₂CH₂N), 20.9 (CH₃CH₂CH₂CH₂N), 14.3

(calcd. 174.1125).

Ethyl 2-(1-benzhydryl-3-hydroxyazetidin-3-yl)acetate (11e)

GP1 was followed using LiHMDS in methyl tert-butyl ether

(2.0 ml, 1.9 mmol), EtOAc (0.19 ml, 1.9 mmol) and 1-

benzhydrylazetidin-3-one 11a (424 mg, 1.70 mmol) in THF

(2.0 ml). The crude product was puriﬁed by ﬂash column

chromatography on silica (eluent: pentane/Et₂O 3:1 + 2%

diethylmethylamine) to afford the desired compound as

amorphous white solid (448 mg, 80%).¹H NMR (500 MHz,

CDCl₃): δ (ppm) = 7.47–7.11 (m, 10H, CH_ar), 4.39 (s, 1H,

NCHC_ar), 4.17 (q, J = 7.1 Hz, 2H, COOCH₂CH₃), 3.79 (s,

1H, OH), 3.34–3.17 (m, 2H, NCH₂COH), 3.09–2.98 (m,

2H, NCH₂COH), 2.91 (s, 2H, CH₂COO), 1.28 (t, J =

7.2 Hz, 3H, COOCH₂CH₃); ¹³C NMR (101 MHz, CDCl₃): δ

(ppm) = 172.9 (COOCH₂CH₃), 142.3 (NCHCCHCHCH),

128.5 (NCHCCHCHCH), 127.6 (NCHCCHCHCH), 127.3

(NCHCCHCHCH), 77.9 (NCHC_ar), 67.6 (NCH₂COH),

65.2 (NCH₂COH), 61.1 (COOCH₂CH₃), 43.0 (CH₂COO),

(CH₃CH₂CH₂CH₂N); IR (ﬁlm): ν = 3427, 2930, 2360, 2342,

̃

1583, 1569, 1420, 1108, 912, 686 cm⁻¹; HREIMS m/z (pos):

187.1193 C₉H₁₇NO₃(calcd. 187.1203).

Ethyl 2-(1-benzyl-3-hydroxypyrrolidin-3-yl)acetate (12e)

GP1 was followed using LiHMDS in MTBE (2.1 ml,

2.0 mmol), EtOAc (0.20 ml, 2.0 mmol) and 1-

benzylpyrrolidin-3-one 12a (371 mg, 2.10 mmol) in THF

(1.0 ml). The crude product was puriﬁed by ﬂash column

chromatography on silica (eluent: pentane/EtOAc 6:4 + 2%

diethylmethylamine) to afford the desired compound as

pale-yellow oil (499 mg, 93%). ¹H NMR (500 MHz,

CDCl₃): δ (ppm) = 7.26 (m, 5H, CH_ar), 4.17 (q, J = 7.1 Hz,

14.3 (COOCH₂CH₃); IR (KBr): ν = 3404, 2952, 1718,

̃

Medicinal Chemistry Research

2H, COOCH₂CH₃), 3.64 (s, 2H, C_arCH₂N), 3.58 (br s, 1H,

OH), 2.80–2.74 (m, 1H, NCH₂CH₂), 2.71–2.54 (m, 5H,

NCH₂CH₂+ NCH₂COH + NCH₂COH + CH₂COO),

2.01–1.94 (m, 1H, NCH₂CH₂), 1.93–1.85 (m, 1H,

NCH₂CH₂), 1.26 (t, J = 7.1 Hz, 3H, COOCH₂CH₃); ¹³C

NMR (126 MHz, CDCl₃): δ (ppm) = 172.8 (COOCH₂CH₃),

139.0 (NCH₂CCHCHCH), 128.9 (NCH₂CCHCHCH),

128.4 (NCH₂CCHCHCH), 127.1 (NCH₂CCHCHCH), 66.6

(NCH₂COH), 60.9 (COOCH₂CH₃), 60.3 (NCH₂C_ar), 52.9

(NCH₂CH₂), 44.5 (CH₂COO), 39.4 (NCH₂CH₂), 14.3

NCH₂CH₂CH₂CH₃); ¹³C NMR (101 MHz, D₂O + NaOD):

δ (ppm) = 180.2 (COOCH₂CH₃), 78.1 (COH), 65.7

(NCH₂COH), 55.9 (NCH₂CH₂CH₂CH₃), 52.7 (NCH₂CH₂

COH), 47.1 (CH₂COO), 38.5 (NCH₂CH₂COH), 29.7

(NCH₂CH₂CH₂CH₃), 20.3 (NCH₂CH₂CH₂CH₃), 13.4

(NCH₂CH₂CH₂CH₃); IR (KBr): ν = 3426, 2965, 2876,

̃

2360, 1589, 1395, 1094, 1014, 741, 669 cm⁻¹; HRESIMS

m/z (pos): 202.1439 C₁₀H₂₀NO₃(calcd. 202.1438).

1-Benzyl-3-hydroxypiperidine-3-carboxamide (13b)

(COOCH₂CH₃); IR (ﬁlm): ν̃ = 3512, 2978, 2796, 1732,

1372, 1189, 1029, 911, 740, 699 cm⁻¹_;HREIMS m/z (pos):

1-Benzylpiperidin-3-one hydrochloride 13a (571 mg,

2.50 mmol) was solved in dry CH₂Cl₂(4.0 ml). Freshly

distilled triethylamine (0.85 ml, 6.1 mmol) was added and

the brownish suspension was placed in an ultrasound bath

for 15 min. After addition of trimethylsilyl cyanide

(0.80 ml, 6.3 mmol) the reaction mixture was stirred for

48 h, diluted with CH₂Cl₂(10.0 ml), ﬁltered through a paper

ﬁlter and reduced in vacuum. The oily residue was solved in

CH₂Cl₂(7.0 ml) and cooled to 0 °C. Concentrated sulfuric

acid (0.70 ml, 13 mmol) was added and the biphasic mixture

was stirred for 2 h at rt, after which the mixture was cooled

to 0 °C, diluted with H₂O (5.0 ml) and alkalized with 25%

ammonium hydroxide solution. Potassium sodium tartrate

(0.50 g) was added and the mixture was extracted ﬁve times

with CH₂Cl₂(20.0 ml). The combined organic phases were

dried over MgSO₄and reduced in vacuum. The crude

product was puriﬁed by ﬂash column chromatography on

silica (eluent: ethyl acetate + 3% triethylamine) to afford

the desired compound as amorphous off-white solid

(567 mg, 96%)_.¹H NMR (400 MHz, 1,1,2,2-tetra-

chloroethane-d2, 80 °C): δ (ppm) = 7.61 (br s, 1H, NH₂),

7.44–7.29 (m, 5H, CH_ar), 5.46 (br s, 1H, NH₂), 3.92 (br s,

1H, OH), 3.64 (s, 2H, NCH₂C_ar), 2.74 (dd, J = 11.3, 1.0 Hz,

1H, NCH_axH_eqCOH), 2.63–2.46 (m, 3H, NCH_axH_eqCOH +

NCH_axH_eqCH₂CH₂+ NCH_axH_eqCH₂CH₂), 1.96 (dddd, J =

13.0, 7.8, 5.1, 1.0 Hz, 1H, NCH₂CH₂CH_axH_eq), 1.89–1.57

(m, 3H, NCH₂CH₂CH_axH_eq+ NCH₂CH_axH_eqCH₂+ NCH₂

CH_axH_eqCH₂); ¹³C NMR (101 MHz, 1,1,2,2-tetra-

chloroethane-d2, 80 °C): δ (ppm) = 176.8 (CNH₂), 137.2

(NCH₂CCHCHCH), 128.9 (NCH₂CCHCHCH), 128.3

(NCH₂CCHCHCH), 127.3 (NCH₂CCHCHCH), 71.9

(NCH₂COH), 62.6 (NCH₂C_ar), 60.3 (NCH₂COH), 52.7

(NCH₂CH₂CH₂), 33.6 (NCH₂CH₂CH₂), 21.5 (NCH₂

263.1525 C₁₅H₂₁NO₃(calcd. 263.1516).

Ethyl 2-(1-butyl-3-hydroxypyrrolidin-3-yl)acetate (12f)

GP2 was followed using 12e (290 mg, 1.10 mmol), palladium

on charcoal (10% Pd, 117 mg, 0.110 mmol) and butyr-

aldehyde (0.30 ml, 3.3 mmol) in EtOH (3.5 ml). The crude

product was puriﬁed by ﬂash column chromatography on

silica gel (eluent: pentane/Et₂O 7:3 + 3% diethylmethyla-

mine) to afford the desired compound as colourless oil

(195 mg, 77%).¹H NMR (400 MHz, CDCl₃): δ (ppm) =

4.18 (q, J = 7.2 Hz, 2H, COOCH₂CH₃), 3.54 (br s,

1H, OH), 2.81–2.61 (m, 4H, CH₂COO + NCH₂CH₂COH +

NCH₂COH), 2.57–2.38 (m, 4H, NCH₂CH₂COH +

NCH₂COH + NCH₂CH₂CH₂CH₃), 2.01–1.92 (m, 1H,

NCH₂CH₂COH), 1.86 (m, 1H, NCH₂CH₂COH), 1.51–1.40

(m, 2H, NCH₂CH₂CH₂CH₃), 1.38–1.25 (m, 5H, COOCH₂

CH₃+ NCH₂CH₂CH₂CH₃), 0.91 (t, J = 7.3 Hz, 3H, NCH₂

CH₂CH₂CH₃); ¹³C NMR (126 MHz, CDCl₃): δ (ppm) =

172.8 (COOCH₂CH₃), 77.6 (COH), 67.0 (NCH₂COH), 60.9

(COOCH₂CH₃), 56.2 (NCH₂CH₂CH₂CH₃), 53.1 (NCH₂

CH₂COH), 44.6 (CH₂COO), 39.4 (NCH₂CH₂COH), 30.9

(NCH₂CH₂CH₂CH₃), 20.9 (NCH₂CH₂CH₂CH₃), 14.3

(COOCH₂CH₃), 14.2 (NCH₂CH₂CH₂CH₃); IR (ﬁlm): ν =

̃

3442, 2958, 2797, 1735, 1465, 1370, 1190, 1031, 946,

904 cm⁻¹HREIMS m/z (pos): 229.1673 C₁₂H₂₃NO₃(calcd.

;

229.1672).

2-(1-Butyl-3-hydroxypyrrolidin-3-yl)acetic acid (12g)

GP3 was followed using 12f (181 mg, 0.790 mmol) and

barium hydroxide octahydrate (500 mg, 1.58 mmol) in

EtOH/H₂O 1:1 (8.0 ml), 6 h, rt. The desired compound

was obtained as amorphous off-white solid (153 mg,

96%). ¹H NMR (400 MHz, D₂O + NaOD): δ (ppm) =

3.90–3.66 (m, 2H, NCH₂COH + NCH₂CH₂COH),

3.46–3.14 (m, 4H, NCH₂COH + NCH₂CH₂COH +

NCH₂CH₂CH₂CH₃), 2.80–2.59 (m, 2H, CH₂COO),

2.36–2.03 (m, 2H, NCH₂CH₂COH + NCH₂CH₂COH),

1.78–1.62 (m, 2H, NCH₂CH₂CH₂CH₃), 1.39 (h, J =

7.4 Hz, 2H, NCH₂CH₂CH₂CH₃), 0.93 (t, J = 7.4 Hz, 3H,

CH₂CH₂); IR (KBr): ν = 3457, 3409, 2790, 1663, 1274,

̃

1206, 1020, 924, 735, 665 cm⁻¹; HREIMS m/z (pos):

234.1361 C₁₃H₁₈N₂O₂(calcd. 234.1363).

1-Butyl-3-hydroxypiperidine-3-carboxamide (13c)

GP2 was followed using 1-benzyl-3-hydroxypiperidine-3-

carboxamide 13b (300 mg, 1.30 mmol), palladium on

charcoal (10% Pd, 138 mg, 0.130 mmol) and butyraldehyde

Medicinal Chemistry Research

(0.29 ml, 3.20 mmol) in EtOH (3.5 ml). The crude product

was puriﬁed by ﬂash column chromatography on silica

(eluent: EtOAc + 3% triethylamine) to afford the desired

obtained as amorphous white solid (155 mg, 79%). ¹H

NMR (500 MHz, MeOD) δ = 4.00 (t, J = 5.7 Hz, 1H,

CHOH), 3.16–3.09 (m, 2H, OHCCH₂CH₂N), 2.97 (m, 2H,

NCH₂CH₂CH₂CH₃), 2.05 (dtd, J = 14.2, 7.1, 5.5 Hz, 1H,

OHCHCH₂), 1.95 (dq, J = 14.5, 6.2 Hz, 1H, OHCHCH₂),

1.65 (m, 2H, CH₃CH₂CH₂), 1.43 (m, 2H, CH₃CH₂CH₂),

0.99 (t, J = 7.4 Hz, 3H, CH₃CH₂CH₂ppm); ¹³C NMR

(126 MHz, MeOD) δ = 180.0 (COOH), 72.0 (COOHCH),

49.8 (NCH₂CH₂CH₂CH₃), 46.8 (OHCHCH₂CH₂), 31.7

1

compound as amorphous white solid (238 mg, 93%). H

NMR (400 MHz, 1,1,2,2-tetrachloroethane-d2, 80 °C): δ

(ppm) = 7.65 (br s, 1H, NH₂), 5.35 (br s, 1H, NH₂), 3.96 (br

s, 1H, OH), 2.63 (d, J = 11.4 Hz, 1H, NCH_axH_eqCOH),

2.48–2.28 (m, 5H, CH₃CH₂CH₂CH₂N + NCH_axH_eqCOH +

NCH_axH_eqCH₂CH₂OH + NCH_axH_eqCH₂CH₂OH),

1.86–1.78 (m, 1H, NCH₂CH₂CH_axH_eqCOH), 1.76–1.57 (m,

2H, NCH₂CH_axH_eqCH₂COH + NCH₂CH_axH_eqCH₂COH),

1.54–1.37 (m, 3H, CH₃CH₂CH₂CH₂N + NCH₂CH₂CH_ax-

H_eqCOH), 1.34–1.20 (m, 2H, CH₃CH₂CH₂CH₂N), 0.88 (t,

J = 7.3 Hz, 3H, CH₃CH₂CH₂CH₂N); ¹³C NMR (101 MHz,

(OHCHCH₂CH₂),

29.4

(NCH₂CH₂CH₂CH₃),

20.8

(NCH₂CH₂CH₂CH₃), 13.9 (NCH₂CH₂CH₂CH₃) ppm; IR

(KBr): ṽ = 3411, 1651, 1359, 1335, 1103, 820 cm⁻¹

;

HRESIMS m/z (pos): 176.1281 C₈H₁₈NO₃(calcd.

176.1287).

1,1,2,2-tetrachloroethane-d2, 80 °C):

δ

(ppm) = 177.4

(CNH₂), 72.1 (NCH₂COH), 61.0 (NCH₂COH), 58.1

(CH₃CH₂CH₂CH₂N), 53.1 (NCH₂CH₂CH₂COH), 34.2

(NCH₂CH₂CH₂COH), 29.0 (NCH₂CH₂CH₂OH), 21.4

(CH₃CH₂CH₂CH₂N), 14.1 (CH₃CH₂CH₂CH₂N);_.IR (KBr):

Methyl 2-{2-[(tert-butyldiphenylsilyl)oxy]ethoxy}-4-(1,3-

dioxoisoindolin-2-yl)butanoate (14d)

GP6 was followed using 14c (369 mg, 1.20 mmol), tert-

butyl(2-iodoethoxy)diphenylsilane (805 mg, 1.70 mmol),

Ag₂CO₃(1.32 g, 4.80 mmol), toluene (2.0 ml), 4d. The

desired compound was obtained as yellow oil (500 mg,

76%). ¹H NMR (500 MHz, CD₂Cl₂) δ = 7.83–7.77 (m, 2H,

NCOC_arCH_arCH_ar), 7.73–7.66 (m, 6H, CH_ar), 7.47–7.36 (m,

6H, CH_ar), 4.02 (dd, J = 8.8, 3.8 Hz, 1H, NCH₂CH₂CH),

3.86–3.70 (m, 5H, NCH₂, SiOCH₂CH₂), 3.68 (s, 3H,

COOCH₃), 3.54 (ddd, J = 9.7, 6.2, 4.2 Hz, 1H,

SiOCH₂CH₂), 2.11 (dtd, J = 14.4, 7.3, 3.8 Hz, 1H,

NCH₂CH₂), 2.08–2.00 (m, 1H, NCH₂CH₂), 1.04 (s, 9H,

CCH₃) ppm; ¹³C NMR (126 MHz, CD₂Cl₂) δ = 172.9

(COOCH₃), 168.7 (NCO), 136.2 (C_ar), 134.5 (C_ar), 134.2

(C_ar), 132.7 (C_ar), 130.2 (C_ar), 128.2 (C_ar), 123.6 (C_ar), 77.8

(NCH₂CH₂CH), 72.4 (SiOCH₂CH₂), 63.9 (SiOCH₂CH₂),

52.3 (COOCH₃), 35.1 (NCH₂), 32.2 (NCH₂CH₂), 27.1

(CCH₃), 19.6 (CCH₃) ppm; IR (ﬁlm): ṽ = 2932, 1770,

1468, 1396, 1112, 823, 738, 703 cm⁻¹; HRESIMS m/z

(pos): 568.2125 C₃₁H₃₅NO₆SiNa (calcd. 568.2126).

ν̃ = 3462, 2955, 2805, 1682, 1454, 1399, 1137, 1019, 917,

659 cm⁻¹; HREIMS m/z (pos): 200.1519 C₁₀H₂₀N₂O₂

(calcd. 200.1525).

1-Butyl-3-hydroxypiperidine-3-carboxylic acid (13d)

GP3 was followed using 1-butyl-3-hydroxypiperidine-3-

carboxamide 13c (80 mg, 0.40 mmol) and barium hydroxide

octahydrate (254 mg, 0.801 mmol) in EtOH/H₂O 1:1

(10.0 ml), 5 h, reﬂux. The desired compound was obtained

as colourless semi-solid (76 mg, 94%). ¹H NMR (400 MHz,

D₂O + NaOD): δ (ppm) = 2.49 (br d, J = 10.8 Hz, 1H,

NCH_axH_eqCH₂CH₂COH), 2.38 (d, J = 12.0 Hz, 1H,

NCH_axH_eqCOH), 2.06–1.90 (m, 3H, CH₃CH₂CH₂CH₂N +

NCH_axH_eqCOH), 1.72–1.61 (m, 1H, NCH_axH_eqCH₂CH₂-

COH), 1.47–1.20 (m, 4 H, NCH₂CH₂CH_axH_eqCOH +

NCH₂CH₂CH_axH_eqCOH + NCH₂CH_axH_eqCH₂COH + NCH₂

CH_axH_eqCH₂COH), 1.15–1.03 (m, 2H, CH₃CH₂CH₂CH₂N),

0.92 (h, J = 7.3 Hz, 2H, CH₃CH₂CH₂CH₂N), 0.54 (t, J =

7.3 Hz, 3H, CH₃CH₂CH₂CH₂N); ¹³C NMR (101 MHz,

D₂O + NaOD): δ (ppm) = 182.1 (COO), 74.3 (NCH₂COH),

59.6 (NCH₂COH), 57.9 (CH₃CH₂CH₂CH₂N), 52.8

(NCH₂CH₂CH₂COH), 32.2 (NCH₂CH₂CH₂COH), 27.2

(CH₃CH₂CH₂CH₂N), 20.3 (NCH₂CH₂CH₂COH), 20.2

(CH₃CH₂CH₂CH₂N), 13.3 (CH₃CH₂CH₂CH₂N); IR (KBr):

Methyl 4-(1,3-dioxoisoindolin-2-yl)-2-(2-hydroxyethoxy)

butanoate (14e)

GP7 was followed using 14d (442 mg, 0.810 mmol), THF/

pyridine 9:1 (10.0 ml), HF-pyridine (116 mg, 4.05 mmol).

The desired compound was obtained as yellow oil (190 mg,

1

ν

̃

= 3414, 2961, 2874, 1609, 1388, 1179, 1120, 1016, 949,

76%). H NMR (500 MHz, CDCl₃) δ = 7.93–7.80 (m, 2H,

719 cm⁻¹_;HREIMS m/z (pos): 201.1365 C₁₀H₁₉NO₃(calcd.

C_arCH_arCH_ar), 7.80–7.66 (m, 2H, C_arCH_arCH_ar), 3.99 (ddd,

J = 13.9, 8.8, 5.0 Hz, 1H, NCH₂), 3.92 (dd, J = 9.9, 3.2 Hz,

1H, NCH₂CH₂CH), 3.81 (dt, J = 14.0, 5.5 Hz, 1H, NCH₂),

3.76–3.67 (m, 6H, OCH₃, CHOCH₂CH₂OH), 3.63–3.56 (m,

1H, CHOCH₂CH₂OH), 2.94 (t, J = 5.7 Hz, 1H, OH), 2.19

(dddd, J = 14.3, 8.7, 5.5, 3.2 Hz, 1H, NCH₂CH₂), 2.08 (ddt,

J = 14.9, 9.78, 5.25, 5.25 Hz, 1H, NCH₂CH₂) ppm; ¹³C

NMR (126 MHz, CDCl₃) δ = 173.0 (COOCH₃), 168.6

201.1359).

4-(Butylamino)-2-hydroxybutanoic acid (14b)

GP4 was followed using 14a (134 mg, 1,12 mmol), EtOH

(2.4 ml), KOH (56,1 mg, 2.25 mmol), 1-bromobutane

(139 mg, 1.01 mmol). The desired compound was

Medicinal Chemistry Research

(NCO), 134.3 (C_arCH_arCH_ar), 132.2 (C_arCH_arCH_ar), 123.5

(C_arCH_arCH_ar), 76.7 (NCH₂CH₂CH), 72.7 (CHOCH₂-

CH₂OH), 62.0 (CHOCH₂CH₂OH), 52.3 (OCH₃), 34.5

(NCH₂CH₂), 32.0 (NCH₂CH₂) ppm; IR (ﬁlm): ṽ = 3464,

1771, 1760, 1700, 1438, 1398, 1148, 721 cm⁻¹; HRESIMS

m/z (pos): 330.0946 C₁₅H₁₇NO₆Na (calcd. 330.0948).

ppm; IR (KBr): ṽ = 1606, 1505, 1463, 1249, 1175, 1034, 828,

735 cm⁻¹; HRESIMS m/z (neg): 494.2185 C₂₈H₃₂NO₇(calcd.

494.2184).

4-(Butylamino)-3-hydroxybutanoic acid (15b)

GP4 was followed using 15a (238 mg, 2.00 mmol), EtOH

(2.4 ml), KOH (264 mg, 4.00 mmol), 1-bromobutane (249 mg,

1.80 mmol). The desired compound was obtained as amor-

phous white solid (263 mg, 75%). ¹H NMR (500 MHz,

MeOD) δ = 4.11 (dtd, J = 8.1, 6.2, 3.9 Hz, 1H,

NCH₂CHCH₂), 3.09 (dd, J = 12.5, 3.8 Hz, 1H, NCH₂CHCH₂),

3.02–2.93 (m, 3H, NCH₂CHCH_2,NCH₂CH₂CH₂CH₃), 2.46

(dd, J = 15.6, 6.2 Hz, 1H, COOHCH₂), 2.42 (dd, J = 15.6,

Methyl 4-{1, 3-dioxoisoindolin-2-yl}-2-{2-[tris(4-

methoxyphenyl)methoxy]ethoxy}butanoate (14f)

GP8 was followed using 14e (461 mg, 1.50 mmol), DMF (1

drop), pyridine (4.0 ml), 4,4′,4″-trimethoxytrithyl chloride

(1.03 g, 2.70 mmol). The desired compound was obtained

as a yellow oil (789 mg, 82%). ¹H NMR (400 MHz, CDCl₃)

δ = 7.75–7.80 (m, 2H, NCOC_arCH_arCH_ar), 7.73–7.64 (m,

2H, NCOC_arCH_arCH_ar), 7.37–7.28 (m, 6H, CH₃OC_arCH-

_arCH_ar), 6.86–6.77 (m, 6H, CH₃OC_arCH_arCH_ar), 4.04 (dd,

J = 7.6, 5.0 Hz, 1H, NCH₂CH₂CH), 3.88 (t, J = 6.9 Hz, 2H,

NCH₂), 3.79 (s, 10H, C_arOCH₃, CHOCH₂CH₂), 3.72 (s, 3H,

COOCH₃), 3.59 (ddd, J = 10.3, 6.2, 4.4 Hz, 1H, CHOCH₂

CH₂), 3.22 (ddd, J = 10.3, 6.2, 4.3 Hz, 1H, CHOCH₂CH₂),

3.14 (ddd, J = 10.2, 5.8, 4.4 Hz, 1H, CHOCH₂CH₂),

2.30–1.88 (m, 2H, NCH₂CH₂) ppm; ¹³C NMR (101 MHz,

CDCl₃) δ = 172.7 (COOCH₃), 168.3 (NCO), 158.4

(CH₃OC_arCH_arCH_arC), 136.9 (CH₃OC_arCH_arCH_arC), 134.0

(NCOC_arCH_arCH_ar), 132.3 (NCOC_arCH_arCH_ar), 129.9 (CH₃

OC_arCH_arCH_arC), 123.3 (NCOC_arCH_arCH_ar), 113.2 (CH₃

OC_arCH_arCH_arC), 85.8 (OCC_ar), 77.6 (NCH₂CH₂CH), 70.5

(CHOCH₂CH₂), 63.2 (CHOCH₂CH₂), 55.3 (C_arOCH₃),

52.1 (COOCH₃), 34.9 (NCH₂CH₂), 31.9 (NCH₂CH₂) ppm;

IR (ﬁlm): ṽ = 1748, 1700, 1607, 1508, 1398, 1249, 1176,

1034 cm⁻¹; HRESIMS m/z (pos): 678.2091 C₃₇H₃₇NO₉

(calcd. 678.210).

6.2 Hz,

1H,

COOHCH₂),

1.75–1.54

(m,

2H,

NCH₂CH₂CH₂CH₃), 1.43 (m, 2H, NCH₂CH₂CH₂CH₃), 0.99

(t, J = 7.4 Hz, 3H, NCH₂CH₂CH₂CH₃) ppm; ¹³C NMR

(126 MHz, MeOD) δ = 178.7 (COOH), 65.9 (CHOH), 53.9

(OHCHCH₂N) 49.2 (NCH₂CH₂CH₂CH₃), 43.9 (COOHCH₂),

29.4 (NCH₂CH₂CH₂CH₃), 20.9 (NCH₂CH₂CH₂CH₃), 13.9

(NCH₂CH₂CH₂CH₃) ppm; IR (KBr): ṽ = 3223, 1629, 1564,

1359, 1251, 1074 cm⁻¹; HRESIMS m/z (pos): 176.1280

C₈H₁₈NO₃(calcd. 176.1287).

Methyl 4-(1,3-dioxoisoindolin-2-yl)-3-hydroxybutanoate

(15c)

GP5a was followed using 4-amino-3-hydroxybutanoic acid

15a (1.25 g, 10.3 mmol), phthalic anhydride (1.56 g,

10.3 mmol), MeOH (80.0 ml) and 2 M HCl in Et₂O

(80.0 ml). The desired compound was obtained as amor-

phous white solid (2.00 g, 74%). ¹H NMR (500 MHz,

CDCl₃) δ = 7.83–7.91 (m, 2H, NCOC_arCH_arCH_ar),

7.80–7.70 (m, 2H, NCOC_arCH_arCH_ar), 4.37 (ddq, J = 8.56,

7.11, 4.53, 4.49 Hz, 1H, NCH₂CHCH₂), 3.89 (dd, J = 14.1,

7.1 Hz, 1H, NCH₂CHCH₂), 3.79 (dd, J = 14.1, 4.5 Hz, 1H,

NCH₂CHCH₂), 3.72 (s, 3H, OCH₃), 3.20 (d, J = 5.1 Hz,

1H, OH), 2.61 (dd, J = 16.5, 4.0 Hz, 1H, NCH₂CHCH₂),

2.53 (dd, J = 16.5, 8.3 Hz, 1H, NCH₂CHCH₂) ppm; ¹³C

NMR (126 MHz, CDCl₃) δ = 172.4 (COOCH₃), 168.7

(NCO), 134.3 (NCOC_arCH_arCH_ar), 132.1 (NCOC_arCH-

_arCH_ar), 123.6 (NCOC_arCH_arCH_ar), 66.8 (NCH₂CHCH₂),

52.1 (OCH₃), 43.0 (NCH₂CHCH₂), 39.0 (NCH₂CHCH₂)

ppm; IR (KBr): ṽ = 3450, 1773, 1700, 1610, 1395, 1206,

4-Amino-2-{2-[tris(4-methoxyphenyl)methoxy]ethoxy}

butanoic acid (14g)

GP9a was followed using 14f (192 mg, 0.300 mmol), MeOH

(3.0 ml), 12 M NaOH (0.05 ml), 1,2-diaminoethane (126 mg,

2.10 mmol). The desired compound was obtained as amor-

phous white solid (102 mg, 69%). ¹H NMR (400 MHz, 0.1 M

NaOD/MeOD) δ = 7.48–7.04 (m, 6H, CH₃OC_arCH_arCH_ar),

6.99–6.66 (m, 6H, CH₃OC_arCH_arCH_ar), 3.87 (dd, J = 8.1,

4.4 Hz, 1H, NCH₂CH₂CH), 3.77 (s, 10H, CHOCH₂CH₂OC,

OCH₃), 3.45 (ddd, J = 10.9, 7.2, 3.9 Hz, 1H, CHOCH₂-

CH₂OC), 3.27 (m, 1H, CHOCH₂CH₂OC), 3.19 (m, 1H,

CHOCH₂CH₂OC), 2.85–2.68 (m, 2H, NCH₂CH₂), 1.96–1.74

(m, 2H, NCH₂CH₂) ppm; ¹³C NMR (101 MHz, 0.1 M NaOD/

MeOD) δ = 180.9 (COOH), 159.9 (CC_arCH_arCH_arCOCH₃),

138.1 (CC_arCH_arCH_arCOCH₃), 131.0 (CC_arCH_arCH_arCOCH₃),

114.1 (CC_arCH_arCH_arCOCH₃), 87.2 (CC_arCH_arCH_arCOCH₃),

82.1 (NCH₂CH₂CH), 70.4 (CHOCH₂CH₂C), 64.4 (CHOCH₂

CH₂C), 55.8 (OCH₃), 40.1 (NCH₂CH₂), 37.5 (NCH₂CH₂)

1024, 717 cm⁻¹

;

HRESIMS m/z (pos): 286.0687

C₁₃H₁₃NO₅Na (calcd. 286.0691).

Methyl 3-{2-[(tert-butyldiphenylsilyl)oxy]ethoxy}-4-{-(1,3-

dioxoisoindolin-2-yl})butanoate (15d)

GP6 was followed using 15c (369 mg, 1.20 mmol), tert-butyl

(2-iodoethoxy)diphenylsilane (662 mg, 1.68 mmol), Ag₂CO₃

(1.32 g, 4.80 mmol), toluene (15 ml), 4d. The desired

Medicinal Chemistry Research

1

compound was obtained as a yellow oil (537 mg, 82.0%). H

NCOC_arCH_arCH_ar), 7.32–7.18 (m, 6H, CH₃OC_arCH_arCH_ar),

6.86–6.72 (m, 6H, CH₃OC_arCH_arCH_ar), 4.13 (dq, J = 7.2,

5.6 Hz, 1H, NCH₂CHH₂), 3.89 (dd, J = 14.1, 5.5 Hz, 1H,

NCH₂CHCH₂), 3.81 (dd, J = 14.0, 5.7 Hz, 1H, NCH₂CHH₂),

3.77 (s, 9H, C_arOCH₃), 3.76 (ddd, J = 10.2, 5.6, 4.4 Hz, 1H,

OCH₂CH₂OCC_ar), 3.64 (ddd, J = 10.2, 5.6, 4.5 Hz, 1H,

OCH₂CH₂OCC_ar), 3.59 (s, 3H, COOCH₃), 3.10 (ddd, J =

12.2, 5.6, 4.3 Hz, 1H, OCH₂CH₂OCC_ar), 3.07 (ddd, J = 12.2,

5.6, 4.2 Hz, 1H, OCH₂CH₂OCC_ar), 2.62 (dd, J = 16.0, 7.1 Hz,

1H, NCH₂CHCH₂), 2.56 (dd, J = 16.0, 5.4 Hz, 1H,

NCH₂CHCH₂) ppm; ¹³C NMR (101 MHz, CD₂Cl₂) δ = 171.8

(COOCH₃), 168.8 (NCO), 158.9 (CH₃OC_ar), 137.4

(CC_arCH_ar), 134.6 (NCOC_arCH_arCH_ar), 132.6 (NCOC_ar), 130.3

(CH₃OC_arCH_arCH_ar), 123.7 (NCOC_arCH_arCH_ar), 113.5

NMR (500 MHz, CDCl₃) δ = 7.83–7.77 (m, 2H, NCO-

C_arCH_arCH_ar), 7.73–7.68 (m, 2H, NCOC_arCH_arCH_ar),

7.67–7.62 (m, 4H, CH_ar), 7.43–7.32 (m, 6H, CH_ar), 4.14 (dq,

J = 7.4, 5.5 Hz, 1H, NCH₂CHCH₂), 3.90–3.84 (m, 2H,

NCH₂CHCH₂), 3.78 (dt, J = 9.7, 4.9 Hz, 1H, OCH₂CH₂Si),

3.75–3.71 (m, 2H, OCH₂CH₂Si), 3.66 (dt, J = 9.6, 4.5 Hz, 1H,

OCH₂CH₂Si), 3.61 (s, 3H, OCH₃), 2.60 (dd, J = 16.1, 7.5 Hz,

1H, NCH₂CHCH₂), 2.55 (dd, J = 16.1, 5.5 Hz, 1H,

NCH₂CHCH₂), 1.00 (s, 9H ppm, SiCCH₃) ppm; ¹³C NMR

(126 MHz, CDCl₃) δ = 171.5 (COOCH₃), 168.4 (NCO), 135.7

(C_ar), 134.1 (C_ar), 133.8 (C_ar), 132.1 (C_ar), 129.7 (C_ar), 127.7

(C_ar), 123.5 (C_ar), 74.5 (NCH₂CHCH₂), 71.5 (OCH₂CH2OSi),

63.5 (OCH₂CH₂Si), 51.8 (OCH₃), 40.3 (NCH₂CHCH₂), 38.6

(NCH₂CHCH₂), 26.9 (SiCCH₃), 19.3 (SiC) ppm; IR (ﬁlm):

ṽ = 2932, 1774, 1770, 1469, 1428, 1396, 1361, 1281, 1192,

1112, 823, 738 cm⁻¹; HRESIMS m/z (pos): 568.2132

C₃₁H₃₅NO₆SiNa (calcd. 568.2131).

(CH₃OC_arCH_arCH_ar),

86.1

(CC_arCH_arCH_ar),

75.1

(NCH₂CHCH₂), 70.3 (OCH₂CH₂OCC_ar), 63.7 (OCH₂-

CH₂OCC_ar), 55.7 (C_arOCH₃), 52.1 (COOCH₃), 41.0

(NCH₂CHCH₂), 38.9 (NCH₂CHCH₂) ppm; IR (ﬁlm): ṽ =

1773, 1607, 1507, 1396, 1249, 1175, 1033, 828 cm⁻¹; HRE-

SIMS m/z (pos): 662.2365 C₃₇H₃₇NO₉Na (calcd. 662.2366).

Methyl 4-(1,3-dioxoisoindolin-2-yl)-3-(2-hydroxyethoxy)

butanoate (15e)

4-Amino-3-{2-[tris(4-methoxyphenyl)methoxy]ethoxy}

butanoic acid (15g)

GP7 was followed using 15d (442 mg, 0.81 mmol), THF/

pyridine 9:1 (10.0 ml), HF-pyridine (116 mg, 4.05 mmol).

The desired compound was obtained as yellow oil (206 mg,

83%). ¹H NMR (500 MHz, CD₂Cl₂) δ = 7.92–7.80 (m, 2H,

NCOC_arCH_arCH_ar), 7.80–7.71 (m, 2H, NCOC_arCH_arCH_ar),

4.10 (dq, J = 9.0, 4.5 Hz, 1H, NCH₂CHCH₂), 3.87 (dd, J =

14.3, 4.9 Hz, 1H, NCH₂CHCH₂), 3.80 (dd, J = 14.3,

4.5 Hz, 1H, NCH₂CHCH₂), 3.73 (ddd, J = 10.3, 5.9,

3.1 Hz, 1H, OCH₂CH₂OH), 3,66 (s, 3H, OCH₃), 3.65 (ddd,

J = 10.3, 5.9, 2.8 Hz, 1H, OCH₂CH₂OH), 3.60 (ddd, J =

12.3, 6.1, 2.8 Hz, 1H, OCH₂CH₂OH), 3.56 (ddd, J = 12.2,

6.0, 3.0, 1H, OCH₂CH₂OH), 2.76 (t, J = 6.3 Hz, 1H, OH),

2.59 (dd, J = 16.4, 4.3 Hz, 1H, NCH₂CHCH₂), 2.52 (dd,

J = 16.4, 8.7 Hz, 1H, NCH₂CHCH₂) ppm; ¹³C NMR

(126 MHz, CD₂Cl₂) δ = 172.3 (COOCH₃), 169.1 (NCO),

134.7 (NCOCarCHarCH_rar), 132.5 (NCOC_arCH_arCH_ar),

123.8 (NCOC_arCH_arCH_ar), 75.3 (NCH₂CHCH₂), 72.6

(OCH₂CH₂OH), 62.3 (OCH₂CH₂OH), 52.3 (OCH₃), 40.9

(NCH₂CHCH₂), 38.6 (NCH₂CHCH₂) ppm; IR (ﬁlm): ṽ =

GP9b was followed using 15f (141 mg, 0.220 mmol), MeOH

(3.0 ml), 12 M NaOH (0.05 ml), 1,2-diaminoethane (92,6 mg,

1.54 mmol). The desired compound was obtained as amor-

1

phous white solid (75 mg, 69%). H NMR (400 MHz, 0.1 M

NaOD/MeOD) δ = 7.36–7.21 (m, 6H, CH₃OC_arCH_arCH_ar),

6.90–6.69 (m, 6H, CH₃OC_arCH_arCH_ar), 3.82–3.72 (m, 11H,

CH₂CHCH₂, OCH₃, CHOCH₂CH₂OC), 3.63 (ddd, J = 10.35,

6.16, 3.94 Hz, 1H, CHOCH₂CH₂OC), 3.28–3.09 (m, 2H,

CHOCH₂CH₂OC), 2.81 (dd, J = 13.4, 3.5 Hz, 1H,

NCH₂CHCH₂), 2.64 (dd, J = 13.4, 7.2 Hz, 1H, NCH₂CHCH₂),

2.51 (dd, J = 14.2, 6.1 Hz, 1H, NCH₂CHCH₂), 2.25 (dd, J =

14.2, 7.3 Hz, 1H, NCH₂CHCH₂) ppm; ¹³C NMR (101 MHz,

0.1 M NaOD/MeOD) δ = 180.2 (COOH), 159.8 (CC_arCH_ar

CH_arCOCH₃), 138.0 (CC_arCH_arCH_arCOCH₃), 130.9 (CC_ar

CH_arCH_arCOCH₃), 114.1 (CC_arCH_arCH_arCOCH₃), 87.1 (CC_ar

CH_arCH_arCOCH₃), 80.8 (NCH₂CHCH₂), 70.1 (CHOCH₂

CH₂C), 64.6 (CHOCH₂CH₂C), 56.0 (, OCH₃), 46.2

(NCH₂CH), 42.2 (NCH₂CHCH₂) ppm; IR (KBr): ṽ = 3375,

1606, 1505, 1463, 1249, 1175, 1034, 828, 735 cm⁻¹; HRE-

SIMS m/z (neg): 494.2196 C₂₈H₃₃NO₇(calcd. 494.2184).

3472, 2951, 1773, 1770, 1467, 1397, 112, 725 cm⁻¹

;

HRESIMS m/z (pos): 330.0949 C₁₅H₁₇NO₆Na (calcd.

330.0954).

Methyl 4-{1,3-dioxoisoindolin-2-yl}-3-{2-[tris(4-

methoxyphenyl)methoxy]ethoxy}butanoate (15f)

Biological evaluation

HEK cells

GP8 was followed using 15e (338 mg, 1.10 mmol), DMF (1

drop), pyridine (4.0 ml), 4,4′,4″-trimethoxytrithyl chloride

(753 mg, 1.98 mmol). The desired compound was obtained as

yellow oil (520 mg, 74%). H NMR (400 MHz, CD₂Cl₂) δ =

7.89–7.76 (m, 2H, NCOC_arCH_arCH_ar), 7.76–7.65 (m, 2H,

HEK293 cells were purchased from the American Type Cul-

ture Collection (ATCC) speciﬁed as ATCC-CRL-1573 (Lot

57954093). The stably mGAT1, mGAT2, mGAT3 or mGAT4

expressing HEK293 cell lines were generated as described

1

Medicinal Chemistry Research

Scheme 3 Synthesis of the cyclic hydroxyamino acids 11d, 11g, 12d and 13d. Reagents and conditions: a 1. Trimethylsilyl cyanide (2.5 eq),

CH₂Cl₂, rt, 48 h; 2. sulfuric acid (5.2 eq), CH₂Cl₂, 0 °C–rt, 2 h. b 1. LiHMDS (1.0 eq), ethyl acetate (1.0 eq), methyl tert-butyl ether, −78 °C,

25 min, 2. 11a/12a, THF, −78 °C to −10 °C. c H₂(10 bar), n-butyraldehyde (2.5 eq), 10% palladium on charcoal (0.1 eq), rt, 16 h; d barium

hydroxide octahydrate (2.0–2.4 eq), EtOH/H₂O 1:1, rt or reﬂux

previously (Kragler et al. 2005, 2008). These cell lines were

cultivated till reaching conﬂuencies >85% (up to passage 25)

and immediately used after detachment and washing.

respectively. Reaction of 11a and 13a with tetramethylsilyl

cyanide followed by acidic hydrolysis of the thus to be formed

TMS-protected cyanohydrines should furnish the correspond-

ing α-hydroxycarboxamides 11b and 13b. Likewise, reaction

of the cyclic aminoketones 11a and 12a with lithium 1-

ethoxyethen-1-olate should lead to the β-hydroxyesters 11e and

12e. Deprotection of the amino nitrogen atom of 11b, 11e, 12e,

and 13b, followed by introduction of a n-butyl rest via

reductive amination and hydrolysis of the carboxamide and

ester function, respectively, should ﬁnally furnish the desired

free amino acids 11d, 11g, 12g, and 13d.

[³H]GABA uptake assays

The [³H]GABA uptake assays were performed as reported

(Kragler et al. 2005, 2008) in a 96‐well plate format with

intact HEK293 cells expressing mGAT1, mGAT2, mGAT3

and mGAT4, respectively.

MS Binding Assays

Synthesis of the cyclic α-hydroxycarboxamide 11b

was performed according to literature (Lamb 2008). The

same reaction sequence was applied for the synthesis of

its ring-expanded analogue 13b. Hence, the N-

benzylpyrrolid-3-one 13a was reacted with trimethylsi-

lyl cyanide to give the respective TMS-protected cya-

nohydrine. Due to the lability of the TMS ether function,

the TMS-protected cyanohydrine was not puriﬁed and

characterized, but used for the next reaction step, the

hydrolysis with concentrated sulfuric acid. This gave the

corresponding α-hydroxycarboxmide 13b in an excellent

yield of 96% over both reaction steps. The

β-hydroxyesters 11e and 12e were obtained in good

yields of 80–93% from the cyclic aminoketones 11a and

12a by reaction with lithium 1-ethoxyethen-1-olate,

which was generated from ethyl acetate and LiHMDS at

low temperature (Scheme 3).

The MS Binding Assays were performed as reported

(Zepperitz et al. 2006) with mGAT1 membrane prepara-

tions obtained from a stable HEK293 cell line and NO711

as unlabelled marker in competitive binding experiments.

Results and discussion

Synthesis

Synthesis of the cyclic N-butylamino acid derivatives

For the synthesis of the cyclic N-butylhydroxyamino acids

11d, 11g, 12g, and 13d we intended to start from the benzyl- or

benzhydryl-protected cyclic aminoketones 11a, 12a and 13a,

Medicinal Chemistry Research

Next, the carboxamide and ester derivatives 11b, 11e, 12e

and 13b were subjected to deprotection of the nitrogen atom

and subsequent n-butylation, accomplished in a single reaction

step by exposing the compounds to hydrogen (10 bar) in pre-

sence of palladium on cabon and 2.0–2.4 equivalents of n-

butyraldehyde. This furnished the respective N-butyl deriva-

tives 11c, 11f, 12f, and 13c in yields of 77–93%. Finally, after

basic hydrolysis and workup (barium hydroxide, carbon

dioxide workup), the free amino acids 11d, 11g, 12g, and 13d

were hence obtained in yields of 87–96% (Scheme 3).

derivatives 7b, 14b, and 15b (see chapter 3 “biological

evaluation”, Table 3), also the respective amino acid deri-

vatives with an tris(4-methoxyphenyl)methyloxyethyl

attached to the hydroxy function of the parent compounds

should be included in this study.

As starting compounds for this synthesis of the target

compounds 7g, 14g, and 15g, the derivatives 7c, 14c, and

15c seemed well suited, as they should allow a selective

functionalization of the OH group as the carboxylic acid

and the amino moieties in 7g, 14g, and 15g are protected in

form of ester and phthalimidic moieties, respectively. The

preparation of 14c has been described in literature (Farkas

et al. 2009), the synthesis of 7c and 15c should be

accomplished in an analogous manner. Based on these

starting materials, 7c, 14c, and 15c, in the next steps ﬁrst a

2-hydroxyethyl residue should be attached to the free OH

function to serve as linker to the lipophilic domain of the

target compound. The trityl based lipophilic moiety should

be introduced only after that, as the reaction conditions

required for the formation of the ﬁrst ether function were

thought to cause side reactions if the terminal trityl moiety

were already present. Deprotection of the carboxylic acid

and the amino group in 7f, 14f, and 15f should ﬁnally lead

to the target compounds 7g, 14g, and 15g.

Synthesis of the acyclic N-butylamino acid

derivatives

For the synthesis of the N-butyl derivatives 7b, 14b, and

15b from the cyclic hydroxyamino acids 7a, 14a, and

15a, a specialized procedure developed for the mono-

butylation of β-alanine (Santimukul and Perez 2011) was

followed. When according to this procedure 7a, 14a, and

15a were treated with n-bromobutane in a mixture of

methanol and water under reﬂux the desired test com-

pounds 7b, 14b, and 15b were obtained in yields of

75–79% (Scheme 4).

Synthesis of O-alkylated hydroxyamino acid

derivatives

The synthesis of 14c was accomplished according to

literature (Farkas et al. 2009) by ﬁrst reacting 14a with

phthalic anhydride to protect the terminal amino group as

phthalimide moiety, followed by etheriﬁcation of the

carboxyl group to give the corresponding carboxylic acid

ester, the overall product being 14c. Applying the same

Since the unsubstituted hydroxyamino acids 7a, 14a, and

15a had been found to exhibit higher inhibitory potencies at

all GAT subtypes than the corresponding N-butyl

Scheme 4 N-Butylation of the acyclic hydroxyamino acids 7a, 14a, and 15a, furnishing 7b, 14b, and 15b. Reagents and conditions: a 1-

bromobutane (0.9 eq), KOH (2.0 eq), EtOH/H₂O, rt, 16 h

Medicinal Chemistry Research

Table 3 Results of the biological evaluation

Entry

Compound

Binding afﬁnity (pK_iSEM)^a

GABA uptake inhibition (pIC₅₀SEM)^b

mGAT1

mGAT2

mGAT3

mGAT4

1

16

3.36 0.02

2.43 0.03

86%

4.11 0.08

2.33 0.05

101%

63%

3.23

81%

3.32 0.04

4.78 0.14

3.26 0.10

60%

2

7a

3.38 0.11

92%

4.87 0.05

3.54 0.04

63%

3

7b

4

7g

85%

57%

5

11c

11d

11f

11g

12f

12g

13c

13d

14a

14b

14g

15a

15b

15g

101%

97%

83%

105%

3.38 0.08

91%

92%

96%

6

89%

72%

85%

7

106%

93%

66%

82%

88%

8

79%

78%

80%

99%

9

88%

104%

94%

96%

86%

71%

10

11

12

13

14

15

16

17

18

93%

3.17

103%

105%

103%

97%

109%

96%

84%

79%

90%

104%

3.25 0.02

59%

85%

91%

3.61 0.05

107%

61%

4.99 0.08

79%

4.73 0.10

3.21 0.08

94%

4.64 0.03

107%

78%

66%

73%

2.31

4.06 0.08

100%

67%

4.19 0.01

76%

4.41 0.09

3.59 0.07

54%

4.37 0.14

3.42 0.12

3.95

94%

74%

67%

^aResults of the MS Binding Assays are given as pK_iSEM. Percent values represent remaining speciﬁc NO711 binding in presence of 100 µM test

compound

^bResults of the [³H]GABA uptake assays are given as pIC₅₀SEM. Percent values represent remaining [³H]GABA uptake in presence of 100 µM test

compound

Medicinal Chemistry Research

procedure to 7a and 15a, the analogous compounds 7c

and 15c could be obtained in yields of 73% (7c) and 72%

(15c) (Scheme 5).

amino acids 7g, 14g, and 15g could be obtained by sub-

jecting the thus obtained crude reaction product without

prior isolation to heating with 1,2-diaminoethane. This

furnished the desired target compounds 7g, 14g, and 15g in

yields of 69–83% over both reaction steps (Scheme 6).

The required hydroxylethylation of the OH function

could be realized by treatment of 7c, 14c, and 15c with 2-

iodoethoxy-TBDPS in the presence of Ag₂CO₃furnishing

the corresponding ethers 7d, 14d, and 15d in good yields of

76–82%. Removal of the TBDPS protecting group by

treatment with HF-pyridine yielded in the free alcohols 7e,

14e, and 15e (76–84%), which upon reaction with 4,4′,4″-

trimethoxytrityl chloride, gave the trityl derivatives 7f, 14f,

and 15f (74–90%, Scheme 6).

The ﬁnal deprotection of the amino and the carboxylic

acid function of 7f, 14f, and 15f in order to obtain the free

amino acids 7g, 14g, and 15g was ﬁrst attempted in a two-

step reaction sequence. Hydrazinolysis of the phthalimide

moiety should liberate the terminal amino group, and sub-

sequently hydrolysis of the methyl ester function under

alkaline conditions (NaOH) the carboxylic acid moiety.

Unfortunately, when the primary amine was formed in the

ﬁrst reaction step, it immediately reacted with the methyl

ester function leading to the formation of the corresponding

lactame, which could not be cleaved again without

destruction of the molecule. Hence, the sequence of the

deprotection was altered applying ﬁrst NaOH to hydrolyse

the methyl ester function. Thereby, also the phthaloyl group

protecting the amino function was partially cleaved leading

to the corresponding phthalamide moieties. Still, the free

Biological evaluation

The amino acids 7a, 7b, 7g, 11d, 11g, 12g, 13d, 14a, 14b,

14g, 15a, 15b, 15g, as well as the carboxamide and ester

derivatives 11c, 11f, 12f, and 13c were tested for their inhi-

bitory potencies on the four GABA transporter subtypes

mGAT1–4 in a [³H]GABA uptake assay previously devel-

oped by our group (Kragler et al. 2008) The tests were per-

formed in a standardized manner in triplicates using HEK293

cell lines, each expressing one of the four GAT subtypes. In

addition, binding afﬁnities towards mGAT1 were examined

employing a standardized MS Binding Assay with NO711 as

native MS marker (Zepperitz et al. 2006). The results are

summarized in Table 3. Inhibitory potencies and binding

afﬁnities of the tested compounds are represented as pIC₅₀

and pK_i, respectively. Each test compound was characterized

in three independent experiments performed in triplicates and

the standard error of mean (SEM) is given. If the determi-

nation of the pIC₅₀value proved not feasible due to low

inhibitory potency, as percentage of the remaining [³H]

GABA uptake at 100 µM concentration of the test compound.

Correspondingly, the percentage of remaining MS marker is

Scheme 5 Synthesis of the protected amino acid derivatives 7c, 14c, and 15c. Reagents and conditions: a 1. Phthalic anhydride (1.0 eq), 140 °C,

30 min; 2. MeOH, 2 M HCl in Et₂O, rt

Medicinal Chemistry Research

Scheme 6 Synthesis of acyclic amino acid derivatives comprising a 4,4′,4″-trimethoxytrityl moiety, which is linked to the 2- or 3-position of the

carbon chain via an OCH₂CH₂O spacer (7g, 14g and 15g). Reagents and conditions: a Ag₂CO₃(4.0 eq), tert-butyl(2-iodoethoxy)diphenylsilane

(1.4 eq), toluene, reﬂux; b HF-pyridine (5.0 eq), THF/pyridine 9:1, 0 °C–rt; c 4,4′,4″-trimethoxytrityl chloride (1.8 eq.), pyridine, dimethylfor-

mamide (cat.), rt; d 1. 1 M NaOH (2.0 eq), MeOH, rt, 16 h; 2. 1,2-diaminoethane (7.0 eq), microwave, 140 °C, 16 h

given in cases when the tested compound caused only a minor

reduction of the MS marker binding.

entry 1), which can be considered as a reasonable reference

point since nipecotic acid constitutes the amino acid partial

structure of many important GAT inhibitors including tia-

gabin (4, Table 1, entry 5) and (S)-SNAP-5114 (5, Table 1,

entry 6). Interestingly, 7b exerts a somewhat higher inhi-

bitory potency at mGAT3 (pIC₅₀= 3.54 0.04) than at

mGAT4, whereas the activity at mGAT1-2 can be con-

sidered negligible ([³H]GABA at 100 µM = 101% and

95%, respectively). This selectivity pattern deviates sig-

niﬁcantly from that of the reference compound 16, which

As can be seen from the data in Table 3, the unsub-

stituted α- and β-hydroxyamino acids 7a, 14a, and 15a,

which served as the starting material for the synthesis of the

N-butyl derivatives 7b, 14b, and 15b, exhibit considerable

inhibitory activities at most of the GAT subtypes, with the

pIC₅₀values ranging from 4.06 0.08 (15a, mGAT1, Table

3, entry 16) to 4.99 0.08 (14a, mGAT1, Table 3, entry

13). Only for 7a at mGAT1 (pIC₅₀= 2.33 0.05, Table 3,

entry 2) and at mGAT2 (pIC₅₀= 3.38 0.11, Table 3, entry

2) and 14a (pIC₅₀= 3.25 0.02, Table 3, entry 13) at

mGAT2 distinctly lower pIC₅₀values were found.

exerts its highest inhibitory potency at mGAT1 (pIC₅₀

4.11 0.08) while being less active at mGAT2 (pIC₅₀

3.23) and mGAT3 ([³H]GABA at 100 µM = 81%).

=

As compared with the unsubstituted α- and

β-hydroxyamino acids 7a, 14a, and 15a the N-butyl deri-

vatives 7b, 14b, and 15b are characterized by lower inhi-

bitory activity throughout, which might indicate that the

binding pose in which the amino nitrogen atom is oriented

towards the cytosol, as postulated by molecular modelling

experiments performed by Wein et al. (Wein et al. 2016) is

energetically disfavoured. As reported in Table 3, entry 3,

N-butylisoserine (7b) shows a pIC₅₀value of 3.26 0.10 at

mGAT4, which is on par with the value reported for race-

mic N-butylnipecotic acid 16 (pIC₅₀= 3.32 0.04, Table 3,

For the homologue of 7b, compound 14b, formally

derived from 7b by inserting a methylene group at the end

of the carboxyl acid carbon chain, the potency at mGAT4

has decreased distinctly, i.e. to a value below the identiﬁ-

cation threshold ([³H]GABA at 100 µM = 107%, Table 3,

entry 14). The biological activity at mGAT3 on the other

hand is only slightly reduced from 3.54 0.04 for 7b to

3.21 0.08 for 14b. The effects exerted at mGAT1 and

mGAT2 are slightly higher as compared with 7b, with the

remaining [³H]GABA uptake for 14b amounting to 79%

(mGAT1) and 59% (mGAT2), respectively.

Medicinal Chemistry Research

By contrast, if the elongation of the carbon chain is

performed by inserting a methylene group between the

carbon carrying the OH function and the carboxylic acid

group (15b, Table 3, entry 17), the pIC₅₀value at mGAT4

increases nominally from 3.26 0.10 for 7b to 3.42 0.12

for 15b, while the effect exerted at mGAT3 remains unal-

tered (15b: pIC₅₀= 3.59 0.07, 7b: pIC₅₀= 3.54 0.04).

As is the case with N-butylisoserine (7b), 15b exhibits

no determinable inhibition at mGAT1 ([³H]GABA at

100 µM = 100%), whereas a slight effect at mGAT2 is

found ([³H]GABA at 100 µM = 76%).

Unexpectedly, rigidization of the N-butylisoserine (7b)

molecule by linking the C-2 atom with the nitrogen atom

via a methylene bridge, resulting in azetidine heterocycle

11d, leads to a compound which exerts its highest inhibitory

potency at mGAT2 (pIC₅₀= 3.38 0.08, Table 3, entry 6).

At the same time the effects of 11d at the other GATs are

minor, the [³H]GABA uptake amounting to 72% (mGAT3),

85% (mGAT4), and 89% (mGAT1). Formal enlargement of

the azetidine ring present in 11d to a piperidine ring causes

the inhibitory potency at mGAT2 to completely vanish

(13d, [³H]GABA at 100 µM = 104%, Table 3, entry 12). In

addition, the activity exerted at the other GAT subtypes is

extremely low, the values for the remaining [³H]GABA

uptake at 100 µM being 96% (mGAT1), 85% (mGAT3),

and 91% (mGAT4), respectively. As compared with its

desoxy analogoue N-butylnipecotic acid (16, Table 3, entry

1), this constitutes a distinct decline of inhibitory potency at

all GATs, hence demonstrating that the introduction of a

hydroxy group into the 3-position of the nipecotic acid

scaffold is associated with a strong reduction of biological

activity.

minor inhibitory potency at mGAT1-3, with the values for

the remaining [³H]GABA uptake at 100 µM lying between

78% (mGAT2) and 80% (mGAT3) and the inhibitory

potency at mGAT4 being negligible ([³H]GABA uptake at

100 µM = 99%, Table 3, entry 8). By contrast, the open-

chain analogue 15b exerted reasonable inhibitory potencies

at mGAT3 (pIC₅₀= 3.59 0.07, Table 3, entry 17) and at

mGAT4 (pIC₅₀= 3.42 0.12).

Linking the C-3 atom and the amino nitrogen atom of

15b via a C₂-bridge, resulting in a pyrrolidine substructure,

is likewise accompanied by a complete loss of inhibitory

potency at mGAT3-4, the values for the remaining [³H]

GABA uptake being nominally 103% and 105%, respec-

tively (12g, Table 3, entry 10). However, the compound

exerts some activity at mGAT2 (pIC₅₀= 3.17). For

mGAT1, a value of 94% remaining GABA uptake at

100 µM was determined, which is consistent with the par-

ent compound 15b not showing any effect at this subtype

(15b, remaining [³H]GABA uptake = 100%, Table 3,

entry 17).

11f and 12f, the ester derivatives of the cyclic amino

acids 11g and 12g, were also tested for their inhibitory

potency at mGAT1–4. As shown in Table 3, entry 7, the

azetidine derivative 11f exhibits some activity at mGAT1

(remaining [³H]GABA uptake = 66%), whereas the effect

at mGAT2-4 is less pronounced (91%, 82% and 88%,

respectively). 12f (Table 3, entry 9), which is the ester

derivative of 12g, is characterized by minor or negligible

inhibitory activity at all GAT subtypes, the values of the

remaining [³H]GABA uptake being 104% (mGAT1),

96% (mGAT2), 86% (mGAT3) and 71% (mGAT4),

respectively.

11c and 13c, the carboxamide derivatives of the cyclic

N-butyl-α-hydroxyamino acids 11d and 13d, were also

tested for their inhibitory potential at all GAT subtypes.

11c, comprising an azetidine heterocyle, is characterized by

marginal or non-detectable inhibitory potencies (Table 3,

entry 5), the values of the remaining [³H]GABA uptake at

100 µM test compound concentration ranging from 83%

(mGAT1) to nominally 105% (mGAT2). Likewise, 13c

(Table 3, entry 11), which features a piperidine ring, redu-

ces the [³H]GABA uptake to 84% (mGAT2), 79%

(mGAT3) and 90% (mGAT4), whereas it appears to be

completely inactive at mGAT1 at 100 µM, the [³H]GABA

uptake amounting nominally to 109%.

Exhibiting pIC₅₀values of 3.59 0.07 at mGAT3 and

3.42 0.12 at mGAT4, 15b showed the highest inhibitory

activity of the tested open-chain N-butylhydroxyamino

acids (Table 3, entry 17). Hence, analogues of 15b that have

the structure of the molecule rigidized by integrating the

amino nitrogen atom and parts of the carbon chain into an

azetidine (11g) and pyrrolidine ring (12g), respectively,

seem of interest. However, 11g was found to exert only

Since both the cyclic and acyclic N-butyl derivatives of

7a, 14a, and 15a were found to exhibit distinctly lower

inhibitory potencies than the parent compounds con-

sistently, derivatives of 7a, 14a, and 15a were synthesized

which have the alcohol function linked to a C₂spacer

bearing a 4,4′,4″-trimethoxytrityloxy moiety. In theory, this

would allow the amino acid subunit to keep the more

favourable orientation towards the intracellular space, while

at the same time enabling interactions between the lipo-

philic domain and the extracellular vestibule, which are

thought to signiﬁcantly increase inhibitory potency and

selectivity in case of nipecotic acid derivatives such as (S)-

SNAP-5114 (5, Table 1, entry 6). Unfortunately, the

introduction of the lipophilic domain into the scaffold of 7a,

14a, and 15a caused a reduction of inhibitory potency. For

the derivatives resulting from this modiﬁcation (7g, 14g,

and 15g) the values for the [³H]GABA uptake at 100 µM

test compound concentration remained at >50% at all

transporter subtypes, equating to pIC₅₀values <4.0. More-

over, 7g and 15g were found to display only negligible

subtype selectivity. In particular, 7g reduced the [³H]GABA

Medicinal Chemistry Research

uptake to 63% (mGAT1), 57% (mGAT2), 63% (mGAT3)

and 60% (mGAT4), respectively (Table 3, entry 4). Like-

wise, the remaining [³H]GABA uptake in presence of

100 µM 15g ranged from 54% (mGAT3) to 67% (mGAT1

and mGAT2). For mGAT4, a pIC₅₀value just short below

4.0 was determined (pIC₅₀= 3.95, Table 3, entry 18). 14g

(Table 3, entry 15) was found to reduce the [³H]GABA

uptake to 78% (mGAT1), 66% (mGAT2), 94% (mGAT3)

and 73% (mGAT4), respectively.

For all test substances the binding afﬁnities at mGAT1

were found to be very low as compared with the reference

compound 16 (pK_i= 3.36 0.02, Table 3, entry 1), the only

exception being 14a (pK_i= 3.61 0.05, Table 3, entry 11).

linking the alcohol function to a C₂spacer bearing a 4,4′,4″-

trimethoxytrityloxy moiety, which is a common structural

motif of mGAT4 inhibitors such as (S)-SNAP-5114. In

theory, this would allow the amino acid subunit to adapt the

more favourable binding pose, while at the same time

enabling interactions between the target and the lipophilic

domain. Unfortunately, the resulting compounds displayed

poor inhibitory potency and selectivity as compared with

the unsubstituted hydroxyamino acids and, even more so, as

compared with (S)-SNAP-5114.

Acknowledgements Open Access funding provided by Projekt DEAL.

Compliance with ethical standards

Conﬂict of interest The authors declare that they have no conﬂict of

interest.

Conclusion

A series of cyclic and acyclic hydroxyamino acid deriva-

tives was synthesized and biologically evaluated for their

potential as amino acid subunits in GAT inhibitors. Among

the compounds synthesized for this study, we identiﬁed 1-

butyl-3-hydroxyazetidine-3-carboxylic acid and, even more

so, 2-(1-butyl-3-hydroxypyrrolidin-3-yl)acetic acid to be

selective and moderately potent inhibitors of mGAT2, with

the respective pIC₅₀values amounting to 3.17 and 3.38

0.08, respectively.

Whereas the cyclic and hence more rigid N-

butylhydroxyamino acids displayed only weak inhibitory

potency at mGAT3 and mGAT4, moderate activity was

observed in case of some acyclic compounds tested for this

study. In particular, 4-(butylamino)-3-hydroxybutanoic acid

(pIC₅₀= 3.42 0.12) and N-butylisoserine (pIC₅₀= 3.26

0.10) were found to be equal to N-butylnipecotic acid

(pIC₅₀= 3.32) in terms of inhibitory potency at mGAT4,

while being distinctly stronger mGAT3 inhibitors with the

pIC₅₀values amounting to 3.59 0.07 and 3.54 0.04,

respectively (N-butylnipecotic acid: [³H]GABA uptake at

100 µM = 81%). At the same time, both compounds exert

no identiﬁable inhibitory effect at mGAT1 under the test

conditions ([³H]GABA uptake at 100 µM = 100% and

101%, respectively), hence deviating strongly from the

reference compound N-butylnipecotic acid, which con-

stitutes a potent inhibitor of this GAT subtype (mGAT1:

pIC₅₀= 4.11 0.08). 4-(Butylamino)-3-hydroxybutanoic acid

and N-butylisoserine might thus be suitable alternatives to

the nipecotic acid subunit in mGAT3/4 inhibitors.

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Article Doi

Synthesis and biological evaluation of α- and β-hydroxy substituted amino acid derivatives as potential mGAT1–4 inhibitors

DOI: 10.1007/s00044-020-02548-x

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Full text of DOI:10.1007/s00044-020-02548-x

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