Chemoenzymatic Synthesis and Pharmacological Characterization of Functionalized Aspartate Analogues As Novel Excitatory Amino Acid Transporter Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00700

Aspartate (Asp) derivatives are privileged compounds for investigating the roles governed by excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we report the synthesis of various Asp derivatives with (cyclo)alkyloxy and (

Full text of DOI:10.1021/acs.jmedchem.8b00700

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Article
Chemoenzymatic synthesis and pharmacological
characterization of functionalized aspartate analogues
as novel excitatory amino acid transporter inhibitors
Haigen Fu, Jielin Zhang, Pieter Tepper, Lennart Bunch, Anders Jensen, and Gerrit J. Poelarends
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00700 • Publication Date (Web): 16 Jul 2018
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Chemoenzymatic synthesis and pharmacological characterization
of functionalized aspartate analogues as novel excitatory
amino acid transporter inhibitors
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a
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Haigen Fu, Jielin Zhang, Pieter G. Tepper, Lennart Bunch,
b,
a,
Anders A. Jensen, * and Gerrit J. Poelarends *
a
Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of
Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands
b
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, DKꢀ2100 Copenhagen OE, Denmark
*
Address correspondence to Prof. Dr. Gerrit J. Poelarends. Tel: +31ꢀ503633354; Eꢀmail:
g.j.poelarends@rug.nl.
*
Address correspondence to Dr. Anders A. Jensen. Tel: +45ꢀ35333260; Eꢀmail:
aaj@sund.ku.dk.
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ABSTRACT
Aspartate (Asp) derivatives are privileged compounds for investigating the roles governed by
excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we
report the synthesis of various Asp derivatives with (cyclo)alkyloxy and (hetero)aryloxy
substituents at Cꢀ3. Their pharmacological properties were characterized at the EAAT1ꢀ4
subtypes. The Lꢀthreoꢀ3ꢀsubstituted Asp derivatives 13a-e and 13g-k were nonꢀsubstrate
inhibitors, exhibiting pan activity at EAAT1ꢀ4 with IC50 values ranging from 0.49 to 15 ꢁM.
Comparisons between (DLꢀthreo)ꢀ19a-c and (DLꢀerythro)ꢀ19a-c Asp analogues confirmed that
the threo configuration is crucial for the EAAT1ꢀ4 inhibitory activities. Analogues (3b-e) of
LꢀTFBꢀTBOA (3a) were shown to be potent EAAT1ꢀ4 inhibitors, with IC50 values ranging
from 5ꢀ530 nM. Hybridization of the nonselective EAAT inhibitor LꢀTBOA with
EAAT2ꢀselective inhibitor WAYꢀ213613 or EAAT3ꢀpreferring inhibitor NBIꢀ59159 yielded
compounds 8 and 9, respectively, which were nonꢀselective EAAT inhibitors displaying
considerably lower IC50 values at EAAT1ꢀ4 (11ꢀ140 nM) than those displayed by the
respective parent molecules.
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KEYWORDS:
Asymmetric synthesis, biocatalysis, aspartate derivative, EAATs, inhibitor.
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INTRODUCTION
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LꢀGlutamate (Glu) is the major excitatory neurotransmitter in the mammalian central nervous
1
ꢀ5
system (CNS), where it mediates numerous physiological and pathophysiological processes.
However, accumulation of high levels of extracellular Glu may lead to hyperꢀactivity in the
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glutamatergic system and neuronal injury. Five subtypes of excitatory amino acid
transporters (termed EAAT1ꢀ5 in humans) have been identified in glial cells (predominantly
EAAT1,2) and neurons (predominantly EAAT3ꢀ5), where they are key players in the
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regulation of glutamatergic transmission. EAAT2 is the major contributor to this, as it is
estimated to be responsible for over 90% of total extracellular Glu uptake in the brain, while
EAAT1 and EAAT3 are also widely expressed in the CNS. Notably, EAAT4 and EAAT5 are
almost specifically located in cerebellum and the retina, respectively. Malfunction of EAAT
function has been implicated in many neurological disorders, such as Alzheimer’s disease,
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epilepsy, amyotrophic lateral sclerosis, and Huntington’s disease. However, in contrast to the
considerable medicinal chemistry efforts in the fields of ionotropic and metabotropic Glu
4
receptors, the EAATs have received much less attention as putative drug targets.
One of the most important scaffolds for the development of EAAT ligands is the
endogenous substrate Lꢀaspartate (1, LꢀAsp, Figure 1B). Represented by
Lꢀthreoꢀ3ꢀbenzyloxyaspartate (2, LꢀTBOA, Figure 1B), 3ꢀaryloxy substituted Asp analogues
were identified as the first class of nonꢀtransportable EAAT inhibitors by Shimamoto and
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ꢀ11
coworkers.
None of these analogues exhibited substantial selectivity or preference towards
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different EAAT subtypes. Further development of the 3ꢀaryloxy substituted Asp analogues as
pharmacological tools in neurobiological research led to the identification of
(
Lꢀthreo)ꢀ3ꢀ[3ꢀ[4ꢀ(trifluoromethyl)benzoylamino]benzyloxy]aspartate (3a, LꢀTFBꢀTBOA,
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12ꢀ14
Figure 1B), the most potent EAAT inhibitor reported to date.
Interestingly, elimination of
the ether oxygen in LꢀTBOA (2) gives the analogue Lꢀthreoꢀ3ꢀbenzylaspartate (4, Lꢀ3ꢀBA,
Figure 1B), which displayed a 10ꢀfold preference as an inhibitor for EAAT3 over EAAT1 and
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EAAT2. Most recently, [3ꢀ(trifluoromethyl)phenyl]sulfonamideꢀLꢀaspartate (5, Figure 1B)
was found to be a potent EAAT2 inhibitor exhibiting over 30ꢀfold selectivity for EAAT2 over
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EAAT1 and EAAT3. In addition, other medicinal chemistry efforts have focused on
4ꢀcarboxylateꢀmodified Asp analogues, which resulted in the discovery of
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N ꢀ[4ꢀ(2ꢀbromoꢀ4,5ꢀdifluorophenoxy)phenyl]ꢀLꢀasparagine
(6,
WAYꢀ213613)
as
a
17ꢀ19
4
EAAT2ꢀselective nonsubstrate inhibitor
and N ꢀ(9Hꢀfluorenꢀ2ꢀyl)ꢀLꢀasparagine (7,
19,20
NBIꢀ59159) as an EAAT3ꢀpreferring inhibitor (Figure 1B).
The fact that derivatization of the Asp structure has yielded several important classes of
EAAT inhibitors (Figure 1B) encouraged us to further explore the potential of this scaffold in
this field (Figure 1A). Here, we describe the chemoenzymatic asymmetric synthesis of a
series of novel Asp derivatives comprising (cyclo)alkyloxy and (hetero)aryloxy substituents at
the Cꢀ3 position, using an engineered variant of methylaspartate ammonia lyase
21,22
(MALꢀL384A) as the biocatalyst.
In addition, we report the synthesis of two Asp
derivatives by hybridization of the nonselective EAAT inhibitor LꢀTBOA (2) with the
EAAT2ꢀselective inhibitor WAYꢀ213613 (6) or the EAAT3ꢀpreferring inhibitor NBIꢀ59159 (7).
The pharmacological properties of the newly synthesized Asp derivatives, as well as those of
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previously synthesized analogues and homologues of LꢀTFBꢀTBOA, have been characterized
at stable cell lines expressing the human EAAT1ꢀ3 and rat EAAT4 and the structureꢀactivity
relationships of these analogues elucidated.
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Figure 1. EAAT inhibitors derived from the
LꢀAsp scaffold. (A) Structures of chemoenzymatic
products and designed hybrid compounds. (B) Structures of previously developed EAAT inhibitors.
RESULTS AND DISCUSSION
Chemoenzymatic synthesis of 3-substituted Asp analogues
The asymmetric synthesis of Asp derivatives with alkyloxy or aryloxy substituents at the Cꢀ3
position has proven to be challenging. Shimamoto and coworkers firstly reported the synthesis
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of LꢀTBOA (2) through an elaborate 11ꢀstep procedure. A rapid chemoenzymatic
methodology for asymmetric synthesis of 2 and its analogues by using MALꢀL384A as
21,22
biocatalyst has been developed in our group.
In order to further explore the substrate
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scope and synthetic usefulness of MALꢀL384A, a series of 2ꢀsubstituted fumarate derivatives
with various cycloalkyloxy (12a-f), heteroaryloxy (12g-j) or alkyloxy (12k) substituents were
prepared through the addition of appropriate alcohols to dimethyl acetylenedicarboxylate (10),
followed by hydrolysis of the methyl esters (Table 1). Interestingly, compounds 12a-e and
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12g-k were accepted as substrates by MALꢀL384A, giving excellent conversions (60ꢀ98%)
and yielding the corresponding amino acid products in 26ꢀ58% isolated yield (Table 1). Hence,
MALꢀL384A has a remarkably broad substrate scope, allowing the addition of ammonia to a
wide variety of fumarate derivatives, providing a powerful synthetic tool for the preparation
of
valuable
3ꢀsubstituted
aspartic
acids.
The
most
bulky
compound,
2ꢀcyclooctylmethoxyfumarate (12f), was not accepted as substrate by MALꢀL384A.
The amino acid product 13a, which is representative for the series of chemoenzymatically
prepared 3ꢀcycloalkyloxy substituted aspartic acids, was identified as the desired threo
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diastereomer (de = 97%, Figure S1) by comparison of its HꢀNMR signals and Jꢀcoupling
values to those of an authentic standard with known Lꢀthreo configuration and chemically
synthesized DLꢀthreo and DLꢀerythro stereoisomers (Scheme 1). To determine the absolute
configuration of product 13a, HPLC analysis on a chiral stationary phase was conducted by
using corresponding reference molecules with known Lꢀthreo or DLꢀthreo configuration. This
analysis revealed that chemoenzymatically produced 13a was present as a single enantiomer
with exclusively the Lꢀthreo configuration (ee >99%, Figure S62). Similarly, the
representative chemoenzymatic products 13g and 13k were also identified as the desired
Lꢀthreo isomers with excellent de (>98%) and ee (>99%) values (Table 1, Figures S2, S3, S63
and S64). Although the relative configurations of products 13b-e and 13h-j were not
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determined by comparison to authentic standards, we assume the relative configurations to be
threo for all enzymatic products on the basis of analogy.
Table 1. Threeꢀstep chemoenzymatic synthesis of 3ꢀsubstituted Asp analogues.
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a
b
c
e
Conv. (yield )
%]
de
ee
[%]
Entry
R
Substrate Product
[
[%]
f
1
2
3
4
12a
12b
12c
12d
13a
13b
13c
13d
88 (31)
89 (58)
85 (39)
90 (56)
97 (threo)
>99 (
L
ꢀthreo)
d
d
d
g
>98 (threo)
>98 (threo)
>98 (threo)
n.d.
n.d.
n.d.
d
5
12e
13e
60 (26)
>98 (threo)
n.d.
ꢀꢀꢀ
6
7
8
9
12f
12g
12h
12i
13f
13g
13h
13i
0
ꢀꢀꢀ
f
98 (47)
98 (48)
98 (35)
98 (39)
83 (47)
>98 (threo)
>99 (
Lꢀthreo)
d
>98 (threo)
>98 (threo)
>98 (threo)
n.d.
n.d.
n.d.
d
d
10
12j
12k
13j
13k
f
11
>98 (threo)
>99 (Lꢀthreo)
a
1
Conversions were determined by comparing H NMR signals of substrates and corresponding
b
c
products. Yield of isolated product after cation exchange chromatography. The diastereomeric excess
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d
(de) was determined by H NMR. The purified products were tentatively assigned the threo
e
configuration on the basis of analogy. The enantiomeric excess (ee) was determined by HPLC on a
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chiral stationary phase using authentic standards with known
Lꢀthreo and DLꢀthreo configuration
f
(Figures S62ꢀS64). Absolute configuration of products 13a, 13g and 13k were determined
1
unambiguously by comparison of H NMR and chiral HPLC data to those of authentic samples with
g
known DLꢀerythro, DLꢀthreo, and
L
ꢀthreo configuration. n.d., not determined.
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Synthesis of (DL-threo)- and (DL-erythro)-3-substituted Asp derivatives
To confirm the importance of the relative configuration of 3ꢀsubstituted Asp derivatives for
inhibition of EAATs, representative compounds with DLꢀthreo and DLꢀerythro configurations
were synthesized according to the route given in Scheme 1. The building block (DLꢀthreo)-16
2
3
was prepared based on the Sharpless aminohydroxylation procedure as previously reported.
Compound (DLꢀthreo)-17, with the Nꢀprotective group transferred from Cbz to Boc, was
achieved by hydrogenolysis of (DLꢀthreo)-16 using H /Pd/C followed by Bocꢀprotection in an
2
excellent yield (2 steps, 83%). To facilitate Oꢀalkylation of 17 with the proper RBr, the strong
base NaH was used to deprotonate the hydroxyl group of 17 which provided the desired
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(DLꢀthreo)-18 with an isolated yield of 11ꢀ53%. In addition, a small amount of epimerized
product (DLꢀerythro)-18 was observed and separated by flash column chromatography in 4ꢀ11%
isolated yield. Subsequently, global deprotection of (DLꢀthreo)-18 was conducted via
treatment with trifluoroacetic acid followed by hydrolysis of the methyl esters with LiOH,
providing the desired final product (DLꢀthreo)-19 with 34ꢀ39% isolated yield over two steps.
Following the same procedure, compound (DLꢀerythro)-19 was obtained in 34ꢀ46% yield.
Notably, (DLꢀthreo)-19 and (DLꢀerythro)-19 are not only valuable molecules for exploring the
stereochemistryꢀactivity relationship of 3ꢀsubstituted Asp analogues as EAAT inhibitors, but
also for determining the stereochemistry of the chemoenzymatically prepared products.
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Scheme 1. Synthesis of (DLꢀthreo)ꢀ and (DLꢀerythro)ꢀ3ꢀsubstituted Asp analogues.
Design and synthesis of hybrid compounds 8 and 9
LꢀTBOA (2) is a potent nonselective EAAT inhibitor, while WAYꢀ213613 (6) and NBIꢀ59159
17ꢀ20
(7) are EAAT2ꢀselective and EAAT3ꢀpreferring inhibitors, respectively (Figure 1B).
We
envisioned that hybridization of LꢀTBOA (2) with WAYꢀ213613 (6) or NBIꢀ59159 (7) would
result in a synergistic effect on inhibitory activity at the EAATs. Thus, two hybrid compounds
8 and 9 were designed by integrating 2 with 6 or 7 (Figure 1A), and their synthesis was
achieved according to the route presented in Scheme 2. The multiꢀgram scale
chemoenzymatic synthesis of 2 (LꢀTBOA, de >98%, ee >99%), starting from commercially
available dimethyl acetylenedicarboxylate 10 and using MALꢀL384A as biocatalyst, has been
24
previously reported. Selective monoꢀesterification at the 4ꢀcarboxylate of 2, which was
accomplished under ambient condition using one equivalent of SOCl in dry methanol,
delivered intermediate 20 without the need for purification. The 1ꢀcarboxylate of compound
2
3 2
20 was subsequently protected by a tertꢀbutyl group via treatment with BF ꢀEt O/tBuOAc,
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
providing compound 21 with 87% isolated yield over two steps. Starting from 21, the chiral
building block 23 was achieved in two successive reactions including Bocꢀprotection and
selective hydrolysis of the 4ꢀmethyl ester with LiOH (2 steps, 42%). The desired Asp
derivatives 8 and 9 were obtained via amidation of 23 with appropriate amine 24 or 25 by
using EDCI/HOBT as condensation reagents followed by global deprotection in TFA/DCM
with 14ꢀ32% isolated yield over two steps.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Synthesis of hybrid compounds 8 and 9. The parent compounds for the blue and black parts
of compound 8 are WAYꢀ213613 (6) and LꢀTBOA (2), respectively, whereas the parent compounds for
the blue and black parts of compound 9 are NBIꢀ59159 (7) and LꢀTBOA (2), respectively.
Pharmacological characterization of the Asp derivatives at EAAT1-4
The pharmacological properties of the aspartate derivatives 13a-e, 13g-k, (DLꢀthreo)ꢀ19a-c
and (DLꢀerythro)ꢀ19a-c, the hybrid molecules 8 and 9, and selected EAAT reference ligands
were determined at HEK293 cell lines stably expressing human EAAT1, EAAT2 and EAAT3
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(
hEAAT1, hEAAT2 and hEAAT3, respectively) and at a tsA201 cell line stably expressing rat
3
25
EAAT4 (rEAAT4) in a conventional [ H]ꢀDꢀAsp uptake assay. The rank order and absolute
values of the IC50 values exhibited by the reference EAAT substrates LꢀGlu, LꢀAsp and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Lꢀthreo)ꢀ3ꢀhydroxyaspartate (LꢀTHA) and the reference EAAT nonꢀsubstrate inhibitors
3
DLꢀTBOA and WAYꢀ213613 at the four EAATs in the [ H]ꢀDꢀAsp uptake assay were in good
10,11,17,25ꢀ27
agreement with previously reported pharmacological properties (Table 2).
Overall, the 3ꢀsubstituted Asp derivatives 13a-e and 13g-k were all relatively potent
inhibitors of the four EAATs, and even though most of them displayed slightly lower IC50
values at hEAAT1 and hEAAT2 compared to hEAAT3 and rEAAT4, they were essentially
nonꢀselective inhibitors (Table 2). The Asp derivatives displayed comparable
highꢀnanomolar/lowꢀmicromolar IC50 values at the four transporter subtypes, the differences
between the highest and lowest IC50 values in the compound series being 5.5ꢀ, 4.9ꢀ, 13ꢀ and
9
.4ꢀfold at hEAAT1, hEAAT2, hEAAT3 and rEAAT4, respectively. Interestingly, the
(
Lꢀthreo)ꢀ3ꢀsubstituted Asp derivatives 13a-e and 13g-k and (DLꢀthreo)ꢀ3ꢀsubstituted Asp
derivatives 19a-c were equipotent with DLꢀTBOA (Table 2). Based on direct comparisons
between racemic Asp analogues (DLꢀthreo)ꢀ19a-c and (DLꢀerythro)ꢀ19a-c, it is clear that the
(DLꢀthreo)ꢀAsp analogues were significantly more potent inhibitors than the corresponding
(DLꢀerythro)ꢀAsp analogues at all four EAAT subtypes (Table 2). Notably, the enantiopure
Asp derivatives (Lꢀthreo)ꢀ13a,g,k only displayed slightly lower IC50 values than their racemic
counterparts (DLꢀthreo)ꢀ19a-c (Table 2). These results are in line with previous observations
that LꢀTBOA and LꢀTFBꢀTBOA were more potent inhibitors of EAATs than their
11,28
corresponding (
Lꢀerythro)ꢀdiastereomers.
These structureꢀactivity relationships thus
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
demonstrate that essentially every (cyclo)alkyloxy and (hetero)aryloxy substituent in the
3ꢀposition of Asp yields a potent EAAT ligand and also confirm that the relative threo
configuration of 3ꢀsubstituted Asp analogues is crucial for their inhibitory activities at EAATs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The retained EAAT inhibitory activity in the Asp analogues 13a-e and 13g-k regardless
of the size of their respective 3ꢀsubstituent contrasts the substantial gradual reduction in
EAAT activity observed upon the introduction of 4ꢀsubstituents of increasing size into
2
9,30
Glu.
It seems reasonable to ascribe these SAR differences to the substituents in the
3ꢀsubstituted Asp analogue and the 4ꢀsubstituted Glu analogue projecting out into different
regions of the EAAT substrate binding pocket. Perhaps more interesting, the complete lack of
subtypeꢀselectivity or –preference exhibited by all of the analogues in this study (13a-e and
13g-k) differs remarkably from our recent findings for a series of sulfonamido functionalized
3ꢀsubstituted aspartate analogues that comprises both EAAT1ꢀpreferring and EAAT2ꢀselective
16
inhibitors. It seems that even when comparing different 3ꢀsubstituted Asp analogues,
different functionalities (an ether vs a sulfonamide group) in this position of the Asp molecule
will result in the substituents being projected out into different binding pocket regions, which
again is reflected in markedly different subtypeꢀselectivity profiles across the EAATs.
Whereas Asp (both
LꢀAsp and DꢀAsp) and its derivative (Lꢀthreo)ꢀ3ꢀhydroxy Asp
(LꢀTHA) are substrates for EAAT1ꢀ4, the (DLꢀthreo)ꢀ3ꢀbenzyloxy Asp (DLꢀTBOA) analogue
10,11,17,25ꢀ27
is a nonꢀsubstrate inhibitor of all four EAAT subtypes.
Since the series of Asp
analogues in the present study comprised analogues with 3ꢀsubstituents covering a
considerable spectrum in terms of sizes, we decided to test these analogues for both substrate
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9
and nonꢀsubstrate inhibitor activity at the hEAAT1ꢀ3 cell lines in the fluorescenceꢀbased
FLIPR Membrane Potential Blue (FMP) assay. This assay measures the membrane potential
changes induced in the cell lines by the substrate translocation and the concomitant
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
+
coꢀtransport of Na , H and the counterꢀtransport of K mediated by the EAAT. In a previous
study we have found the assay to be capable of distinguishing substrates from nonꢀsubstrate
2
5
inhibitors within a series of reference EAAT ligands. None of the 3ꢀsubstituted Asp
analogues (13a-e and 13g-k) were substrates at hEAAT1ꢀ3, as they did not evoke a significant
increase in the fluorescence levels in the assay when applied on their own (all at
concentrations of 300 ꢁM) at hEAAT1ꢀ, hEAAT2ꢀ and hEAAT3ꢀHEK293 cell lines (data not
shown). In contrast, preincubation and coꢀapplication of the analogues (all at concentrations
of 300 ꢁM) together with LꢀGlu EC80 completely eliminated the Gluꢀinduced response at all
three transporters (data not shown), and thus all 3ꢀsubstituted Asp analogues were
nonꢀsubstrate inhibitors of the EAATs. Given that
findings clearly demonstrate that the borderline between when the Asp analogue can be
transported by the EAAT or not lies right between the 3ꢀhydroxy substituent ( ꢀTHA) and
ꢀsubstituents such as cyclopropyl (13a), propꢀ2ꢀyn (13k) and larger groups in the series of
Asp analogues tested here (Figure 1, Table 2).
LꢀTHA is a substrate for EAAT1ꢀ4, these
L
3
The IC50 values displayed by the TBOA/WAYꢀ213613 hybrid, compound 8, at hEAAT1,
3
hEAAT2, hEAAT3 and rEAAT4 in the [ H]ꢀ
D
ꢀAsp uptake assay were 190ꢀ, 53ꢀ, 40ꢀ and
16ꢀfold lower than those of DLꢀTBOA and 78ꢀ, 2.0ꢀ, 20ꢀ and 11ꢀfold lower than those of
WAYꢀ213613 (6), respectively (Table 2, Figure 2). Thus, introduction of a benzyloxy group
into the 3ꢀposition of WAY213613 converts this EAAT2ꢀselective inhibitor into a
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nonꢀselective but much more potent EAAT inhibitor. Hybrid compound 9, derived from
TBOA (2) and NBIꢀ59159 (7), displayed IC50 values of 12, 47, 55 and 80 nM and was thus
180ꢀ, 40ꢀ, 69ꢀ and 29ꢀfold more potent as inhibitor at hEAAT1, hEAAT2, hEAAT3 and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
rEAAT4, respectively, than DLꢀTBOA. Since we did not test NBIꢀ59159 (7) in the [ H]ꢀDꢀAsp
uptake assay, we cannot make a direct comparison between the inhibitory potencies displayed
by 9 and those of this other parent compound. However, compared to the reported inhibitory
19
data of NBIꢀ59159 (7), the hybrid analogue 9 displayed 83ꢀ, 30ꢀ and 1.6ꢀfold increased
inhibitory potencies at EAAT1, EAAT2 and EAAT3, respectively (Table 2, Figure 2).
Analogously to the observation for the hybrid analogue 8 and its parent compound
WAYꢀ213613, introducing a benzyloxy group at the 3ꢀposition of NBIꢀ59159 converted this
EAAT3ꢀpreferring inhibitor into a nonꢀselective but considerably more potent EAAT inhibitor.
The chemoenzymatic synthesis of LꢀTFBꢀTBOA (3a) and its four derivatives 3b-e was
24
12
reported elsewhere. In agreement with the original study of LꢀTFBꢀTBOA, the
chemoenzymatically prepared compound 3a was found to be a potent EAAT inhibitor
displaying IC values of 3.6, 10, 120 and 40 nM at hEAAT1, hEAAT2, hEAAT3 and rEAAT4,
5
0
respectively, thus displaying some preference for hEAAT1,2 over hEAAT3 (Table 2).
Whereas substitution of the pꢀCF group in LꢀTFBꢀTBOA with oꢀCF (3b) resulted in
significantly decreased inhibitory potencies at the EAATs, the mꢀCF analogue (3c) displayed
3
3
3
comparable IC50 values as LꢀTFBꢀTBOA (3a) at all four transporter subtypes. While extension
of the functional group at the Cꢀ3 position in LꢀTFBꢀTBOA with one carbon (3d) resulted in
5ꢀ10ꢀfold increased IC50 values at hEAAT1, hEAAT2 and rEAAT4, the homologue retained
the inhibitory potency of LꢀTFBꢀTBOA at hEAAT3. Interestingly, extension of the Cꢀ3
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functional group with two carbons resulted in a homologue (3e) with similar inhibitory
properties at the four EAATs as LꢀTFBꢀTBOA (Table 2).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Pharmacological properties of EAAT reference ligands, 3ꢀsubstituted aspartate analogues and
3
hybrid compounds at hEAAT1, hEAAT2, hEAAT3 and rEAAT4 in the [ H]ꢀ
Dꢀaspartate uptake assay.
IC50 values are given in µM with pIC50 ± S.E.M. in brackets, and the number of independent
experiments (n) are given in superscript behind each pIC50 ± S.E.M. value.
H
n
N
R
O
R
O
O
O
H
COOH
NH2
N
COOH
NH2
COOH
NH2
HOOC
R
HOOC
O
3
-substituted Asp analogues
Hybrid compounds
L-TFB-TBOA analogues
13a-k and 19a-c
8 and 9
3a-e
hEAAT1
hEAAT2
hEAAT3
IC50 (µM)
rEAAT4
IC50 (µM)
No.
R
IC50 (µM)
IC50 (µM)
(n)
(n)
(n)
(n)
[
pIC50 ± S.E.M.]
[pIC50 ± S.E.M.]
[pIC50 ± S.E.M.]
[pIC50 ± S.E.M.]
Reference EAAT ligands
(
4)
(4)
(3)
(3)
(4)
(3)
(3)
(3)
(3)
(3)
L
L
L
ꢀGlu
11 [4.94 ± 0.07]
9.3 [5.03 ± 0.11]
3.9 [5.41 ± 0.15]
2.1 [5.68 ± 0.09]
52 [4.28 ± 0.02]
32 [4.48 ± 0.09]
32 [4.49 ± 0.07]
20 [4.71 ± 0.13]
13 [4.88 ± 0.06]
(
3)
ꢀAsp
11 [4.96 ± 0.07]
(
3)
ꢀTHA
9.6 [5.02 ± 0.03]
9.6 [5.02 ± 0.08]
3.8 [5.42 ± 0.06]
1.9 [5.73 ± 0.05]
2.8 [5.55 ± 0.09]
2.3 [5.64 ± 0.07]
1.5 [5.97 ± 0.10]
(4)
(4)
(4)
(3)
(3)
(3)
DLꢀTBOA
1.9 [5.72 ± 0.08]
(3)
(3)
WAYꢀ213613 (6)
0.86 [6.07 ± 0.01]
0.071 [7.15±0.05]
Lꢀthreoꢀ3ꢀsubstituted Asp analogues
(3)
(3)
(3)
(3)
(4)
(4)
(3)
(3)
(3)
(3)
1
1
1
1
3a
3b
3c
3d
3.4 [5.46 ± 0.07]
1.4 [5.86 ± 0.02]
3.1 [5.51 ± 0.05]
2.1 [5.67 ± 0.04]
2.7 [5.57 ± 0.04]
2.6 [5.59 ± 0.05]
8.3 [5.08 ± 0.02]
3.0 [5.52 ± 0.01]
2.4 [5.61 ± 0.07]
1.6 [5.79 ± 0.02]
(
(
3)
4)
(3)
(4)
(4)
1.7 [5.78 ± 0.06]
1.4 [5.86 ± 0.06]
0.69 [6.16 ± 0.07]
0.94 [6.03 ± 0.07]
0.49 [6.31 ± 0.06]
(4)
0.62 [6.20 ± 0.05]
(3)
(3)
(3)
(3)
13e
2.2 [5.65 ± 0.11]
2.4 [5.61 ± 0.04]
11 [4.98 ± 0.06]
7.8 [5.11 ± 0.01]
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
(3)
1
3g
1.6 [5.79 ± 0.03]
1.2 [5.91 ± 0.06]
1.3 [5.90 ± 0.06]
0.92 [6.03 ± 0.04]
0.77 [6.11 ± 0.04]
0.76 [6.12 ± 0.04]
0.83 [6.08 ± 0.05]
0.86 [6.06 ± 0.04]
5.0 [5.31 ± 0.06]
3.3 [5.48 ± 0.07]
3.1 [5.51 ± 0.07]
7.8 [5.11 ± 0.05]
(
3)
(3)
13h
1.2 [5.93 ± 0.02]
1.2 [5.92 ± 0.03]
1.6 [5.79 ± 0.02]
(3)
(3)
1
1
3i
3j
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(
3)
3)
(3)
3.0 [5.52 ± 0.05]
2.7 [5.57 ± 0.06]
(3)
(3)
(3)
13k
1.6 [5.79 ± 0.02]
0.84 [6.07 ± 0.04]
15 [4.82 ± 0.07]
Racemic DLꢀthreoꢀ and DLꢀerythroꢀ3ꢀsubstituted Asp analogues
3.0
[5.53
±
(
3)
(3)
(3)
DLꢀthreo-19a
5.8 [5.24 ± 0.03]
3.7 [5.43 ± 0.04]
16 [4.80 ± 0.07]
(3)
0.04]
(
3)
(3)
(3)
(3)
(3)
DLꢀerythroꢀ19a
DLꢀthreo-19b
DLꢀerythroꢀ19b
DLꢀthreo-19c
87 [4.06 ± 0.12]
2.4 [5.61 ± 0.04]
29 [4.53 ± 0.06]
6.9 [5.16 ± 0.11]
75 [4.13 ± 0.10]
1.6 [5.80 ± 0.05]
97 [4.01 ± 0.12]
~300 [~3.5]
(3)
(3)
(3)
1.6 [5.80 ± 0.07]
4.3 [5.37 ± 0.07]
(
3)
(3)
(3)
(3)
33 [4.48 ± 0.10]
31 [4.51 ± 0.04]
~100 [~4.0]
(
4)
(4)
(4)
(3)
2.8 [5.56 ± 0.04]
1.6 [5.81 ± 0.05]
13 [4.90 ± 0.07]
(
3)
(3)
(3)
(3)
DLꢀerythroꢀ19c
99 [4.00 ± 0.05]
82 [4.09 ± 0.04]
Hybrid analogues
0.036 [7.4 ±0.11]
28 [4.55 ± 0.06]
~100 [~4.0]
(
4)
(4)
(4)
(4)
(4)
8
9
0.011 [7.97 ± 0.10]
0.012 [7.93 ± 0.05]
0.094 [7.03 ± 0.08]
0.055 [7.26 ± 0.09]
0.14 [6.86 ± 0.09]
0.080
[7.10
(
3)
(4)
0.047 [7.33±0.12]
(4)
±
0.10]
L
ꢀTFBꢀTBOA analogues
(
3)
(3)
(4)
(4)
(4)
(4)
(3)
(3)
3
3
3
3
3
a
b
c
n=0, pꢀCF
3
3
0.0036 [8.45 ± 0.09]
0.010 [8.00±0.14]
0.084 [7.07±0.06]
0.017 [7.78±0.07]
0.11 [6.97 ± 0.03]
0.018 [7.74±0.04]
0.12 [6.93 ± 0.11]
0.53 [6.28 ± 0.08]
0.29 [6.54 ± 0.03]
0.13 [6.89 ± 0.03]
0.11 [6.98 ± 0.04]
0.040 [7.40±0.11]
0.17 [6.77 ± 0.09]
0.071 [7.15±0.05]
0.19 [6.71 ± 0.03]
0.044 [7.35±0.05]
(4)
(4)
(4)
(4)
(4)
(4)
n=0, oꢀCF
0.031 [7.51 ± 0.13]
0.0061 [8.21 ± 0.03]
(
4)
(4)
(4)
(4)
n=0, mꢀCF
3
(
4)
4)
d
e
n=1, pꢀCF
3
0.021 [7.68 ± 0.02]
(
n=2, pꢀCF
0.0051 [8.9 ± 0.07]
3
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9
1
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2
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0
1
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9
0
1
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9
0
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8
9
0
1
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4
5
6
7
8
9
0
Figure 2. The functional properties of the hybrid analogues 8 and 9 as EAAT inhibitors.
Representative concentrationꢀinhibition curves for 8, 9, DLꢀTBOA (parent compound of both 8 and 9)
and WAYꢀ213613 (6, parent compound of 8) at hEAAT1ꢀHEK293, hEAAT2ꢀHEK293,
3
hEAAT3ꢀHEK293 and rEAAT4ꢀtsA201 cells in the [ H]ꢀ
D
ꢀAsp uptake assay. Data are from a
representative specific experiment and given as mean ± S.D. values (based on duplicate
determinations).
CONCLUSION
We have presented the design, synthesis and pharmacological characterization of an elaborate
series of 3ꢀsubstituted Asp analogues as inhibitors of hEAAT1, hEAAT2, hEAAT3 and
rEAAT4. Inspired by the potential of the Asp scaffold for the development of EAAT
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1
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
inhibitors, various new Asp derivatives with (cyclo)alkyloxy and (hetero)aryloxy substituents
at the Cꢀ3 position were synthesized using a key stereoselective enzymatic step. Remarkably,
all these Asp derivatives were found to be potent nonꢀsubstrate pan inhibitors of the EAATs.
The functional properties exhibited by the Asp analogues also provide insight into the relation
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
between ligand structure and EAAT transport. Whereas Asp and (
Lꢀthreo)ꢀ3ꢀhydroxy Asp
(
LꢀTHA) are substrates of EAATs, it is clear from this series that basically any Asp derivative
with a substituent at Cꢀ3 larger than a hydroxyl group will be a nonꢀsubstrate inhibitor. Since
all these compounds are Asp derivatives, it is reasonable to assume that they function as
2
5
competitive inhibitors, like DLꢀTBOA, and act through the substrate binding site in the
transporters. On the other hand, judging from the fact that all 3ꢀsubstituted Asp analogues
comprising a wide range of substituents displayed comparable inhibitory potencies, the
3ꢀsubstituent either does not contribute substantially to this inhibitory potency by forming
interactions with residues in the substrate binding site of the transporter or alternatively all of
the 3ꢀsubstituents in the tested analogues are able to form similar interactions. Finally, our
results confirm that the threo configuration of the 3ꢀsubstituted Asp analogue is crucial for its
high inhibitory activities at EAATs.
Unique EAAT inhibitors were developed by hybridization of the nonꢀselective EAAT
inhibitor
LꢀTBOA (2) with the EAAT2ꢀselective inhibitor WAYꢀ213613 or the
EAAT3ꢀpreferring inhibitor NBIꢀ59159 to yield hybrid analogues 8 and 9, respectively.
Compounds 8 and 9 displayed significantly higher inhibitory activities at EAATs than both of
their respective parent structures, although the hybridization in both cases leads to pan
inhibitors of EAAT1ꢀ4. Thus, while the additional interactions with the transporter formed by
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9
the benzyloxy group at the 3ꢀposition in 8/9 in both cases leads to increased inhibitory
potency, it also seems to induce a binding conformation of the Asp analogue that dictates a
different spatial orientation of the 4ꢀ(2ꢀbromoꢀ4,5ꢀdifluorophenoxy)phenyl/(9Hꢀfluorenꢀ2ꢀyl)
substituent than in WAYꢀ213613/NBIꢀ59159. We propose that this and other hybridization
strategies could advance future design and development of more potent EAAT inhibitors.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We recently reported the synthesis and evaluation of
LꢀTFBꢀTBOAꢀbased inhibitors of
the prokaryotic aspartate transporter GltTk with photoꢀcontrolled activity, allowing the remote,
31
reversible and spatiotemporally resolved regulation of transport. Work is in progress to
convert the hybrid compounds 8 and 9, which were shown here to be highly potent
nonꢀselective EAAT inhibitors, into lightꢀcontrolled glutamate transporter inhibitors by
introducing a photoswitchable azobenzene moiety.
EXPERIMENTAL SECTION
Chemicals and general methods. All chemicals were purchased from commercial sources
unless otherwise specified. All the solvents were of analytical reagent grade and were used
1
13
19
without further purification. H NMR, C NMR and F NMR analysis were performed on a
Bruker Avance III 400/500 MHz spectrometer. High resolution mass spectra (HRMS) were
recorded on a LTQ Orbitrap XL. Flash chromatography was performed on a GRACE X2
system with silica gel (200−300 mesh) purchased from Merck. High performance liquid
chromatography (HPLC) analysis was performed with a Shimadzu LCꢀ10AT HPLC with a
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Shimadzu SPꢀM10A ELSD detector and a Shimadzu SPDꢀM10A photodiode array detector.
Analytical HPLC was performed using a Kinetex C18 column (150 x 4.6 mm, 5 ꢁm) with
2
5ꢀ95% MeCN gradient in H O (0.5% TFA) as mobile phase. The purity of all the target
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds was determined to be >95%. Chiral HPLC analysis for compound 13g was
perfomed using Nucleosil chiralꢀ1 column (250 x 4 mm, 5 ꢁm) with 0.5 mM aqueous CuSO4
as mobile phase. Chiral HPLC analysis for compounds 13a and 13k was perfomed using
CROWNPAK CRꢀI (+) column (150 x 3 mm, 5 ꢁm) with isocratic MeCN/H
TFA) as mobile phase.
2
O (98%, v/v, 0.5%
Synthesis of hybrid compounds 8 and 9
(L-threo)-2-Amino-3-(benzyloxy)-4-[[4-(2-bromo-4,5-difluorophenoxy)phenyl]amino]-4-
oxobutanoic acid (8)
To a stirred solution of 26 (68 mg, 0.1 mmol, see below) in dry DCM (2 mL), in an iceꢀbath,
was added trifluoroacetic acid (2 mL) dropwise. After the complete addition of trifluoroacetic
acid, the iceꢀbath was removed and the reaction was allowed to proceed at room temperature
for further 6 h. After completion of the starting material, the solvent was removed in vacuo to
1
provide pure 8 as a trifluoroacetate salt (brown solid, 34 mg, yield 52%). H NMR (500 MHz,
D
2
O/DMSOꢀ
d
6
):
δ
7.74 (dd,
J
= 9.8, 8.3 Hz, 1H), 7.43 – 7.41 (m, 2H), 7.32 – 7.25 (m, 5H),
= 4.0 Hz, 1H), 4.16
168.1, 166.4, 152.4, 149.5 (dd, = 8.2,
= 372.3, 13.9 Hz), 136.9, 134.2, 128.2
7.10 – 7.02 (m, 1H), 6.89 – 6.88 (m, 2H), 4.62 – 4.56 (m, 2H), 4.46 (d,
J
13
(d,
J
= 3.9 Hz, 1H); C NMR (126 MHz, DMSOꢀd6):
δ
J
3.8 Hz), 148.5 (dd, = 374.9, 13.9 Hz), 148.6 (dd, J
J
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9
(
2C), 128.0 (2C), 127.9, 122.0, 121.9, 117.8 (2C), 115.3 (d,
J
= 21.4 Hz), 110.2 (d,
J
δ
= 20.2
ꢀ73.51
1
9
Hz), 108.6 (dd,
J = 7.6, 3.8 Hz), 76.8, 72.1, 54.1; F NMR (376 MHz, DMSOꢀd ):
6
(s), ꢀ134.99 (dt,
J
= 20.6, 10.1 Hz), ꢀ141.25 (dt, J = 23.2, 9.0 Hz). HRMS: calcd. for
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
C
23
H20BrF
2
N
2
O
5
[M+H] : 521.0518, found: 521.0520. HPLC: purity 98%, retention time 9.0
min.
(L-threo)-4-[(9H-fluoren-2-yl)amino]-2-amino-3-(benzyloxy)-4-oxobutanoic acid (9)
Hybrid compound 9 was prepared from 27 (100 mg, 0.18 mmol, see below) following a
procedure similar to that used for compound 8. The title product was obtained as a white solid
1
(32 mg, 35%). H NMR (500 MHz, DMSOꢀ
d
6
):
= 13.4, 7.9 Hz, 2H), 7.42 (d,
.27 (m, 2H), 4.70 (d, = 11.7 Hz, 1H), 4.63 (d,
.06 (bs, 1H), 3.92 (s, 2H); C NMR (126 MHz, Methanolꢀ
δ
10.35 (s, 1H), 7.99 (s, 1H), 7.84 (t,
= 7.3 Hz, 2H), 7.38 – 7.34 (m, 3H), 7.32 –
= 4.1 Hz, 1H), 4.59 (d, = 11.7 Hz, 1H),
): 169.5, 168.4, 145.4, 144.5,
J = 7.8
Hz, 2H), 7.58 (dd,
J
J
7
4
1
J
J
J
13
d
4
δ
42.4, 140.0, 138.7, 137.7, 137.4, 129.7 (2C), 129.6, 127.9, 127.7, 126.0, 121.0, 120.6, 120.5,
+
1
18.6, 107.3, 77.7, 74.6, 56.1, 37.7. HRMS: calcd. for C24
H
23
N
2
O
4
[M+H] : 403.1652, found:
4
03.1653. HPLC: purity 98%, retention time 8.4 min.
Synthesis of dimethyl 2-substituted fumarate derivatives 11a-k. The synthesis of the
dimethyl 2ꢀsubstituted fumarate derivatives 11a-k was based on a previously published
22,32
procedure.
Briefly, to a stirred solution of dimethyl acetylenedicarboxylate (10, 568 mg, 4
mmol) in dichloromethane (30 mL) was added DABCO (45 mg, 0.4 mmol) followed by the
appropriate alcohol (5 mmol). The reaction mixture was stirred at room temperature for 12 h.
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2
2
2
2
2
2
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3
3
3
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5
6
The solvent was removed under reduced pressure to give the crude product as a dark brown
oil. The trans/cis isomers of the products were separated by flash chromatography (silica gel)
using 5% EtOAc/Petroleum ether (boiling point 40 to 60 °C) as eluent. The preferred
transꢀisomers 11a-k (i.e., the fumarate derivatives) were used in further experiments.
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8
9
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0
Dimethyl 2-(cyclopropylmethoxy)fumarate (11a)
1
Clear oil. 230 mg (27% yield). H NMR (500 MHz, CDCl
3
):
δ
6.11 (s, 1H), 3.84 (d,
J
= 7.2
Hz, 2H), 3.72 (s, 3H), 3.63 (s, 3H), 1.16 – 1.08 (m, 1H), 0.49 – 0.45 (m, 2H), 0.21 – 0.18 (m,
13
2
H); C NMR (126 MHz, CDCl
3
):
δ
164.5, 163.4, 154.1, 109.6, 78.4, 52.6, 51.4, 10.6, 3.0
(2C).
Dimethyl 2-(cyclobutylmethoxy)fumarate (11b)
1
Clear oil. 164 mg (18% yield). H NMR (500 MHz, CDCl
3
):
δ
6.11 (s, 1H), 4.00 (d,
J
= 6.7
Hz, 2H), 3.77 (s, 3H), 3.68 (s, 3H), 2.69 – 2.61 (m, 1H), 2.04 – 1.97 (m, 2H), 1.87 – 1.76 (m,
13
4
H); C NMR (126 MHz, CDCl
3
): δ 164.7, 163.4, 154.5, 108.9, 77.6, 52.7, 51.5, 35.1, 24.5
(2C), 18.3.
Dimethyl 2-(cyclopentylmethoxy)fumarate (11c)
1
Clear oil. 136 mg (14% yield). H NMR (500 MHz, CDCl
3
):
δ
6.15 (s, 1H), 3.96 (d,
J
= 6.9
Hz, 2H), 3.82 (s, 3H), 3.74 (s, 3H), 2.34 – 2.25 (m, 1H), 1.81 – 1.75 (m, 2H), 1.62 – 1.53 (m,
13
4
H), 1.35 – 1.29 (m, 2H); C NMR (126 MHz, CDCl
3
): δ 164.9, 163.7, 154.7, 108.6, 78.0,
52.9, 51.8, 39.8, 29.2 (2C), 25.6 (2C).
Dimethyl 2-(cyclohexylmethoxy)fumarate (11d)
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9
1
3
Clear oil. 130 mg (13% yield). H NMR (500 MHz, CDCl ): δ 6.13 (s, 1H), 3.88 (d, J = 6.4
Hz, 2H), 3.82 (s, 3H), 3.74 (s, 3H), 1.84 – 1.66 (m, 6H), 1.30 – 1.14 (m, 3H), 1.05 – 0.97 (m,
13
2
H); C NMR (126 MHz, CDCl
3
):
δ
165.0, 163.7, 154.9, 108.1, 79.4, 52.9, 51.8, 38.5, 29.6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2C), 26.6, 25.9 (2C).
Dimethyl 2-(cycloheptylmethoxy)fumarate (11e)
1
Clear oil. 83 mg (8% yield). H NMR (500 MHz, CDCl
3
):
δ
6.12 (s, 1H), 3.85 (d,
J
= 6.6 Hz,
2H), 3.81 (s, 3H), 3.72 (s, 3H), 1.93 – 1.86 (m, 1H), 1.83 – 1.77 (m, 2H), 1.69 – 1.63 (m, 2H),
13
1
.58 – 1.54 (m, 2H), 1.51 – 1.39 (m, 4H), 1.27 – 1.20 (m, 2H); C NMR (126 MHz, CDCl
3
):
δ
164.9, 163.6, 154.7, 108.3, 79.2, 52.9, 51.7, 39.9, 30.7 (2C), 28.6 (2C), 26.5 (2C).
Dimethyl 2-(cyclooctylmethoxy)fumarate (11f)
1
Clear oil. 56 mg (5% yield). H NMR (500 MHz, CDCl
3
):
δ
6.12 (s, 1H), 3.85 (d,
J
= 6.6 Hz,
2
1
H), 3.82 (s, 3H), 3.73 (s, 3H), 1.97 – 1.91 (m, 1H), 1.74 – 1.65 (m, 4H), 1.62 – 1.46 (m, 8H),
13
3
.36 – 1.28 (m, 2H); C NMR (126 MHz, CDCl ): δ 164.9, 163.7, 154.8, 108.2, 79.6, 52.9,
51.7, 38.2, 29.1 (2C), 27.0 (2C), 26.5, 25.5 (2C).
Dimethyl 2-(thiophen-3-ylmethoxy)fumarate (11g)
1
Light yellow solid. 275 mg (27% yield). H NMR (500 MHz, CDCl
3
):
= 5.0, 1.3 Hz, 1H), 6.23 (s, 1H), 5.22 (s, 2H), 3.82
s, 3H), 3.74 (s, 3H); C NMR (126 MHz, CDCl ): 164.7, 163.4, 153.6, 137.2, 127.6, 126.3,
24.6, 110.4, 70.2, 53.0, 51.8.
δ 7.35 – 7.34 (m, 1H),
7
.30 (dd, J = 5.0, 3.0 Hz, 1H), 7.16 (dd, J
13
(
3
δ
1
Dimethyl 2-(thiophen-2-ylmethoxy)fumarate (11h)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Light yellow solid. 310 mg (38% yield). H NMR (500 MHz, CDCl
3
):
δ
7.33 (dd,
J
= 5.1, 1.2
Hz, 1H), 7.08 (d,
J
= 3.4 Hz, 1H), 6.98 (dd,
J
= 5.1, 3.5 Hz, 1H), 6.28 (s, 1H), 5.37 (s, 2H),
): 164.7, 163.4, 153.0, 138.1, 128.53,
13
3
1
.83 (s, 3H), 3.75 (s, 3H); C NMR (126 MHz, CDCl
3
δ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
27.4, 126.9, 111.5, 69.2, 53.0, 51.9.
Dimethyl 2-(furan-3-ylmethoxy)fumarate (11i)
1
Light yellow solid. 345 mg (36% yield). H NMR (500 MHz, CDCl
3
):
= 1.2 Hz, 1H), 6.24 (s, 1H), 5.10 (s, 2H), 3.82 (s,
): 164.7, 163.5, 153.5, 143.6, 141.8, 120.8,
δ 7.46 (dd, J = 1.7, 0.9
Hz, 1H), 7.39 (t,
J
= 1.7 Hz, 1H), 6.49 (d, J
13
3
H), 3.74 (s, 3H); C NMR (126 MHz, CDCl
3
δ
110.6 (2C), 66.6, 53.0, 51.8.
Dimethyl 2-(furan-2-ylmethoxy)fumarate (11j)
1
Light yellow solid. 382 mg (40% yield). H NMR (500 MHz, CDCl
3
):
= 3.3, 1.8 Hz, 1H), 6.29 (s, 1H), 5.17 (s, 2H),
.83 (s, 3H), 3.73 (s, 3H); C NMR (126 MHz, CDCl ): 164.6, 163.4, 153.0, 149.7, 143.7,
111.4, 111.3, 110.6, 66.7, 53.0, 51.7.
δ 7.42 (dd, J = 1.9, 0.9
Hz, 1H), 6.41 (d,
J = 3.3 Hz, 1H), 6.35 (dd, J
13
3
3
δ
Dimethyl 2-(prop-2-yn-1-yloxy)fumarate (11k)
1
Clear oil. 295 mg (37% yield). H NMR (500 MHz, CDCl
3
):
δ
6.35 (s, 1H), 4.90 (d,
J
= 2.5
):
13
Hz, 2H), 3.84 (s, 3H), 3.75 (s, 3H), 2.55 (t,
J
= 2.5 Hz, 1H); C NMR (126 MHz, CDCl
3
δ
1
64.5, 163.2, 152.0, 111.6, 82.9, 77.1, 60.4, 53.1, 52.0.
Synthesis of 2-substituted fumaric acid derivatives 12a-k.
To a stirred solution of dimethyl
2
ꢀsubstituted fumarate 11 (11a, 214 mg, 1.0 mmol; 11b, 150 mg, 0.66 mmol; 11c, 121 mg,
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2
3
4
5
6
7
8
9
0
.50 mmol; 11d, 115 mg, 0.45 mmol; 11e, 70 mg, 0.26 mmol; 11f, 56 mg, 0.20 mmol; 11g,
112 mg, 0.44 mmol; 11h, 87 mg, 0.34 mmol; 11i, 62 mg, 0.26 mmol; 11j, 70 mg, 0.30 mmol;
1k, 198 mg, 1.0 mmol; respectively) in EtOH (2 mL) was added 2 M NaOH (2 mL), and the
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reaction mixture was heated to reflux for 2 h. After completion of the hydrolysis, the reaction
mixture was allowed to cool to room temperature followed by removing the EtOH under
vacuum. For compounds 11a-f and 11k, the resulting aqueous layer was acidified with HCl (1
M) until white precipitates appeared (pH 1). The precipitates were filtered and dried under
vacuum to provide pure products 12a-f and 12k as white solids. For compounds 12g-j, the
resulting aqueous solutions were directly used for the next enzymatic step after adjusting the
pH of the solution to pH 9.5 with 1 M HCl.
2-(Cyclopropylmethoxy)fumaric acid (12a)
1
White solid. 150 mg (81% yield). H NMR (500 MHz, DMSOꢀ
d
6
):
δ
6.02 (s, 1H), 3.85 (d,
J
=
1
3
7.1 Hz, 2H), 1.13 – 1.07 (m, 1H), 0.51 – 0.47 (m, 2H), 0.25 – 0.22 (m, 2H); C NMR (126
MHz, DMSOꢀ ): 165.4, 164.2, 153.8, 109.8, 76.8, 10.6, 3.0 (2C).
d
6
δ
2-(Cyclobutylmethoxy)fumaric acid (12b)
1
White solid. 110 mg (84% yield). H NMR (500 MHz, Methanolꢀ
d
4
):
δ
6.07 (s, 1H), 4.02 (d,
J
13
=
5.6 Hz, 2H), 2.68 – 2.62 (m, 1H), 2.04 – 2.00 (m, 2H), 1.91 – 1.83 (m, 4H); C NMR (126
4
MHz, Methanolꢀd ): δ 167.9, 165.8, 156.5, 110.0, 78.2, 36.6, 25.5 (2C), 19.2.
2-(Cyclopentylmethoxy)fumaric acid (12c)
1
White solid. 77 mg (72% yield). H NMR (500 MHz, Methanolꢀd4):
δ 6.00 (s, 1H), 3.90 (d, J
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
=
7.0 Hz, 2H), 2.24 – 2.15 (m, 1H), 1.72 – 1.66 (m, 2H), 1.57 – 1.47 (m, 4H), 1.31 – 1.23 (m,
1
3
4
2H); C NMR (126 MHz, Methanolꢀd ): δ 168.0, 165.8, 156.7, 109.3, 78.4, 41.0, 30.1 (2C),
26.4 (2C).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-(Cyclohexylmethoxy)fumaric acid (12d)
1
White solid. 78 mg (76% yield). H NMR (500 MHz, DMSOꢀd6):
δ
5.93 (s, 1H), 3.81 (d,
J
=
1
3
6.4 Hz, 2H), 1.74 – 1.56 (m, 6H), 1.24 – 1.10 (m, 3H), 1.00 – 0.92 (m, 2H); C NMR (126
MHz, DMSOꢀ ): 165.8, 164.6, 154.8, 108.5, 78.2, 38.3, 29.3 (2C), 26.3, 25.6 (2C).
d
6
δ
2-(Cycloheptylmethoxy)fumaric acid (12e)
1
White solid. 42 mg (67% yield). H NMR (500 MHz, Methanolꢀ
d
4
):
δ
6.05 (s, 1H), 3.86 (d,
J
=
6.4 Hz, 2H), 1.88 – 1.78 (m, 3H), 1.70 – 1.65 (m, 2H), 1.60 – 1.43 (m, 6H), 1.29– 1.23 (m,
1
3
2H); C NMR (126 MHz, Methanolꢀ
4
d ): δ 168.0, 165.8, 156.5, 109.1, 79.6, 41.1, 31.7 (2C),
29.7 (2C), 27.5 (2C).
2-(Cyclooctylmethoxy)fumaric acid (12f)
1
White solid. 20 mg (39% yield). H NMR (500 MHz, Methanolꢀ
d
4
):
δ
6.03 (s, 1H), 3.82 (d,
J
=
6.7 Hz, 2H), 1.93 – 1.86 (m, 1H), 1.72 – 1.64 (m, 4H), 1.59 – 1.44 (m, 8H), 1.33 – 1.27 (m,
1
3
2H); C NMR (126 MHz, Methanolꢀ
4
d ): δ 168.0, 165.8, 156.6, 109.0, 80.1, 39.3, 30.2 (2C),
28.0 (2C), 27.7, 26.5 (2C).
Disodium 2-(thiophen-3-ylmethoxy)fumarate (12g)
1
6
H NMR (500 MHz, DMSOꢀd ): δ 7.53 – 7.50 (m, 2H), 7.15 (dd, J = 4.9, 1.3 Hz, 1H), 6.05 (s,
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2
3
4
5
6
7
8
9
13
1
H), 5.10 (s, 2H); C NMR (126 MHz, DMSOꢀ
6
d ): δ 165.3, 164.0, 153.5, 137.7, 127.6, 126.6,
124.2, 110.2, 69.1.
Disodium 2-(thiophen-2-ylmethoxy)fumarate (12h)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
H NMR (500 MHz, D O): δ 7.42 (dd, J = 5.0, 1.3 Hz, 1H), 7.08 – 7.05 (m, 1H), 7.00 (dd, J =
1
3
5
.1, 3.5 Hz, 1H), 5.91 (s, 1H), 5.14 (s, 2H); C NMR (126 MHz, D O):
δ
173.9, 170.9, 152.4,
2
1
38.7, 128.6, 127.5, 126.9, 114.4, 66.6.
Disodium 2-(furan-3-ylmethoxy)fumarate (12i)
1
H NMR (500 MHz, D
2
O):
δ
7.50 (dd,
J
= 1.6, 0.8 Hz, 1H), 7.44 (t,
J
= 1.9 Hz, 1H), 6.49 (s,
13
1
H), 5.89 (s, 1H), 4.85 (s, 2H); C NMR (126 MHz, D
2
O): δ 174.0, 171.0, 152.7, 143.6,
1
42.0, 120.8, 113.8, 110.6, 63.8.
Disodium 2-(furan-2-ylmethoxy)fumarate (12j)
1
H NMR (500 MHz, D
.36 (dd,
170.8, 152.2, 150.0, 143.8, 114.6, 111.2, 110.5, 64.2.
2
O):
δ
7.45 (dd,
J
= 1.9, 0.8 Hz, 1H), 6.38 (dd,
J
= 3.3, 0.8 Hz, 1H),
O): 173.8,
13
6
J
= 3.3, 1.9 Hz, 1H), 5.91 (s, 1H), 4.89 (s, 2H); C NMR (126 MHz, D
2
δ
2
-(Prop-2-yn-1-yloxy)fumaric acid (12k)
1
White solid. 142 mg (83% yield). H NMR (500 MHz, DMSOꢀ
d
6
):
δ
6.14 (s, 1H), 4.82 (d,
J =
13
2
.5 Hz, 2H), 3.63 (t,
J
= 2.5 Hz, 1H); C NMR (126 MHz, DMSOꢀ
d
6
): δ 165.1, 163.7, 151.8,
1
11.7, 79.4, 78.4, 59.1.
Enzymatic synthesis of 3-substituted aspartic acid derivatives 13a-k. Reaction mixtures
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
consisted of 2ꢀsubstituted fumaric acid derivatives 12 (12a, 21 mg, 0.11 mmol; 12b, 40 mg,
0.20 mmol; 12c, 42 mg, 0.20 mmol; 12d, 28 mg, 0.12 mmol; 12e, 23 mg, 0.095 mmol; 12f,
1
2 mg, 0.047 mmol; 12g, 0.44 mmol; 12h, 0.34 mmol; 12i, 0.26 mmol; 12j, 0.30 mmol; 12k,
.7 mg, 0.056 mmol; respectively) in buffer (5 M NH /NH Cl and 20 mM MgCl , pH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
3
4
2
adjusted to pH 9.5). The reaction was started by addition of freshly purified MALꢀL384A
21,22
mutant (0.01 mol%),
and the reaction mixture was incubated at room temperature for 24 h.
After completion of the reaction, the enzyme was inactivated by heating to 70 °C for 10 min.
1
The progress and conversion yield of the enzymatic reaction was monitored by H NMR
spectroscopy by comparing signals of substrates and corresponding products. For a typical
purification procedure, the precipitated enzyme was removed by filtration. The filtrate was
acidified with 1 M HCl to pH 1 and loaded onto a column packed with cationꢀexchange resin
(
10 g of Dowex 50W X8, 50ꢀ100 mesh), which was preꢀtreated with 2 M aqueous ammonia
4 column volumes), 1 M HCl (2 column volumes) and distilled water (4 column volumes).
(
The column was washed with distilled water (2 column volumes) and the desired product was
eluted with 2 M aqueous ammonia (4 column volumes). The ninhydrinꢀpositive fractions were
collected and lyophilized to yield the product as ammonium salt.
(L-threo)-3-(Cyclopropylmethoxy)aspartate (13a)
1
White solid. 7 mg (conversion 88%, isolated yield 31%). H NMR (500 MHz, D
2
O):
δ
J
4.38 (d,
= 10.7,
J
= 2.5 Hz, 1H), 3.98 (d, J = 2.5 Hz, 1H), 3.45 (dd, J = 10.7, 7.0 Hz, 1H), 3.28 (dd,
1
3
7
.2 Hz, 1H), 1.05 – 0.97 (m, 1H), 0.53 – 0.46 (m, 2H), 0.20 – 0.15 (m, 2H); C NMR (126
MHz, D
2
O): 176.1, 171.6, 77.4, 75.7, 56.6, 9.5, 2.6, 2.1. HRMS: calcd. for C H14NO
δ
8 5
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2
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5
6
7
8
9
+
[
M+H] : 204.0866, found: 204.0861. HPLC: purity 96%, retention time 4.2 min. Comparison
1
1
of the H NMR data of 13a with the H NMR data of chemically prepared racemic
threo)ꢀ19a and (DL erythro)ꢀ19a showed that the de of product 13a was 97% (Figure S1).
(DL
ꢀ
ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chiral HPLC analysis: CROWNPAK CRꢀI (+) 150 x 3 mm. Phase A: ACN+0.5% TFA, phase
B: H O+0.5% TFA, A/B = 98:2. Flow rate 0.4 mL/min, column temperature 25 °C, detected
2
R
by ELSD at 35 °C, t = 2.8 min, ee >99% (Figure S62).
3
-(Cyclobutylmethoxy)aspartate (13b)
1
White solid. 25 mg (conversion 89%, isolated yield 58%). H NMR (500 MHz, D
(d, = 2.4 Hz, 1H), 3.95 (d, = 2.4 Hz, 1H), 3.60 (dd, = 9.8, 6.8 Hz, 1H), 3.39 (dd,
.3 Hz, 1H), 2.56 – 2.47 (m, 1H), 2.01 – 1.95 (m, 2H), 1.86 – 1.77 (m, 2H), 1.68 – 1.61 (m,
2
O):
δ
4.24
J
J
J
J
= 9.8,
7
2
1
3
H); C NMR (126 MHz, D
2
O):
δ
176.4, 171.7, 78.3, 75.6, 56.5, 34.1, 24.5, 24.2, 18.0.
+
HRMS: calcd. for C
9
H
16NO
5
[M+H] : 218.1023, found: 218.1022. HPLC: purity 98%,
retention time 6.5 min.
3
-(Cyclopentylmethoxy)aspartate (13c)
1
White solid. 19 mg (conversion 85%, isolated yield 39%). H NMR (500 MHz, D O):
δ
4.28
= 9.4,
.7 Hz, 1H), 2.17 – 2.11 (m, 1H), 1.70 – 1.63 (m, 2H), 1.56 – 1.47 (m, 4H), 1.22 – 1.13 (m,
2
(d, J = 2.5 Hz, 1H), 3.98 (d, J = 2.5 Hz, 1H), 3.54 (dd, J = 9.4, 7.0 Hz, 1H), 3.25 (dd, J
7
2
13
H); C NMR (126 MHz, D
2
O):
δ
176.2, 171.5, 78.2, 75.7, 56.4, 38.5, 28.9, 28.7, 24.9 (2C).
+
HRMS: calcd. for C10
H
18NO
5
[M+H] : 232.1180, found: 232.1180. HPLC: purity 97%,
retention time 9.3 min.
29
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