β-Sulfonamido Functionalized Aspartate Analogues as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity Profiles Arising from Subtle Structural Differences

Article

pubs.acs.org/jmc

β‑Sulfonamido Functionalized Aspartate Analogues as Excitatory

Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity

Proﬁles Arising from Subtle Structural Diﬀerences

Jacob C. Hansen, Walden E. Bjørn-Yoshimoto, Niels Bisballe, Birgitte Nielsen, Anders A. Jensen,

and Lennart Bunch*

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,

Universitetsparken 2, Copenhagen Ø 2100, Denmark

S

* Supporting Information

ABSTRACT: In this study inspired by previous work on 3-

substituted Asp analogues, we designed and synthesized a total

of 32 β-sulfonamide Asp analogues and characterized their

pharmacological properties at the excitatory amino acid

transporter subtypes EAAT1, EAAT2, and EAAT3. In addition

to several potent EAAT inhibitors displaying IC₅₀values ∼1

μM at all three subtypes, this elaborate structure−activity

relationship also identiﬁed analogues exhibiting distinct

preferences or selectivities for speciﬁc transporter subtypes.

Introduction of two ﬂuorine atoms on the phenyl ring yielded analogue 4y that displayed an IC₅₀of 0.8 μM at EAAT1 with a 14-

and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the m-CF₃-phenyl analogue 4r was a potent selective

EAAT2-inhibitor (IC₅₀= 2.8 μM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion,

even small structural diﬀerences in these β-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity

proﬁles.

and cellular distribution of the ﬁve EAAT subtypes in the CNS

is likely to be important for their regulation of glutamatergic

neurotransmission, just as selective modulation of the Glu

uptake through diﬀerent transporter subtypes may have

diﬀerent impact on glutamatergic neurotransmission, both

when it comes to the characteristics and levels of the induced

eﬀects but also in terms of the CNS regions aﬀected.

The potential in EAATs as therapeutic targets has so far not

been explored to the extent as is the case in the Glu receptor

ﬁelds,¹¹and the limited selection of potent and truly subtype-

selective EAAT modulators identiﬁed to date constitutes an

obstacle to the delineation of the physiological roles and the

therapeutic potential of each of the ﬁve transporters. Medicinal

chemistry development in the EAAT ﬁeld has mainly been

based on the endogenous transporter substrates Glu and (S/R)-

aspartate (Asp).⁶Several nonselective substrates and com-

petitive nonsubstrate inhibitors of the transporters have

emerged from these eﬀorts, ^DL-threo-β-benxyloxyaspartate

(TBOA)^12,13being the prototypic nonselective EAAT inhibitor.

Interestingly, EAAT2-selectivity has emerged from quite

diﬀerent modiﬁcations to the Glu and Asp structures, as

EAAT2-selective inhibitors include the conformationally

restricted Glu analogue dihydrokainic acid (DHK),^14,15the 4-

substituted Glu analogue (2S,4R)-2-amino-4-(3-(2,2-dipheny-

INTRODUCTION

■

(S)-Glutamate (Glu, Figure 1A) is the major excitatory

neurotransmitter in the central nervous system (CNS), where

it controls and regulates an immense number of diﬀerent

processes. Because of this abundance and the importance of

glutamatergic neurotransmission for virtually all central

functions, hyperactivity or hypoactivity in these systems are

believed to be at the core of numerous cognitive, psychiatric,

neurotoxic, and neurodegenerative disorders. Thus, the

considerable therapeutic interest in glutamatergic targets is

rooted in the possibility that these disorders as well as others

not based in glutamatergic dysfunction potentially could be

treated through this neurotransmitter system.¹⁻⁴

The synaptic uptake of Glu following its release into the

synaptic cleft is mediated by the ﬁve members of the excitatory

amino acid transporter (EAAT) family, EAAT1−5.⁵⁻¹⁰These

high-aﬃnity Glu transporters are expressed in the plasma

membranes of glia cells (predominantly EAAT1,2) and neurons

(predominantly EAAT3−5). EAAT2 is the major EAAT

subtype estimated to be responsible for >90% of total Glu

uptake in the brain, with EAAT1 and EAAT3 also being

abundantly distributed throughout the CNS. In contrast,

EAAT4 and EAAT5 are almost exclusively expressed in

cerebellar Purkinje cells and in the retina, respectively, and

these subtypes also exhibit distinct transporter/channel proper-

ties compared to the other three EAATs and thus are believed

to act predominantly as Cl⁻channels.^5,6,8The distinct regional

Received: July 18, 2016

A

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Chemical structures of: (A) Endogenous EAAT substrates Glu and Asp. (B) Competitive EAAT inhibitors TBOA,¹²18,²⁵14,¹⁷DHK,^14,15

and 13.¹⁶(C) Noncompetitive EAAT1 inhibitors 15,²¹16,²²and 17.²⁴

Figure 2. Chemical structures of rationally designed β-functionalized Asp analogues 1, 2, 3-A/B, and 4a.

lethylamino)-3-oxopropyl)pentanedioic acid (13),¹⁶and the

functionalized asparagine analogue N-[4-(2-bromo-4,5-

diﬂuorophenoxy)phenyl]-^L-asparagine (14, WAY-213613)¹⁷

(Figure 1B). Although 4-methyl-Glu is a substrate of EAAT1

and a nonsubstrate inhibitor of EAAT2,3 and thus exhibits

functional EAAT1-selectivity,^18,19the only truly selective

EAAT1 inhibitors reported to date are the noncompetitive

inhibitors 2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-

5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (15,

UCPH-101),^20,212-amino-4-(4-methyl)-7-(naphthalen-1-yl)-5-

oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (16,

UCPH-102),^22,23and most recently (Z)-4-chloro-3-(5-((3-(2-

ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)methyl)-

furan-2-yl)benzoic acid (17)²⁴(Figure 1C). Truly subtype-

selective modulators of EAAT3−5 have yet to be discovered,

but interestingly the β-substituted Asp analogue ^L-threo-β-

benzylaspartate (18, ^L-β-BA)²⁵has been reported to display a

10-fold preference as an inhibitor of EAAT3 over EAAT1,2.

Over the past decade, we have applied various approaches in

our eﬀorts to discover subtype-selective EAAT li-

gands.^{6,16,19,21,26}In the present study, we were inspired by

the previously reported work on 3-substituted Asp analogues as

EAAT inhibitors, where the initial discovery of the nonselective

EAAT substrate ^L-threo-β-hydroxyaspartic acid^15,27has led to

subsequent development of the nonselective EAAT inhibitor

TBOA^12,13and the more potent and EAAT1,2-preferring

inhibitor TFB-TBOA^28,29as well as to the EAAT3-preferring

inhibitor 18.²⁵To explore the potential in the 3-substituted Asp

analogue as a scaﬀold for EAAT ligands further, we have

designed and synthesized a series of β-amino-Asp analogues

and characterized their pharmacological properties at EAAT1−

3. On the basis of this elaborate structure−activity relationship

(SAR) study, we report that even small structural diﬀerences in

the 3-substituents of these analogues give rise to dramatically

diﬀerent functional proﬁles at EAAT1−3.

RESULTS AND DISCUSSION

■

In view of the stereochemical match (threo)³⁰and structural

similarity of Asp analogues TBOA and 18, we hypothesized

that the functionality in the β-position of Asp plays a decisive

role for the observed EAAT-selectivity proﬁle and that this

diﬀerence in pharmacology is likely to arise from diﬀerential

spatial orientation of the aryl group in the substrate binding

pocket of the EAAT. We therefore designed new series of β-

amino-Asp analogues 1−4a (Figure 2) to explore the

orientation of the aryl group as well as its distance from the

Asp scaﬀold on inhibitory activity.

Synthesis of 1, 2, 3-A/B, and 4a. The apparently

important orientation of the phenyl ring in the Asp analogues

18 and TBOA urged us to reduce the degree of rotational

freedom between the Asp backbone and the aromatic ring. To

install this structural restriction, we opted for the incorporation

B

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 1. Synthesis of β-Functionalized Asp Analogues 1, 2, 3-A/B, and 4a

a

Reagents and conditions: (a) MeOH, H₂SO₄, reﬂux, quant; (b) TrCl, TEA, DCM, 75% (2 steps); (c) 6, LHMDS, THF, 30 min, −30 °C, then NBS

100 min, −30 to −5 °C; (d) NaN₃, DMF, 56% (2 steps); (e) PPh₃, THF, 79%; (f) phenylacetylene, sodium ascorbate, CuSO₄·5H₂O, H₂O/t-BuOH;

(g) PhCOCl, TEA, DCM; (h) PhOCOCl, TEA, DCM; (i) PhSO₂Cl, TEA, DCM; (j) MeI, K₂CO₃, DMF; (k) 6 M HCl, reﬂux, 1 h, preparative

HPLC.

of an sp²hybridized nitrogen atom in the β-position of Asp.

Furthermore, insertion of an amino group would provide a

versatile scaﬀold, allowing for the synthesis of a variety of

derivatives. We therefore nominated β-azido Asp 8 to be a key

intermediate in the synthesis of the new β-functionalized Asp

analogues.

Because of the apparent lack of reactivity of the azidation

reagents toward the enolate, the direct azidation strategy was

abandoned. Instead, we turned to the more reactive

bromination reagent that could aﬀord the desired 3-bromo

Asp 7, which could then undergo nucleophilic substitution with

−

the sterically less demanding N₃. Reaction of NBS with the

It has been reported, that diﬀerentiation of the Re and Si face

of the in situ formed enolate of N-trityl protected Asp ensures

diastereoselective insertion of an electrophile at the 3-postion

of Asp.³¹A key feature in this stereoselectivity is countercation

chelation and temperature. Using LHMDS, the diastereomeric

ratio of 1:11 (S,S:S,R) is obtained, whereas for KHMDS, a 99:1

(S,S:S,R) ratio is achieved. Because the threo-diastereomer

(TBOA) is the pharmacologically active diastereomer of this

class, we planned a direct insertion of nitrogen functionality at

the β-position using KHMDS. Conversely, indirect insertion of

nitrogen could be pursued, i.e., suitable S_N2 substitution

reaction on the erythro intermediate prepared with the use of

lithium hexamethyldisilazide (LHMDS).

Initial attempts to functionalize the β-position of Asp were

aimed at azidation of dimethyl (2S)-N-(triphenylmethyl)-

aspartate 6 using potassium hexamethyldisilazide (KHMDS)

(2.1 equiv) and trisyl-N₃(2.5 equiv) (Scheme 1). Applying low

temperature chelation (−78 °C) failed to produce any

conversion. Likewise, elevation of the temperature after

addition of electrophilic azidation reagent to 7 °C did not

oﬀer any of the desired azide 8. Increasing the amount of added

electrophile and base, for both KHMDS and LHMDS, similarly

failed. Conﬁdent that the enolate was successfully formed (by

quenching with D₂O), we attempted the reaction with less

sterically hindered tosyl-N₃, however, this was also unsuccess-

ful.

lithium enolate of 6 (generated with LHMDS) was unreactive

at −78 °C but gave full conversion with dr 1:0.3 when warming

to rt. Repeating these conditions with Br₂as the electrophile

produced a complex mixture of products, even at −78 °C. We

therefore examined the temperature range of reactivity for NBS

and found it to be −48 °C to −30 °C under these reaction

conditions. Realizing that we could not obtain full diaster-

eoselective control in this reaction step, we decided to aim for a

1:1 mixture of diastereomers. Gratifyingly, full conversion was

achieved at −30 to −5 °C to give bromide 7 in diastereomeric

ratio 2:3, which was used without further puriﬁcation.

Conversion of crude bromide 7 to azide 8 proceeded smoothly

with NaN₃in anhyd DMF in quantitative yield. To access

triazole 1, azide 8 cleanly underwent copper catalyzed Huisgen

cycloaddition, followed by hydrolysis and puriﬁcation by

preparative HPLC to give target compound 1 in 14% yield

with dr 0.5:1. Amide 2, carbamates 3A-A/B, and sulfonamide

4a (Figure 2) were synthesized by ﬁrst reducing azide 8 to

amine 9 under Staudinger conditions followed by reaction with

benzoyl chloride, phenyl chloroformate, and phenylsulfonyl

chloride, respectively (Scheme 1).

Pharmacological Characterization of 1, 2, 3-A/B, and

4a at EAAT1−3. The pharmacological properties of the four

Asp analogues 1, 2, 3-A/B, and 4a were characterized at stable

EAAT1-, EAAT2-, and EAAT3-HEK293 cells in a conventional

[³H]-D-Asp uptake assay,¹⁴and the results are given in Table 1.

C

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 1. Pharmacological Properties of Previously Reported Asp Analogues and β-Sulfonamide Asp Analogues at Stable

EAAT1-, EAAT2-, and EAAT3-HEK293 Cell Lines in the [³H]-D-Asp Uptake Assay (IC₅₀Values are Given in μM with pIC₅₀

SEM in Brackets (n = 3−5))

a

Because the concentration−inhibition curves for the compound were not completed within the concentration range tested, the IC₅₀value was

estimated based on the ﬁtted curve.

While the 4′-phenyl triazole analogue 1 was inactive as inhibitor

at EAAT1−3, the amide 2 displayed weak inhibitory activity at

EAAT2 (IC₅₀∼ 70 μM) while being inactive at EAAT1,3 at

concentrations up to 300 μM. As for the two diastereomeric

carbamate analogues, 3-A displayed negligible inhibitory activity

at EAAT1−3, whereas the other diastereomer 3-B showed

weak inhibitory activity at EAAT1 (IC₅₀∼ 70 μM) and even

weaker activity at EAAT2,3. Interestingly, the sulfonamide

analogue 4a displayed signiﬁcant inhibitory activity at the three

transporter subtypes, with IC₅₀values ranging from 2.0 to 6.8

μM.

Stereochemistry of 4a. To address stereochemical

reference to 18 and TBOA, the diastereomeric mixture 4a

was separated by preparative HPLC to aﬀord the single

diastereomers 4a-A and 4a-B. When characterized functionally

at EAAT1−3 in the [³H]-D-Asp uptake assay, distinct

pharmacological proﬁles were disclosed for the respective

diastereomers. Diastereomer 4a-A displayed negligible inhib-

itory activity at EAAT1−3, whereas 4a-B exhibited inhibitory

potencies in the high nanomolar/low micromolar range across

the three subtypes (IC₅₀values of 0.80−2.4 μM, Table 2 and

Figure 3). To assign the absolute stereochemistry at the β-

position of diastereomers 4a-A and 4a-B, they were function-

alized with PhSO₂Cl at the α-amino group to form the

corresponding disulfonamido derivatives (Scheme 2). Hereby,

only the erythro-diastereomer would acquire a plane of

symmetry (meso compound) and thus not be optically active.

The disulfonamido derivative of the pharmacologically inactive

diastereomer 4a-A was without optical rotatory power and

hence assigned the erythro conﬁguration, while the disulfona-

mido derivative of the pharmacologically active diastereomer

4a-B was optically active and hence unambiguously assigned

the ^L-threo isomer (Scheme 2).

Design, Synthesis, and Pharmacological Character-

ization of Analogues of 4a. Given the interesting

pharmacological properties of the sulfonamide analogue 4a-B,

this series was expanded to investigate the inﬂuence of

substitution on the aryl ring (Table 2). The synthesis of all

28 new analogues followed the strategy outline in Scheme 1 for

the synthesis of 4a, and the pharmacological characterization

was performed at the EAAT1-, EAAT2-, and EAAT3-HEK293

cell lines in the [³H]-D-Asp uptake assay.¹⁴

In comparison with lead structure 4a, the 1-napthyl (4b) and

the 2-naphtyl (4c) analogues both displayed reduced inhibitory

activities at EAAT1−3 (IC₅₀values of 6.5−65 μM).

Interestingly though, expansion of the aromatic system in 1-

naphthyl Asp derivative 4b induced a 5-fold preference for

EAAT2 over EAAT1,3. Increasing the distance between the

D

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 3. Concentration−inhibition curves for TBOA and β-sulfonamide Asp analogues 4a-B, 4d-B, 4r, and 4ac at stable EAAT1-, EAAT2-, and

EAAT3-HEK293 cell lines in the [³H]-D-Asp uptake assay. Data are given as mean SEM (n = 3−5).

a

Scheme 2. Assignment of Absolute Conﬁgurations of the Diastereomers Phenylsulfonamides 4a-A and 4a-B

a

(a) PhSO₂Cl, NaOH, DIPEA, acetone

E

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 2. Pharmacological Properties of β-sulfonamide Asp Analogues at Stable EAAT1-, EAAT2-, and EAAT3-HEK293 Cell

Lines in the [³H]-D-Asp Uptake Assay

F

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 2. continued

G

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 2. continued

a

Because the concentration−inhibition curves for the compound were not completed within the concentration range tested, the IC₅₀value was

estimated based on the ﬁtted curve.

Asp backbone and the aromatic group in the benzyl derivative

4aa also failed to increase the inhibitory potency (IC₅₀values of

49−76 μM), and as with the lead 4a, no subtype selectivity was

observed for this analogue.

the substrate binding pocket in the EAAT. Hence, the

pentaﬂuorophenyl derivative 4d was synthesized.³²As for

lead structure 4a, the diastereomers of 4d were easily separated

into diastereomer 4d-A and diastereomer 4d-B. In agreement

with lead structure 4a, the inhibitory activity resided only in the

single diastereomer 4d-B, which was shown to be an EAAT1-

preferring inhibitor, exhibiting an IC₅₀value of 16 μM at

EAAT1 and 6-fold and 10-fold lower inhibitory potencies at

EAAT2 and EAAT3, respectively (Table 2 and Figure 3).

Considering the diﬀerences in inhibitory potencies across

EAAT1−3, the putative π−cation is either manifested diﬀer-

ently between EAAT subtypes, or alternatively the diﬀerence in

the pharmacological proﬁles of 4d-B and 4a-B is a direct eﬀect

of the ﬂuorine substitution.

To gain further insight into the impact that pentaﬂuorination

of the phenyl ring had on EAAT potency and subtype

selectivity, the mono-, di-, and triﬂuoro analogues 4e, 4y, and

4z were synthesized. Analogously to pentaﬂuorinated analogue

4d-B, the 2,4,5-triﬂuorinated analogue 4z displayed preference

for EAAT1 over EAAT2 and EAAT3 (9- and 4-fold,

respectively), and moreover it exhibited a 6-fold increase in

inhibitory potency for EAAT1 compared to 4d-B. Interestingly,

the 3,4-diﬂuorinated analogue 4y again incrementally increased

inhibitory potency toward EAAT1 by 3-fold (IC₅₀0.80 μM),

and furthermore this analogue exhibited an even more

pronounced preference for EAAT1 over EAAT2 (14-fold)

and EAAT3 (9-fold) (Table 2). The 4-ﬂuoro analogue 4e

displayed similar inhibitory potency at EAAT1 (IC₅₀1.6 μM) as

Extending the study of the aromatic group to include

heteroaromatic analogues 4ab and 4ac gave further insight into

the biologically active space for this ligand series. The two

analogues were essentially nonselective inhibitors of EAAT1−3,

with the 2-thiophene analogue 4ac being roughly equipotent

with 4a-B at the transporters. Interestingly, however, 4ac

displayed a slightly lower (∼3-fold) IC₅₀value at EAAT3 than

at EAAT1,2 (Table 2 and Figure 3). Although this diﬀerence is

too small to classify the analogue as an EAAT3-preferring

inhibitor, the fairly high inhibitory potency and EAAT1−3

proﬁle of 4ac suggests that it could be a potential lead structure

for the future development of EAAT3-preferring or -selective

inhibitors.

The synthesis and pharmacological characterization of the N-

methylated analogue 4ad again demonstrated that subtle

chemical changes to a ligand may completely eliminate

pharmacological activity. The analogue 4ad was without

inhibitory activity at all three EAATs (IC₅₀values >300 μM).

Rotational restriction of the phenyl ring by introduction of two

ortho-chloro atoms, compound 4x, also decreased the inhibitory

EAAT potency signiﬁcantly (IC₅₀values of 50−300 μM) in

comparison to the lead structure 4a.

We were intrigued to investigate the possibility of a π−cation

interaction between the phenyl ring in the lead structure 4a and

H

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 4. (A) Superimposition of binding mode of TBOA (black) when crystallized in Glt_Ph(PDB code: 2nww) with the two most favorable binding

modes of 4a-B (magenta and green) obtained by docking into a homology model of EAAT1 built from the Glt_Ph/TBOA crystal structure.³⁶(B)

Binding of magenta 4a-B in EAAT1. (C) Binding of green 4a-B in EAAT1.

4y, but in contrast to 4y it was essentially nonselective at the

three EAAT subtypes (IC₅₀values of 1.6−4.7 μM). Thus, the

di-, tri-, and pentaﬂuoro derivatives 4y, 4z, and 4d-B all

displayed similar degrees of selectivity for EAAT1 over

EAAT2,3, whereas reducing the number of ﬂuorine atoms

increased the inhibitory potency of the analogues at all three

transporter subtypes. However, because the EAAT1-preference

of the 3,4-diﬂuoro analogue 4y was eliminated in the 4-ﬂuoro

analogue 4e, we concluded that a ﬂuoro atom in the 3-position

could be essential for this preference. The study of the four

ﬂuorinated analogues did not disclose clear evidence for a π−

cation interaction, as no correlation of reduced potency with

reducing number of ﬂuoro atoms was observed.³³

To explore in greater details the eﬀects of substituents on the

aromatic ring, we then synthesized a number of ortho-, meta-,

and para-substituted analogues (Table 2). The ortho-Cl

analogue 4t and ortho-Me analogue 4u showed similar

pharmacological characteristics as the lead 4a, with low

micromolar inhibitory potencies at all three transporters.

The para-position was ﬁrst probed with the para-CF₃

analogue 4l, which interestingly displayed a signiﬁcant loss of

EAAT activity (IC₅₀values of 30−100 μM). In view of the high

inhibitory potencies exhibited by para-ﬂuoro analogue 4e (IC₅₀

values of 1.6−4.7 μM) and para-Cl analogue 4f (IC₅₀values of

0.32−3.1 μM), we synthesized the para-bromo analogue 4g.

This analogue, however, displayed a similar pharmacological

proﬁle (IC₅₀values of 0.35−5.0 μM) to these smaller halide

analogues. It was unclear why the para-CF₃analogue 4l showed

weak inhibitory EAAT potency despite having an electro-

negativity between those of Cl and Br.³⁴Thus, we synthesized

derivatives 4o, 4m, and 4j to characterize the eﬀects of

resonance electron withdrawing. The bulky sulfone analogue 4j

was essentially inactive at EAAT1−3, indicating a possible

spatial restriction in the para-position. The conjugated ketone

analogue 4o displayed weak, nonselective EAAT inhibition

(IC₅₀values of 43−100 μM), while the para-NO₂derivative 4m

exhibited moderate inhibitory activity at EAAT1 (IC₅₀13 μM)

and a small preference for this subtype over EAAT2,3 (Table

2). Thus, inhibitory potency seems not exclusively to be a result

of electronic eﬀects, and other factors may play a deﬁning role

as well. This lack of correlation is further exempliﬁed when

examining electron donating derivatives 4k and 4n. While both

analogues display low micromolar inhibitory potencies at

EAAT1 (IC₅₀values of 4.0 and 2.3 μM, respectively), the

aniline analogue 4n exhibits equipotent inhibition at EAAT2

and EAAT3, whereas the anisole analogue 4k loses inhibitory

potency at these subtypes (IC₅₀values of ∼30 μM). Finally, in

an attempt to deﬁne the spatial limitations of the para-

substituent, analogues 4h, 4i, and 4p were synthesized. Here,

the steric bulk of the para-tert-butyl substituent in 4i almost

completely eliminated EAAT activity, analogously to what was

observed with the para-sulfone analogue 4j. As with the

sterically similar halides, the para-Me analogue 4h displayed

potent, albeit nonselective, EAAT inhibition. Highly interest-

ingly, the sterically demanding para-biphenyl analogue 4p

displayed high inhibitory potency at EAAT1 and EAAT2 (IC₅₀

values of 2.3 and 0.50 μM, respectively) and 10−40-fold lower

potency at EAAT3. This selectivity proﬁle is somewhat similar

to that exhibited by the 2-naphthyl analogue 4c (Table 2), and

it demonstrates that the EAAT protein is ﬂexible when stressed

by a para-substituent substantial in size.

In regard to the meta-position, the meta-CF₃substituted

analogue 4r displayed a highly interesting selectivity proﬁle at

EAAT1−3, being a potent inhibitor of EAAT2 (IC₅₀2.8 μM)

with 30- and 50-fold selectivity toward EAAT1 (IC₅₀∼ 100

μM) and EAAT3 (IC₅₀∼ 150 μM), respectively (Table 2 and

Figure 3). Interesting, the 3,5-dimethyl analogue 4v exhibited

5−9-fold preference for EAAT2 over EAAT1,3, and thus the

selectivity proﬁles of these two analogues strongly suggested

that the meta-position in the phenyl ring is a hot spot for

EAAT2-selectivity. Supplementing these ﬁndings was the

complete loss of EAAT activity observed for the meta-COOH

analogue 4s and the fact that the meta-Cl analogue 4q was a

potent EAAT1,2-preferring inhibitor. Hence, the identity of the

substituent introduced in the meta-position of the phenyl ring

in the β-sulfonamide Asp analogue is clearly of key importance

for its functional properties, both when it comes to its basic

EAAT activity but also in terms of whether it exhibits EAAT2-

selectivity or not.

Pharmacological Characterization of Selected β-

Sulfonamide Asp Analogues at Ionotropic Glutamate

Receptors. The β-sulfonamide Asp analogues 4a-A, 4a-B, 4b,

4c, 4e, 4h, 4v, 4r, 4f, 4q, and 4t were characterized in

radioligand binding assays to native ionotropic glutamate

receptors (rat synaptosomes).³⁵None of the 11 analogues

exhibited signiﬁcant binding aﬃnity to AMPA receptors

([³H]AMPA displacement), kainic acid receptors ([³H]kainic

acid displacement), or NMDA receptors ([³H]-(R,Z)-2-amino-

I

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

4-(phosphonomethyl)hept-3-enoic acid ([³H]CGP39653) dis-

placement) when tested at concentrations up to 100 μM.

Modeling. Homology models of EAAT1−3 were built from

the crystal structure of Glt_Phcrystallized with TBOA (PDB

code: 2nww)³⁶based on an alignment of the amino acid

sequences of the three human transporters and that of Glt_Ph

(Supporting Information). Subsequently, a docking study was

performed of the selected ligands 4a-B (nonselective), 4d-B

(EAAT1-preferring), 4r (EAAT2-selective), 4p (EAAT1,2-

preferring), 4ac (nonselective), and 4ad (inactive). Unfortu-

nately, this in silico study did not shed further light on the

observed diﬀerences in pharmacological proﬁles of the

analogues with respect to amino acid residues diﬀering between

the substrate binding pocket in EAAT1−3. However, two

energetically similar binding modes of 4a-B were disclosed

(Figure 4). The one binding mode of 4a-B (Figure 4A,B,

magenta) very much resembles the binding mode of TBOA in

the Glt_Phcrystal structure, showing occupancy of a large area in

space. The other binding mode of 4a-B (Figure 4A,C, green)

orients the phenyl ring into a diﬀerent cavity of the substrate

binding pocket. Future SAR studies of the β-sulfonamide Asp

analogue scaﬀold may unequivocally conclude which of the two

binding modes is the biologically active one or whether both

binding modes are possible with preference depending on the

speciﬁc analogue.

Moreover, considering the relatively limited SAR work focused

on the meta-position in the phenyl ring of the β-sulfonamide

Asp analogue performed in the present study, it seems plausible

that future medicinal chemistry development based on 4r as

lead could provide analogues with further increased inhibitory

potencies and/or degrees of EAAT2-selectivity.

This SAR study also elucidates the existence of an adjacent

vestibule to the substrate binding site in the EAAT that holds

considerable promise for future development of transporter

ligands. Collectively, the EAAT1-preferences and EAAT2-

selectivities exhibited by analogues in this series, the EAAT3-

preference reported for 18,²⁵and the completely nonselective

EAAT inhibition mediated by TBOA^14,15and other analogues

from this series illustrate the apparently immense potential for

functional diversity in Asp analogues with 3-substituents

projecting into this adjacent vestibule. Although we were

unable to rationalize the molecular basis for the diﬀerent

selectivity proﬁles displayed by the β-sulfonamide Asp

analogues in our in silico study, we propose that Asp analogues

comprising 3-substituents with even slightly diﬀerent spatial

orientations could exhibit very diﬀerent selectivity proﬁles at

the EAATs, and future studies applying this strategy could thus

perhaps help to supplement the presently limited selection of

pharmacological tools in the ﬁeld.

EXPERIMENTAL SECTION

■

CONCLUSION

Chemistry. All reactions involving dry solvents or sensitive agents

were performed under an argon atmosphere, and glassware was ﬂame-

dried under vacuum prior to use. Commercially available chemicals

were used without further puriﬁcation with the exception of N-

bromosuccinimide (NBS), which was recrystallized from water and

lyophilized to produce a white ﬂaky solid. DCM, THF, and DMF were

dried using a SG WATER solvent puriﬁcation system (commercialized

by Pure Process Technology). MeOH was dried by standing over 4 Å

molecular sieves for a minimum of 48 h. Triethylamine (TEA) was

puriﬁed by fractional distillation, followed by standing over 4 Å

molecular sieves. Reactions were monitored by analytical thin-layer

chromatography (TLC, Merck silica gel 60 F₂₅₄aluminum sheets) or

by HPLC.

Flash chromatography was carried out using Merck silica gel 60A

(40−63 μm). For dry column vacuum chromatography (DCVC),

Merck silica gel 60 (15−40 μm) and a standard setup was used.

¹H NMR spectra were recorded at 400 or 600 MHz and ¹³C NMR

spectra at 100 or 150 MHz on a Bruker Avance III or Bruker Avance

III HD, respectively. Chemical shifts (δ) are reported in ppm relative

to TMS. For ¹³C NMR in D₂O was added 1% MeOD as internal

reference. ¹⁹F NMR spectra were recorded using CFCl₃as reference

standard.

HPLC was performed using a Dionex UltiMate 3000 pump and

photodiode array detector (210 and 254 nm, respectively) installed

with an XTerra MS C 18 3.5 μ m, 4.6 mm × 150 mm column, using a

5 → 95% MeCN gradient in H₂O containing 0.1% TFA. For HPLC

control, data collection, and data handling, Chromeleon software v.

6.80 was used. Preparative HPLC was carried out on an Agilent Prep

HPLC systems with Agilent 1100 series pump, Agilent 1200 series

diode array, multiple wavelength detector (G1365B), and Agilent

PrepHT High Performance Preparative Cartridge Column (Zorbax,

300 SB-C18 Prep HT, 21.2 mm × 250 mm, 7 μm). LC-MS spectra

were recorded using either an Agilent 1200 series solvent delivery

system equipped with an autoinjector coupled to an Agilent 6400

series triple quadrupole mass spectrometer equipped with an

electrospray ionization source or Waters Aquity UPLC-MS with dual

wavelength detection with electrospray ionization. Gradients of 5%

aqueous MeCN + 0.1% HCO₂H (solvent A) and 95% aqueous MeCN

+ 0.05% HCO₂H (solvent B) were employed. IR spectra were

recorded on a PerkinElmer 801 spectrophotometer. Optical rotation

was measured using a PerkinElmer 241 spectrometer, with Na lamp

■

In the present study, we have explored new β-functionalized

Asp analogues (Table 1) as inhibitors of the EAATs. An

elaborate SAR study on the new and potent β-sulfonamide Asp

scaﬀold 4a was carried out by the synthesis of 28 analogues, and

thereby new pharmacological tools for the EAATs were

identiﬁed. Furthermore, we have highlighted the existence of

an adjacent vestibule to the substrate binding site in the

transporters that could be highly interesting for future ligand

development.

Several analogues of 4a-B were potent nonselective inhibitors

which displayed IC₅₀values in the high-nanomolar/low-

microlar range at all three transporters (4f, 4g, and 4h, Table

2). However, the main objective of this study was to discover

subtype-selective or subtype-preferring EAAT inhibitors, and

introduction of ﬂuorine atoms on the phenyl ring did indeed

yield analogues characterized by preference for EAAT1 over

EAAT2,3 (4d-B, 4z, and 4y). The 3,4-diﬂuoro analogue 4y, a

potent EAAT1 inhibitor (IC₅₀0.8 μM) displaying 14- and 9-

fold preference for this subtype over EAAT2 and EAAT3,

respectively, is particularly interesting, as it to the best of our

knowledge is the most EAAT1-selective competitive non-

substrate inhibitor to be published to date. Even though its

EAAT1-preference admittedly pales in comparison with the

>300-fold EAAT1-selectivity exhibited by the noncompetitive

inhibitors 15²¹and 16,²²we nevertheless ﬁnd 4y a promising

lead for future development of truly EAAT1-selective inhibitors

acting through the substrate-binding site in the transporter.

Another highly interesting analogue to come out of the SAR

study was 4r, obtained by installation of a triﬂuoromethyl group

in the meta-position of the phenyl ring. 4r is a potent EAAT2

inhibitor (IC₅₀2.8 μM) characterized by ≥30-fold selectivity

toward EAAT1 and EAAT3. While this EAAT2-selectivity is

substantially lower than that previously reported for WAY-

213613, it is roughly comparable to those exhibited by DHK

and 13, and it should also be noted that 4r is 10−30-fold more

potent at EAAT2 than these two latter inhibitors.¹⁴⁻¹⁷

J

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(D line, 589 nm). Compounds were dried under high vacuum or

freeze-dried using a Holm & Halby, Heto LyoPro 6000 freeze drier.

The purity of compounds submitted for pharmacological character-

ization was determined by LCMS to be >95%.

16 h under argon. The reaction mixture was diluted with H₂O and

extracted three times with Et₂O. The combined organic extracts were

concentrated in vacuo and directly suspended in 6 M HCl (5 mL) and

subsequently reﬂuxed for 4 h. The cooled aqueous reaction mixture

was washed twice with Et₂O, concentrated in vacuo, and puriﬁed twice

by preparative HPLC. The combined were evaporated, and 1 M HCl

(5 mL) was added and evaporated. This was repeated three times to

give the HCl salt of 1 (13 mg, 14% from 8, diastereomeric ratio 0.5:1).

General Procedure for Synthesis and Puriﬁcation of

Sulfonamide Aspartate Analogues (General Procedure A). In

a representative reaction, a dry, argon-ﬁlled 20 mL vial, ﬁtted with

screw-cap and PTFE/silicone septum and magnetic stirrer, for 18 h

was charged with dimethyl (2S)-3-amino-N-(triphenylmethyl)-

aspartate 9 (200 mg, 478 μmol) and anhydrous DCM (6 mL). To

the stirred solution was added TEA (100 μL, 717 μmol) and the

desired arylsulfonyl chloride, respectively. The mixture was stirred at

ambient temperature for 20 h and then diluted with DCM (2 mL).

The reaction mixture was washed once with 1 M HCl and three times

with saturated NaHCO₃by addition of the aqueous solution to the

vial, agitating, and removing the aqueous layer with a pipet. The

organic phase was reduced under a stream of air, followed by

evaporation in vacuo. The residue was used in the following step

without further puriﬁcation.

1

Diastereomer 1: H NMR (400 MHz, DMSO-d₆) δ: 8.80 (broad s,

3H), 8.65 (s, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.46 (t, J = 7.6 Hz, 2H),

7.40−7.30 (m, 1H), 6.21 (d, J = 4.4 Hz, 1H), 4.85 (d, J = 4.4 Hz, 1H).

Diastereomer 2: ¹H NMR (400 MHz, DMSO-d₆) δ: 8.80 (s, 3H), 8.51

(s, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.46 (t, J = 7.6 Hz, 2H), 7.40−7.30

(m, 1H), 6.18 (d, J = 3.7 Hz, 1H), 4.91 (d, J = 3.7 Hz, 1H).

Diastereomer 1: ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.5, 166.5,

146.4, 130.3, 129.1, 128.3, 125.4, 123.2, 61.3, 53.0. Diastereomer 2:

¹³C NMR (101 MHz, DMSO-d₆) δ: 167.4, 166.4, 146.2, 130.4, 129.1,

128.2, 125.3, 123.6, 61.2, 52.8. LCMS (ES+) m/z 277.0 [M + H]⁺,

C₁₂H₁₃N₄O₄requires 277.1.

The crude, protected sulfonamide aspartate analogue was

suspended in 6 M HCl (5 mL). The capped vial was stirred vigorously

at 100 °C for 4.5 h and then allowed to cool to room temperature. The

reaction mixture was then washed three times with Et₂O and three

times with DCM, and the aqueous phase was concentrated in vacuo.

The residue was taken up in a minimum of H₂O (0.1% TFA) and

puriﬁed on a preparative HPLC. Fractions were analyzed by LC-MS or

NMR (0.5 mL sample from tube + 0.05 mL D₂O) and collected

accordingly. The combined fractions were sequentially concentrated

and then added 1 M HCl (5 mL), after which the compounds were

lyophilized.

Alternative Procedure for the Synthesis and Puriﬁcation of

Sulfonamide Aspartate Analogues (General Procedure B). In

cases of large amounts of arylsulfonate byproduct, an alternative

procedure was used. The desired aspartate analogue was synthesized

and hydrolyzed as described in general procedure A. Following the

wash of the aqueous hydrolysate with Et₂O and DCM, the solution

was subjected to ion exchange chromatography. A ﬁlter with a sintered

glass frit was ﬁlled with Amberlite IRA-420 (2 cm × 7 cm). The funnel

was assembled with a separation funnel and an adapter with side arm,

resembling a setup for dry column vacuum chromatography. The

column was pre-equilibrated with 1 M HCl until the eﬄuent was at pH

= 1. The hydrolysate was charged to the resin and passed through the

column with 1 M HCl (20 mL) by applying a very slight vacuum. The

resulting eﬄuent was collected in a 100 mL round-bottom ﬂask,

evaporated, and puriﬁed by preparative HPLC.

(2S)-3-Benzamidoaspartic Acid (2). Dimethyl (2S)-3-amino-N-

(triphenylmethyl)aspartate 9 (150 mg, 358 μmol) was dissolved in

anhydrous DCM (4.5 mL) in a dry ﬂask under argon. The solution

was cooled to 0 °C followed by addition of TEA (75 μL, 537 μmol)

and benzoyl chloride (50 μL, 430 μmol), and the solution was allowed

to reach ambient temperature. The solution was stirred for 3 days,

evaporated, and resuspended in 5 mL of 6 M HCl and reﬂuxed for 4 h.

The resulting mixture was concentrated in vacuum and puriﬁed by

preparative HPLC (combined fractions were evaporated, 1 M HCl (5

mL) added, and concentrated). This was repeated three times to

produce the HCl salt to give 2 as a colorless solid (26 mg, 29% from 9,

diastereomeric ratio 0.5:1). Diastereomer 1: ¹H NMR (600 MHz,

D₂O) δ: 7.73 (ddd, J = 8.3, 6.6, 1.3 Hz, 2H), 7.59−7.53 (m, 1H), 7.45

(td, J = 7.9, 1.4 Hz, 2H), 5.24 (d, J = 3.5 Hz, 1H), 4.66 (d, J = 3.5 Hz,

1H). Diastereomer 2: ¹H NMR (600 MHz, D₂O) δ: 7.73 (ddd, J = 8.3,

6.6, 1.3 Hz, 2H), 7.59−7.53 (m, 1H), 7.45 (td, J = 7.9, 1.4 Hz, 2H),

5.16 (d, J = 3.5 Hz, 1H), 4.57 (d, J = 3.5 Hz, 1H). Diastereomer 1: ¹³C

NMR (151 MHz, D₂O) δ: 172.60, 171.52, 169.88, 133.82, 133.10,

129.74, 128.48, 56.19, 53.79. Diastereomer 2: ¹³C NMR (151 MHz,

D₂O) δ: 172.25, 171.95, 170.50, 133.86, 132.97, 129.79, 128.38, 55.28,

53.58. LCMS (ES+) m/z 253.2 [M + H]⁺, C₁₁H₁₃N₂O₅requires 253.1.

(2S)-3-((Phenoxycarbonyl)amino)aspartic Acid (3-A). Dimethyl

(2S)-3-amino-N-(triphenylmethyl)aspartate 9 (150 mg, 358 μmol)

was dissolved in 4.5 mL of anhydrous DCM in a dry ﬂask under argon

followed by addition of TEA (75 μL, 537 μmol) and phenyl

chloroformate (54 μL, 439 μmol). The reaction mixture was stirred at

room temperature for 18 h and then concentrated in vacuo. The crude

residue was suspended in 6 M HCl (5 mL) and reﬂuxed for 4 h. The

cooled mixture was washed twice with Et₂O, concentrated, and

puriﬁed by preparative HPLC to yield single diastereomer (22 mg,

23% from 9) after sequential evaporation from 5 mL of 1 M HCl

(three times). One single diastereomer: ¹H NMR (400 MHz, DMSO-

d₆) δ: 8.66 (s, 3H), 8.34 (d, J = 9.4 Hz, 1H), 7.45−7.36 (m, 2H),

7.28−7.20 (m, 1H), 7.20−7.12 (m, 2H), 4.85 (dd, J = 9.4, 3.4 Hz,

1H), 4.40 (d, J = 3.8 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ:

169.0, 167.6, 154.8, 150.8, 129.3, 125.3, 121.6, 53.9, 53.8. LCMS (ES

+) m/z 269.2 [M + H]⁺, C₁₁H₁₂N₂O₆requires 269.1.

Symmetrization of 4a-A and 4a-B for Elucidation of

Absolute Stereochemistry. Each of the puriﬁed and separated

diastereomers, 4a-A and 4a-B (3.15 mg, 9.7 μmol), were dissolved in

NaOH (29 μL, 1 M aq, 29 μmol) in separate Eppendorf tubes at 0 °C.

Benzenesulfonyl chloride (1.4 μL, 10.7 μmol), N,N-diisopropylethyl-

amine (1.9 μL, 10.7 μmol), and acetone (10 μL) were added, and the

reaction mixture was allowed to warm to room temperature over 2 h

and stirred for an additional 16 h. Each of the reaction mixtures were

then diluted with 1 mL of H₂O and puriﬁed by preparative HPLC to

produce 11 (2.05 mg, 49%, from 4a-A) and 12 (inseparable 2:3 ratio

with benzenesulfonate, from 4a-B). Optical rotation was measured in

triplicate for each of the samples, and only 12 was able to perform

rotation (in 1 mL of distilled MeOH, at 20 °C; cuvette, 10 cm, α =

(2S)-3-((Phenoxycarbonyl)amino)aspartic Acid (3-B). Diaster-

eomer 3-B was isolated as impure fractions from preparative HPLC

in 3-A. These concentrated fractions were subsequently puriﬁed by a

second preparative HPLC run to give 3-B (15 mg, 16% from 9) after

three sequential evaporations from 1 M HCl (5 mL). One single

1

0.03 for 12). 11: H NMR (600 MHz, CD₃OD) δ: 7.86−7.82 (m,

4H), 7.61 (td, J = 7.3, 1.3 Hz, 2H), 7.55−7.51 (m, 4H), 4.24 (s, 2H).

12: ¹H NMR (600 MHz, CD₃OD) δ: 7.84−7.79 (m, 4H), 7.59 (dd, J

= 7.4, 1.2 Hz, 2H), 7.54−7.47 (m, 4H), 4.41 (s, 2H). 11: ¹³C NMR

1

diastereomer: H NMR (400 MHz, DMSO-d₆) δ: 8.75 (s, 3H), 8.31

(151 MHz, CD₃OD) δ: 170.6, 141.7, 133.8, 130.1, 128.4, 59.6. 12: ¹³

C

(d, J = 9.3 Hz, 1H), 7.47−7.34 (m, 2H), 7.29−7.17 (m, 1H), 7.15−

7.07 (m, 2H), 4.77 (dd, J = 9.3, 4.6 Hz, 1H), 4.37 (d, J = 4.6 Hz, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ: 169.3, 168.2, 154.4, 150.8, 129.4,

125.4, 121.6, 53.8, 53.1. LCMS (ES+) m/z 269.0 [M + H]⁺,

C₁₁H₁₂N₂O₆requires 269.1.

NMR (151 MHz, CD₃OD) δ: 170.7, 141.8, 133.8, 130.0, 128.2, 59.3.

(2S)-3-(4-Phenyl-1H-1,2,3-triazol-1-yl)aspartic Acid (1). Dimethyl

(2S)-3-azido-N-(triphenylmethyl)aspartate (8) (150 mg, 338 μmol)

and phenylacetylene (78 μL, 709 μmol) were suspended in H₂O/t-

BuOH (1.35 mL 1:1 v/v) in a vial. Sodium ascorbate (33 μL, 1 M aq,

34 μmol) was added, followed by CuSO₄·5H₂O (114 μL, 0.3 M aq, 34

μmol), and the reaction mixture was stirred at ambient temperature for

(2S,3R)-3-(Phenylsulfonamido)aspartic Acid (4a-A). General

1

procedure A. Yield 17 mg, 15%. H NMR (400 MHz, DMSO-d₆) δ:

8.66 (br s, 3H), 8.54 (d, J = 9.2 Hz, 1H), 7.80−7.77 (m, 2H), 7.65−

K

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.58 (m, 1H), 7.54 (m, 2H), 4.48 (dd, J = 9.0, 3.4 Hz, 1H), 4.15 (d, J =

3.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 168.7, 167.9, 140.6,

133.0, 129.2, 126.9, 55.5, 54.3. LCMS (ES+) m/z 289.0 [M + H]⁺,

C₁₀H₁₃N₂O₆S requires 289.0.

(2S)-3-((4-Chlorophenyl)sulfonamido)aspartic Acid (4f). General

procedure A. Yield from 9: 46 mg, 30%. Diastereomer 1: H NMR

1

(600 MHz, DMSO-d₆) δ: 8.64 (s, 3H), 7.88−7.79 (m, 2H), 7.69−7.63

(m, 2H), 4.44 (m, 1H), 4.16 (d, J = 4.1 Hz, 1H). Diastereomer 2: ¹H

NMR (600 MHz, DMSO-d₆) δ: 8.64 (s, 3H), 7.88−7.79 (m, 2H),

7.69−7.63 (m, 2H), 4.41 (broad s, 1H), 4.25−4.21 (m, 1H).

Diastereomer 1: ¹³C NMR (151 MHz, DMSO-d₆) δ: 168.5, 167.8,

139.2, 137.6, 129.1, 128.7, 55.3, 54.1. Diastereomer 2: ¹³C NMR (151

MHz, DMSO-d₆) δ: 167.7, 167.1, 139.0, 137.6, 129.1, 128.9, 55.7,

54.4. LCMS (ES+) m/z 323.0 [M + H]⁺, C₁₀H₁₂ClN₂O₆S requires

323.0.

(2S,3S)-3-(Phenylsulfonamido)aspartic Acid (4a-B). General

1

procedure A. Yield 9 mg, 8%. H NMR (400 MHz, DMSO-d₆) δ:

8.66 (br s, 3H), 8.31 (d, J = 9.4 Hz, 1H), 7.84−7.81 (m, 2H), 7.65−

7.58 (m, 1H), 7.54 (m, 2H), 4.50 (dd, J = 9.0, 3.7 Hz, 1H), 4.09 (br s,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 168.0, 167.3, 140.2, 133.1,

129.3, 127.1, 55.8, 54.9. LCMS (ES+) m/z 289.0 [M + H]⁺,

C₁₀H₁₃N₂O₆S requires 289.0.

(2S)-3-(Naphthalene-1-sulfonamido)aspartic Acid (4b). General

procedure A. Yield from 9: 47 mg, 29%. Diastereomer 1: H NMR

(2S)-3-((4-Bromophenyl)sulfonamido)aspartic Acid (4g). General

procedure B; 10 mol % 4-bromobenzenesulfonate. Yield from 9: 29

mg, 17%. Diastereomer 1: ¹H NMR (400 MHz, D₂O) δ: 7.83 (s, 4H),

4.67 (d, J = 3.1 Hz, 1H), 4.53 (d, J = 3.1 Hz, 1H). Diastereomer 2: ¹H

NMR (400 MHz, D₂O) δ: 7.83 (s, 4H), 4.53 (d, J = 4.8 Hz, 1H), 4.37

(d, J = 4.3 Hz, 1H). Diastereomer 1 ¹³C NMR (151 MHz, D₂O) δ:

170.3, 169.5, 137.8, 133.5, 129.6, 129.3, 56.8, 56.0. Diastereomer 2:

¹³C NMR (151 MHz, D₂O) δ: 171.2, 170.2, 137.9, 133.6, 129.6, 129.3,

56.6, 55.9. LCMS (ES+) m/z 367.0 [M + H]⁺, 369.0 [m + 2 + H]⁺,

C₁₀H₁₂BrN₂O₆S requires 367.0 and 369.0.

1

(600 MHz, DMSO-d₆) δ: 8.74 (dd, J = 8.6, 1.1 Hz, 1H), 8.49 (broad s,

3H), 8.24 (d, J = 8.2 Hz, 1H), 8.20 (dd, J = 7.4, 1.2 Hz, 1H), 8.11−

8.05 (m, 1H), 7.72 (dt, J = 8.4, 1.4 Hz, 1H), 7.68−7.66 (m, 1H), 7.63

(t, J = 8.1 Hz, 1H), 4.43 (broad s, 1H), 4.19 (broad s, 1H).

1

Diastereomer 2: H NMR (600 MHz, DMSO-d₆) δ: 8.67−8.62 (m,

1H), 8.47 (s, 3H), 8.24 (d, J = 8.2 Hz, 1H), 8.16 (dd, J = 7.3, 1.2 Hz,

1H), 8.08 (m, 1H), 7.74−7.70 (m, 1H), 7.69−7.65 (m, 1H), 7.66−

7.62 (m, 1H), 4.45−4.41 (m, 1H), 4.17 (d, J = 4.0 Hz, 1H).

Diastereomer 1 (d1) and diastereomer 2 (d2): ¹³C NMR (151 MHz,

DMSO-d₆) δ: 168.4 (d2), 167.7 (d2), 167.7 (d1), 167.1 (d1), 135.0,

134.9, 134.3, 134.3, 133.8, 128.9, 128.8, 128.7, 127.8, 127.8, 127.8,

127.7, 126.8, 126.8, 125.1, 124.8, 124.3, 55.5 (d1), 55.3 (d2), 54.8

(d1), 54.0 (d2). LCMS (ES+) m/z 339.0 [M + H]⁺, C₁₄H₁₅N₂O₆S

requires 339.1.

(2S)-3-((4-Methylphenyl)sulfonamido)aspartic Acid (4h). General

1

procedure A. Yield from 9: 33 mg, 23%. Diastereomer 1: H NMR

(600 MHz, DMSO-d₆) δ: 8.35 (br s, 3H), 7.72 (d, J = 8.4 Hz, 2H),

7.40−7.35 (m, 2H), 4.41−4.29 (m, 1H), 4.17−4.12 (m, 1H), 2.38 (s,

1

3H). Diastereomer 2: H NMR (600 MHz, DMSO-d₆) δ: 8.35 (br s,

3H), 7.72 (d, J = 8.4 Hz, 2H), 7.40−7.35 (m, 2H), 4.41−4.29 (m,

1H), 4.11 (d, J = 4.0 Hz, 1H), 2.38 (s, 3H). Diastereomer 1: ¹³C NMR

(151 MHz, DMSO-d₆) δ: 167.7, 167.2, 143.0, 137.1, 129.4, 126.9,

55.7, 54.6, 21.0. Diastereomer 2: ¹³C NMR (151 MHz, DMSO-d₆) δ:

168.5, 167.9, 143.0, 137.4, 129.4, 126.8, 55.3, 54.1, 21.0. LCMS (ES+)

m/z 303.1 [M + H]⁺, C₁₁H₁₅N₂O₆S requires 303.1.

(2S)-3-((4-(tert-Butyl)phenyl)sulfonamido)aspartic Acid (4i). Gen-

eral procedure B; 12 mol % 4-tert-butylbenzenesulfonate. Yield from 9:

40 mg, 24%. Diastereomer 1: ¹H NMR (400 MHz, D₂O) δ: 7.87 (dd, J

= 8.8, 2.4 Hz, 2H), 7.72 (dd, J = 8.8, 2.5 Hz, 2H), 4.65 (d, J = 3.1 Hz,

1H), 4.50 (d, J = 3.1 Hz, 1H), 1.36 (s, 9H). Diastereomer 2: ¹H NMR

(400 MHz, D₂O) δ: 7.87 (dd, J = 8.8, 2.4 Hz, 2H), 7.72 (dd, J = 8.8,

2.5 Hz, 2H), 4.47 (d, J = 4.5 Hz, 1H), 4.35 (d, J = 4.4 Hz, 1H), 1.36 (s,

9H). Diastereomer 1: ¹³C NMR (151 MHz, D₂O) δ: 170.3, 169.6,

159.3, 135.4, 127.9, 127.5, 56.8, 56.1, 35.6, 31.1. Diastereomer 2: ¹³C

NMR (151 MHz, D₂O) δ: 171.2, 170.2, 159.4, 135.4, 127.9, 127.5,

56.6, 55.9, 35.6, 31.1. LCMS (ES+) m/z 345.1 [M + H]⁺,

C₁₄H₂₁N₂O₆S requires 345.1.

(2S)-3-(Naphthalene-2-sulfonamido)aspartic Acid (4c). General

1

procedure A. Yield from 9: 22 mg, 14%. Diastereomer 1: H NMR

(600 MHz, DMSO-d₆) δ: 8.51 (s, 3H), 8.43 (d, J = 1.7 Hz, 1H), 8.17−

8.13 (m, 1H), 8.12 (d, J = 8.8 Hz, 1H), 8.05 (dt, J = 8.1, 1.6 Hz, 1H),

7.88 (dd, J = 8.7, 1.9 Hz, 1H), 7.73−7.70 (m, 1H), 7.69−7.66 (m,

1H), 4.46−4.43 (m, 1H), 4.24 (d, J = 3.1 Hz, 1H). Diastereomer 2: ¹H

NMR (600 MHz, DMSO-d₆) δ: 8.51 (s, 3H), 8.45 (d, J = 1.5 Hz, 1H),

8.17−8.13 (m, 1H), 8.11 (d, J = 8.7 Hz, 1H), 8.05 (dt, J = 8.1, 1.6 Hz,

1H), 7.84 (dd, J = 8.7, 1.9 Hz, 1H), 7.73−7.70 (m, 1H), 7.69−7.66

(m, 1H), 4.48 (d, J = 4.1 Hz, 1H), 4.18 (d, J = 4.2 Hz, 1H).

Diastereomer 1: ¹³C NMR (151 MHz, DMSO-d₆) δ: 167.7, 167.2,

137.1, 134.3, 131.5, 129.2, 129.0, 128.8, 127.8, 127.6, 127.5, 122.8,

55.7, 54.5. Diastereomer 2: ¹³C NMR (151 MHz, DMSO-d₆) δ: 168.5,

167.8, 137.2, 134.3, 131.5, 129.3, 129.1, 128.8, 127.8, 127.6, 127.5,

122.5, 55.4, 54.1. LCMS (ES+) m/z 339.1 [M + H]⁺, C₁₄H₁₅N₂O₆S

requires 339.1.

(2S)-3-((Perﬂuorophenyl)sulfonamido)aspartic Acid (4d-A). Gen-

eral procedure A. Yield from 9: 7.0 mg, 4%. One single diastereomer:

¹H NMR (600 MHz, D₂O) δ: 4.74−4.69 (m, 2H). ¹³C NMR (151

MHz, D₂O) δ: 169.6, 168.6, 145.7, 145.6, 144.0, 143.8, 138.8, 137.2,

114.2, 56.4, 55.1. ¹⁹F NMR (376 MHz, D₂O) δ: −136.56 to −136.71

(m), −145.07 (tt, J = 21.1, 7.4 Hz), −159.57 to −159.75 (m). LCMS

(ES+) m/z 379.0 [M + H]⁺, C₁₀H₈F₅N₂O₆S requires 379.0.

(2S)-3-((Perﬂuorophenyl)sulfonamido)aspartic Acid (4d-B). Gen-

eral procedure A. Yield from 9: 4.3 mg, 2%. One single diastereomer:

¹H NMR (400 MHz, D₂O) δ: 4.90 (d, J = 3.5 Hz, 1H), 4.58 (d, J = 3.5

Hz, 1H). ¹³C NMR (151 MHz, D₂O) δ: 169.7, 168.7, 145.6, 145.5,

143.9, 143.8, 138.8, 137.2, 114.5, 55.4, 54.5. ¹⁹F NMR (376 MHz,

D₂O) δ: −137.13 to −137.28 (m), −145.03 (tt, J = 21.3, 7.5 Hz),

−159.56 to −159.74 (m). LCMS (ES+) m/z 379.0 [M + H]⁺,

C₁₀H₈F₅N₂O₆S requires 379.0.

(2S)-3-((4-(Methylsulfonyl)phenyl)sulfonamido)aspartic Acid (4j).

1

General procedure B. Yield from 9: 23 mg, 13%. Diastereomer 1: H

NMR (400 MHz, DMSO-d₆) δ: 8.16−8.10 (m, 2H), 8.10−8.04 (m,

2H), 4.52−4.45 (m, 1H), 4.22 (d, J = 4.1 Hz, 1H), 3.29 (s, 3H).

1

Diastereomer 2: H NMR (400 MHz, DMSO-d₆) δ: 8.16−8.10 (m,

2H), 8.10−8.04 (m, 2H), 4.52−4.45 (m, 1H), 4.35−4.28 (m, 1H),

3.29 (s, 3H). Diastereomer 1: ¹³C NMR (151 MHz, DMSO-d₆) δ:

167.6, 166.9, 144.7, 144.2, 128.0, 127.8, 55.2, 54.2, 43.2. Diastereomer

2: ¹³C NMR (151 MHz, DMSO-d₆) δ: 168.3, 167.6, 144.9, 144.2,

127.8, 127.7, 55.7, 53.8, 43.2. LCMS (ES+) m/z 367.1 [M + H]⁺,

C₁₁H₁₅N₂O₈S₂requires 367.0.

(2S)-3-((4-Methoxyphenyl)sulfonamido)aspartic Acid (4k). Gen-

eral procedure A. Yield from 9: 43 mg, 28%. Diastereomer 1: ¹H NMR

(600 MHz, D₂O) δ: 7.81 (dd, J = 8.9, 3.0 Hz, 2H), 7.07 (dd, J = 9.1,

3.1 Hz, 2H), 4.73 (d, J = 3.1 Hz, 1H), 4.52 (d, J = 3.1 Hz, 1H), 3.85 (s,

3H). Diastereomer 2: ¹H NMR (600 MHz, D₂O) δ: 7.81 (dd, J = 9.0,

3.1 Hz, 2H), 7.07 (dd, J = 9.1, 3.0 Hz, 2H), 4.55 (d, J = 4.1 Hz, 1H),

4.45 (d, J = 4.1 Hz, 1H), 3.86 (s, 3H). Diastereomer 1: ¹³C NMR (151

MHz, D₂O) δ: 170.3, 169.3, 164.2, 130.4, 130.2, 115.6, 56.7, 56.6, 55.9.

Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 171.2, 170.0, 164.3,

130.4, 130.2, 115.6, 56.7, 56.4, 55.7. LCMS (ES+) m/z 319.1 [M +

H]⁺, C₁₁H₁₅N₂O₇S requires 319.1.

(2S)-3-((4-Fluorophenyl)sulfonamido)aspartic Acid (4e). General

1

procedure A. Yield from 9: 44 mg, 30%. Diastereomer 1: H NMR

(600 MHz, DMSO-d₆) δ: 8.55 (broad s, 3H), 7.94−7.84 (m, 2H),

7.45−7.39 (m, 2H), 4.44−4.36 (m, 1H), 4.22 (dd, J = 3.1 Hz, 1H).

1

Diastereomer 2: H NMR (600 MHz, DMSO-d₆) δ: 8.55 (s, 3H),

7.94−7.84 (m, 2H), 7.45−7.39 (m, 2H), 4.44−4.36 (m, 1H), 4.15 (dd,

J = 4.2, 1.6 Hz, 1H). Diastereomer 1: ¹³C NMR (151 MHz, DMSO-

d₆) δ: 167.7, 167.2, 165.1, 165.1, 136.5, 136.5, 130.0, 130.0, 116.0,

116.0, 55.7, 54.4. Diastereomer 2: ¹³C NMR (151 MHz, DMSO-d₆) δ:

168.5, 167.8, 163.5, 163.5, 136.7, 129.9, 129.8, 116.2, 116.2, 55.3, 54.1.

¹⁹F NMR (376 MHz, DMSO-d₆) δ: −106.6. LCMS (ES+) m/z 307.0

[M + H]⁺, C₁₀H₁₂FN₂O₆S requires 307.0.

(2S)-3-((4-(Triﬂuoromethyl)phenyl)sulfonamido)aspartic Acid

(4l). General procedure B. Yield from 9: 52 mg, 31%. Diastereomer

1: ¹H NMR (400 MHz, D₂O) δ: 8.10 (d, J = 8.2 Hz, 2H), 7.97 (d, J =

8.8 Hz, 2H), 4.67 (d, J = 4.1 Hz, 1H), 4.48 (d, J = 4.1 Hz, 1H).

L

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

Diastereomer 2: H NMR (400 MHz, D₂O) δ: 8.10 (d, J = 8.2 Hz,

Hz, 1H), 8.13−8.09 (m, 1H), 8.04 (dd, J = 8.0, 2.1 Hz, 1H), 7.83 (td, J

= 7.9, 2.4 Hz, 1H), 4.42 (d, J = 4.0 Hz, 1H), 4.29−4.25 (m, 1H).

Diastereomer 2: ¹H NMR (600 MHz, DMSO-d₆) δ: 8.74 (s, 3H), 8.13

(d, J = 1.7 Hz, 1H), 8.13−8.09 (m, 1H), 8.04 (dd, J = 8.0, 2.1 Hz, 1H),

7.83 (td, J = 7.9, 2.4 Hz, 1H), 4.48 (m, 1H), 4.15 (d, J = 4.0 Hz, 1H).

Diastereomer 1: ¹³C NMR (151 MHz, DMSO-d₆) δ: 167.7, 167.2,

141.3, 130.8, 130.4, 129.9, 129.6, 129.4, 129.4, 129.2, 126.2, 124.4,

123.9, 123.8, 123.8, 123.8, 122.6, 120.8, 55.9, 54.3. Diastereomer 2:

¹³C NMR (151 MHz, DMSO-d₆) δ: 168.5, 168.0, 167.7, 167.2, 141.6,

130.8, 130.5, 129.9, 129.7, 129.4, 129.4, 129.4, 129.2, 126.2, 124.4,

2H), 7.96 (d, J = 8.7 Hz, 2H), 4.76 (m, inter alia 1H), 4.63 (d, J = 3.1

Hz, 1H). Diastereomer 1: ¹³C NMR (151 MHz, D₂O) δ: 170.8, 169.9,

142.6, 135.5, 135.3, 128.7, 127.6, 127.5, 126.9, 125.1, 123.3, 121.5,

56.3, 55.7. Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 170.0,

169.1, 142.4, 135.5, 135.2, 128.7, 127.5, 127.5, 126.9, 125.1, 123.3,

121.5, 56.7, 55.7. ¹⁹F NMR (376 MHz, D₂O) δ: −63.1. LCMS (ES+)

m/z 357.0 [M + H]⁺, C₁₁H₁₂F₃N₂O₆S requires 357.0.

(2S)-3-((4-Nitrophenyl)sulfonamido)aspartic Acid (4m). General

procedure B. Yield from 9: 32 mg, 20%. Diastereomer 1: H NMR

(400 MHz, D₂O) δ: 8.48−8.39 (m, 2H), 8.19−8.12 (m, 2H), 4.79 (m,

inter alia 1H), 4.66 (d, J = 3.1 Hz, 1H). Diastereomer 2: H NMR

(400 MHz, D₂O) δ: 8.44 (d, J = 9.0 Hz, 2H), 8.15 (d, J = 8.9 Hz, 2H),

4.71 (d, J = 3.9 Hz, 1H), 4.50 (d, J = 3.9 Hz, 1H). Diastereomer 1: ¹³C

NMR (151 MHz, D₂O) δ: 170.0, 169.0, 151.4, 144.7, 129.5, 125.5,

56.7, 55.6. Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 170.8,

169.8, 151.4, 144.8, 129.5, 125.6, 56.3, 55.6. LCMS (ES+) m/z 334.1

[M + H]⁺, C₁₀H₁₂N₃O₈S requires 334.0.

1

123.6, 123.6, 123.5, 123.5, 122.6, 120.8, 55.4, 54.1. Diastereomer 1: ¹⁹

F

1

NMR (376 MHz, DMSO-d₆) δ: −61.36 (d, J = 1.4 Hz). Diastereomer

2: ¹⁹F NMR (376 MHz, DMSO-d₆) δ: −61.34 (d, J = 1.3 Hz). LCMS

(ES+) m/z 357.0 [M + H]⁺, C₁₁H₁₂F₃N₂O₆S requires 357.0.

(2S)-3-((3-Carboxyphenyl)sulfonamido)aspartic Acid (4s). Using a

modiﬁed general procedure A, 9 (125 mg, 0.30 mmol) was dissolved

in dry CH₂Cl₂(3 mL) under argon gas. Triethylamine (125 μL, 0.90

mmol) was added, and after 5 min of stirring, 3-cyanobenzenesulfonyl

chloride (91 mg, 0.45 mmol) was added. The reaction mixture was

stirred overnight and evaporated. Without further puriﬁcation, the

crude was treated with 6 M HCl (11.5 mL) and stirred in a closed vial

overnight at 110 °C. The reaction mixture was washed with Et₂O (3 ×

3 mL) and CH₂Cl₂(3 × 3 mL). The solvent was evaporated and

puriﬁed by preparative HPLC to yield (2S)-3-((3-carboxyphenyl)-

sulfonamido)aspartic acid (19 mg, 0.06 mmol, 19%). Diastereomer 1:

¹H NMR (400 MHz, D₂O) δ: 8.47 (t, J = 1.8 Hz, 1H), 8.29 (dt, J =

7.9, 1.4 Hz, 1H), 8.15 (ddd, J = 8.0, 2.0, 1.1 Hz, 1H), 7.74 (t, J = 7.9

Hz, 1H), 4.76 (d, J = 3.1 Hz, 1H), 4.61 (d, J = 3.1 Hz, 1H).

(2S)-3-((4-Aminophenyl)sulfonamido)aspartic Acid (4n). General

procedure A; 18 mol % triethylammonium chloride. Yield from 9 (150

mg, 0.358 mmol): 29 mg, 26%. Diastereomer 1: ¹H NMR (600 MHz,

D₂O) δ: 8.03−8.01 (m, 2H), 7.59−7.56 (m, 2H), 4.76 (d, J = 3.1 Hz,

1

1H), 4.61 (d, J = 3.1 Hz, 1H). Diastereomer 2: H NMR (600 MHz,

D₂O) δ: 8.02−8.00 (m, 2H), 7.59−7.56 (m, 2H), 4.66 (d, J = 4.0 Hz,

1H), 4.49 (d, J = 3.9 Hz, 1H). Diastereomer 1: ¹³C NMR (151 MHz,

D₂O) δ: 170.0, 168.9, 139.0, 136.7, 130.1, 124.7, 56.5, 55.6.

Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 170.7, 169.7, 139.0,

136.9, 130.1, 124.7, 56.2, 55.5. LCMS (ES+) m/z 303.9 [M + H]⁺,

C₁₀H₁₄N₃O₆S requires 304.1.

1

Diastereomer 2: H NMR (400 MHz, D₂O) δ: 8.48 (t, J = 1.8 Hz,

(3S)-2-((4-Acetylphenyl)sulfonamido)-3-aminoaspartic Acid (4o).

1H), 8.30 (dt, J = 7.8, 1.4 Hz, 1H), 8.15 (ddd, J = 8.0, 2.0, 1.1 Hz, 1H),

7.75 (t, J = 7.9 Hz, 1H), 4.62 (d, J = 4.2 Hz, 1H), 4.44 (d, J = 4.2 Hz,

1H). LCMS (ES+) m/z 333.1 [M + H]⁺, C₁₁H₁₃N₂O₈S requires 333.0.

(2S)-3-((2-Chlorophenyl)sulfonamido)aspartic Acid (4t). General

General procedure A. Yield from 9 (150 mg, 0.358 mmol): 6.5 mg, 6%.

1

Diastereomer 1: H NMR (600 MHz, D₂O) δ: 8.17−8.14 (m, 2H),

8.05−8.02 (m, 2H), 4.78−4.77 (m, inter alia 1H), 4.63 (d, J = 3.1 Hz,

1

procedure A. Yield from 9: 59 mg, 38%. Diastereomer 1: H NMR

1H), 2.71 (s, 3H). Diastereomer 2: H NMR (600 MHz, D₂O) δ:

(600 MHz, DMSO-d₆) δ: 8.47 (broad s, 3H), 7.98−7.93 (m, 1H),

7.68−7.63 (m, 2H), 7.56−7.50 (m, 1H), 4.49 (d, J = 3.9 Hz, 1H), 4.30

(d, J = 4.0 Hz, 1H). Diastereomer 2: ¹H NMR (600 MHz, DMSO-d₆)

δ: 8.47 (broad s, 3H), 8.02 (dd, J = 7.5, 1.3 Hz, 1H, 7.68−7.63 (m,

2H), 7.56−7.50 (m, 1H), 4.60 (d, J = 3.1 Hz, 1H), 4.22 (d, J = 3.2 Hz,

1H). Diastereomer 1: ¹³C NMR (151 MHz, DMSO-d₆) δ: 168.3,

167.8, 137.4, 134.3, 131.6, 131.2, 130.2, 127.5, 55.6, 53.9.

Diastereomer 2: ¹³C NMR (151 MHz, DMSO-d₆) δ: 168.1, 167.2,

137.6, 134.3, 131.7, 131.1, 130.4, 127.5, 55.9, 55.0. LCMS (ES+) m/z

323.0 [M + H]⁺, C₁₀H₁₂ClN₂O₆S requires 323.0.

8.17−8.14 (m, 2H), 8.05−8.02 (m, 2H), 4.69 (d, J = 4.1 Hz, 1H), 4.50

(d, J = 4.1 Hz, 1H), 2.71 (s, 3H). Diastereomer 1: ¹³C NMR (151

MHz, D₂O) δ: 203.3, 169.9, 169.0, 142.9, 141.2, 130.2, 128.4, 56.6,

55.7, 27.5. Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 203.3,

170.7, 169.7, 143.1, 141.3, 130.3, 128.4, 56.2, 55.6, 27.5. LCMS (ES+)

m/z 330.8 [M + H]⁺, C₁₂H₁₅N₂O₇S requires 331.0.

(3S)-2-([1,1′-Biphenyl]-4-sulfonamido)-3-aminoaspartic Acid

(4p). General procedure B; 6 mol % triethylammonium chloride.

Yield from 9 (150 mg, 0.358 mmol): 25 mg, 19%. Diastereomer 1: ¹H

NMR (600 MHz, methanol-d₄/MeCN-d₃1:1) δ: 8.00 (d, J = 8.5 Hz,

2H), 7.82 (d, J = 8.4 Hz, 2H), 7.71−7.67 (m, 2H), 7.49 (t, J = 7.7 Hz,

2H), 7.43 (t, J = 7.3 Hz, 1H), 4.59 (d, J = 2.9 Hz, 1H), 4.44 (d, J = 2.8

(2S)-3-((2-Methylphenyl)sulfonamido)aspartic Acid (4u). General

procedure A. Yield from 9 (250 mg, 0.597 mmol): 38 mg, 21%.

1

Diastereomer 1: H NMR (600 MHz, D₂O) δ: 7.92 (dd, J = 7.9, 1.0

Hz, 1H). Diastereomer 2: H NMR (600 MHz, methanol-d₄/MeCN-

Hz, 1H), 7.59−7.53 (m, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.40−7.34 (m,

d₃1:1) δ: 7.98 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.3 Hz, 2H), 7.71−7.67

(m, 2H), 7.49 (t, J = 7.7 Hz, 2H), 7.43 (t, J = 7.3 Hz, 1H), 4.54 (d, J =

4.1 Hz, 1H), 4.34 (d, J = 4.1 Hz, 1H). Diastereomer 1: ¹³C NMR (151

MHz, methanol-d₄/MeCN-d₃1:1) δ: 168.9, 168.1, 147.2, 140.5, 139.2,

130.2, 129.7, 129.2, 128.6, 128.3, 57.0, 56.2. Diastereomer 2: ¹³C NMR

(151 MHz, methanol-d₄/MeCN-d₃1:1) δ: 170.1, 168.8, 147.2, 140.5,

139.5, 130.2, 129.7, 129.1, 128.6, 128.3, 56.6, 55.9. LCMS (ES+) m/z

364.9 [M + H]⁺, C₁₆H₁₇N₂O₆S requires 365.1.

1H), 4.70 (d, J = 3.2 Hz, 1H), 4.44 (d, J = 3.3 Hz, 1H), 2.64 (s, 3H).

1

Diastereomer 2: H NMR (600 MHz, D₂O) δ: 7.91 (dd, J = 8.0, 1.0

Hz, 1H), 7.59−7.55 (m, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.40−7.36 (m,

1H), 4.49 (d, J = 4.2 Hz, 1H), 4.43 (d, J = 4.2 Hz, 1H), 2.59 (s, 3H).

Diastereomer 1: ¹³C NMR (151 MHz, D₂O) δ: 170.1, 169.0, 139.1,

136.0, 135.0, 133.8, 130.4, 127.2, 56.2, 55.7, 20.5. Diastereomer 2: ¹³C

NMR (151 MHz, D₂O) δ: 170.9, 169.6, 138.8, 136.2, 135.1, 133.8,

130.4, 127.3, 55.9, 55.3, 20.3. LCMS (ES+) m/z 303.1 [M + H]⁺,

C₁₁H₁₅N₂O₆S requires 303.1.

(2S)-3-((3-Chlorophenyl)sulfonamido)aspartic Acid (4q). General

1

procedure A. Yield from 9: 41 mg, 27%. Diastereomer 1: H NMR

(2S)-3-((3,5-Dimethylphenyl)sulfonamido)aspartic Acid (4v).

General procedure A. Yield from 9: 23 mg, 15%. Diastereomer 1:

¹H NMR (600 MHz, DMSO-d₆) δ: 8.33 (broad s, 3H), 7.43−7.41 (m,

2H), 7.30−7.26 (m, 1H), 4.35−4.30 (m, 1H), 4.08 (d, J = 4.3 Hz,

(600 MHz, DMSO-d₆) δ: 8.69 (s, 3H), 7.85 (t, J = 1.9 Hz, 1H), 7.73

(ddt, J = 8.1, 2.2, 1.1 Hz, 2H), 7.61 (t, J = 8.0 Hz, 1H), 4.45 (d, J = 4.1

1

Hz, 1H), 4.14 (d, J = 4.0 Hz, 1H). Diastereomer 2: H NMR (600

MHz, DMSO-d₆) δ: 8.69 (s, 3H), 7.88 (t, J = 1.9 Hz, 1H), 7.77 (ddt, J

= 7.8, 2.7, 1.2 Hz, 2H), 7.61 (t, J = 8.0 Hz, 1H), 4.41 (d, J = 2.9 Hz,

1H), 4.25 (d, J = 3.0 Hz, 1H). Diastereomer 1: ¹³C NMR (151 MHz,

DMSO-d₆) δ: 168.5, 167.9, 142.2, 133.5, 132.6, 130.9, 126.5, 125.4,

55.4, 54.1. Diastereomer 2: ¹³C NMR (151 MHz, DMSO-d₆) δ: 167.7,

167.2, 142.0, 133.5, 132.6, 131.0, 126.7, 125.5, 55.9, 54.4. LCMS (ES

+) m/z 323.0 [M + H]⁺, C₁₀H₁₂ClN₂O₆S requires 323.0.

1

1H), 2.34 (s, 6H). Diastereomer 2: H NMR (600 MHz, DMSO-d₆)

δ: 8.33 (br s, 3H), 7.55−7.43 (m, 2H), 7.30−7.26 (m, 1H), 4.29 (br s,

1H), 4.17 (br s, 1H), 2.34 (s, 6H). Diastereomer 1: ¹³C NMR (151

MHz, DMSO-d₆) δ: 168.5, 167.9, 139.9, 138.4, 134.1, 124.2, 55.3,

54.1, 20.7. Diastereomer 2: ¹³C NMR (151 MHz, DMSO-d₆) δ: 167.7,

167.2, 139.7, 138.4, 134.1, 124.4, 55.8, 54.6, 20.7. LCMS (ES+) m/z

317.1 [M + H]⁺, C₁₂H₁₇N₂O₆S requires 317.1.

(2S)-3-((3-(Triﬂuoromethyl)phenyl)sulfonamido)aspartic Acid

(4r). General procedure B. Yield from 9: 27 mg, 16%. Diastereomer

(2S)-3-((2,6-Dichlorophenyl)sulfonamido)aspartic Acid (4x). Gen-

1

1: H NMR (600 MHz, DMSO-d₆) δ: 8.74 (s, 3H), 8.17 (d, J = 1.8

eral procedure B. Yield from 9: 52 mg, 30%. Diastereomer 1: ¹H NMR

M

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(600 MHz, D₂O) δ: 7.62−7.59 (m, 2H), 7.54−7.50 (m, 1H), 4.70 (d,

D₂O) δ: 7.88 (dd, J = 5.0, 1.1 Hz, 1H), 7.80−7.75 (m, 1H), 7.23−7.21

(m, 1H), 4.74 (d, J = 3.1 Hz, 1H), 4.63 (d, J = 3.1 Hz, 1H).

Diastereomer 1: ¹³C NMR (151 MHz, D₂O) δ: 171.0, 169.9, 138.6,

135.4, 135.0, 129.0, 56.6, 55.7. Diastereomer 2: ¹³C NMR (151 MHz,

D₂O) δ: 170.2, 169.2, 138.6, 135.2, 134.7, 129.1, 56.9, 55.8. LCMS (ES

+) m/z 294.8 [M + H]⁺, C₈H₁₁N₂O₆S₂requires 295.0.

1

J = 3.5 Hz, 1H), 4.69 (d, J = 3.4 Hz, 1H). Diastereomer 2: H NMR

(600 MHz, D₂O) δ: 7.62−7.59 (m, 2H), 7.54−7.50 (m, 1H), 4.71 (d,

J = 4.4 Hz, 1H), 4.49 (d, J = 4.4 Hz, 1H). Diastereomer 1: ¹³C NMR

(151 MHz, D₂O) δ: 170.2, 169.1, 135.4, 135.0, 133.7, 132.5, 56.7, 55.7.

Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 170.8, 169.6, 135.3,

134.9, 134.0, 132.5, 56.4, 55.2. LCMS (ES+) m/z 357.0 [M + H]⁺,

C₁₀H₁₁Cl₂N₂O₆S requires 357.0.

(2S)-3-(N-Methylphenylsulfonamido)aspartic Acid (4ad). Amine

9 (80 mg, 0.191 mmol) was dissolved in anhyd DCM (2.4 mL) and

added TEA (40 μL, 0.287 mmol), followed by phenyl sulfonyl chloride

(37 μL, 0.287 mmol) and stirred 20 h. Reaction mixture was

subsequently diluted with DCM, washed with 1 M HCl, satd

NaHCO₃, and then brine. The organic partition was dried over

MgSO₄, ﬁltered, and concentrated. The crude was dissolved in anhyd

DMF (0.46 mL), cooled to 0 °C and K₂CO₃(78 mg, 0.573 mmol)

added, followed by dropwise addition of methyl iodide (50 μL, 3.82 M,

0.191 mmol). Reaction was stirred for 3 h at 0 °C, and an additional

equivalent of methyl iodide was added. Reaction was diluted with H₂O

and EtOAc, washed four times with H₂O and brine, and dried over

MgSO₄, followed by ﬁltration and evaporated. Crude was reﬂuxed in 6

M HCl (3 mL) for 4 h, cooled and washed four times with Et₂O and

evaporated. Desired compound 4ad was isolated by puriﬁcation on

(2S)-3-((3,4-Diﬂuorophenyl)sulfonamido)aspartic Acid (4y). Gen-

eral procedure B. Yield from 9: 45 mg, 29%. Diastereomer 1: ¹H NMR

(400 MHz, D₂O) δ: 7.93−7.86 (m, 1H), 7.81−7.76 (m, 1H), 7.57−

7.49 (m, 1H), 4.70 (d, J = 3.1 Hz, 1H), 4.56 (d, J = 3.1 Hz, 1H).

1

Diastereomer 2: H NMR (400 MHz, D₂O) δ: 7.93−7.86 (m, 1H),

7.81−7.76 (m, 1H), 7.57−7.49 (m, 1H), 4.58 (d, J = 4.2 Hz, 1H), 4.41

(d, J = 4.2 Hz, 1H). Diastereomer 1: ¹³C NMR (151 MHz, D₂O) δ:

170.3, 169.4, 155.3, 155.3, 153.5, 153.5, 151.6, 150.1, 135.6, 135.6,

135.6, 135.6, 125.8, 125.8, 125.7, 119.6, 119.5, 118.1, 118.0, 56.8, 55.9.

Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 171.2, 170.1, 155.2,

155.2, 153.6, 153.6, 151.7, 150.0, 135.7, 135.7, 135.7, 135.7, 125.8,

125.8, 125.8, 119.6, 119.5, 118.1, 118.0, 56.6, 55.9. Diastereomer 1: ¹⁹F

NMR (376 MHz, D₂O) δ: −129.36 (d, J = 20.6 Hz), −134.74 (d, J =

20.9 Hz). Diastereomer 2: ¹⁹F NMR (376 MHz, D₂O) δ: −129.23 (d,

J = 20.9 Hz), −134.64 (d, J = 20.4 Hz). LCMS (ES+) m/z 325.0 [M +

H]⁺, C₁₀H₁₁F₂N₂O₆S requires 325.0.

1

preparative HPLC to yield (5.1 mg, 9%). Diastereomer 1: H NMR

(600 MHz, D₂O) δ: 7.94−7.92 (m, 2H), 7.79−7.74 (m, 1H), 7.69−

7.64 (m, 2H), 5.04 (d, J = 8.3 Hz, 1H), 4.54 (d, J = 8.3 Hz, 1H), 2.94

(s, 3H). Diastereomer 2: ¹H NMR (600 MHz, D₂O) δ: 7.94−7.92 (m,

2H), 7.79−7.74 (m, 1H), 7.69−7.64 (m, 2H), 5.22 (d, J = 5.8 Hz,

1H), 4.68 (d, J = 5.8 Hz, 1H), 2.94 (s, 3H). Diastereomer 1: ¹³C NMR

(151 MHz, D₂O) δ: 169.9, 169.5, 136.6, 135.2, 130.3, 128.7, 60.6, 53.4,

33.3. Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 170.2, 169.9,

137.1, 135.0, 130.3, 128.3, 60.4, 54.1, 33.0. LCMS (ES+) m/z 302.9

[M + H]⁺, C₁₁H₁₅N₂O₆S requires 303.1.

(2S)-3-((2,4,5-Triﬂuorophenyl)sulfonamido)aspartic Acid (4z).

General procedure A. Yield from 9 (150 mg, 0.358 mmol): 48 mg,

1

39%. Diastereomer 1: H NMR (400 MHz, D₂O) δ: 7.77−7.68 (m,

1H), 7.32−7.21 (m, 1H), 4.69 (d, J = 3.7 Hz, 1H), 4.47 (d, J = 3.6 Hz,

1

1H). Diastereomer 2: H NMR (400 MHz, D₂O) δ: 7.77−7.68 (m,

1H), 7.32−7.21 (m, 1H), 4.72 (d, J = 3.1 Hz, 1H), 4.60 (d, J = 3.1 Hz,

1H). Diastereomer 1: ¹³C NMR (151 MHz, D₂O) δ: 170.6, 169.4,

156.8, 156.7, 156.7, 155.5, 155.1, 155.1, 155.0, 153.8, 147.8, 147.7,

146.2, 146.1, 123.5, 123.4, 119.5, 119.4, 108.8, 108.7, 108.5, 56.0, 55.4.

Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 169.9, 168.6, 156.8,

156.8, 156.7, 155.5, 155.1, 155.1, 155.0, 153.8, 147.8, 147.7, 146.2,

146.1, 123.3, 123.2, 119.6, 119.4, 108.9, 108.7, 108.5, 56.6, 55.3.

Diastereomer 1: ¹⁹F NMR (376 MHz, D₂O) δ: −110.08 (dd, J = 15.1,

9.4 Hz), −123.54 (dd, J = 21.4, 9.6 Hz), −140.33 (t, J = 15.2 Hz).

Diastereomer 2: ¹⁹F NMR (376 MHz, D₂O) δ: −109.38 (dd, J = 15.1,

9.4 Hz), −123.63 (dd, J = 21.3, 9.5 Hz), −140.39 (t, J = 15.1 Hz).

LCMS (ES+) m/z 343.0 [M + H]⁺, C₁₀H₁₀F₃N₂O₆S requires 343.0.

(2S)-3-((Phenylmethyl)sulfonamido)aspartic Acid (4aa). General

procedure B; 9 mol % phenylmethanesulfonate. Yield from 9 (150 mg,

Dimethyl (2S)-3-Bromo-N-(triphenylmethyl)aspartate (7). Follow-

ing the optimized procedure for the synthesis, dimethyl (2S)-N-

(triphenylmethyl)aspartate (6) (1.00 g, 2.48 mmol) was charged to a

ﬂame-dried, argon-purged 100 mL round-bottom ﬂask ﬁtted with

septum and dissolved in anhydrous THF (40 mL) at ambient

temperature, under argon. Once dissolved, the ﬂask was wrapped in tin

foil and cooled to −30 °C for 10 min. Lithium hexamethyldisilazide

(2.7 mL 1 M in THF, 2.73 mmol) was added over 30 s, and the

resulting mixture was stirred at −30 °C for 30 min. N-

Bromosuccinimide (500 mg, 2.73 mmol) was added in one portion

swiftly to the reaction mixture, which was then allowed to warm to −5

°C over the course of 40 min and maintained at −5 °C for an

additional hour. The reaction was quenched by the addition of 15 mL

of satd NH₄Cl, warmed to rt, and concentrated in vacuo. The mixture

was extracted twice with EtOAc, the combined organic phases were

washed with H₂O and brine, dried over MgSO₄, ﬁltered, and

concentrated in vacuo, yielding a brownish oil (diastereomeric ratio

2:3, conversion by NMR), which was used without further puriﬁcation

due to instability. Diastereomer 1: ¹H NMR (600 MHz, chloroform-d)

δ: 7.50−7.47 (m, 6H), 7.30−7.27 (m, 6H), 7.22−7.18 (m, 3H), 4.25

(d, J = 4.1 Hz, 1H), 3.93 (dd, J = 10.3, 4.2 Hz, 1H), 3.78 (s, 3H), 3.33

1

0.358 mmol): 22 mg, 20%. Diastereomer 1: H NMR (600 MHz,

D₂O) δ: 7.51−7.45 (m, 5H), 4.61 (d, J = 3.5 Hz, inter alia 1H), 4.63−

1

4.56 (m, 2H), 4.28 (d, J = 3.3 Hz, 1H). Diastereomer 2: H NMR

(600 MHz, D₂O) δ: 7.51−7.45 (m, 5H), 4.65 (d, J = 2.9 Hz, 1H),

4.63−4.56 (m, 2H), 4.53 (d, J = 2.9 Hz, 1H). Diastereomer 1: ¹³C

NMR (151 MHz, D₂O) δ: 170.1, 169.2, 131.9, 130.1, 129.9, 129.0,

60.0, 56.6, 55.9. Diastereomer 2: ¹³C NMR (151 MHz, D₂O) δ: 171.6,

170.8, 131.9, 130.1, 129.9, 128.9, 59.8, 57.2, 56.3. LCMS (ES+) m/z

302.9 [M + H]⁺, C₁₁H₁₅N₂O₆S requires 303.1.

1

(2S)-3-(Pyridine-3-sulfonamido)aspartic Acid (4ab). General

(s, 3H), 3.27 (d, J = 10.4 Hz, 1H). Diastereomer 2: H NMR (600

procedure A. Yield from 9 (150 mg, 0.358 mmol): 25 mg, 24%.

Diastereomer 1: H NMR (600 MHz, D₂O) δ: 9.33 (d, J = 2.0 Hz,

1H), 9.04 (dd, J = 5.8, 1.5 Hz, 1H), 8.98 (dt, J = 8.2, 1.8 Hz, 1H), 8.24

MHz, chloroform-d) δ: 7.47−7.45 (m, 6H), 7.29−7.27 (m, 6H),

7.22−7.18 (m, 3H), 4.24 (d, J = 6.2 Hz, 1H), 4.10 (dd, J = 10.5, 6.2

Hz, 1H), 3.74 (s, 3H), 3.31 (s, 3H), 3.06 (d, J = 10.5 Hz, 1H).

Dimethyl (2S)-3-Azido-N-(triphenylmethyl)aspartate (8). Crude

dimethyl (2S)-3-bromo-N-(triphenylmethyl)aspartate (7) (theoret-

ically 7.62 mmol) was dissolved in anhydrous DMF (33 mL) under an

argon atmosphere in a tin foil covered ﬂask, and NaN₃(536 mg, 11,43

mmol) was added in one portion. The solution was stirred for 16 h at

rt, then diluted with EtOAc and washed with H₂O until a clear organic

phase was obtained (4−5 times). The organic phase was washed with

brine, dried over MgSO₄, ﬁltered, and concentrated. The resulting

crude could be used without puriﬁcation or puriﬁed by ﬂash column

chromatography (heptane/EtOAc 8:1, 2% TEA) to give 8 as a white,

sticky, amorphous syrup (1,90 g, 56% over two steps corrected yield as

the amorphous nature of the compound inseparably trapped heptane,

diastereomeric ratio 1:1.6). IR (neat): 2113, 1743, 1208, 707 cm⁻¹.

Diastereomer 1: ¹H NMR (600 MHz, chloroform-d) δ: 7.50−7.46 (m,

1

(dd, J = 8.3, 5.7 Hz, 1H), 4.82 (d, J = 3.1 Hz, 1H), 4.73 (d, J = 3.0 Hz,

1

1H). Diastereomer 2: H NMR (600 MHz, D₂O) δ: 9.33 (d, J = 2.0

Hz, 1H), 9.04 (dd, J = 5.8, 1.5 Hz, 1H), 8.98 (dt, J = 8.2, 1.8 Hz, 1H),

8.24 (dd, J = 8.3, 5.7 Hz, 1H), 4.84 (d, J = 3.4 Hz, 1H), 4.54 (d, J = 3.4

Hz, 1H). Diastereomer 1: ¹³C NMR (151 MHz, D₂O) δ: 169.2, 168.0,

146.3, 144.1, 141.6, 139.0, 127.9, 56.0, 54.5. Diastereomer 2: ¹³C NMR

(151 MHz, D₂O) δ: 170.0, 169.0, 146.3, 144.0, 141.6, 139.3, 127.9,

55.4, 54.7. LCMS (ES+) m/z 289.9 [M + H]⁺, C₉H₁₂N₃O₆S requires

290.0.

(2S)-3-(Thiophene-2-sulfonamido)aspartic Acid (4ac). General

procedure A. Yield from 9 (150 mg, 0.358 mmol): 14 mg, 13%.

1

Diastereomer 1: H NMR (600 MHz, D₂O) δ: 7.90 (dd, J = 5.0, 1.1

Hz, 1H), 7.80−7.75 (m, 1H), 7.23−7.21 (m, 1H), 4.64 (d, J = 4.2 Hz,

1

1H), 4.45 (d, J = 4.1 Hz, 1H). Diastereomer 2: H NMR (600 MHz,

N

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

6H), 7.31−7.27 (m, 6H), 7.24−7.17 (m, 3H), 4.04 (dd, J = 8.2, 4.2

Hz, 1H), 3.72 (s, 3H), 3.71 (d, J = 4.3 Hz, 1H), 3.39 (s, 3H), 3.19 (d, J

= 8.9 Hz, 1H). Diastereomer 2: ¹H NMR (600 MHz, chloroform-d) δ:

7.50−7.46 (m, 6H), 7.31−7.26 (m, 6H), 7.23−7.15 (m, 3H), 3.90 (dd,

J = 10.9, 3.2 Hz, 1H), 3.82 (d, J = 3.4 Hz, 1H), 3.78 (s, 3H), 3.26 (s,

3H), 3.11 (d, J = 11.0 Hz, 1H). Diastereomer 1: ¹³C NMR (151 MHz,

CDCl₃) δ: 170.9, 168.4, 145.4, 128.9, 128.3, 127.0, 71.5, 65.7, 59.0,

52.8, 52.6. Diastereomer 2: ¹³C NMR (151 MHz, CDCl₃) δ: 171.5,

168.9, 145.3, 128.9, 128.1, 126.8, 71.3, 64.4, 59.2, 53.0, 52.5.

Sequence alignment of EAAT1−3 with Glt_Ph(PDB code:

2nww) (PDF)

3D coordinates of Figure 4 (PDB format) (PDB)

Molecular formula strings (CSV)

AUTHOR INFORMATION

■

Corresponding Author

*Phone: +45 35 33 62 44. E-mail: lebu@sund.ku.dk.

Dimethyl (2S)-3-Amino-N-(triphenylmethyl)aspartate (9). Di-

methyl (2S)-3-azido-N-(triphenylmethyl)aspartate (8) (2.47 g, 5.56

mmol) was dissolved in 66 mL of anhydrous THF in an argon-ﬁlled

ﬂask. Triphenylphosphine (2.19 g, 8.34 mmol) was added in one

portion, followed by the addition of H₂O (150 μL, 8.34 mmol), and

the slowly eﬀervescing solution was allowed to stir at ambient

temperature for 18 h. The reaction mixture was concentrated in vacuo

and puriﬁed by ﬂash column chromatography (heptane/EtOAc 8:1 to

4:1 with 5% TEA) to yield 9 as a viscous syrup (1.83 g, 79%, 1:1.7

diastereomeric ratio). The product was immediately stored at −18 °C

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the Danish Medical Research Council, Lundbeck

Foundation, the Novo Nordisk Foundation, the Augustinus

Foundation, and the Carlsberg Foundation for ﬁnancial

support.

1

due to instability. Diastereomer 1: H NMR (600 MHz, CDCl₃) δ:

7.52−7.49 (m, 6H), 7.29−7.23 (m, 6H), 7.21−7.15 (m, 3H), 3.82 (dd,

J = 9.8, 4.4 Hz, 1H), 3.65 (s, 3H), 3.40 (d, J = 4.6 Hz, 1H), 3.30 (s,

3H), 3.14 (d, J = 9.8 Hz, 1H). Diastereomer 2: H NMR (600 MHz,

ABBREVIATIONS USED

■

1

CNS, central nervous system; dr, diastereomeric ratio; EAAT,

excitatory amino acid transporter; KHMDS, potassium

hexamethyldisilazide; LHMDS, lithium hexamethyldisilazide;

TEA, triethylamine; Tr, trityl; TBOA, threo-benzyloxy

aspartate; EBOA, erythro-benzyloxy aspartate; DHK, dihydro-

kainic acid; DIPEA, diisopropylethyl amine

CDCl₃) δ: 7.48−7.45 (m, 6H), 7.28−7.23 (m, 6H), 7.21−7.15 (m,

3H), 3.77 (dd, J = 11.1, 2.7 Hz, 1H), 3.72 (s, 3H), 3.59 (d, J = 2.7 Hz,

1H), 3.22 (s, 3H), 2.98 (d, J = 11.0 Hz, 1H). Diastereomer 1: ¹³C

NMR (151 MHz, CDCl₃) δ: 173.4, 172.2, 145.8, 128.9, 128.1, 126.7,

71.4, 59.5, 58.4, 52.2, 52.0. Diastereomer 2: ¹³C NMR (151 MHz,

CDCl₃) δ: 173.9, 172.9, 145.6, 129.0, 128.0, 126.7, 70.8, 59.3, 58.1,

52.5, 52.1.

In Silico Study. The modeling study was performed using the

software package MOE 2015 (Molecular Operating Environment,

Chemical Computing Group) using the built-in mmﬀ94x force ﬁeld

and the GB/SA continuum solvation model. Docking: The receptor

proteins were prepared for docking by running the algorithm

Protonate 3D. Docking studies were performed using the Rigid Fit

algorithm under standard setup with the mmﬀ94x force ﬁeld, solvation

set to distance, and ligand atoms selected as docking site. Homology

models: the amino acid sequences of human EAAT1-3 were aligned

with that of the bacterial homolog Glt_Ph(PDB code: 2nww). After this

a small segment present in the EAATs but not in Glt_Phwas truncated

from the EAAT sequences (see Supporting Information for details).

Homology models were build on the 3D coordinates of Glt_Ph(PDB

code: 2nww) by applying parameters from standard setup (see

Supporting Information for details).

REFERENCES

■

(1) Moghaddam, B.; Javitt, D. From Revolution to Evolution: The

Glutamate Hypothesis of Schizophrenia and Its Implication for

Treatment. Neuropsychopharmacology 2012, 37, 4−15.

(2) Machado-Vieira, R.; Henter, I. D.; Zarate, C. A., Jr. New Targets

for Rapid Antidepressant Action. Prog. Neurobiol. 2015, 10.1016/

j.pneurobio.2015.12.001.

́

(3) Gonzalez, J.; Jurado-Coronel, J. C.; Avila, M. F.; Sabogal, A.;

Capani, F.; Barreto, G. E. NMDARs in Neurological Diseases: A

Potential Therapeutic Target. Int. J. Neurosci. 2015, 125, 315−327.

(4) Finlay, C.; Duty, S. Therapeutic Potential of Targeting Glutamate

Receptors in Parkinson’s Disease. J. Neural Transm. 2014, 121, 861−

880.

(5) Danbolt, N. C. Glutamate Uptake. Prog. Neurobiol. 2001, 65, 1−

Cell Culture and [³H]-D-Aspartate Uptake Assay. All cell

culture reagents were from ThermoFisher Scientiﬁc (Waltham, MA).

Stable EAAT1-, EAAT2-, and EAAT3-HEK293 cell lines established as

described previously¹⁴were maintained in DMEM with high glucose

and GlutaMAX supplemented with 5% dialyzed FBS, 100 U/mL

penicillin, 100 μg/mL streptomycin, and 1 mg/L G418, and the uptake

assay was performed essentially as previously described.¹⁴Brieﬂy, cells

were seeded in 96-well OptiPlates (PerkinElmer, Boston, MA),

reaching conﬂuence after 16−24 h, where they were washed in 100 μL

of room temperature uptake buﬀer (Hank’s Buﬀered Saline Solution

(ThermoFisher) supplemented with 20 mM HEPES, 1 mM CaCl₂,

and 1 mM MgCl₂(all from Sigma, St. Louis, MO), pH 7.4). Cells were

then incubated for 3 min at room temperature in 50 μL of uptake

buﬀer with 100 nM [³H]-D-aspartate (PerkinElmer) and various

concentrations of the test compounds, washed twice in ice-cold uptake

buﬀer before 150 μL of MicroScint20 (PerkinElmer) was added, and

the plates were shaken vigorously for ≥1 h before reading in a

TopCount NXT (PerkinElmer).

105.

(6) Bunch, L.; Erichsen, M. N.; Jensen, A. A. Excitatory Amino Acid

Transporters as Potential Drug Targets. Expert Opin. Ther. Targets

2009, 13, 719−731.

(7) Jiang, J.; Amara, S. G. New Views of Glutamate Transporter

Structure and Function: Advances and Challenges. Neuropharmacology

2011, 60, 172−181.

(8) Vandenberg, R. J.; Ryan, R. M. Mechanisms of Glutamate

Transport. Physiol. Rev. 2013, 93, 1621−1657.

(9) Jensen, A. A.; Fahlke, C.; Bjørn-Yoshimoto, W. E.; Bunch, L.

Excitatory amino acid transporters: recent insights into molecular

mechanisms, novel modes of modulation and new therapeutic

possibilities. Curr. Opin. Pharmacol. 2015, 20, 116−123.

(10) Grewer, C.; Gameiro, A.; Rauen, T. SLC1 Glutamate

Transporters. Pfluegers Arch. 2014, 466, 3−24.

(11) Traynelis, S. F.; Wollmuth, L. P.; McBain, C. J.; Menniti, F. S.;

Vance, K. M.; Ogden, K. K.; Hansen, K. B.; Yuan, H.; Myers, S. J.;

Dingledine, R. Glutamate Receptor Ion Channels: Structure,

Regulation, and Function. Pharmacol. Rev. 2010, 62, 405−496.

(12) Shimamoto, K.; Lebrun, B.; Yasuda-Kamatani, Y.; Sakaitani, M.;

Shigeri, Y.; Yumoto, N.; Nakajima, T. DL-Threo-Beta-Benzyloxyas-

partate, a Potent Blocker of Excitatory Amino Acid Transporters. Mol.

Pharmacol. 1998, 53, 195−201.

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b01066.

(13) Lebrun, B.; Sakaitani, M.; Shimamoto, K.; Yasuda-Kamatani, Y.;

Nakajima, T. New β-Hydroxyaspartate Derivatives Are Competitive

O

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Blockers for the Bovine Glutamate/Aspartate Transporter. J. Biol.

Chem. 1997, 272, 20336−20339.

Human Glutamate Transporter Subtypes 1, 2, and 3. J. Med. Chem.

2006, 49, 172−178.

(14) Jensen, A. A.; Brauner-Osborne, H. Pharmacological Character-

(27) Kanai, Y.; Hediger, M. A. Primary Structure and Functional

Characterization of a High-Affinity Glutamate Transporter. Nature

1992, 360, 467−471.

̈

ization of Human Excitatory Amino Acid Transporters EAAT1,

EAAT2 and EAAT3 in a Fluorescence-Based Membrane Potential

Assay. Biochem. Pharmacol. 2004, 67, 2115−2127.

(15) Arriza, J.; Fairman, W.; Wadiche, J.; Murdoch, G.; Kavanaugh,

M.; Amara, S. Functional Comparisons of Three Glutamate Trans-

porter Subtypes Cloned from Human Motor Cortex. J. Neurosci. 1994,

14, 5559−5569.

(16) Sagot, E.; Jensen, A. A.; Pickering, D. S.; Pu, X.; Umberti, M.;

Stensbøl, T. B.; Nielsen, B.; Assaf, Z.; Aboab, B.; Bolte, J.; Gefflaut, T.;

Bunch, L. Chemo-Enzymatic Synthesis of (2S,4R)-2-Amino-4-(3-(2,2-

Diphenylethylamino)-3-Oxopropyl)pentanedioic Acid: A Novel Se-

lective Inhibitor of Human Excitatory Amino Acid Transporter

Subtype 2. J. Med. Chem. 2008, 51, 4085−4092.

(28) Leuenberger, M.; Ritler, A.; Simonin, A.; Hediger, M. A.;

Lochner, M. Concise Asymmetric Synthesis and Pharmacological

Characterization of All Stereoisomers of Glutamate Transporter

Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes.

ACS Chem. Neurosci. 2016, 7, 534−539.

(29) Shimamoto, K.; Sakai, R.; Takaoka, K.; Yumoto, N.; Nakajima,

T.; Amara, S. G.; Shigeri, Y. Characterization of Novel L-Threo-

-Benzyloxyaspartate Derivatives, Potent Blockers of the Glutamate

Transporters. Mol. Pharmacol. 2004, 65, 1008−1015.

(30) Shimamoto, K.; Shigeri, Y.; Yasuda-Kamatani, Y.; Lebrun, B.;

Yumoto, N.; Nakajima, T. Syntheses of Optically Pure β-

Hydroxyaspartate Derivatives as Glutamate Transporter Blockers.

Bioorg. Med. Chem. Lett. 2000, 10, 2407−2410.

(31) Mavencamp, T. L.; Rhoderick, J. F.; Bridges, R. J.; Esslinger, C.

S. Synthesis and Preliminary Pharmacological Evaluation of Novel

Derivatives of L-β-Threo-Benzylaspartate as Inhibitors of the Neuronal

Glutamate Transporter EAAT3. Bioorg. Med. Chem. 2008, 16, 7740−

7748.

(17) Dunlop, J.; McIlvain, H. B.; Carrick, T. A.; Jow, B.; Lu, Q.;

Kowal, D.; Lin, S.; Greenfield, A.; Grosanu, C.; Fan, K.; Petroski, R.;

Williams, J.; Foster, A.; Butera, J. Characterization of Novel Aryl-Ether,

Biaryl, and Fluorene Aspartic Acid and Diaminopropionic Acid

Analogs as Potent Inhibitors of the High-Affinity Glutamate

Transporter EAAT2. Mol. Pharmacol. 2005, 68, 974−982.

(18) Vandenberg, R. J.; Mitrovic, A. D.; Chebib, M.; Balcar, V. J.;

Johnston, G. A. R. Contrasting Modes of Action of Methylglutamate

Derivatives on the Excitatory Amino Acid Transporters, EAAT1 and

EAAT2. Mol. Pharmacol. 1997, 51, 809−815.

(32) Meadows, E. S.; De Wall, S. L.; Barbour, L. J.; Gokel, G. W.

Alkali Metal Cation - π Interactions Observed by Using a Lariat Ether

Model System. J. Am. Chem. Soc. 2001, 123, 3092−3107.

(33) Zhong, W.; Gallivan, J. P.; Zhang, Y.; Li, L.; Lester, H. A.;

Dougherty, D. A. From Ab Initio Quantum Mechanics to Molecular

Neurobiology: A Cation-Pi Binding Site in the Nicotinic Receptor.

Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 12088−12093.

(19) Alaux, S.; Kusk, M.; Sagot, E.; Bolte, J.; Jensen, A. a; Brauner-

̈

Osborne, H.; Gefflaut, T.; Bunch, L. Chemoenzymatic Synthesis of a

Series of 4-Substituted Glutamate Analogues and Pharmacological

Characterization at Human Glutamate Transporters Subtypes 1−3. J.

Med. Chem. 2005, 48, 7980−7992.

(34) True, J. E.; Thomas, T. D.; Winter, R. W.; Gard, G. L.

Electronegativities from Core-Ionization Energies: Electronegativities

of SF5 and CF3. Inorg. Chem. 2003, 42, 4437−4441.

(20) Abrahamsen, B.; Schneider, N.; Erichsen, M. N.; Huynh, T. H.;

Fahlke, C.; Bunch, L.; Jensen, A. A. Allosteric Modulation of an

Excitatory Amino Acid Transporter: The Subtype-Selective Inhibitor

UCPH-101 Exerts Sustained Inhibition of EAAT1 through an

Intramonomeric Site in the Trimerization Domain. J. Neurosci. 2013,

33, 1068−1087.

(21) Jensen, A. A.; Erichsen, M. N.; Nielsen, C. W.; Stensbøl, T. B.;

Kehler, J.; Bunch, L. Discovery of the First Selective Inhibitor of

Excitatory Amino Acid Transporter Subtype 1. J. Med. Chem. 2009, 52,

912−915.

(22) Erichsen, M. N.; Huynh, T. H. V.; Abrahamsen, B.; Bastlund, J.

F.; Bundgaard, C.; Monrad, O.; Bekker-Jensen, A.; Nielsen, C. W.;

Frydenvang, K.; Jensen, A. A.; Bunch, L. Structure−Activity Relation-

ship Study of First Selective Inhibitor of Excitatory Amino Acid

Transporter Subtype 1:2-Amino-4-(4-Methoxyphenyl)-7-(Naphtha-

len-1-Yl)-5-Oxo-5,6,7,8-Tetrahydro-4 H -Chromene-3-Carbonitrile

(UCPH-101). J. Med. Chem. 2010, 53, 7180−7191.

(35) Assaf, Z.; Larsen, A. P.; Venskutonyte, R.; Han, L.; Abrahamsen,

̇

B.; Nielsen, B.; Gajhede, M.; Kastrup, J. S.; Jensen, A. a; Pickering, D.

S.; Frydenvang, K.; Gefflaut, T.; Bunch, L. Chemoenzymatic Synthesis

of New 2,4-Syn-Functionalized (S)-Glutamate Analogues and

Structure-Activity Relationship Studies at Ionotropic Glutamate

Receptors and Excitatory Amino Acid Transporters. J. Med. Chem.

2013, 56, 1614−1628.

(36) Boudker, O.; Ryan, R. M.; Yernool, D.; Shimamoto, K.; Gouaux,

E. Coupling Substrate and Ion Binding to Extracellular Gate of a

Sodium-Dependent Aspartate Transporter. Nature 2007, 445, 387−

393.

(23) Haym, I.; Huynh, T. H. V; Hansen, S. W.; Pedersen, M. H. F.;

Ruiz, J. A.; Erichsen, M. N.; Gynther, M.; Bjørn-Yoshimoto, W. E.;

Abrahamsen, B.; Bastlund, J. F.; Bundgaard, C.; Eriksen, A. L.; Jensen,

A. A.; Bunch, L. Bioavailability Studies and in Vitro Profiling of the

Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1)

Inhibitor UCPH-102. ChemMedChem 2016, 11, 403−419.

(24) Hansen, S. W.; Erichsen, M. N.; Fu, B.; Bjørn-Yoshimoto, W. E.;

Abrahamsen, B.; Hansen, J. C.; Jensen, A. A.; Bunch, L. Identiﬁcation

of a New Class of Selective Excitatory Amino Acid Transporter

Subtype 1 (EAAT1) Inhibitors Followed by a Structure−Activity

Relationship Study. J. Med. Chem. 2016, 59, 10.1021/acs.jmed-

chem.6b01058.

(25) Esslinger, C. S.; Agarwal, S.; Gerdes, J.; Wilson, P. A.; Davis, E.

S.; Awes, A. N.; O’Brien, E.; Mavencamp, T.; Koch, H. P.; Poulsen, D.

J.; Rhoderick, J. F.; Chamberlin, A. R.; Kavanaugh, M. P.; Bridges, R. J.

The Substituted Aspartate Analogue L-Beta-Threo-Benzyl-Aspartate

Preferentially Inhibits the Neuronal Excitatory Amino Acid Trans-

porter EAAT3. Neuropharmacology 2005, 49, 850−861.

(26) Bunch, L.; Nielsen, B.; Jensen, A. a; Brauner-Osborne, H.

̈

Rational Design and Enantioselective Synthesis of (1R,4S,5R,6S)-3-

azabicyclo[3.3.0]octane-4,6-Dicarboxylic Acid - a Novel Inhibitor at

P

DOI: 10.1021/acs.jmedchem.6b01066

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

β-Sulfonamido Functionalized Aspartate Analogues as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity Profiles Arising from Subtle Structural Differences

DOI: 10.1021/acs.jmedchem.6b01066

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.6b01066

Full text of DOI:10.1021/acs.jmedchem.6b01066

Products guided by the article

R&D Labs maybe for 116393-72-7

Relevant to this article

Hot Product