2-Amino-4-bis(aryloxybenzyl)aminobutanoic acids: A novel scaffold for inhibition of ASCT2-mediated glutamine transport Dedicated to the memory of Eric S. Dawson, Ph.D

Article abstract of DOI:10.1016/j.bmcl.2015.12.031

Herein, we report the discovery of 2-amino-4-bis(aryloxybenzyl)aminobutanoic acids as novel inhibitors of ASCT2(SLC1A5)-mediated glutamine accumulation in mammalian cells. Focused library development led to two novel ASCT2 inhibitors that exhibit significantly improved potency compared with prior art in C6 (rat) and HEK293 (human) cells. The potency of leads reported here represents a 40-fold improvement over our most potent, previously reported inhibitor and represents, to our knowledge, the most potent pharmacological inhibitors of ASCT2-mediated glutamine accumulation in live cells. These and other compounds in this novel series exhibit tractable chemical properties for further development as potential therapeutic leads.

Full text of DOI:10.1016/j.bmcl.2015.12.031

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1044–1047
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Amino-4-bis(aryloxybenzyl)aminobutanoic acids: A novel scaffold
for inhibition of ASCT2-mediated glutamine transport
Michael L. Schulte a,b,c, Alexandra B. Khodadadi a,b, Madison L. Cuthbertson d, Jarrod A. Smith e,f
,
H. Charles Manning a,b,c,g,h,i,j,
⇑
a Vanderbilt Center for Molecular Probes, Vanderbilt University Medical Center, Nashville, TN 37232, United States
b Vanderbilt University Institute of Imaging Science (VUIIS), Vanderbilt University Medical Center, Nashville, TN 37232, United States
c Department of Radiology and Radiological Sciences, Vanderbilt University Medical Center, Nashville, TN 37232, United States
d Hume-Fogg Academic High School, Metropolitan Nashville Public Schools, Nashville, TN 37203, United States
e Vanderbilt Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, United States
f Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, United States
g Vanderbilt-Ingram Cancer Center (VICC), Vanderbilt University Medical Center, Nashville, TN 37232, United States
h Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232, United States
i Department of Neurosurgery, Vanderbilt University Medical Center, Nashville, TN 37232, United States
j Department of Chemistry, Vanderbilt University, Nashville, TN 37232, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the discovery of 2-amino-4-bis(aryloxybenzyl)aminobutanoic acids as novel inhibitors
of ASCT2(SLC1A5)-mediated glutamine accumulation in mammalian cells. Focused library development
led to two novel ASCT2 inhibitors that exhibit significantly improved potency compared with prior art in
C6 (rat) and HEK293 (human) cells. The potency of leads reported here represents a 40-fold improvement
over our most potent, previously reported inhibitor and represents, to our knowledge, the most potent
pharmacological inhibitors of ASCT2-mediated glutamine accumulation in live cells. These and other
compounds in this novel series exhibit tractable chemical properties for further development as potential
therapeutic leads.
Received 6 October 2015
Revised 3 December 2015
Accepted 10 December 2015
Available online 11 December 2015
Dedicated to the memory of Eric S. Dawson,
Ph.D
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
ASCT2
SLC1A5
Glutamine
Cancer
Metabolism
Cancer cells exhibit an altered metabolic profile compared to
normal cells. In addition to increased utilization of glucose, they
can become highly dependent on the amino acid glutamine. Mam-
malian cells can regulate glutamine import and export through an
evolutionarily redundant series of cell surface transporters. A
sodium-dependent transporter of glutamine, ASCT2 (gene symbol
SLC1A5), stands out as a major transporter for glutamine uptake
making it a promising target for probe development. In addition
to being the primary glutamine transporter in cancer, SLC1A5
expression is associated with oncogenic MYC1,2 and KRAS,3,4
suggesting its relevance in many clinically important tumors,
including those of the lung, colon, and pancreas.5–7 Fuchs and
co-workers demonstrated that SLC1A5 antisense RNA triggered
apoptosis in human hepatocellular carcinoma cells.8 Furthermore,
Hassanein et al. more recently reported that SLC1A5 was expressed
in 95% of squamous cell carcinomas (SCC), 74% of adenocarcinomas
(ADC), and 50% of neuroendocrine tumors. In those studies, siRNA
down-regulation of ASCT2 in lung cancer cells resulted in signifi-
cant growth inhibition.9 Collectively, these studies suggest devel-
opment of small molecules capable of inhibiting ASCT2 activity
could be promising as precision cancer medicines.
To date, few compounds that target ASCT2 have been reported
and most are derivatives of endogenous ASCT2 substrates. As an
early entrant to the field, in 2004 Esslinger and co-workers
described a series of glutamine analogs that explored pKa effects
on the amide NH bond to probe the ASCT2 amino acid binding site
through the addition of electron-donating and electron-withdraw-
ing aryl groups to the terminal amide of glutamine. The best com-
pound,
L-c-glutamyl-p-nitroanilide (GPNA, compound 1),
exhibited modest potency in the low millimolar range and no
observations were made regarding steric requirements for binding
to ASCT2.10 Our group was able to expand upon this class of
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.12.031
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

M. L. Schulte et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1044–1047
1045
inhibitors by exploring the steric requirements for binding to
ASCT2 and found that while SAR was flat, 2-substituted glutamy-
lanilides were preferred. We described N-(2-(morpholinomethyl)
phenyl)-L-glutamine (compound 2) as a novel glutamyl-anilide
with three-fold improved activity against ASCT2 compared to
GPNA.11 In 2011, Albers et al. described a series of ASCT2 inhibitors
based on the ASCT2 substrate serine. Using an in silico approach
along with experimental validation, they found that side chain
aromaticity was required for high-affinity interaction. This lead to
the discovery of O-(4-phenylbenzoyl)-
inhibitor of ASCT2 with an apparent affinity of 30
L
-serine (compound 3), an
l
M.12 In 2012,
Figure 2. Chemical optimization plan for hit compound (S)-2-amino-4-(bis(2-(4-
fluorophenoxy)benzyl)amino)butanoic acid via iterative parallel synthesis.
Oppedisano et al. identified the first small molecule lacking an
amino acid that blocked glutamine uptake in ASCT2 reconstituted
in proteoliposomes (compound 4). This series of 1,2,3-dithiazoles
likely inhibited uptake of glutamine non-competitively through
formation of mixed sulfides at Cys-207 or Cys-210 with the most
potent compound exhibiting an IC50 of 3.7 l
M (Fig. 1).13
To continue our efforts toward novel ASCT2 inhibitors, we
focused our work around elaborating the glutamylanilide scaffold,
particularly the amide linker. After screening multiple iterations of
this scaffold, we arrived at a series of substituted 2,4-diaminobu-
tanoic acids. While there was no SAR within a library of 4-N-
mono-substituted derivatives, the resultant library, generated via
reductive amination, afforded small amounts of 4-N-disubstituted
products. We were able to cleanly isolate these byproducts and test
their ability to block ASCT2-mediated 3H-glutamine uptake. Sur-
prisingly, these compounds demonstrated up 10 fold better activ-
ity against ASCT2 compared with GPNA and had tractable
structural elements for further development. While there were
many areas on this new scaffold where SAR could be investigated,
we chose to focus on the distal aromatic rings and the linker region
connecting it (Fig. 2).
Figure 3. Two-step synthesis of 2-amino-4-bis(aryloxybenzyl)-aminobutanoic
acids. Reagents and conditions: (a) RCHO, NaBH(OAc)3, CH2Cl2, rt, 12 h. (b) HCl,
dioxane, 40 °C, 5 h.
Table 1
Structures and activities of phenoxybenzyl analogs
We developed
2-amino-4-bis(aryloxybenzyl)aminobutanoic acids. (Fig. 3). This
synthesis required just two steps starting from N-Boc- -2,4-
a facile synthetic scheme to yield target
L
diaminobutyric acid tert-butyl ester hydrochloride and afforded
products in overall yields ranging from 52% to 75%. Similar to the
initial lead compound, libraries focused on 2-amino-4-bis(aryloxy-
benzyl)aminobutanoic acids with 2 identical functional groups on
the 4-N position.
Screening of the synthesized amino acids 5–11 (Table 1), in
both C6 (rat) and HEK293 (human) cell lines revealed that com-
pounds 6, 7, and 11 displayed roughly equal potencies in both cells
lines.14 Interestingly, other compounds in this series, (Table 1,
compounds 8 and 10) demonstrated some preference for blocking
Compound R=
IC50 (rat)
M)
SEM
M)
IC50
(human)
(lM)
SEM
M)
(l
(l
(l
5
6
1.3
0.7
57.2
11.9
20.8
0.4
8.7
0.5
2.8
7
24.3
26.0
33.8
11.5
4.0
8
1.8
36.3
10.5
68.2
0.6
7.2
9
10.9
2.7
2.2
10
11
141.7
59.6
14.2
4.9
10.3
All IC50 values are reported as the mean of at least 3 biological replicates.
glutamine transport in the rat cell line. Our most potent compound
in the rat cell line (Table 1, compound 5) also exhibited this pref-
erence and blocked ASCT2-mediated glutamine uptake in C6 cells
Figure 1. Structures of previously reported ASCT2 inhibitors: GPNA (1) and an
improved glutamylanilde (2), A potent inhibitor derived from the ASCT2 substrate
serine (3), and a small molecule inhibitor of ASCT2 lacking an amino acid moiety
(4).
with an IC50 of 1.3 lM.

1046
M. L. Schulte et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1044–1047
Table 2
Structures and activities of benzyloxybenzyl analogs
Compound R =
IC50 (rat)
M)
SEM
M)
IC50
(human)
SEM
M)
Compound
M)
R =
IC50 (rat)
M)
SEM
M)
IC50
(human)
SEM
M)
(
l
(
l
(
l
(
l
(
l
(
l
(
l
(l
M)
(lM)
12
13
14
15
16
17
5.1
1.0
7.2
0.5
18
19
20
21
22
23
86.4
3.3
13.0
1.4
2.3
1.1
8.5
5.2
17.5
7.9
3.0
18.4
12.7
20.1
41.7
9.0
3.3
1.6
3.8
5.0
3.3
40.1
50.8
33.7
25.6
9.6
4.8
16.1
9.7
2.0
0.1
1.9
11.2
8.4
13.7
48.3
69.1
17.6
1.7
10.8
11.5
3.4
17.3
22.5
All IC50 values are reported as the mean of at least 3 biological replicates.
Figure 4. Structures and activities of the most potent compounds in C6 cells (compound 5) and HEK293 cells (compound 12).

M. L. Schulte et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1044–1047
1047
In summary, we report a novel series of 2-amino-4-bis(aryl-
oxybenzyl)aminobutanoic acids as inhibitors of cellular glutamine
uptake via ASCT2. Members of this series represent some of the
most potent inhibitors of ASCT2-mediated glutamine uptake
reported to date. Evaluation of the chemical series within the con-
text of ligand docking to a homology model of human ASCT2
revealed reasonable compatibility with the ASCT2 binding site
based on SurflexDock Total Scores. Based upon our data, we antic-
ipate that compounds with the greatest potency may interact with
multiple structural elements within the ASCT2 binding site, includ-
ing the amino acid zwitterion binding site and the adjacent
hydrophobic pocket. Efforts to further investigate SAR and opti-
mize binding as well as selectivity within this scaffold are ongoing
and advances from these studies will be reported in due course.
Acknowledgments
This work was supported by the National Institutes of Health
(5P50-CA95103, 5P30-DK058404), the Kleberg Foundation, the
Vanderbilt Center for Molecular Probes (VCMP), and the VICC Tho-
racic Oncology Program. The authors gratefully acknowledge Sam
A. Saleh for assisting with the chemical synthesis. Ping Zhao and
Allie Fu are acknowledged for assistance with compound
screening.
Figure 5. Docking of the most potent compound (Table 2, entry 1) into ASCT2
homology model. The compound 5 (colored sticks) fits the homology model (blue
surface) generated for the inhibited form of human ASCT2 and is consistent with
the displacement of a key loop region (right).
To further investigate the SAR of this scaffold, we prepared a
library of 2-amino-4-bis(benzyloxybenzyl)aminobutanoic acids
with various substitutions around the distal aromatic ring. Overall,
we found that this addition of a rotatable bond between the two
aromatic groups improved activity against glutamine uptake in
both C6 and HEK293 cells. Our two most potent compounds
(Table 2, compounds 12 and 19) blocked ASCT2-mediated glu-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.12.
031.
tamine uptake with potencies of 5.1 and 3.3
lM in C6 cells and
7.2 and 7.9 M in HEK293 cells respectively. One compound in
l
References and notes
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ASCT2 are shown in Figure 4.
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