116
M. L. Schulte et al. / Bioorg. Med. Chem. Lett. 25 (2015) 113–116
16. General procedure for the synthesis of NÓ—glutamylanilides: To a microwave vial
containing a solution of Boc- -glutamic acid tert-butyl ester (0.165 mmol,
1.0 equiv) and HATU (0.165 mmol, 1.0 equiv) in DMF (1.65 mL) was added the
amine followed by DIPEA (57.5 L, 2.0 equiv). The vial was sealed and heated
20). Furthermore, 4 novel compounds among the series exhibited
potencies equivalent to GPNA. Full Concentration response curves
for compounds 4, 5, and 20 are shown in Figure 2.
L
l
under microwave irradiation for 30 min at 120 °C. Upon completion, the
reaction was partitioned between water and CH2Cl2, extracted 3Â with CH2Cl2,
dried over anhydrous Na2SO4, and concentrated under vacuum. Compounds
were purified via reverse phase chromatography (5–95% acetonitrile/water) to
afford the N-Boc-glutamylanilide-tert-butyl esters. The compounds were
transferred to vials followed by the addition of 2.0 mL of 4.0 M HCl in
dioxane. The reaction stirred at 40 °C for 4 h. The reactions were concentrated
under vacuum to afford the title compounds which were used without further
purification.
Biologically active compounds were also evaluated computa-
tionally in the open human ASCT2 model. The best scoring poses
for the most potent compounds identified demonstrated a compat-
ible fit with the human ASCT2 model and, interestingly, a tendency
to exhibit points of interaction with both the amino acid zwitterion
binding site and an adjacent hydrophobic pocket (Fig. 3).22
In summary, we report three novel Nc-glutamylanilides as
17. The compound was prepared according to the general procedure. 1H NMR
(400 MHz, CD3OD) d (ppm): 7.85 (d, J = 7.9 Hz, 1H); 7.62–7.50 (m, 3H); 4.19–
4.09 (m, 5H); 3.78–3.71 (m, 4H); 3.05–2.89 (m, 2H); 2.45–2.27 (m, 2H). 13C
NMR (100 MHz, CD3OD) d (ppm): 175.69; 171.37; 132.17; 132.07; 129.32;
127.35; 123.22; 73.56; 72.45; 62.18; 55.93; 53.24; 43.75; 32.65; 26.59.
19. Live-cell glutamine uptake assays featuring HEK293 cells were carried out in
96 well plates (CulturPlate-96, Perkin Elmer). Cells were plated at a density of
35,000 cells per well 24 h prior to carrying out the assay. Each set of conditions
was carried out in at least triplicate. For the assay, cells were washed three
inhibitors of cellular glutamine uptake via ASCT2 with modestly
greater potency than GPNA. Evaluation of this chemical series
within the context 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
anticipate that compounds with the greatest potency may interact
with multiple structural elements within the ASCT2 binding site,
including the amino acid zwitterion binding site and the adjacent
hydrophobic pocket. Ongoing efforts employing a combination of
these effects may lead to compounds with even greater potency.
times with 100 lL of assay buffer at pH 6.0 (containing 137 mM NaCl, 5.1 mM
KCl, 0.77 mM KH2PO4, 0.71 mM MgSO.47H2O, 1.1 mM CaCl2, 10 mM
D-glucose,
Uniquely, previous work in the Nc-glutamylanilide series sug-
and 10 mM HEPES). 3H-glutamine (500 nM) in the same buffer was added
concomitantly with inhibitor and allowed to incubate for 15 min at 37 °C.
Following incubation period, the 3H-glutamine/inhibitor is removed and the
cells were washed three times with buffer. The cells were then lysed by the
gested that reduction of the glutamine amide pKa was required
for ASCT2 inhibition;10 we did not observe this trend in our study.
Advances from these studies will be reported in due course.
addition of 50 lL 1 M NaOH. For reading, 150 lL of scintillation fluid
(Microscint 40, Perkin Elmer) was added and the plates were counted on a
scintillation counter (Topcount, Perkin Elmer). Fifty percent inhibitory
Acknowledgment
concentrations (IC50) were calculated (6 concentrations) using GraphPad
Prism version 6.01 for Windows, GraphPad Software, San Diego California
replicates.
The authors acknowledge research support from the Kleburg
foundation.
20. The compound was prepared according to the general procedure. 1H NMR
(400 MHz, CD3OD) d (ppm): 8.62 (d, J = 8.1 Hz, 1H); 8.12 (d, J = 8.1 Hz, 1H); 8.06
(d, J = 7.8 Hz, 1H); 8.01 (dd, J1 = 7.9 Hz, J2 = 1.4 Hz, 1H); 7.59 (td, J1 = 7.1 Hz,
J2 = 1.2 Hz, 1H); 7.52 (qd, J1 = 8.4 Hz, J2 = 1.5 Hz, 2H); 7.28 (td, J1 = 7.0 Hz,
J2 = 1.1 Hz, 1H); 4.16 (t, J = 6.5 Hz, 1H); 2.95–2.84 (m, 2H); 2.47–2.29 (m, 2H).
13C NMR (100 MHz, CD3OD) d (ppm): 171.05; 170.06; 168.39; 152.56; 137.00;
133.31; 131.55; 129.77; 126.62; 125.90; 123.87; 122.36; 121.42; 121.12;
119.91; 52.00; 32.95; 25.57.
References and notes
7. Kaira, K.; Sunrose, Y.; Arakawa, K.; Sunaga, N.; Shimizu, K.; Tominaga, H.;
Oriuchi, N.; Nagamori, S.; Kanai, Y.; Oyama, T.; Takeyoshi, I. Histopathology,
2014, accepted for publication.
21. The compound was prepared according to the general procedure. 1H NMR
(400 MHz, CD3OD)
d (ppm): 7.64 (d, J = 7.61, 1H); 7.56 (td, J1 = 7.6 Hz,
J2 = 1.1 Hz, 1H); 7.46–7.40 (m, 2H); 4.39 (s, 2H); 4.13 (t, J = 6.6 Hz, 1H); 4.05
(dd, J1 = 12.7 Hz, J2 = 2.4 Hz, 2H); 3.82 (t, J = 12.1 Hz, 2H); 3.42 (d, J = 12.3 Hz);
2.96–2.81 (m, 2H); 2.41–2.24 (m, 2H). 13C NMR (100 MHz, CD3OD) d (ppm):
173.34; 169.98; 136.88; 133.03; 131.06; 127.37; 127.19; 124.24; 63.59; 56.59;
51.89; 51.52; 51.48; 30.99; 25.26.
22. A model of an inhibitor-bound conformation of human ASCT2 was used as a
target for ligand docking of proposed compounds in the 2-substituted Nc-
glutamylanilide series using SurflexDock v.2.706 from Biopharmics (Jain et al. J.
Med. Chem. 2003, 46, 499–511) as implemented in Tripos’ SYBYL-X v2.1
(Certera, 1699 South Hanley Rd. St. Louis, MO 63144-2917; http://
for their potency in inhibition of the uptake of 3H-glutamine in a plate-based
assay. Compounds with potency values equal to or better than the inhibitor
GPNA were retained and assessed for their fit into the homology model of
human ASCT2 to facilitate design of further ligand series in an attempt to
discover structure activity relationships for development of potent inhibitors
of ASCT2-mediated transport of 3H-glutamine. Two-dimensional structures for
all ligands were generated in ChemDraw and imported into Tripos Sybyl for
conversion into three-dimensional structures using CONCORD and docking
using SurflexDock (referenced above). Figures for docked complexes were
generated and ray-traced using PyMol (The PyMOL Molecular Graphics System,
Version 1.5.0.4, Schrödinger, LLC.).