An efficient synthetic route tol-γ-methyleneglutamine and its amide derivatives, and their selective anticancer activity

Article abstract of DOI:10.1039/d0ra08249j

In cancer cells, glutaminolysis is the primary source of biosynthetic precursors, fueling the TCA cycle with glutamine-derived α-ketoglutarate. The enhanced production of α-ketoglutarate is critical to cancer cells as it provides carbons for the TCA cycle to produce glutathione, fatty acids, and nucleotides, and contributes nitrogens to produce hexosamines, nucleotides, and many nonessential amino acids. Efforts to inhibit glutamine metabolism in cancer using amino acid analogs have been extensive.l-γ-Methyleneglutamine was shown to be of considerable biochemical importance, playing a major role in nitrogen transport inArachisandAmorphaplants. Herein we report for the first time an efficient synthetic route tol-γ-methyleneglutamine and its amide derivatives. Many of thesel-γ-methyleneglutamic acid amides were shown to be as efficacious as tamoxifen or olaparib at arresting cell growth among MCF-7 (ER⁺/PR⁺/HER2^?), and SK-BR-3 (ER^?/PR^?/HER2⁺) breast cancer cells at 24 or 72 h of treatment. Several of these compounds exerted similar efficacy to olaparib at arresting cell growth among triple-negative MDA-MB-231 breast cancer cells by 72 h of treatment. None of the compounds inhibited cell growth in benign MCF-10A breast cells. Overall,N-phenyl amides andN-benzyl amides, such as3,5,9, and10, arrested the growth of all three (MCF-7, SK-BR-3, and MDA-MB-231) cell lines for 72 h and were devoid of cytotoxicity on MCF-10A control cells;N-benzyl amides with an electron withdrawing group at theparaposition, such as5and6, inhibited the growth of triple-negative MDA-MB-231 cells commensurate to olaparib. These compounds hold promise as novel therapeutics for the treatment of multiple breast cancer subtypes.

Full text of DOI:10.1039/d0ra08249j

RSC Advances
View Article Online
View Journal | View Issue
PAPER
An efficient synthetic route to L-g-
methyleneglutamine and its amide derivatives, and
Cite this: RSC Adv., 2021, 11, 7115
their selective anticancer activity†
Md Imran Hossain,a Ajit G. Thomas,b Fakhri Mahdi,a Amna T. Adam,a
a
b
Nicholas S. Akins,
Morgan M. Woodard,a Jason J. Paris,a Barbara S. Slusher
a
*
and Hoang V. Le
In cancer cells, glutaminolysis is the primary source of biosynthetic precursors, fueling the TCA cycle with
glutamine-derived a-ketoglutarate. The enhanced production of a-ketoglutarate is critical to cancer cells
as it provides carbons for the TCA cycle to produce glutathione, fatty acids, and nucleotides, and
contributes nitrogens to produce hexosamines, nucleotides, and many nonessential amino acids. Efforts
to inhibit glutamine metabolism in cancer using amino acid analogs have been extensive. L-g-
Methyleneglutamine was shown to be of considerable biochemical importance, playing a major role in
nitrogen transport in Arachis and Amorpha plants. Herein we report for the first time an efficient
synthetic route to L-g-methyleneglutamine and its amide derivatives. Many of these L-g-
methyleneglutamic acid amides were shown to be as efficacious as tamoxifen or olaparib at arresting
cell growth among MCF-7 (ER+/PR+/HER2ꢀ), and SK-BR-3 (ERꢀ/PRꢀ/HER2+) breast cancer cells at 24 or
72 h of treatment. Several of these compounds exerted similar efficacy to olaparib at arresting cell
growth among triple-negative MDA-MB-231 breast cancer cells by 72 h of treatment. None of the
compounds inhibited cell growth in benign MCF-10A breast cells. Overall, N-phenyl amides and N-
benzyl amides, such as 3, 5, 9, and 10, arrested the growth of all three (MCF-7, SK-BR-3, and MDA-MB-
231) cell lines for 72 h and were devoid of cytotoxicity on MCF-10A control cells; N-benzyl amides with
an electron withdrawing group at the para position, such as 5 and 6, inhibited the growth of triple-
negative MDA-MB-231 cells commensurate to olaparib. These compounds hold promise as novel
therapeutics for the treatment of multiple breast cancer subtypes.
Received 26th September 2020
Accepted 1st February 2021
DOI: 10.1039/d0ra08249j
rsc.li/rsc-advances
organs.1 Many of these functions are done via the formation of
glutamate from glutamine. In cancer cells, glutaminolysis is the
1. Introduction
primary source of biosynthetic precursors, fueling the TCA cycle
with glutamine-derived a-ketoglutarate.3,4 The enhanced
production of a-ketoglutarate is critical to cancer cells as it
provides carbons for the citric acid cycle to produce glutathione,
fatty acids, and nucleotides, and contributes nitrogens to
produce hexosamines, nucleotides, and many nonessential
amino acids.5
Efforts to inhibit glutamine metabolism in cancer using
amino acid analogs have been extensive.6 There are a number of
naturally occurring glutamine analogues, such as azaserine,6
acivicin,6 and 6-diazo-5-oxo-L-norleucine (DON)7 (Fig. 1), that
are inhibitors of glutaminase,8,9 NAD synthase,10 CTP synthe-
tase,11 FGAR aminotransferase,12 and many other glutamine-
dependent enzymes.5 These compounds have been shown to
suppress the growth of a variety of tumors and demonstrate
their activity in some clinical trials.6,7 However, they have also
demonstrated variable degrees of gastrointestinal toxicity,
myelosuppression, and neurotoxicity, due to their non-
selectivity. In recent years, considerable interest has focused
While glucose is the primary nutrient for the maintenance and
promotion of cell function, glutamine and glutamate are
considered to be equally important.1 Glutamine participates in
numerous functional activities in cells, including being
a substrate for protein synthesis, ureogenesis in the liver, and
for hepatic and renal gluconeogenesis.1 Glutamine has been
shown to be a precursor for neurotransmitter synthesis,
nucleotide and nucleic acid synthesis, and glutathione
production.2 In addition, glutamine is an oxidative fuel for the
immune system, a major source of nitrogen for purine and
pyrimidine biosynthesis, and a nitrogen transporter between
aDepartment of BioMolecular Sciences, Research Institute of Pharmaceutical Sciences,
School of Pharmacy, University of Mississippi, Mississippi 38677, USA. E-mail: hle@
olemiss.edu
bJohns Hopkins Drug Discovery, Department of Neurology, Johns Hopkins University
School of Medicine, Baltimore, MD 21205, USA
† Electronic supplementary information (ESI) available: More experimental
procedures, NMR spectroscopy, and cell assay data. See DOI: 10.1039/d0ra08249j
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 7115–7128 | 7115

View Article Online
RSC Advances
Paper
playing a major role in nitrogen transport in Arachis and
Amorpha plants.18,19 Compound 2 was later shown to exhibit
strong central nervous system (CNS) inhibitory activity20 as well
as to be 10 times more potent than L-glutamate as a depolarizing
agent on the newborn rat spinal cord.21 The syntheses of
racemic mixture of 1 and racemic mixture of 2 were reported in
1955 (Scheme 1),13,14 starting from diethyl 1-formamidobut-3-
yne-1,1-dicarboxylate (11) that had been prepared in advance
from another publication.22 The synthesis of the biological
relevant isomer 2 was reported several times in the literature by
a reaction of homochiral aziridine-2-carboxylates with stabi-
lized Wittig reagents,23 a 15-step synthesis starting from N-Boc-
L-aspartate acid g-benzyl ester,21 a reduction of the enaminone
product of the reaction between tert-butyl-N-Boc-pyroglutamate
and the Bredereck reagent,24 or a synthetic route via a reaction
between the lithium lactam enolate of ethyl N-Boc-
pyroglutamate and the Eschenmoser's salt.25 In addition, the
synthesis of C3-deuterium-labeled L-g-methyleneglutamic acid
2 from L-pyroglutamic acid was also reported.26 However, no
synthesis of L-g-methyleneglutamine 1, which is the biological
relevant isomer, nor further research on its biological activity
has been reported. Herein we report an efficient synthetic route
to L-g-methyleneglutamine (1) and its amide derivatives (3–10,
Fig. 2). These compounds were evaluated for their anticancer
activity on three different breast cancer cell lines: MCF-7 (ER+/
PR+/HER2ꢀ), SK-BR-3 (ERꢀ/PRꢀ/HER2+), and triple negative
MDA-MB-231. The compounds were also evaluated for their
activity on a non-cancerous breast cell line, MCF-10A, as
a control.
Fig.
derivatives.
1 Structures of important naturally occurring glutamine
2. Results and discussion
2.1. Synthesis of L-g-methyleneglutamine and its amide
Scheme 1 Synthesis of racemic mixture of 1 and racemic mixture of
2.13,14
derivatives
In our initial attempt to synthesize L-g-methyleneglutamine (1),
we decided to base part of the synthetic route on the previously
reported synthesis of the racemic mixture of 1,13,14 going
through the formation of the corresponding phthalimido-
anhydride 15 (Fig. 3). We started the synthetic route with the
commercially available L-pyroglutamic acid (12). The carboxylic
acid and amide groups of 12 were rst protected by ethyl ester
and Boc group, respectively. Then, the methylene group was
introduced at C4 via a a-methylenation of the sterically
hindered carbonyl group27 to give 13. Hydrolysis of 13 with
LiOMe opened the lactam ring; however, an esterication also
on the discovery of new agents that selectively target glutamine-
consuming processes, as well as the development of methods
directed at specic nodes of glutamine metabolism.
L-g-Methyleneglutamine (1) and L-g-methyleneglutamic acid
(2) (Fig. 1) were rst isolated from groundnut seedling (Arachis
hypogaea) in 1952.13,15 These compounds were also later found
in several other quite unrelated species, including tulip bulbs
(Tulipa gerneriana),16 hops,17 and Amorpha fruticosa (a species of
owering plant in the legume family, Fabaceae).18 Both 1 and 2
were shown to be of considerable biochemical importance,
Fig. 2 Structures of L-g-methyleneglutamine (1) and its amide derivatives (3–10).
7116 | RSC Adv., 2021, 11, 7115–7128
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
Fig. 3 Our original synthetic plan for L-g-methyleneglutamine (1).
occurred at the newly formed acid group and gave us 14.
Hydrolysis of 14 with 9 M HBr resulted in 2 in a mixture with
other side products that were very difficult to purify 2 from. We
then used the crude mixture of 2 to react with phthalic anhy-
dride to try to synthesize the phthalimido-anhydride 15. Despite Scheme 3 Synthesis of L-g-methyleneglutamine amide derivatives 3–
8.
our effort with many different reaction conditions, the forma-
tion of 15 was not detected. Therefore, our initial synthetic plan
to synthesize 1, which is shown in Fig. 3, was abandoned.
(3, 9, 10) and non-aromatic secondary amides (4, 5, 6, 7) to
tertiary amide (8), was quickly generated using the common
intermediate 19 and the corresponding amines. The amide
coupling protocol was observed to accommodate a wide range
of functional groups. Protected amides 22–29 were synthesized
via the published amide coupling protocol29 using 19 and the
corresponding amines (21) (Scheme 3). The removal of the tert-
butyl and Boc protecting groups using TFA was less accommo-
dating. While the desired deprotected amides 3–8 were ob-
tained in good yields from treating the corresponding 22–27
with TFA (Scheme 3), the desired deprotected 4-substituted
phenyl amides 9–10 were not formed. Upon treating with TFA,
the corresponding 28–29 quickly underwent cyclization,
producing 30 instead (Scheme 4). This cyclization turned out to
be an example that follows the 5-exo-trig of Baldwin's rules for
ring closure reactions.30,31 When a milder acid, H3PO4, was used,
some 9–10 were detected by LC-MS, but 30 was still the major
product. When a neutral deprotection condition, ZnBr2 in DCM,
was used, the desired deprotected amides 9–10 were obtained in
We then decided to utilize an installment of the amide group
directly from the corresponding carboxylic acid using an amide
coupling protocol in our synthetic route to 1. We also decided to
utilize tert-butyl group as the protecting group for the carboxylic
acid, so that we can use an acid to remove it later, instead of
a base. We were successful in synthesizing 1 from the
commercially available L-pyroglutamic acid (12) (Scheme 2). The
carboxylic acid and amide groups of 12 were rst protected by
tert-butyl ester and Boc group, respectively, giving 17. Then, the
methylene group was introduced at C4 via a a-methylenation of
the sterically hindered carbonyl group27 to give 18 in 66% yield.
The cyclic amide ring was selectively opened with LiOH28 to
afford the common intermediate 19 in 70% yield. Amide was
then installed to 19 from ammonium chloride using a pub-
lished amide coupling protocol,29 resulting in 20 in 85% yield.
Treatment of 20 with TFA removed both the tert-butyl and Boc
protecting groups, affording the desired L-g-methyleneglut-
amine 1 in 80% yield.
In a similar fashion, a library of L-g-methyleneglutamine
amide derivatives (3–10, Fig. 2), varying from aromatic amides
Scheme 2 Synthesis of L-g-methyleneglutamine 1.
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 7115–7128 | 7117

View Article Online
RSC Advances
Paper
glutamine uptake.32 Olaparib was chosen because it is one of
a few FDA-approved chemotherapeutics for triple-negative
breast cancer.
When assessed in MCF-7 cells, six compounds exerted
a signicant inhibition of cell growth lasting at least 72 h, apart
from tamoxifen and olaparib: 3, 5, 7, 8, 9, and 10 (Fig. 4A–A0).
Compared to vehicle-treated cells, compounds 5 and 9 signi-
cantly inhibited MCF-7 growth at any concentration assessed at
72 h (0.32 mM–320 mM; p ¼ 0.0002–0.05; Fig. 4A0). Similarly,
compound 3 signicantly inhibited MCF-7 growth by 72 h when
administered at 1 mM or any greater concentration (with the
exception of 100 mM; p ¼ 0.0008–0.05; Fig. 4A0). Compounds 7,
8, and 10 exerted concentration-dependent effects to inhibit
MCF-7 growth at 72 h (7 inhibiting only at 10 mM, 8 inhibiting
only at 10 and 100 mM, and 10 inhibiting only at 320 mM; p ¼
0.02–0.05; Fig. 4A0). Only compounds 7 and 10 exerted early
inhibition of MCF-7 growth as measured at 24 h (p ¼ 0.03–0.05;
Fig. 4A). When compared to positive controls, compounds 5, 9,
and 10 exerted potency that did not differ from tamoxifen or
olaparib at 72 h (Table 1). Conversely, compounds 1, 3, 4, and 6
were signicantly less potent than tamoxifen (p ¼ 0.0008–0.02),
and compounds 7 and 8 were less potent than tamoxifen or
olaparib at 72 h (p ¼ 0.01–0.04; Table 1). No signicant differ-
ences in potency were observed at 24 h (Table 1).
All compounds exerted an apparent broad capacity to inhibit
growth in SK-BR-3 cells (Fig. 4B–B0), but this was driven by
cytotoxicity for most compounds (including the positive
control, tamoxifen as described in Section 2.3. below). By 72 h,
compounds 4, 7, and 8 signicantly inhibited SK-BR-3 growth at
any concentration assessed (0.32 mM–320 mM; p < 0.0001–0.02;
Fig. 4B0). Similarly, compound 3, 6, 9, and 10 signicantly
inhibited SK-BR-3 growth at all but the lowest concentration
assessed (1 mM–320 mM; p < 0.0001–0.04; Fig. 4B0). Compound 5
signicantly inhibited growth at 3.2 mM or greater concentra-
tions (p < 0.0001), and 1 signicantly inhibited growth at 10 mM
or greater concentrations (p ¼ 0.0002; Fig. 4B0). All compounds
showed the capacity for early inhibition of growth at 24 h (p ¼
0.01–0.05) with the exceptions of 4 and 5 (Fig. 4B). In compar-
ison to positive controls, compounds 5, 6, and 9 exerted potency
that did not differ from tamoxifen or olaparib at 24 h (Table 1),
all other compounds were signicantly less potent than
tamoxifen and/or olaparib at this time-point (p ¼ 0.002–0.04;
Table 1). However, by 72 h, all compounds were equipotent to
tamoxifen (except 1 which was less potent; p < 0.0001), and
compounds 3, 4, 7, 8, and 10 were equipotent to olaparib (all
others were signicantly less potent; p < 0.0001–0.04; Table 1).
As expected, the triple-negative MDA-MB-231 cell line
provoked the greatest variance in compound effectiveness
(Fig. 4C–C0) and potency (Table 1). Aer 24 h, only the highest
concentration (320 mM) of compounds 1, 4, 7, 8, 9, and 10
signicantly inhibited MDA-MB-231 growth (p ¼ 0.009–0.04;
Fig. 4C). Several concentrations of 6 signicantly inhibited
growth at this time-point (3.2, 32, and 320 mM; p ¼ 0.0003–0.02;
Fig. 4C). Neither 3 nor 5 signicantly inuenced MDA-MB-231
Scheme 4 Deprotection of compounds 28 and 29 via acidic and
neutral conditions. The formation of 30 turned out to be an example
that follows the 5-exo-trig of Baldwin's rules for ring closure reactions.
11–13% yields aer purication via both silica gel ash column
and HPLC.
We suspected that the cyclization of 28 and 29 upon depro-
tection in acidic conditions was due to the electron withdrawing
effect of the substituted phenyl groups. We decided to make the
corresponding protected amide with 3,5-dichloroaniline (31,
Scheme 5) to test its cyclization in a neutral deprotection
condition. However, only the cyclized product 30 was obtained.
The desired amide 31 was detected in trace amount and
observed to be very unstable. It is very likely that 31 was formed
from the coupling reaction, but then quickly underwent cycli-
zation, even in the coupling reaction condition. We then tried
the reaction again with several other coupling reaction condi-
tions using different amide coupling reagents, including HATU,
EDC, HOBt. Similar results were observed with 30 being the
major product and 31 being detected only in trace amounts.
2.2. Evaluation of L-g-methyleneglutamine and its amide
derivatives for their anticancer activity
In order to assess their capacity for inhibition of tumor growth,
compounds 1 and 3–10 were screened for 24 h and 72 h growth
of MCF-7 (ER+/PR+/HER2ꢀ), SK-BR-3 (ERꢀ/PRꢀ/HER2+), and
MDA-MB-231 (triple negative) breast cancer cell lines. Inhibi-
tion of cell growth was assessed as the proportional change
from vehicle-treated cells (negative controls) (Fig. 4 and Table
1). Compound potency was also assessed in comparison to
tamoxifen and olaparib (positive controls). Tamoxifen was
chosen given its capacity to suppress the proliferation of not
only ER+ cancers, but also ER-negative cells via the inhibition of
growth aer only 24
h (Fig. 4C). However, by 72 h,
compounds 4 and 7 signicantly inhibited MDA-MB-231 growth
at 10 mM, 100 mM, or 320 mM concentrations (p ¼ 0.009–0.04),
Scheme 5 Result of the coupling reaction between 19 and 3,5-
dichloroaniline.
7118 | RSC Adv., 2021, 11, 7115–7128
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
Fig. 4 Proportional change from vehicle-treated MCF-7 (A–A0), SK-BR-3 (B–B0), MDA-MB-231 (C–C0), or MCF-10A (D–D0) cells after 24 h (left)
or 72 h (right) exposure to a concentration-response regimen (0.32–320 mM) of compounds 1, 3–10, or positive controls (tamoxifen or olaparib).
Dotted line indicates vehicle-treated control values. * significantly different from vehicle-treated control, p # 0.05.
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 7115–7128 | 7119

View Article Online
RSC Advances
Paper
Table 1 Concentrations that produced half-maximal inhibition of cell growth [log(IC50 [mM]) ꢁ SEM] after 24 h or 72 h exposure to a dose–
response regimen (0.32–320 mM) of compounds 1, 3–10, or positive controls (tamoxifen or olaparib). Significant leftward shifts are indicated with
bold font
Growth inhibition [log(IC50 [mM]) ꢁ SEM]
MCF-7
24 h
SK-BR-3
24 h
MDA-MB-231
24 h
MCF-10A
24 h
72 h
72 h
72 h
72 h
Tamoxifen
Olaparib
1
3
4
5
6
7
2.46 ꢁ 0.24
>2.50
1.62 ꢁ 0.14
2.03 ꢁ 0.22
2.79 ꢁ 0.39a
2.40 ꢁ 0.18a
2.29 ꢁ 0.22a
1.93 ꢁ 0.24
2.73 ꢁ 0.36a
2.62 ꢁ 0.24a,b
2.78 ꢁ 0.28a,b
1.88 ꢁ 0.21
2.32 ꢁ 0.22
ꢀ0.06 ꢁ 0.25
0.21 ꢁ 0.27
0.24 ꢁ 0.19
1.73 ꢁ 0.12
1.62 ꢁ 0.13
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
0.02 ꢁ 0.19
1.05 ꢁ 0.16a,b
0.22 ꢁ 0.19
2.21 ꢁ 0.15
2.19 ꢁ 0.14
2.66 ꢁ 0.30
3.03 ꢁ 0.42
3.41 ꢁ 0.94
2.77 ꢁ 0.21
2.66 ꢁ 0.21
2.36 ꢁ 0.24
2.64 ꢁ 0.28
2.62 ꢁ 0.25
2.60 ꢁ 0.25
0.90 ꢁ 0.23a,b
1.57 ꢁ 0.29a
1.87 ꢁ 0.36a,b
0.59 ꢁ 0.32
2.15 ꢁ 0.19
2.77 ꢁ 0.17a,b
2.91 ꢁ 0.18a,b
2.81 ꢁ 0.12a,b
2.53 ꢁ 0.18a,b
2.57 ꢁ 0.18a,b
2.97 ꢁ 0.15a,b
2.91 ꢁ 0.14a,b
2.62 ꢁ 0.12a,b
2.85 ꢁ 0.15a,b
2.89 ꢁ 0.22a,b
2.68 ꢁ 0.14a,b
2.54 ꢁ 0.29a
2.34 ꢁ 0.17a
2.81 ꢁ 0.15a,b
2.84 ꢁ 0.15a,b
2.81 ꢁ 0.16a,b
2.94 ꢁ 0.16a,b
ꢀ0.25 ꢁ 0.28
0.65 ꢁ 0.16b
0.50 ꢁ 0.18b
ꢀ0.24 ꢁ 0.28
ꢀ0.21 ꢁ 0.22
0.68 ꢁ 0.22b
0.60 ꢁ 0.21
1.11 ꢁ 0.27
2.50 ꢁ 0.35a,b
2.13 ꢁ 0.30a,b
1.06 ꢁ 0.27
8
9
10
1.90 ꢁ 0.27a,b
a
Signicantly different from tamoxifen. b Signicantly different from olaparib, p # 0.05.
and compounds 5, 6, 8, 9, and 10 caused signicant inhibition tamoxifen and/or olaparib, 9 was signicantly more potent at
at the highest concentration (320 mM; p ¼ 0.003–0.04; Fig. 4C0). 24 h and 72 h (p ¼ 0.03–0.05), whereas 1, 3, and 6 were signif-
Compounds 1 and 3 inhibited MDA-MB-231 growth only at the icantly more potent only at 72 h (p ¼ 0.01–0.04; Table 2).
100 mM concentration (p ¼ 0.02–0.04; Fig. 4C0). Compared to Compounds 4 and 7 were less potent than olaparib at
olaparib, only 1 was equipotent when assessed at 24 h, all other promoting 72 h MCF-7 cytotoxity (p ¼ 0.0003–0.009; Table 2).
compounds exerted signicantly reduced potency at 24 h or 72 h
(p < 0.001–0.005; Table 1).
The greatest observations of cytotoxicity occurred in SK-BR-3
cells and were found in response to most compounds
As a control, all compounds were assessed against benign (including the control compound, tamoxifen). In SK-BR-3 cells,
MCF-10A breast cells (Fig. 4D–D0). None of the compounds some early concentration-dependent increases in cytotoxicity
assessed signicantly inhibited the growth of MCF-10A cells were observed for compounds 3 (100 mM; p ¼ 0.04), 6 (3.2 and 32
(Fig. 4D–D0; Table 1). However, signicant increases were mM; p ¼ 0.03–0.05), 7 (100 mM; p ¼ 0.05), and olaparib (10 mM; p
observed at 24 h with compounds 1, 5, and 6 (p ¼ 0.0005–0.04; ¼ 0.01) compared to 24 h vehicle treatment (Fig. 5B). Moreover,
Fig. 4D) as well as at 72 h with compounds 1, 3, 5, 7, 8, and 10 (p several compounds demonstrated signicantly more cytotoxic
¼ 0.001–0.05; Fig. 4D0).
potency aer 24 h than did tamoxifen or olaparib, including 1,
3, 4, 6, 8, and 9 (p < 0.0001–0.001; Table 2). Aer 72 h, all
compounds with the exceptions of
7 and 8 exerted
2.3. Evaluation of L-g-methyleneglutamine and its amide
derivatives for their cytotoxic activity
concentration-dependent increases in cytotoxicity, including 1
(10–320 mM; p ¼ 0.0002–0.002), 3 (1–320 mM; p ¼ 0.0006–0.04), 4
(0.32–10, 100–320 mM; p ¼ 0.002–0.05), 5 (10–320 mM; p ¼ 0.002–
0.04), 6 (0.32–100 mM; p < 0.0001–0.02), 9 (3.2–320 mM; p ¼
0.005–0.05), 10 (1–320 mM; p ¼ 0.003–0.04), tamoxifen (3.2, 32–
100 mM; p ¼ 0.002–0.03), and olaparib (3.2–3.2, 100–320 mM; p <
0.0001–0.02; Fig. 5B0). However, only 1 exerted more cytotoxic
potency than tamoxifen or olaparib (p ¼ 0.001–0.02; Table 2).
When assessed in MDA-MB-231 cells aer 24 h, all
compounds with the exceptions of 3, 8, and tamoxifen exerted
some concentration-dependent toxicity including 1 (32 and 320
mM; p ¼ 0.002–0.05), 4 (320 mM; p ¼ 0.02), 5 (320 mM; p ¼ 0.01), 6
(320 mM; p ¼ 0.02), 7 (320 mM; p ¼ 0.05), 9 (10 mM; p ¼ 0.03), 10
(32 mM; p ¼ 0.04), and olaparib (320 mM; p ¼ 0.009; Fig. 5C).
Compound 1 exerted signicantly more potent cytotoxic effects
than did olaparib (p ¼ 0.05), whereas all other compounds were
less potent than tamoxifen and/or olaparib (p < 0.0001–0.04;
Table 2). By 72 h, the highest concentrations of compounds 3
(100 mM; p ¼ 0.04), 5 (320 mM; p ¼ 0.001), 6 (320 mM; p ¼ 0.02), 9
(320 mM; p ¼ 0.003), tamoxifen (100–320 mM; p ¼ 0.0004–0.004),
To assess whether cytotoxicity contributed to reductions in cell
growth, positive controls and compounds were screened for
viability at 24 h and 72 h using the necrosis indicator, propi-
dium iodide (Fig. 5; Table 2).
Compared to vehicle-treated MCF-7 cells, compounds 4 (10
mM; p ¼ 0.05), 5 (32 mM; p ¼ 0.05), and 7 (1, 3.2, and 100 mM; p ¼
0.03–0.04) exerted some early concentration-dependent toxicity
when assessed at 24 h (Fig. 5A). Aer 72 h, compounds 7 (0.32
and 10 mM; p ¼ 0.006–0.009), 8 (1–10 and 100 mM; p ¼ 0.004–
0.04), 9 (32–100 mM; p ¼ 0.02–0.04), and olaparib (3.2–10 and
100 mM; p ¼ 0.007–0.02) signicantly increased toxicity at
multiple concentrations (Fig. 5A0). Only the highest concentra-
tion (320 mM) of compounds 1, 3, 4, 6, and 10 caused a signi-
cant increase in cytotoxicity to MCF-7 cells (p ¼ 0.01–0.05;
Fig. 5A0). Of note, the 100 mM concentration of tamoxifen was
signicantly more toxic to MCF-7 cells than vehicle (p ¼ 0.03;
Fig. 5A0). No signicant increase in cytotoxicity was observed for
compound 5 at 72 h. When compared to the cytotoxic potency of
7120 | RSC Adv., 2021, 11, 7115–7128
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
Fig. 5 Proportional increase from vehicle-treated MCF-7 (A–A0), SK-BR-3 (B–B0), MDA-MB-231 (C–C0), or MCF-10A (D–D0) cells after 24 h (left)
or 72 h (right) exposure to a concentration-response regimen (0.32–320 mM) of compounds 1, 3–10, or positive controls (tamoxifen or olaparib).
* significantly different from vehicle-treated control, p # 0.05.
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 7115–7128 | 7121

View Article Online
RSC Advances
Paper
Table 2 Concentrations that produced half-maximal effect for cellular necrosis [log(EC50 [mM]) ꢁ SEM] after 24 h or 72 h exposure to a dose–
response regimen (0.32–320 mM) of compounds 1, 3–10, or positive controls (tamoxifen or olaparib). Significant leftward shifts are indicated with
bold font
Cytotoxicity [log(EC50 [mM]) ꢁ SEM]
MCF-7
24 h
SK-BR-3
24 h
MDA-MB-231
24 h
MCF-10A
24 h
72 h
72 h
72 h
72 h
Tamoxifen
Olaparib
1
3
4
5
6
7
>2.50
>2.50
2.46 ꢁ 0.20
1.42 ꢁ 0.29
0.47 ꢁ 0.37a
0.31 ꢁ 0.33a
2.34 ꢁ 0.18b
1.74 ꢁ 0.33
1.31 ꢁ 0.29a
>2.50b
2.30 ꢁ 0.31
0.36 ꢁ 0.28a,b
1.97 ꢁ 0.30
>2.50
>2.50
ꢀ0.48 ꢁ 0.52
ꢀ1.22 ꢁ 1.05
0.31 ꢁ 0.36a,b
>2.50
1.42 ꢁ 0.32
1.79 ꢁ 0.32
0.95 ꢁ 0.29b
>2.50a,b
1.49 ꢁ 0.17
1.99 ꢁ 0.19
>2.50a,b
2.43 ꢁ 0.12
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
>2.50
2.18 ꢁ 0.39
ꢀ0.73 ꢁ 1.28a,b
ꢀ0.71 ꢁ 0.75a,b
ꢀ1.01 ꢁ 1.73a,b
>2.50
>2.50
>2.50a,b
>2.50
>2.50
>2.50
1.81 ꢁ 0.35
2.29 ꢁ 0.32
2.20 ꢁ 0.25a,b
2.31 ꢁ 0.28
>2.50
>2.50a,b
>2.50a,b
ꢀ0.41 ꢁ 1.47
ꢀ0.36 ꢁ 0.88
>2.50
2.42 ꢁ 0.25a,b
2.18 ꢁ 0.34a
>2.50a,b
2.42 ꢁ 0.19a,b
2.43 ꢁ 0.19a,b
>2.50a,b
ꢀ0.47 ꢁ 0.57a,b
2.44 ꢁ 0.23
ꢀ1.14 ꢁ 1.38a,b
ꢀ0.74 ꢁ 0.87a,b
2.42 ꢁ 0.32
8
9
10
>2.50
>2.50a,b
>2.50a,b
ꢀ1.22 ꢁ 1.58
>2.50a,b
>2.50a,b
>2.50
>2.50
>2.50
>2.50a,b
>2.50a,b
a
Signicantly different from tamoxifen. b Signicantly different from olaparib, p # 0.05.
and olaparib (320 mM; p ¼ 0.003) were more cytotoxic than without pre-incubation (2 h) with the enzyme. This result sug-
vehicle (Fig. 5C0). However, compounds 1 (10–320 mM; p ¼ gested that these compounds may follow a different mechanism
0.001–0.04), 7 (10–32 and 320 mM; p ¼ 0.0004–0.04), and 8 (10 to that of azaserine, acivicin, and DON in suppressing the
mM; p ¼ 0.03) demonstrated greater toxicity at lower concen- growth of MCF-7, SK-BR-3, and MDA-MB-231 breast cancer
trations (Fig. 5C0). Compounds 4 and 10 did not increase toxicity cells, which is good news as azaserine, acivicin, and DON failed
(Fig. 5C0). When compared to positive controls for potency, all to advance in clinical trials. One possible biological target of
compounds exerted less potent cytotoxicity than did tamoxifen these L-g-methyleneglutamic acid amides is glutamine trans-
or olaparib at this time-point (p < 0.0001–0.03; Table 2).
porters, rather than glutamine-dependent enzymes. Recent
Our criteria for identifying leads required that efficacious studies have highlighted the importance of glutamine trans-
concentrations occur without cytotoxicity. Despite the cytotoxic porters in physiology and cancer cell growth.33,34 Given that
effects observed on cancer cell lines, compounds exerted far less these compounds are unsaturated amides, which are signi-
toxicity when assessed on benign MCF-10A cells (Fig. 5D–D0). cantly less reactive than typical Michael acceptors like unsatu-
Only compounds 4 (0.32–3.2 mM; p ¼ 0.006–0.03) and 5 (320 mM; rated ketones, and that these compounds did not inhibit the
p ¼ 0.001) exerted any cytotoxicity aer 24 h of treatment, as did activity of GLS1 with pre-incubation, and that these compounds
tamoxifen (100–320 mM; p ¼ 0.002–0.005) and olaparib (320 mM; did not signicantly inhibit the growth and exerted far less
p ¼ 0.02; Fig. 5D). Aer 72 h, compounds 1 (3.2 mM; p ¼ 0.03), 5 toxicity when assessed on benign MCF-10A cells, it is very likely
(320 mM; p ¼ 0.005), 10 (100 mM; p ¼ 0.05), tamoxifen (32–320 that these compounds would act as reversible inhibitors of
mM; p ¼ 0.007–0.02), and olaparib (320 mM; p ¼ 0.04) exerted glutamine transporters, rather than irreversible inhibitors.
signicant toxicity compared to vehicle (Fig. 5D0). Future experiments will aim to further characterize lead
Concentration-response shis could not be calculated given compounds to determine their mechanism(s) of action.
that all EC50s exceeded 2.5 logarithmic units (Table 2).
3. Conclusion
2.4. Evaluation of L-g-methyleneglutamine and its amide
derivatives for their possible inhibitory activity on
glutaminase
We have reported for the rst time an efficient synthetic route to
the biologically-relevant L-g-methyleneglutamine (1) and its
As mentioned earlier, azaserine, acivicin, and DON failed to amide derivatives (3–10). These compounds were evaluated for
advance in clinical trials due to their non-selectivity on their anticancer activity on three different breast cancer cell
glutamine-dependent enzymes. We were wondering whether lines: MCF-7 (ER+/PR+/HER2ꢀ), SK-BR-3 (ERꢀ/PRꢀ/HER2+), and
the suppression activity of these compounds on the growth of triple negative MDA-MB-231. The compounds were also evalu-
MCF-7, SK-BR-3, and MDA-MB-231 breast cancer cell lines fol- ated for their activity on a benign cell line, MCF-10A, as
lowed a similar mechanism to that of azaserine, acivicin, and a control. For MCF-7 cancer cells, L-g-methyleneglutamic acid
DON. These compounds were tested for their potential inhibi- amides with secondary amine, like 8, and branched alkyl amine,
tory activity on glutaminase, the enzyme that catalyzes the like 7, are less potent in inhibiting the growth than amides with
conversion of glutamine in the cytosol to glutamate in the primary amine, like 4, 5, and 6, and aromatic amine, like 3, 9,
mitochondria. However, these compounds did not show any and 10. Within each subset, the amines with a stronger electron
inhibitory activity on kidney-type glutaminase (GLS1), with or withdrawing group exhibit better potency. For example, 5 and 6
7122 | RSC Adv., 2021, 11, 7115–7128
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
1
with a uoro and a nitro groups, respectively, at the para posi- referenced to CDCl3 (7.26 ppm for H NMR and 77.16 ppm for
tion of the aromatic ring are more potent than 4. Similarly, 9 is 13C NMR), MeOD (3.31 ppm for 1H NMR and 49.00 ppm for 13
C
more potent than 10, which is more potent than 3. Amides with NMR), or D2O (4.79 ppm for H NMR). 19F NMR spectra were
aromatic amine are more potent than primary amine; for recorded on a Bruker-400 spectrometer. Coupling constants (J)
example 9 is more potent than 5. Notably, compounds 5 and 9 are in Hz. The following abbreviations were used to designate
carry a uorine atom at the para position of the aromatic ring, the multiplicities: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼
and they are the most potent compounds of the series. This may quartet, quint ¼ quintuplet, m ¼ multiplet, br ¼ broad. Melting
be due to the hydrophobic nature of uorine that enhances the points were measured using an OptiMelt automated melting
binding and its potential formation of special interactions with point system. LC-MS were measured using an ACQUITY-Waters
the biological target. These structure–activity relationships micromass (ESCi) system. High-resolution mass spectra
(SARs) are generally also true for the SK-BR-3 and MDA-MB-231 (HRMS) were measured using a Synapt Q-TOF ESI-MS.
cancer cells. Overall, N-phenyl amides and N-benzyl amides,
1
such as 3, 5, 9, and 10, exerted concentration-dependent inhi-
bition of growth by 72 h across all three cancer cell lines.
Additionally, N-benzyl amides with an electron withdrawing
group at the para position, such as 5 and 6, were the only
compounds to inhibit the growth of triple-negative MDA-MB-
231 cells commensurate to olaparib. Unlike azaserine, acivi-
cin, and DON, these compounds did not inhibit GLS1, sug-
gesting that they may follow a different mechanism to that of
azaserine, acivicin, and DON in exerting commensurate cyto-
toxic effects to cancer cell lines, which is good news as azaser-
ine, acivicin, and DON failed to advance in clinical trials. One
possible biological target of these L-g-methyleneglutamic acid
amides is glutamine transporters, rather than glutamine-
dependent enzymes. In support, these amides were largely
devoid of cytotoxicity in benign MCF-10A cells and the modest
toxicity observed at the highest concentrations of 5 and 10 did
not reach that produced by tamoxifen or olaparib. Notably,
some compounds demonstrated an apparent reverse dose-
dependency (exerting greater effects at lower dosing, such as 4
and 7; Fig. 5A–A0). Given that the pharmacodynamic proles are
not yet known, it cannot be ruled out whether greater dosing
produces an upregulation of biological targets, thereby
reducing the efficacy of some compounds at higher
concentrations.
4.2. Synthetic procedures
4.2.1. Di-tert-butyl-(S)-4-methylene-5-oxopyrrolidine-1,2-
dicarboxylate (18). Compound 17 (3.0 g, 10.5 mmol, see the ESI†
for preparation procedure) was dissolved in anhydrous THF (10
mL) under argon and cooled to ꢀ78 ꢂC. LHMDS 1 M solution in
THF (25 mL) was added to the reaction mixture slowly. The
reaction was stirred for 30 min at ꢀ78 ꢂC and then added 2,2,2-
triuoroethyl 2,2,2-triuoroacetate (2.47 g, 12.6 mmol). The
reaction mixture was stirred at ꢀ78 ꢂC for 1.5 h then quenched
with saturated NH4Cl solution. The crude mixture was extracted
twice with dichloromethane. The organic layer was dried over
sodium sulfate and evaporated in vacuo. The crude intermediate
mixture was used for the next step without further purication.
The crude intermediate mixture was dissolved in anhydrous
benzene (80 mL) and was added K2CO3 (3.97 g, 28.75 mmol),
paraformaldehyde (3.5 g), and 18-crown-6 (414 mg, 1.57 mmol)
under argon condition. The reaction mixture was heated at
ꢂ
60 C for 2 hours until the reaction was complete. The solids
were ltered off and the solvents were evaporated. The crude
mixture was puried using silica column (33% ethyl acetate in
hexane) to afford 18 (2.08 g, 66% yield) as a viscous colorless gel.
1H NMR (400 MHz, CDCl3) d 6.16 (d, J ¼ 2.9 Hz, 1H), 5.45 (t, J ¼
2.5 Hz, 1H), 4.43 (dd, J ¼ 10.0, 3.1 Hz, 1H), 2.99 (ddt, J ¼ 17.5,
10.1, 3.0 Hz, 1H), 2.69–2.53 (m, 1H), 1.44 (d, J ¼ 25.6 Hz, 18H);
13C NMR (101 MHz, CDCl3) d 169.9, 165.5, 149.8, 136.8, 120.4,
83.4, 82.3, 56.3, 27.9, 27.9, 27.8; HRMS: m/z calcd for
These compounds hold promise for the development of
novel anticancer therapeutics given their capacity for cancer-
selective necrosis across breast cancer subtypes. Future experi-
ments will aim to further characterize lead compounds to
determine their mechanism(s) of action, including their
capacity to arrest the cell cycle or promote apoptosis, as well as
their pharmacokinetic proles.
C
15H23NO5Cs [M + Cs] 430.0630; found 430.0623.
4.2.2. (S)-5-(tert-Butoxy)-4-((tert-butoxycarbonyl)amino)-2-
methylene-5-oxopentanoic acid (19). Compound 18 (782 mg, 2.6
mmol) was dissolved in THF (30 mL) and added LiOH
(124.5 mg, 5.2 mmol) at rt. The reaction mixture was stirred
overnight at ambient temperature. Aer completion, the reac-
tion mixture was passed through a short silica column and
washed with 20% MeOH in DCM to afford 19 (700 mg, 70%
yield) as a white solid. 1H NMR (400 MHz, MeOD) d 6.23 (s, 1H),
5.68 (s, 1H), 4.24 (dd, J ¼ 9.3, 6.1 Hz, 1H), 2.77 (dd, J ¼ 13.8,
5.9 Hz, 1H), 2.61–2.42 (m, 1H), 1.44 (d, J ¼ 10.7 Hz, 18H); 13C
NMR (101 MHz, MeOD) d 173.1, 169.9, 157.9, 138.3, 128.9, 82.9,
80.5, 55.0, 35.6, 28.8, 28.7, 28.4; LC-MS (ESI) m/z calcd for
4. Experimental section
4.1. General chemical synthesis procedures
All chemicals were obtained from Sigma-Aldrich or Fisher
Scientic and used as received unless specied. All syntheses
were conducted with anhydrous conditions under an atmo-
sphere of argon, using ame-dried glassware and employing
standard techniques for handling air-sensitive materials unless
otherwise noted. All solvents were distilled and stored under an
argon or nitrogen atmosphere before use. 1H NMR and 13C
NMR spectra were recorded on a Bruker-400 and or a Bruker-500
C
15H26N2O6 [M + H] ¼ 316.2; found 316.2.
4.3. General procedure for amide coupling
spectrometer using CDCl3, MeOD, or D2O as the solvent. To a solution of the acid compound (1 equiv.) in anhydrous THF
Chemical shis (d) were recorded in parts per million and in argon condition was added HBTU (1.5 equiv.), Et3N (1.5
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 7115–7128 | 7123

View Article Online
RSC Advances
Paper
equiv.), 4-methylmorpholin (1.5 equiv.), and amine (1.5 equiv.) sticky gel. 1H NMR (400 MHz, CDCl3) d 7.41–7.18 (m, 5H), 7.00
at rt. The reaction mixture was stirred for 3 hours at rt. The solid (s, 1H), 5.73 (s, 1H), 5.59 (d, J ¼ 7.7 Hz, 1H), 5.36 (s, 1H), 4.47 (d,
was ltered off, and the ltrate was concentrated. The crude J ¼ 5.7 Hz, 2H), 4.19 (q, J ¼ 6.8 Hz, 1H), 2.78 (dd, J ¼ 14.1, 5.8 Hz,
mixture was puried via silica column (ethyl acetate and 1H), 2.62 (dd, J ¼ 14.0, 7.6 Hz, 1H), 1.41 (d, J ¼ 13.2 Hz, 18H);
hexane) to afford the amides.
4.3.1. tert-Butyl
13C NMR (101 MHz, CDCl3) d 170.7, 168.0, 155.4, 140.5, 138.0,
(S)-2-((tert-butoxycarbonyl)amino)-4- 128.6, 127.6, 127.2, 121.3, 82.0, 79.5, 53.7, 43.6, 35.5, 28.1, 27.8;
carbamoylpent-4-enoate (20). Compound 20 was synthesized HRMS m/z calcd for C22H32N2O5Cs [M + Cs] 537.1365; found
via the general amide coupling procedure where 4 equivalent of 537.1371.
ammonium chloride was used in place of amine. Yield 85%,
white solid. H NMR (400 MHz, CDCl3) d 5.86 (s, 1H), 5.51 (s, (4). Compound 4 was prepared from 23 using the general
4.4.5. (S)-2-Amino-4-(benzylcarbamoyl)pent-4-enoic acid
1
ꢂ
1H), 4.14 (dt, J ¼ 10.7, 5.4 Hz, 1H), 2.81 (m, 1H), 2.57 (dd, J ¼ deprotection method. Yield 93%, white solid. Mp 182–184 C.
14.0, 8.8 Hz, 1H), 1.45 (d, J ¼ 13.0 Hz, 18H); 13C NMR (101 MHz, 1H NMR (500 MHz, MeOD) d 7.44–7.23 (m, 5H), 5.89 (s, 1H), 5.68
CDCl3 + MeOD) d 173.0, 172.9, 157.9, 141.5, 123.2, 83.0, 80.6, (s, 1H), 4.44 (s, 2H), 2.90 (dd, J ¼ 15.0, 4.7 Hz, 1H), 2.77 (q, J ¼
55.2, 39.0, 28.8, 28.4; LC-MS (ESI) m/z calcd for C15H27N2O5 [M + 7.5 Hz, 1H); 13C NMR (126 MHz, MeOD) d 172.8, 170.6, 138.7,
H] ¼ 315.2; found 315.2.
137.9, 128.7, 127.4, 127.2, 124.2, 54.2, 43.2, 33.7; HRMS m/z
calcd for C13H15N2O3 [M ꢀ H] 247.1083; found [M ꢀ H]
247.1073.
4.4. General method for deprotection of amide compounds
4.4.6. tert-Butyl(S)-2-((tert-butoxycarbonyl)amino)-4-((4-
tert-Butyl and Boc protected amides were dissolved in uorobenzyl)carbamoyl)pent-4-enoate (24). Compound 24 was
a DCM : TFA ¼ 4 : 1 mixture at rt and stirred until all the synthesized from intermediate 19 and p-uorobenzylamine by
starting material was fully consumed. Aer the reaction was following the general amide coupling procedure. Yield 76%,
complete, the solvents were evaporated. The crude mixture was sticky gel. 1H NMR (400 MHz, CDCl3) d 7.36–7.18 (m, 2H), 7.13
puried via silica gel column chromatography (DCM and (d, J ¼ 5.9 Hz, 1H), 7.03–6.90 (m, 2H), 5.77 (s, 1H), 5.54 (d, J ¼
MeOH) or HPLC (H2O and ACN).
7.6 Hz, 1H), 5.36 (s, 1H), 4.44 (d, J ¼ 5.8 Hz, 2H), 4.17 (d, J ¼
(1). 6.9 Hz, 1H), 2.78 (dd, J ¼ 14.1, 6.1 Hz, 1H), 2.61 (dd, J ¼ 13.9,
4.4.1. (S)-2-Amino-4-carbamoylpent-4-enoic
acid
Compound 1 was prepared from 20 by following the general 7.2 Hz, 1H), 1.41 (d, J ¼ 15.2 Hz, 18H); 13C NMR (101 MHz,
deprotection procedure. Yield 80%, white solid. Mp 157–158 ꢂC. CDCl3) d 170.4, 168.0, 163.2, 160.8, 155.6, 140.4, 134.0, 134.0,
1H NMR (400 MHz, DMSO + D2O) d 5.82 (s, 1H), 5.58 (s, 1H), 129.5, 129.4, 121.9, 115.4, 115.2, 82.3, 79.8, 60.3, 53.7, 43.0, 35.9,
3.67–3.57 (m, 1H), 2.75 (dd, J ¼ 14.7, 4.5 Hz, 1H), 2.59–2.48 (m, 28.23, 27.9; LC-MS (ESI) m/z calcd for C22H32FN2O5 [M + H]
1H); NH2 was not observed in DMSO + D2O mixture. 13C NMR 423.2; found 423.2.
(101 MHz, DMSO + D2O) d 171.9, 138.0, 125.2, 53.6, 33.6; LC-MS
(ESI) m/z calcd for C6H11N2O3 [M + H] ¼ 159.1; found 159.1.
4.4.2. tert-Butyl(S)-2-((tert-butoxycarbonyl)amino)-4-
4.4.7. (S)-2-Amino-4-((4-uorobenzyl)carbamoyl)pent-4-
enoic acid (5). Compound 5 was prepared from 24 using the
general deprotection method. Yield 86%, white solid. Mp 189–
(phenylcarbamoyl)pent-4-enoate (22). Compound 22 was 190 ꢂC. 1H NMR (400 MHz, DMSO-d6) d 9.01 (s, 1H), 7.32 (t, J ¼
synthesized from the intermediate 19 and aniline by following 7.0 Hz, 2H), 7.21–7.07 (m, 2H), 5.82 (s, 1H), 5.52 (s, 1H), 4.32 (d, J
the general amide coupling procedure. Yield 43%, sticky gel. 1H ¼ 5.8 Hz, 2H), 3.37 (dd, J ¼ 8.7, 4.4 Hz, 1H), 2.84 (dt, J ¼ 14.6,
NMR (400 MHz, MeOD) d 7.59 (d, J ¼ 8.0 Hz, 2H), 7.37–7.26 (m, 3.2 Hz, 1H), 2.50–2.43 (m, 1H); 13C NMR (101 MHz, DMSO-d6)
2H), 7.17–7.06 (m, 1H), 5.89 (s, 1H), 5.58 (s, 1H), 4.19 (dd, J ¼ d 167.9, 162.3, 159.9, 140.2, 135.6, 129.2, 129.1, 121.8, 114.9,
8.6, 5.2 Hz, 1H), 2.89 (dd, J ¼ 14.0, 5.5 Hz, 1H), 2.68 (dd, J ¼ 14.1, 114.7, 53.4, 41.7, 34.5; 19F NMR (376 MHz, DMSO-d6) d ꢀ116.31;
8.9 Hz, 1H), 1.43 (d, J ¼ 19.5 Hz, 18H); 13C NMR (101 MHz, HRMS m/z calcd for C13H14FN2O3 [M ꢀ H] 265.0988; found
CDCl3) d 170.5, 166.5, 155.8, 141.4, 138.3, 128.8, 124.2, 122.2, 265.0964.
120.1, 82.7, 80.2, 53.5, 36.8, 28.3, 28.0, 28.0; LC-MS (ESI) m/z
calcd for C21H31N2O5 [M + H] ¼ 391.2; found 391.2.
4.4.8. tert-Butyl (S)-2-((tert-butoxycarbonyl)amino)-4-((4-
nitrobenzyl)carbamoyl)pent-4-enoate (25). Compound 25 was
4.4.3. (S)-2-Amino-4-(phenylcarbamoyl)pent-4-enoic acid prepared from the intermediate 19 and p-nitrobenzylamine by
(3). Compound 3 was synthesized from 22 by following the following the general amide coupling procedure. This
general deprotection procedure. Yield 55%, white solid. Mp compound was partially puried and used for next general
1
ꢂ
182–184 C. H NMR (400 MHz, CH3CN + D2O) d 7.59 (d, J ¼ deprotection step.
8.0 Hz, 2H), 7.37–7.26 (m, 2H), 7.17–7.06 (m, 1H), 5.89 (s, 1H),
4.4.9. (S)-2-Amino-4-((4-nitrobenzyl)carbamoyl)pent-4-
5.58 (s, 1H), 4.19 (dd, J ¼ 8.6, 5.2 Hz, 1H), 2.89 (dd, J ¼ 14.0, enoic acid (6). Compound 6 was prepared from 25 using the
5.5 Hz, 1H), 2.68 (dd, J ¼ 14.1, 8.9 Hz, 1H), 1.43 (d, J ¼ 19.5 Hz, general deprotection method. Yield 91%, white solid. Mp 186–
20H).; 13C NMR (126 MHz, CH3CN + D2O) d 172.7, 169.1, 139.45, 188 ꢂC. 1H NMR (400 MHz, DMSO-d6) d 9.26 (d, J ¼ 6.0 Hz, 1H),
139.4, 137.6, 129.1, 125.2, 124.9, 121.6, 54.4, 33.8; LC-MS (ESI) 8.17 (d, J ¼ 8.4 Hz, 2H), 7.56 (d, J ¼ 8.3 Hz, 2H), 5.85 (s, 1H), 5.54
m/z calcd for C12H15N2O3 [M + H] ¼ 235.1; found 235.1.
(s, 1H), 4.46 (d, J ¼ 6.0 Hz, 2H), 3.71–3.01 (m, 4H), 2.83 (dd, J ¼
4.4.4. tert-Butyl
(S)-4-(benzylcarbamoyl)-2-((tert- 14.7, 4.7 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) d 169.9, 168.5,
butoxycarbonyl)amino)pent-4-enoateenoate (23). Compound 23 148.1, 146.8, 140.6, 128.6, 123.8, 122.5, 53.8, 42.5, 34.9. HRMS
was prepared from the intermediate 19 and the benxylamine by m/z calcd for C13H16N3O5 [M + H] 294.1012; found 294.1020.
following the general amide coupling procedure. Yield 70%,
7124 | RSC Adv., 2021, 11, 7115–7128
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
4.4.10. tert-Butyl
RSC Advances
1H), 3.38 (dd, J ¼ 14.7, 4.6 Hz, 1H), 3.20 (dd, J ¼ 14.8, 8.0 Hz,
1H); 13C NMR (126 MHz, D2O + CH3CN) d 172.6, 168.9, 160.6,
158.73, 139.1, 133.7, 133.7, 124.8, 123.6, 123.5, 115.6, 115.4,
54.3, 33.7; 19F NMR (376 MHz, D2O + CH3CN) d ꢀ114.84; LC-MS
(ESI) m/z calcd for C12H14FN2O3 [M + H] ¼ 253.1; found 253.1.
4.4.16. tert-Butyl (S)-2-((tert-butoxycarbonyl)amino)-4-((4-
chlorophenyl)carbamoyl)pent-4-enoate (29). Compound 29
was prepared from intermediate 19 and p-chloroaniline
following the general amide coupling method. This compound
was partially puried and used for the next step.
4.4.17. (S)-2-Amino-4-((4-chlorophenyl)carbamoyl)pent-4-
enoic acid (10). Compound 10 was prepared and puried by
following the same procedure as compound 9. Yield 11%, white
solid. Mp 181–182 C. H NMR (400 MHz, D2O) d 7.91 (d, J ¼
9.1 Hz, 2H), 7.82–7.69 (m, 2H), 6.34 (s, 1H), 6.12 (s, 1H), 4.16
(dd, J ¼ 7.8, 4.4 Hz, 1H), 3.29 (dd, J ¼ 14.8, 4.7 Hz, 1H), 3.11 (dd,
J ¼ 15.1, 7.9 Hz, 1H); 13C NMR (126 MHz, D2O + CH3CN) d 172.5,
168.8, 138.9, 136.2, 129.2, 128.7, 124.9, 122.7, 54.1, 33.5; LC-MS
(ESI) m/z calcd for C12H14ClN2O3 [M + H] ¼ 269.1; found 269.1.
(S)-2-((tert-butoxycarbonyl)amino)-4-
(cyclopropylcarbamoyl)pent-4-enoate (26). Compound 26 was
synthesized from intermediate 19 and cyclopropylamine by
following the general amide coupling method. Yield 76%, sticky
gel. 1H NMR (400 MHz, CDCl3) d 6.78 (s, 1H), 5.71 (s, 1H), 5.47
(d, J ¼ 7.4 Hz, 1H), 5.30 (s, 1H), 4.11 (d, J ¼ 7.0 Hz, 1H), 2.85–
2.66 (m, 2H), 2.58 (dd, J ¼ 14.1, 7.2 Hz, 1H), 1.41 (d, J ¼ 7.7 Hz,
16H), 0.85–0.70 (m, 1H), 0.66–0.42 (m, 2H); 13C NMR (101 MHz,
CDCl3) d 170.7, 169.4, 155.6, 140.5, 121.6, 82.3, 79.8, 53.7, 35.8,
28.2, 27.9, 22.9, 6.3; HRMS calcd for C18H30N2O5Cs [M + Cs]
487.1209; found 487.1215.
4.4.11. (S)-2-Amino-4-(cyclopropylcarbamoyl)pent-4-enoic
acid (7). Compound 7 was prepared from 26 using the general
deprotection method. Yield 95%, white solid. Mp 191–193 C.
1
ꢂ
ꢂ
1H NMR (500 MHz, D2O + MeOD) d 5.71 (s, 1H), 5.58 (s, 1H),
3.73–3.62 (m, 1H), 3.23 (s, 1H), 2.79 (dd, J ¼ 15.0, 4.2 Hz, 1H),
2.63 (dt, J ¼ 16.4, 8.3 Hz, 2H), 0.70 (d, J ¼ 6.8 Hz, 2H), 0.56–0.46
(m, 2H); 13C NMR (126 MHz, D2O + MeOD) d 173.6, 173.6, 140.3,
125.2, 55.9, 35.3, 23.8, 6.7, 6.6; HRMS calcd for m/z C9H13N2O3
[M ꢀ H] 197.0926; found 197.0921.
4.4.12. tert-Butyl
(S)-2-((tert-butoxycarbonyl)amino)-4-
4.5. Evaluation of growth inhibition and viability of cancer
cell lines
(piperidine-1-carbonyl)pent-4-enoate (27). Compound 27 was
prepared from intermediate 19 and piperidine by following the
general amide coupling method. Yield 93%, sticky gel. 1H NMR
(400 MHz, CDCl3) d 5.45 (d, J ¼ 8.1 Hz, 1H), 5.20 (s, 1H), 5.06 (s,
1H), 4.17–4.07 (m, 1H), 3.54–3.37 (m, 4H), 2.71 (dd, J ¼ 14.5,
5.0 Hz, 1H), 2.59 (dd, J ¼ 14.8, 7.6 Hz, 1H), 1.63–1.26 (m, 24H);
13C NMR (101 MHz, CDCl3) d 170.7, 169.6, 155.2, 139.6, 117.7,
81.5, 79.1, 53.3, 35.9, 28.1, 27.8, 27.7, 24.4; HRMS calcd for
Compounds 1 and 3–10 were puried by HPLC (7.8 ꢃ 30 mm, 7
mm, C18, gradient 98% water in acetonitrile to 80% water in
acetonitrile, ow rate 2 mL minꢀ1, retention time 13 min) until
their purities were higher than 95% before being evaluated in
cell assays; purities were measured using a Waters 2695
analytical HPLC system. All cell lines were all obtained from the
American Type Culture Collection (ATCC; Manassas, VA) for the
purpose of this study. All cells used were passaged less than 10
times. Cells were seeded onto 96-well plates at a density of 2 ꢃ
104 cells per well for assessment of growth and cell death. MCF-
7, SK-BR-3, and MDA-MB-231 cells were maintained in DMEM/
F12 media (#11320-033, Life Technologies, Carlsbad, CA) sup-
plemented with 10% heat-inactivated fetal bovine serum (FBS;
#SH30071.03, Thermo Scientic Hyclone, Logan, UT), and 0.5%
antibiotic/antimycotic mixture (#15240-062, Life Technologies).
MCF-10A cells were maintained in sterile, unltered MEBM
growth media supplemented with all components of a MEGM
kit (#CC-3150, Lonza Group Ltd, Switzerland), with exception of
#GA-1000 (gentamycin–amphotericin-B mixture). In addition,
0.5% penicillin–streptomycin mixture (#15-140-163, Thermo
Fisher Scientic, Waltham, MA) and Cholera toxin (100 ng
mLꢀ1; #C8052, Sigma) were added to the media. All compounds
were dissolved in 50% DMSO with the exception of tamoxifen
which was dissolved in 90% EtOH. All compounds were then
diluted to concentration in media (DMSO nal concentration <
0.8%; EtOH nal concentration < 0.6%). Control cells were
incubated with the same concentrations of DMSO or EtOH and
used as a negative control for statistical comparison. Cells were
incubated with all compounds in a concentration-response
regimen (0.32, 1, 3.2, 10, 32, 100, 320 mM) for 24 h or 72 h in
a 37 ꢂC humidied incubator (5% CO2). Media was not changed
over the course of the experiment. On the day of assessment,
a working solution of propidium iodide (ex/em: 536/617 nm)
and Hoechst 33342 (ex/em: 350/461 nm) was prepared by
C
20H34N2O5Cs [M + Cs] 515.1522; found 515.1512.
4.4.13. (S)-2-Amino-4-(piperidine-1-carbonyl)pent-4-enoic
acid (8). Compound 8 was prepared from 27 using the general
deprotection method. Yield 92%, white solid. Mp 53–55 ꢂC. H
1
NMR (400 MHz, MeOD) d 5.54 (s, 1H), 5.34 (s, 1H), 3.77 (dd, J ¼
8.8, 4.5 Hz, 1H), 3.69–3.52 (m, 4H), 2.88 (dd, J ¼ 14.9, 4.6 Hz,
1H), 2.70 (dd, J ¼ 14.9, 8.7 Hz, 1H), 1.80–1.53 (m, 6H); 13C NMR
(101 MHz, MeOD) d 172.9, 171.9, 140.0, 121.3, 55.4, 44.1, 36.6,
27.7, 26.7, 25.5; MS calcd for C11H18N2O3Cs [M + Cs] 359.0372;
found 359.0376.
4.4.14. tert-Butyl (S)-2-((tert-butoxycarbonyl)amino)-4-((4-
uorophenyl)carbamoyl)pent-4-enoate (28). Compound 28 was
prepared from intermediate 19 and p-uoroaniline following
the general amide coupling method. This compound was
partially puried and used for the next step.
4.4.15. (S)-2-Amino-4-((4-uorophenyl)carbamoyl) pent-4-
enoic acid (9). Crude compound 28 (87 mg) was dissolved in
25 mL of DCM and stirred with ZnBr2 (12 equiv.) overnight.
Aer the reaction was complete, the solvent was concentrated,
and the crude product was passed through a silica column,
which was then run in gradient with 1% MeOH in DCM to 100%
MeOH. The partially puried product from the silica column
was further puried by HPLC (7.8 ꢃ 30 mm, 7 mm, C18, gradient
98% water in acetonitrile to 80% water in acetonitrile, ow rate
2 mL minꢀ1, retention time 13 min). Yield 13%, white solid. Mp
183–184 ꢂC. 1H NMR (400 MHz, D2O) d 8.06–7.94 (m, 2H), 7.66–
7.55 (m, 2H), 6.43 (s, 1H), 6.21 (s, 1H), 4.25 (dd, J ¼ 7.9, 4.4 Hz,
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 7115–7128 | 7125

View Article Online
RSC Advances
Paper
diluting stocks in Hank's Balanced Salt Solution (HBSS; 1 : 50 of the compounds. In all cases, the highest concentration tested
dilution for propidium iodide and 1/10 000 for Hoescht). Media was equal to or greater than 100 mM. At the end of the reaction
in the 96-well plates were removed and 100 mL of HBSS con- period, the assay was terminated upon the addition of imid-
taining uorophores was added to each well. Cells were incu- azole buffer (pH 7). 96-well spin columns packed with strong
bated for 15 minutes at 37 ꢂC (5% CO2) and uorescent anion ion-exchange resin was used to separate the substrate and
emissions were read on a CLARIOstar plate reader (BMG Lab- the reaction product. Unreacted [3H]-glutamine was removed by
tech, Cary, NC). Relative uorescent units (RFU) for treatment washing with imidazole buffer. [3H]-Glutamate, the reaction
wells were calculated as a proportion of non-treated control product, was eluted with diluted HCl and analyzed for
wells in order to assess growth (negative controls indicate by radioactivity.
dashed line at 100% in Fig. 4). Viability of the cells was assessed
by calculating the proportion of necrotic cells as a function of
the total cell RFU per well (data depicted as % increase from
Author contributions
negative control wells in Fig. 5). All experiments were inde-
H. V. L. designed the molecules and supervised the overall
pendently replicated 3 times and each treatment was run in
coordination of the research. A. T. A., M. I. H., and M. M. W.
technical duplicate for each experiment.
worked on the original synthetic plan for 1 (Fig. 3). M. I. H.
synthesized compounds 1–10 and studied the formation of 30
(Schemes 2–5). N. S. A. measured melting points of the
4.6. Statistical analyses
To delineate differences in cell growth and necrosis in compounds and took 19F NMR spectra. F. M. and J. J. P. carried
comparison to negative controls (vehicle-treated cells), separate out the evaluation of compounds 1, 3–10 for their antigrowth/
two-way analyses of variance (ANOVA) were conducted with necrotic activity. A. G. T. and B. S. S. carried out the evalua-
treatment exposure time (24 or 72 h) and compound concen- tion of compounds 1, 3–10 for their possible inhibitory activity
tration as the between-subjects factors (see Fig. 4 and 5). For on glutaminase. M. I. H., J. J. P., and H. V. L. wrote the manu-
each compound, simple main effects and planned post hoc script. All authors approved the nal version.
contrasts were conducted to reveal dosing that signicantly
differed from controls. All post hoc comparisons were corrected
for family-wise error and considered signicant when p # 0.05.
To assess comparative changes in potency from positive
controls (tamoxifen and olaparib), median inhibitory and
Conflicts of interest
There are no conicts of interest to declare.
effective concentrations (log IC50, log EC50) were determined via
non-linear regression (sigmoidal curvilinear modeling with
variable slope; Prism 7, GraphPad Soware, La Jolla, CA) using
Acknowledgements
a least-squares t for each treatment group (bottom values Work was supported by the American Association of Colleges of
constrained to 0; see Tables 1 and 2). For each cell type (MCF-7, Pharmacy (2018 New Investigator Award to H. V. L.), National
SK-BR-3, MDA-MB-231, or MCF-10A) and treatment time (24 or Institute on Drug Abuse (R00 DA039791 to J. J. P.), National
72 h), log IC50/log EC50 values were compared to those obtained Institute of General Medical Sciences (P30GM122733 pilot
for tamoxifen and olaparib via extra sum-of-squares F-test. project awards to H. V. L. and J. J. P.), National Cancer Institute
Median shis were considered signicant when p # 0.05.
(R01 CA193895 to B. S. S.), and funds from the Department of
BioMolecular Sciences at the University of Mississippi, School
of Pharmacy. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
these funders.
4.7. Evaluation for possible inhibitory activity on kidney-
type glutaminase
Compounds 1 and 3–10 were puried by HPLC (7.8 ꢃ 30 mm, 7
mm, C18, gradient 98% water in acetonitrile to 80% water in
acetonitrile, ow rate 2 mL minꢀ1, retention time 13 min) until
their purities were higher than 95% before being evaluated in
GLS1 assays; purities were measured using a Waters 2695
analytical HPLC system. A published procedure was followed.35
hKGAd1 construct was prepared from the hKGA cDNA by
deleting the sequence encoded in exon 1 and cloning into
pET15b. Puried human GLS1 (hKGA124–669; 250 nM) was used
as the source of enzyme, and radiolabeled glutamine (L-[3H]-
glutamine) was used as the substrate. The assay was conduct-
ed in the presence and absence of the compound, at rt (with and
without 2 h incubation), in phosphate buffer (pH 8.2). All
compounds were tested on an 7-point concentration response
curve, with 10-fold dilution between concentrations and start-
ing at the highest concentrations determined by the solubility
References
1 P. Newsholme, J. Procopio, M. M. R. Lima, T. C. Pithon-Curi
and R. Curi, Glutamine and Glutamate – Their Central Role
in Cell Metabolism and Function, Cell Biochem. Funct., 2003,
21(1), 1–9.
2 P. Newsholme, M. M. R. Lima, J. Procopio, T. C. Pithon-Curi,
S. Q. Doi, R. B. Bazotte and R. Curi, Glutamine and
Glutamate as Vital Metabolites, Braz. J. Med. Biol. Res.,
2003, 36(2), 153–163.
3 N. S. Akins, T. C. Nielson and H. V. Le, Inhibition of
Glycolysis and Glutaminolysis: An Emerging Drug
Discovery Approach to Combat Cancer, Curr. Top. Med.
Chem., 2018, 18(6), 494–504.
7126 | RSC Adv., 2021, 11, 7115–7128
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
4 J. W. Erickson and R. A. Cerione, Glutaminase: A Hot Spot 17 G. Harris and A. R. Tatchell, Amino Acids and Peptides of
For Regulation Of Cancer Cell Metabolism?, Oncotarget,
2010, 1(8), 734–740.
5 Y. Xiang, Z. E. Stine, J. Xia, Y. Lu, R. S. O'Connor, 18 B. Tschiersch, Uber g-Methylenglutamin Und g-
Hops and Wort III. The Amino Acids of Fresh Hops, J. Inst.
Brew., 1953, 59(5), 371–377.
¨
¨
B. J. Altman, A. L. Hsieh, A. M. Gouw, A. G. Thomas,
P. Gao, L. Sun, L. Song, B. Yan, B. S. Slusher, J. Zhuo,
Methylenglutaminsaure in Keimlingen von Amorpha
fruticosa L, Phytochemistry, 1962, 1(2), 103–105.
L. L. Ooi, C. G. L. Lee, A. Mancuso, A. S. McCallion, A. Le, 19 L. Fowden, The Nitrogen Metabolism of Groundnut Plants:
M. C. Milone, S. Rayport, D. W. Felsher and C. V. Dang,
The
Role
of
g-Methyleneglutamine
and
g-
Targeted Inhibition of Tumor-Specic Glutaminase
Methyleneglutamic Acid, Ann. Bot., 1954, 18(4), 417–440.
Diminishes Cell-Autonomous Tumorigenesis, J. Clin. 20 G. K. Powell and E. E. Dekker, A Modied, High Yield
Invest., 2015, 125(6), 2293–2306.
Procedure for the Synthesis of Unlabeled and 14C-Labeled
4-Methylene-DL-Glutamic Acid, Prep. Biochem. Biotechnol.,
1981, 11(3), 339–350.
6 C. T. Hensley, A. T. Wasti and R. J. DeBerardinis, Glutamine
and Cancer: Cell Biology, Physiology, and Clinical
Opportunities, J. Clin. Invest., 2013, 123(9), 3678–3684.
7 R. A. Shapiro, V. M. Clark and N. P. Curthoys, Inactivation of
Rat Renal Phosphate-Dependent Glutaminase with 6-Diazo-
21 O. Ouerfelli, M. Ishida, H. Shinozaki, K. Nakanishi and
Y. Ohfune, Efficient Synthesis of 4-Methylene-L-Glutamic
Acid and Its Analogues, Synlett, 1993, 1993(6), 409–410.
5-Oxo-L-Norleucine. Evidence for Interaction at the 22 H. Gershon, J. S. Meek and K. Dittmer, Propargylglycine: An
Glutamine Binding Site, J. Biol. Chem., 1979, 254(8), 2835–
2838.
Acetylenic Amino Acid Antagonist, J. Am. Chem. Soc., 1949,
71(10), 3573–3574.
8 K. Thangavelu, C. Q. Pan, T. Karlberg, G. Balaji, 23 J. E. Baldwin, R. M. Adlington and N. G. Robinson,
¨
M. Uttamchandani, V. Suresh, H. Schuler, B. C. Low and
Nucleophilic Ring Opening of Aziridine-2-Carboxylates
with Wittig Reagents; an Enantioefficient Synthesis of
Unsaturated Amino Acids, J. Chem. Soc., Chem. Commun.,
1987, (3), 153–155.
J. Sivaraman, Structural Basis for the Allosteric Inhibitory
Mechanism of Human Kidney-Type Glutaminase (KGA)
and Its Regulation by Raf-Mek-Erk Signaling in Cancer Cell
Metabolism, Proc. Natl. Acad. Sci. U. S. A., 2012, 109(20), 24 C. M. Moody and D. W. Young, Synthesis of Naturally-
7705–7710.
Occurring 4-Alkylideneglutamic Acids, Tetrahedron Lett.,
1993, 34(29), 4667–4670.
9 K. Thangavelu, Q. Y. Chong, B. C. Low and J. Sivaraman,
´
´
Structural Basis for the Active Site Inhibition Mechanism 25 J. Eacquerra, C. Pedregal, I. Mico and C. Najera, Efficient
of Human Kidney-Type Glutaminase (KGA), Sci. Rep., 2014,
4, 3827.
10 R. K. Barclay and M. A. Phillipps, Effects of 6-Diazo-5-Oxol-
Synthesis of 4-Methylene-L-Glutamic Acid and Its
Cyclopropyl Analogue, Tetrahedron: Asymmetry, 1994, 5(5),
921–926.
Norleucine and Other Tumor Inhibitors on the 26 P. Dieterich and D. W. Young, Synthesis of (2S,3S)-[3-2H1]-4-
Biosynthesis of Nicotinamide Adenine Dinucleotide in
Mice, Cancer Res., 1966, 26(2), 282–286.
11 A. Hofer, D. Steverding, A. Chabes, R. Brun and L. Thelander,
Methyleneglutamic
Methyleneglutamic Acid, Org. Biomol. Chem., 2006, 4(8),
1492–1496.
Acid
and
(2S,3R)-[2,3-2H2]-4-
Trypanosoma brucei CTP Synthetase: A Target for the 27 M. V. Riofski, J. P. John, M. M. Zheng, J. Kirshner and
Treatment of African Sleeping Sickness, Proc. Natl. Acad.
Sci. U. S. A., 2001, 98(11), 6412–6416.
D. A. Colby, Exploiting the Facile Release of
Triuoroacetate for the a-Methylenation of the Sterically
Hindered Carbonyl Groups on (+)-Sclareolide and
(ꢀ)-Eburnamonine, J. Org. Chem., 2011, 76(10), 3676–3683.
28 X. Durand, P. Hudhomme, J. A. Khan and D. W. Young, Two
Independent Syntheses of (2S,4S)- and (2S,4R)-[5,5-2H2]-5,50-
Dihydroxyleucine, Tetrahedron Lett., 1995, 36(8), 1351–1354.
29 M. I. Hossain, S. Hanashima, T. Nomura, S. Lethu,
H. Tsuchikawa, M. Murata, H. Kusaka, S. Kita and
K. Maenaka, Synthesis and Th1-Immunostimulatory
Activity of a-Galactosylceramide Analogues Bearing
a Halogen-Containing or Selenium-Containing Acyl Chain,
Bioorg. Med. Chem., 2016, 24(16), 3687–3695.
12 A. I. Grayzel, J. E. Seegmiller and E. Love, Suppression of Uric
Acid Synthesis in the Gouty Human by the Use of 6-Diazo-5-
Oxo-L-Norleucine, J. Clin. Invest., 1960, 39(3), 447–454.
13 P. Wailes, M. C. Whitting and L. Fowden, Synthesis of g-
Methyleneglutamic Acid and g-Methyleneglutamine,
Nature, 1954, 174(4420), 130–131.
14 P. C. Wailes and M. C. Whiting, Researches on Acetylenic
Compounds.
Part
LI.
The
Syntheses
of
g-
Methyleneglutamic Acid and g-Methyleneglutamine, J.
Chem. Soc., 1955, 3636–3641.
15 J. Done and L. Fowden, A New Amino Acid Amide in the
Groundnut Plant (Arachis Hypogaea); Evidence of the 30 J. E. Baldwin, Rules for Ring Closure, J. Chem. Soc., Chem.
Occurrence of Gamma-Methyleneglutamine and Gamma-
Methyleneglutamic Acid, Biochem. J., 1952, 51(4), 451–458.
16 R. M. Zacharius, J. K. Pollard and F. C. Steward, g-
Methyleneglutamine and g-Methyleneglutamic Acid in the
Tulip (Tulipa gesneriana), J. Am. Chem. Soc., 1954, 76(7),
1961–1962.
Commun., 1976, (18), 734–736.
31 J. E. Baldwin, R. C. Thomas, L. I. Kruse and L. Silberman,
Rules for Ring Closure: Ring Formation by Conjugate
Addition of Oxygen Nucleophiles, J. Org. Chem., 1977,
42(24), 3846–3852.
32 V. K. Todorova, Y. Kaufmann, S. Luo and V. S. Klimberg,
Tamoxifen and Raloxifene Suppress the Proliferation of
© 2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 7115–7128 | 7127

View Article Online
RSC Advances
Paper
Estrogen Receptor-Negative Cells through Inhibition of
Glutamine Uptake, Cancer Chemother. Pharmacol., 2011,
67(2), 285–291.
Metabolism in Cancer Cell Growth, Front. Oncol., 2017, 7,
306.
35 K. Shukla, D. V. Ferraris, A. G. Thomas, M. Stathis, B. Duvall,
G. Delahanty, J. Alt, R. Rais, C. Rojas, P. Gao, Y. Xiang,
C. V. Dang, B. S. Slusher and T. Tsukamoto, Design,
Synthesis, and Pharmacological Evaluation of Bis-2-(5-
33 Y. D. Bhutia and V. Ganapathy, Glutamine Transporters in
Mammalian Cells and Their Functions in Physiology and
Cancer, Biochim. Biophys. Acta, Mol. Cell Res., 2016,
1863(10), 2531–2539.
Phenylacetamido-1,2,4-Thiadiazol-2-Yl)Ethyl
Sulde
3
34 M. Scalise, L. Pochini, M. Galluccio, L. Console and
C. Indiveri, Glutamine Transport and Mitochondrial
(BPTES) Analogs as Glutaminase Inhibitors, J. Med. Chem.,
2012, 55(23), 10551–10563.
7128 | RSC Adv., 2021, 11, 7115–7128
© 2021 The Author(s). Published by the Royal Society of Chemistry