Allosteric Glutaminase Inhibitors Based on a 1,4-Di(5-amino-1,3,4-thiadiazol-2-yl)butane Scaffold

Article abstract of DOI:10.1021/acsmedchemlett.6b00060

A series of allosteric kidney-type glutaminase (GLS) inhibitors were designed and synthesized using 1,4-di(5-amino-1,3,4-thiadiazol-2-yl)butane as a core scaffold. A variety of modified phenylacetyl groups were incorporated into the 5-amino group of the two thiadiazole rings in an attempt to facilitate additional binding interactions with the allosteric binding site of GLS. Among the newly synthesized compounds, 4-hydroxy-N-[5-[4-[5-[(2-phenylacetyl)amino]-1,3,4-thiadiazol-2-yl]butyl]-1,3,4-thiadiazol-2-yl]-benzeneacetamide, 2m, potently inhibited GLS with an IC₅₀ value of 70 nM, although it did not exhibit time-dependency as seen with CB-839. Antiproliferative effects of 2m on human breast cancer lines will be also presented in comparison with those observed with CB-839.

Full text of DOI:10.1021/acsmedchemlett.6b00060

Letter
pubs.acs.org/acsmedchemlett
Allosteric Glutaminase Inhibitors Based on a 1,4-Di(5-amino-1,3,4-
thiadiazol-2-yl)butane Scaffold
Sarah C. Zimmermann,†,‡ Emily F. Wolf,† Andrew Luu,† Ajit G. Thomas,† Marigo Stathis,† Brad Poore,§
Christopher Nguyen,§ Anne Le,§ Camilo Rojas,∥ Barbara S. Slusher,†,‡ and Takashi Tsukamoto*,†,‡
†Johns Hopkins Drug Discovery Program, Johns Hopkins University, Baltimore, Maryland 21205, United States
‡Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21205, United States
§Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21231, United States
∥Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, Maryland 21205, United States
S
* Supporting Information
ABSTRACT: A series of allosteric kidney-type glutaminase (GLS) inhibitors
were designed and synthesized using 1,4-di(5-amino-1,3,4-thiadiazol-2-yl)-
butane as a core scaffold. A variety of modified phenylacetyl groups were
incorporated into the 5-amino group of the two thiadiazole rings in an attempt
to facilitate additional binding interactions with the allosteric binding site of
GLS. Among the newly synthesized compounds, 4-hydroxy-N-[5-[4-[5-[(2-
phenylacetyl)amino]-1,3,4-thiadiazol-2-yl]butyl]-1,3,4-thiadiazol-2-yl]-benze-
neacetamide, 2m, potently inhibited GLS with an IC50 value of 70 nM, although it did not exhibit time-dependency as seen with
CB-839. Antiproliferative effects of 2m on human breast cancer lines will be also presented in comparison with those observed
with CB-839.
KEYWORDS: Glutaminase, allosteric inhibition, cancer metabolism
lutaminolysis involves a series of biochemical reactions by
Gwhich glutamine is utilized as a source of nitrogen atoms
and carbon skeletons to create a variety of biologically
important substances. The first step in glutaminolysis is the
hydrolysis of glutamine to glutamate and ammonia, which is
catalyzed by the phosphate-activated glutaminase. Mammalian
tissues express two forms of glutaminase encoded by two
paralogous genes located in distinct chromosomes.1 Liver-type
glutaminase (GLS2) is predominantly found in adult liver,
whereas kidney-type glutaminase (GLS) is widely distributed
throughout extra-hepatic tissues. Two splicing variants derived
from the GLS gene are known as KGA and GAC, which share a
common N-terminal sequence (1−550) but contain unique C-
terminal segments (551−669 for KGA, 551−598 for GAC).2
Although distinct molecular functions of the two splicing
Figure 1. Representative glutaminase inhibitors.
variants have not yet been clearly understood, it appears that
the GAC form of GLS is predominantly upregulated in many
thiadiazol-2-yl)ethyl sulfide (BPTES, Figure 1)7 is another
glutaminase inhibitor structurally distinct from DON. Unlike
DON, BPTES does not contain any reactive chemical group
that might form a covalent bond and is unlikely to cause
toxicity by irreversibly forming covalent adducts with
endogenous proteins. In addition, while DON inhibits both
GLS and GLS2, BPTES selectively inhibits GLS (both KGA
and GAC isoenzymes) over GLS2.8 Moreover, BPTES bears no
proliferating cells, especially rapidly growing malignant cells.3
Inhibition of GLS, therefore, has gained considerable attention
as a new therapeutic approach for the treatment of cancer.
6-Diazo-5-oxo-L-norleucine (DON, Figure 1) is one of the
earliest glutaminase inhibitors,4 though its narrow therapeutic
index highlighted the need for a new class of glutaminase
inhibitors pharmacologically distinct from glutamine mimetics
such as DON. In this regard, compound 968 (Figure 1)5 is one
of the promising new glutaminase inhibitors structurally
dissimilar to glutamine. It remains to be seen whether
successful structural optimization can be carried out using the
benzophenanthridinone lead.6 Bis-2-(5-phenylacetamido-1,3,4-
Received: February 9, 2016
Accepted: March 13, 2016
© XXXX American Chemical Society
A
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a
structural similarity to glutamine, minimizing off-target effects
due to its interaction with other glutamine-related enzymes,
transporters, or receptors. Indeed, preliminary toxicity studies
of BPTES in mice showed no histopathologies in liver, heart,
lung, skeletal muscle, kidney, and brain.9 Further, BPTES did
not show any significant effects on body weight, blood
chemistries, and hematology measurements.9
Scheme 2. Synthesis of 2p−u
BPTES is, however, limited as a therapeutic agent due to its
poor aqueous solubility at any given pH, hindering its ability to
be dosed at therapeutically relevant levels. Recently, we and
other groups have begun exploring a new generation of GLS
inhibitors based on BPTES. Our group has reported medicinal
chemistry efforts on derivatives of BPTES with the primary
objective of improving solubility as compared to BPTES.10
While these analogues were successful in enhancing the
solubility, a significant improvement in glutaminase inhibitory
potency was not achieved. Meanwhile, Calithera Biosciences
developed a BPTES derivative CB-839 (Figure 1)11 containing
a pryridazine ring, which is currently under clinical investigation
in patients with cancer.
Recently published cocrystal structures of GLS in complex
with BPTES uncovered the unique allosteric binding of BPTES
to the dimer interface of GLS tetramer.12,13 The cocrystal
structures revealed that the two phenylacetyl groups at the
edges of the molecule do not participate in any particular
interactions with the allosteric binding site of GLS. The
findings prompted us to modify the phenylacetyl moiety in an
attempt to gain additional interactions with GLS. Herein we
report the design, synthesis, and biological evaluation of
allosteric glutaminase inhibitors based on a 1,4-di(5-amino-
1,3,4-thiadiazol-2-yl)butane scaffold into which a variety of
modified phenylacetyl groups were incorporated at the 5-amino
group.
In the first series of compounds 2a−o, we attached a
phenylacetyl group to one end of the 1,4-di(5-amino-1,3,4-
thiadiazol-2-yl)butane scaffold and incorporated various
modified phenylacetyl groups into the other end of the
scaffold. The general synthetic route from the common
intermediate 1 is illustrated in Scheme 1. HATU-mediated
a
a
Scheme 1. Synthesis of 2a−o
Conditions: (a) DIPEA, HATU, DMF, 0 °C; (b) 2 N NaOH, THF,
rt; (c) for 4r to 6r, DIPEA, HATU, DMF, 0 °C; (d) for 4s to 6s, T3P,
NEt3, DMF, RT; (e) 2.5% LiOH, THF, RT; (f) NH2CSN2H3, POCl3,
90 °C; (g) DIPEA, HATU, DMF, 0 °C; (h) 2 N NaOH, THF, rt.
acetoxyphenylacetamide) derivative 2p was prepared by
coupling 4-acetoxyphenylacetic acid with 5,5′-(butane-1,4-
diyl)bis(1,3,4-thiadiazol-2-amine) 3. Compound 2p was sub-
sequently converted into bisphenol derivative 2q. Additional
derivatives 2r−u containing two distinct modified phenyl-
acetamide groups were synthesized using methyl 5-(5-amino-
1,3,4-thiadiazol-2-yl)pentanoate 5 as a starting material. The
free amino group of 5 was coupled with 3-trifuloromethylox-
yphenylacetic acid 4r using HATU-mediated coupling method
to give the corresponding carboxamide 6r. Preparation of 6s
from 3-pyridylacetic acid 4s and 5 utilized propylphosphonic
anhydride (T3P)14 as a coupling reagent since HATU-
mediated reaction resulted in the low yield of the desired
product. Hydrolysis of the methyl ester of 6s−r afforded 7s−r,
which was subsequently converted into bis(5-amino-1,3,4-
thiadiazole) derivatives 8r−s by condensing with thiosemicar-
bazide in the presence of POCl3. Coupling with 4-
acetoxyohenylacetic acid provided 2r−s, which were further
a
Conditions: (a) DIPEA, HATU, DMF, 0 °C; (b) 2 N NaOH,
MeOH, rt; (c) 2 N NaOH, THF, rt; (d) 4 N HCl, 1,4-dioxane, rt.
coupling was found to be most effective in forming
carboxamide at the 5-amino group of the thiadiazole ring of
1. Compounds 2j−l were further converted into 2m−o,
respectively.
A second series of compounds contain modified phenyl-
acetamide groups at the both ends of the 1,4-di(5-amino-1,3,4-
thiadiazol-2-yl)butane scaffold. As shown in Scheme 2, bis(4-
B
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transformed into the corresponding phenol derivatives 2t−u by
base-mediated hydrolysis.
GLS unit and Glu325 from a diagonally opposite GLS unit in
the vicinity of the phenylacetyl groups of BPTES. Given the
flexible side chain of these residues, it is possible that the
phenolic group of 2m serves as a hydrogen bond acceptor from
the guanidinium group of Arg317 and a donor to the
carboxylate group of Glu325, contributing to the stronger
affinity of 2m to GLS. The phenolic group also appears to
improve the aqueous solubility of 2m (17 μg/mL) as compared
to BPTES (0.14 μg/mL).
All new synthetic compounds were tested for their ability to
inhibit GLS using L-[3H]-glutamine as substrate and human
kidney-type glutaminase (hKGA124−669).10 In this assay,
BPTES and CB-839 inhibited GLS with IC50 values of 3.3 and
0.06 μM, respectively. GLS inhibitory data of the new
analogues 2a−u are summarized in Table 1.
Compounds 2p−u possess modified phenylacetamide groups
at both ends of the 1,4-di(5-amino-1,3,4-thiadiazol-2-yl)butane
scaffold. Although compound 2p bearing two 4-acetoxypheny-
lacetyl groups was only as potent as compound 2a, the bis(4-
hydroxyphenylacetyl) derivative 2q exhibited 10-fold higher
GLS inhibitory potency. While this is consistent with the
improvement seen in the 4-hydroxyphenylacetyl derivative 2m
as compared to 4-acetoxyphenylacetyl derivative 2j, there
appears to be little synergistic effects between the two phenol
moieties of 2q, which would make the compound more potent
than 2m. Nevertheless, compounds containing a 4-hydrox-
yphenylacetyl group (2q, 2t, and 2u) still showed reasonably
potent GLS inhibitory activity, demonstrating the key role
played by the 4-hydroxyphenylacetyl moiety in binding to the
GLS allosteric site.
Table 1. Inhibition of GLS by Compounds 2a−u
a
cmpd
R1
R2
IC50 (μM)
2a
2b
2c
2d
2e
2f
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
1.8 0.1
4-MeOPh
4-PhOPh
4-BnOPh
4-Me2NPh
3-HOPh
0.21 0.04
0.38 0.03
>25
3.0 0.3
0.12 0.01
0.17 0.02
0.07 0.01
0.07 0.00
0.54 0.04
2g
2h
2i
4-bromo-2-pyridyl
2-pyridyl
In order to further characterize the mode of inhibition of
compound 2m, we examined the time dependency of GLS
inhibitory potency. As shown in Figure 2, compound 2m
3-pyridyl
2j
4-AcOPh
4-MeO2CPh
4-BocHNPh
4-HOPh
2k
2l
13
2
0.45 0.04
0.07 0.01
0.11 0.01
2.3 0.3
2m
2n
2o
2p
2q
2r
2s
2t
4-HO2CPh
4-H2NPh
4-AcOPh
4-HOPh
4-AcOPh
4-HOPh
1.2 0.2
0.12 0.02
2.0 0.2
3-CF3OPh
3-pyridyl
3-CF3OPh
3-pyridyl
4-AcOPh
4-AcOPh
4-HOPh
0.12 0.01
0.51 0.06
0.14 0.01
2u
4-HOPh
a
Values are mean SD of at least four experiments.
Modifications at one of the two phenylacetyl groups of 2a
(compounds 2b−o) had varied effects on GLS inhibitory
potency. Among them, four compounds displayed IC50 values
higher than that of 2a. Incorporation of a 4-benzyloxypheny-
lacetyl group (compound 2d) resulted in a significant loss of
inhibitory activity, underscoring the steric limits of the pockets
at the edges of the allosteric binding site. Incorporation of
positively charged substituents such as dimethylamino (com-
pound 2e) and amino (compound 2o) groups also led to
inhibitors with slightly higher IC50 values, while a more
substantial loss of inhibitory potency was seen as a result of
introducing a 4-methoxycarbonyl group (compound 2k). Many
derivatives within this series exhibited improved GLS inhibitory
potency compared to 2a. It is worth noting that most of these
inhibitors possess substituents capable of serving as a hydrogen
bond acceptor, which may be contributing to the stronger
intermolecular interaction with GLS. Compounds 2h, 2i, and
2m displayed IC50 values below 100 nM. Compound 2m is
particularly interesting as its phenolic group can possibly act as
both a hydrogen bond acceptor and a donor. In fact, nearly 8-
fold increase in potency from the corresponding acetoxy
derivative 2j suggests the potentially important role played by
the phenolic hydrogen. Indeed, the cocrystal structure of
BPTES bound to GLS (3VOZ)13 presents Arg317 from one
Figure 2. Effect of preincubation time on GLS inhibitory potency of
CB-839 and compound 2m.
showed no time-dependent increase in GLS inhibitory potency.
In sharp contrast to compound 2m, CB-839 displayed
increasing potency toward GLS over time as previously
reported.11 The precise structural features that produce time-
dependent GLS inhibition have not been clearly elucidated
though it is conceivable that the pyridazine ring unique to CB-
839 may play a critical role in its mode of GLS inhibition.
Antiproliferative effects of 2m were compared to BPTES and
CB-839 in triple negative breast cancer cell lines, MDA-MB-
231 and HCC1806. As shown in Figure 3, the three
compounds exhibited various degrees of antiproliferative
activity in both cell lines. At 72 h of incubation, compound
2m was least effective in both cell lines and displayed
statistically significant growth inhibition only in MDA-MB-
231 at the higher concentration (1000 nM). The superior
effects of CB-839 may be attributed to its time-dependent
C
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Letter
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00060.
Description of synthetic procedures and identification of
compounds; experimental protocols for glutaminase and
antiproliferative assays (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 410-614-0982. E-mail: ttsukamoto@jhmi.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work was supported by NIH grants (R21NS074151 to
T.T., R21CA169757 to A.L., P30MH075673 to B.S.S.,
R01CA19389501 to B.S.S., and F32CA200275 to S.C.Z.) and
a Maryland Innovation Initiative grant (MII 90062191 to A.L.).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
BPTES, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl
sulfide; T3P, propylphosphonic anhydride; DON, 6-diazo-5-
oxo-L-norlucine
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■
Figure 3. Effects of BPTES, CB-839, and compound 2m on (A)
MDA-MB-231 and (B) HCC1806 cell growth. Cell viability assays
were performed in quadruplicate, and error bars represent standard
deviation. Live cell viability was determined using CellTiter-Glo
Luminescent Cell Viability Assay daily. *P < 0.05 compared to the
controls.
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