Design and synthesis of novel tellurodibenzoic acid compounds as kidney-type glutaminase (KGA) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.04.032

Organotellurium compounds have been reported as an immune-modulator sensitizing chemotherapeutics. Herein, we report the design and synthesis of a series of novel tellurodibenzoic acids as mimics of diphenylarsenic acid (DPAA) and potential selective KGA

Full text of DOI:10.1016/j.bmcl.2019.04.032

Accepted Manuscript
Design and Synthesis of Novel Tellurodibenzoic Acid Compounds as Kidney-
Type Glutaminase (KGA) Inhibitors
Wei Hou, Yucheng Zhou, Jingjing Rui, Ruisong Bai, Aman K.K. Bhasin,
Benfang Helen Ruan
PII:
S0960-894X(19)30252-5
https://doi.org/10.1016/j.bmcl.2019.04.032
BMCL 26399
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
4 February 2019
9 April 2019
23 April 2019
Please cite this article as: Hou, W., Zhou, Y., Rui, J., Bai, R., Bhasin, A.K.K., Helen Ruan, B., Design and Synthesis
of Novel Tellurodibenzoic Acid Compounds as Kidney-Type Glutaminase (KGA) Inhibitors, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.04.032
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Design and Synthesis of Novel Tellurodibenzoic Acid
Compounds as Kidney-Type Glutaminase (KGA) Inhibitors
a,1
a,1
1
1
2
Wei Hou , Yucheng Zhou , Jingjing Rui , Ruisong Bai , Aman K.K. Bhasin , Benfang Helen
1
,
Ruan
1
College of Pharmaceutical Science, Institute of Drug Development & Chemical Biology (IDD &CB), Collaborative Innovation
Center of Yangtza River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou,310014, PR China
Department of Chemistry and Centre of Advanced Sciences, Panjab University, Sector 14, Chandigarh, 160014, India
2
ABSTRACT
Organotellurium compounds have been reported as an immune-modulator sensitizing
chemotherapeutics. Herein, we report the design and synthesis of a series of novel
tellurodibenzoic acids as mimics of diphenylarsenic acid (DPAA) and potential selective KGA
inhibitors. Representative compound 3B exhibited potent inhibition of KGA and
glutamine-dependent HCT-116 cells. Stability experiments indicated that 3B has excellent
3 2 2 2
stability under acidic (HCOOH), basic (NH •H O) and oxidative (H O ) conditions, but reacts
with β-ME, DTT and lysine which suggested that compound 3B may interact with cysteine or
lysine residues. Moreover, molecular docking disclosed that compound 3B binds to the allosteric
317
320
321
322
394
325
site of the GAC tetramer containing Arg -Lys -Leu -Phe -Tyr -Glu , which helped to
rationalize the SAR and further design and optimization. Taken together, compound 3B could be
used as a starting point for the development of new KGA inhibitors.
Keywords: K-type Glutaminase; Allosteric Inhibitor; Hexylselen; Synthesis; Biomolecular
interaction assay
Organotellurium compounds have shown early promise as therapeutic agents. Ammonium
tri-chloro(dioxoethylene-O,O'-)tellurate (AS-101) demonstrated immunomodulating properties
1-3
with minimal toxicity. Diethyl-2-phenyl-2-tellurophenyl vinylphosphonate shows antioxidant
4
,5
activity against excitotoxic agents without neurotoxic effects in rat brain. Diaryl tellurides were
6
discovered as efficient retarders of lipid peroxidation. One of the most active organotellurium
compounds, bis(4-aminophenyl) telluride, reacted considerably faster with DPPH than the
corresponding chalcogen elements, selenide or sulfide. Some arylselenium compounds, such as
7
8
9,10
methyl(phenyl)selane, benzeneselenol and their deriviatives, were found to exert antitumor
effects by induction of cell cycle arrest, apoptosis and angiogenesis. Beside these studies, very
little else was reported for novel synthesis of organotellurium compounds or mechanism of action
studies at the molecular level.
Accordingly, we are interested in designing, synthesizing and exploring the biological
activity and chemical reactivity of diphenyl tellurium derivatives. As depicted in Figure 1,
diphenylarsenic acid (DPAA) was reported to be an efficient KGA/GAC allosteric inhibitor.
DPAA downregulated the level of glutaminase
C
(GAC) in HepG2 cells in
a
11
concentration-dependent manner, and GAC/KGA is the initial enzyme of glutaminolysis and has

Corresponding author. Tel: + 86-18357023608; fax: + (0086) 571-88871098; e-mail: ruanbf@zjut.edu.cn,
ruanbf@yahoo.com.
a
These authors contributed equally to this work.

1
2,13
become an attractive target for cancer therapy.
Arsenate with Tellurium would result in a novel KGA/GAC inhibitor. Because diphenylditelluride
We were interested in testing if replacing the
14
(
DPDT) was reported to be a potent neurotoxin disrupting the homeostasis of the cytoskeleton
1
5
and was neurotoxic to mice, which might due to its high lipophilicity and easy penetration of the
blood-brain barrier. To generate a KGA inhibitor with low neurotoxicity, we designed
tellurodibenzoic acid 3 and ditellanediyldibenzoic acid 4 as our synthetic targets to explore their
inhibition of KGA and glutamine-dependent cancer cells. Based on computational modeling, we
envisaged that the dimeric structure may help them to match with the allosteric binding pocket at
the dimer-dimer interface of KGA to inhibit the protein activity.
Figure 1. Structure of DPDT and DPAA, and Molecular design of Tellurodibenzoic acid and
ditellanediyldibenzoic acid Compounds.
The synthesis of compounds 3 and 4 is shown in Figure 2. Commercially available
2
-aminobenzoic acids 1 were used as starting materials. Treating 1 with an aqueous solution of
sodium nitrite in the presence of HCl afforded a solution of the corresponding diazonium salt 2,
1
6
which was then treated with freshly prepared disodium ditelluride solution . To our surprise,
compound 4 was not obtained and 3 was only obtained in relative low yields (10-24%). Perhaps
the diazonium salts were converted to radical species A under reduced conditions, which
subsequently reacted with the ditellanediyldibenzoic acid intermediates to give tellurodibenzoic
1
7
18
acid compounds 3 (Figure 3) . Next we adopted sodium telluride solution to improve the yields
and 3A-3F were obtained in 30-60% yields. Besides, treating 3B with excessive SOCl afforded
2
acyl chloride intermediate 5, which was then converted into esters (6A-C) or amides (6D-F) by
1
reaction with corresponding alcohol or amines. These compounds were characterized by H and
1
3
C NMR spectroscopy, and low and high resolution-mass spectrometry (see Supplementary data
for detail).
Figure 2. Synthesis methods of Compounds 3A-3F, 5 and 6A-F.

Figure 3. Proposed mechanism.
As shown in Table 1, all the synthesized compounds were initially tested for their inhibitory
effects against KGA, GDH and thioredoxin reductase (TrxR), respectively. Generally, all the
compounds showed no inhibitory activity against GDH and TrxR (IC50>66.7 μM). With the
exception of 3D, the tellurodibenzoic acids exhibited moderate to good inhibitory activity against
KGA with IC50 values ranging from 2.4 to 11.3 μM, while the esters and amides showed no
inhibitory activity (6A-F). The unsubstituted phenyl compound 3A inhibited KGA with an IC50
value of 7.0 μM. Introducing the bromo or hydroxy group to the C-4 position resulted in
compounds 3B-C with improved activity, and the methoxy (3F) or iodo (3G) group with
maintained inhibitory activity, while methyl analogue 3H was slightly less active. Among them,
3
B was most potent (IC50 = 2.4 μM), nearly 3-fold more potent than 3A. However, it was found
that the MeO- group at C-5 (3D) or C-6 (3E) position resulted in sharply or slightly reduced
inhibitory activity. These results indicated the C-4 position was a suitable site for further
modification. We then evaluated their antiproliferative activity against glutamine-dependent
19-20
human colon cancer cells (HCT-116).
The results showed that compounds 3B and 3F, which
had better inhibitory effect against KGA, were also more potent in inhibiting the proliferation of
HCT-116 cells, with EC50 values of 5.0 μM and 10.2 μM respectively (Figure 4).
Table 1. Biological Activity of Compounds 3A-H and 6A-F.
a
b
IC50 (μM)
EC50 (μM)
1
2
3
4
Compd.
R
R
R
R
KGA GDH TrxR
HCT-116
17.8
5.0
18.0
>50.0
7.5
10.2
25.6
16.9
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
3
A
H
H
H
-OMe
H
H
H
H
H
H
H
H
H
H
-OMe
H
H
H
H
H
H
Br
-OH
H
H
-OMe
I
-Me
Br
Br
Br
Br
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OMe
-OEt
-OBn
7.0
2.4
6.3
>66.7 >66.7
>66.7 >66.7
>66.7 >66.7
3
B
3
3
C
D
>66.7 >66.7 >66.7
3
E
11.3
7.8
7.4
>66.7 >66.7
>66.7 >66.7
>66.7 >66.7
>66.7 >66.7
3
F
3
3
G
H
10.2
6
A
>66.7 >66.7 >66.7
>66.7 >66.7 >66.7
>66.7 >66.7 >66.7
6
B
6
6
C
D
H
H
H
H
H
H
H
H
-NHMe >66.7 >66.7 >66.7
-NHEt 62.8 >66.7 >66.7
-NHPh >66.7 >66.7 >66.7
6
E
Br
Br
6
F

a
KGA, GDH and TrxR were pre-treated with the tested compounds for 0.5 h. Each experiment was run
for at least three times independently in duplicates.
b
HCT-116 cells were treated with the tested compounds for 5 days. Each experiment was run for at
least three times independently in duplicates.
Figure 4. Biochemical assay: KGA Inhibition by compound 3A-H.
Figure 5. Docking compounds 3B and 3D to the tetrameric GAC (PDB: 3UO9). (A) The
interactions of 3B with the GAC/KGA enzyme, carbon atoms are shown in green, oxygen atoms
in red, nitrogen atoms in blue and tellurium atom in brown. (B) The interactions of 3D with the

GAC/KGA enzyme, carbon atoms are shown in green, oxygen atoms in red, nitrogen atoms in
blue and tellurium atom in brown.
To explore their possible binding mode, we carried out a docking study of 3B and 3D with
2
1
human GAC tetramer (PDB: 3UO9) via Discovery Studio 3.0 molecular docking software
BIOVIA). The proposed binding mode of 3B and 3D in an allosteric binding pocket are illustrated
(
in Figure 5. It was observed that the central diphenyltellane module of 3B fit into the hydrophobic
environment composed with Phe322 and Leu321. Moreover, the carboxyl groups formed
hydrogen bonds with residues Tyr394 (2.89 Å) and Arg317 (2.28 Å), respectively. Whereas
compound 3D with a methoxy-group at C-6 position has a greater dihedral angle between the two
phenyl rings, which disturbed the proper orientation of the C-2 carboxyl groups, thus hampering
the H-bond formation like 3B. 3D was observed to form a single hydrogen bond with residue
Leu321 (1.94 Å). The LibDockScore of 3B and 3D were 87.1292 and 57.3929, respectively.
Besides, the poor activity of esters and amides may be attributed to their increased lipophilicity,
which prevents them from interacting with residues Tyr394 and Arg317. These docking results
were in accord with their potencies, so this binding model may explain the SAR observed above.
Figure 6. Reactivity of organotellurium compounds: compound 3B were incubated with a series
of reagents at 37 °C for 24 h and then aliquots were taken for HPLC analysis. New adducts were
observed in reactions with Lys, DTT, β-Me.
We next tested the chemical stability of compound 3B in vitro. As depicted in Figure 6, 3B was
incubated under a series of conditions, including nucleophilic (Cys, Ser, β-ME, DTT, Lys), neutral
2 3 2 3 2 2
(MeOH, H O), acidic (HCOOH), basic (NH •H O), reductive (NaHSO ) and oxidative (H O ).
The incubations were conducted at 37 °C for 24 h and then aliquots were taken for HPLC analysis.
The results indicated that compound 3B was not changed under most of the tested conditions, such
3 2 2 2
as under the acidic (HCOOH), basic (NH •H O) and oxidative (H O ) conditions, suggesting 3B
has excellent stability. However, additional peaks were observed when β-ME, DTT and Lysine
were employed (Figure 6, labeled with stars), suggesting 3B reacts with β-ME, DTT and lysine.
This reactivity suggests that tellurium-containing compounds may react with lysine residues of a
protein. These results may be helpful information for further drug design and optimization.

In summary, we have designed and synthesized a class of novel tellurodibenzoic acids as
320
317
selective KGA inhibitors which showed binding to the Lys -Arg containing allosteric site
based on computer-modelling. Representative compound 3B exhibited selective inhibition of
KGA and the glutamine-dependent HCT-116 cell line. Chemical stability studies indicated that 3B
3 2 2 2
has excellent stability under acidic (HCOOH), basic (NH •H O) and oxidative (H O ) conditions,
but reacts with β-ME, DTT and lysine which suggested that the tellurium-contained 3B molecules
may interact with lysine residues of a protein. Based on these preliminary results compound 3B
may be used as a starting point for the development of new KGA inhibitors.
Acknowledgments
We appreciate financial support from QinLan Grant award from Hangzhou innovation foundation, China
(Number: H1160492), Start-up funding of Zhejiang University of Technology, China (Number:414800129)
and Zhejiang province Natural Science Foundation/General Project, China (Number: LY19H300002).
Supplementary Material
1
13
Synthesis, H NMR, C NMR, and MS of new compounds and other data associated with this article.
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