Novel 1,3,4-Selenadiazole-Containing Kidney-Type Glutaminase Inhibitors Showed Improved Cellular Uptake and Antitumor Activity

Article abstract of DOI:10.1021/acs.jmedchem.8b01198

Kidney-type glutaminase [KGA/isoenzyme glutaminase C (GAC)] is becoming an important tumor metabolism target in cancer chemotherapy. Its allosteric inhibitor, CB839, showed early promise in cancer therapeutics but limited efficacy in in vivo cancer models. To improve the in vivo activity, we explored a bioisostere replacement of the sulfur atom in bis-2-(5-phenylacetamido-1,2,4-thiadiazol)ethyl sulfide and CB839 analogues with selenium using a novel synthesis of the selenadiazole moiety from carboxylic acids or nitriles. The resulting selenadiazole compounds showed enhanced KGA inhibition, more potent induction of reactive oxygen species, improved inhibition of cancer cells, and higher cellular and tumor accumulation than the corresponding sulfur-containing molecules. However, both CB839 and its selenium analogues show incomplete inhibition of the tested cancer cells, and a partial reduction in tumor size was observed in both the glutamine-dependent HCT116 and aggressive H22 liver cancer xenograft models. Despite this, tumor tissue damage and prolonged survival were observed in animals treated with the selenium analogue of CB839.

Full text of DOI:10.1021/acs.jmedchem.8b01198

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Article
Novel 1,3,4-Selenadiazole Containing Kidney-Type Glutaminase
Inhibitors Showed Improved Cellular Uptake and Antitumor Activity
Zhao Chen, li di, Ning Xu, Jinzhang Fang, Yan Yu, Wei Hou, Haoqiang Ruan, Panpan Zhu, Renchao
Ma, Shiying Lu, Danhui Cao, Rui Wu, Mowei Ni, Wei Zhang, Weike Su, and Benfang Helen Ruan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01198 • Publication Date (Web): 13 Dec 2018
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Novel 1,3,4-Selenadiazole Containing Kidney-Type Glutaminase Inhibitors Showed
Improved Cellular Uptake and Antitumor Activity
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Zhao Chen1a, Di Li 1a, Ning Xu1a, Jinzhang Fang1a, Yan Yu1, Wei Hou1, Haoqiang Ruan1,
Panpan Zhu1, Renchao Ma1, Shiying Lu1, Danhui Cao1, Rui Wu1, Mowei Ni2, Wei Zhang3,
Weike Su1, Benfang Helen Ruan1*
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aEqual first author
1College of Pharmaceutical Science, Collaborative Innovation` Center of Yangtza River Delta
Region Green Pharmaceuticals, IDD & CB, Zhejiang University of Technology, Hangzhou,
China
2 Center for Cancer Research, Zhejiang Cancer Hospital
3 Department of Urology, Tongde Hospital of Zhejiang Province
*Corresponding author
Benfang Helen Ruan (Ph. D)
Professor of Pharmaceutical Science
College of Pharmaceutical Science, Collaborative Innovation Center of Yangtza River Delta
Region, IDD & CB, Green Pharmaceuticals, Zhejiang University of Technology
E-mail: ruanbf@zjut.edu.cn; ruanbf@yahoo.com
Tel: 86-18357023608
Fax: (0086) 571-88871098
¤Current address: 18 Chaowang Road, Xiachengqu, Hangzhou, Zhejiang, China, 310014
Running title: 1,3,4-Selenadiazole containing KGA allosteric inhibitors
Key word: 1,3,4-selenadiazole, kidney type glutaminase inhibitors, KGA, gls1, EZMTT
non-toxic cell viability assay, potency, efficacy, ROS, biomolecular interaction assay,
xenograft model
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ABSTRACT: Kidney-type glutaminase (KGA/isoenzyme glutaminase C [GAC]) is
becoming an important tumor metabolism target in cancer chemotherapy. Its allosteric
inhibitor, CB839, showed early promise in cancer therapeutics but limited efficacy in in vivo
cancer models. To improve the in vivo activity, we explored a bioisostere replacement of the
sulfur atom in bis-2-(5-phenylacetamido-1,2,4-thiadiazol ethyl sulfide (BPTES) and CB-839
analogs with selenium using a novel synthesis of the selenadiazole moiety from carboxylic
acids or nitriles. The resulting selenadiazole compounds showed enhanced KGA inhibition,
more potent induction of reactive oxygen species (ROS), improved inhibition of cancer cells,
and higher cellular and tumor accumulation than the corresponding sulfur-containing
molecules. However, both CB839 and its selenium analogs show incomplete inhibition of the
tested cancer cells, and a partial reduction in tumor size was observed in both the
glutamine-dependent HCT116 and aggressive H22 liver cancer xenograft models. Despite this,
tumor tissue damage and prolonged survival were observed in animals treated with the
selenium analog of CB839.
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INTRODUCTION
Targeting KGA (or GAC)1-3 is expected to selectively inhibit the growth of tumor cells
growth, but not normal cells. This is because, in tumor cells, the Warburg effect increases
glycolysis 200-fold compared to normal cells, but the majority of glucose carbon is converted
to lactate for excretion and cannot be used in the mitochondrial tricarboxylic acid (TCA)
cycle. To compensate for this, cancer cells develop glutamine dependence, and KGA is highly
elevated4-5 to hydrolyze glutamine to glutamate and then to α-ketoglutarate for energy
production throughout the TCA cycle.
Glutaminase has two isoforms with very different structural features: liver-type
glutaminase (LGA) and kidney-type glutaminase (KGA). The glutaminase activity of LGA is
constant, whereas tetrameric KGA (or its isoenzyme GAC) is activated by phosphate6. In
addition, their tissue distributions are different; LGA exists predominantly in the liver, while
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KGA is mainly found in the kidney and brain and is highly elevated in tumors4-5. Interestingly,
in cases of liver cancer, down-regulation of LGA (GLS2 gene) and up-regulation of KGA
(GLS1 gene) were reportedly associated with poor outcome5-6. Therefore, KGA is an
important isoform to target in cancer therapeutics.
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So far, various KGA inhibitors have been developed for cancer, several of which are
shown in Figure 1. The active site inhibitors (Acivicin and DON)7 showed good in vivo
anticancer activity but high toxicity, perhaps due to the lack of selectivity against LGA, which
is important for liver function. Specific targeting of KGA is important to lower its toxic side
effects.
BPTES8-9 is known to be a KGA allosteric inhibitor, and its x-ray co-crystal structure
shows that it binds at the KGA tetramer interface. Since BPTES showed poor solubility (0.01
µM), several series of BPTES derivatives were prepared to improve solubility, including
Compound 69, UPGL0000410, thiazolidine-2,4-dione derivatives11, and CB-83912, as shown in
Figure 1. Thiazolidine-2,4-dione derivatives contain a Michael acceptor and react to KGA
protein residues. They demonstrated good in vivo anti-tumor effects, whereas the other
compounds demonstrated only limited in vivo efficacy. CB-839 was the most potent KGA
allosteric inhibitor; it inhibited a triple-negative breast cancer cell line with an IC50 value of
20–30 nM, but at an oral dose of 200 mg/kg, it achieved only 50% tumor growth suppression
in an in vivo xenograft model12.
To develop more potent KGA inhibitors, we investigated the possibility of improving
KGA inhibition and anticancer activity with the 1,3,4-selenadiazole group. In the co-crystal
structure of KGA and BPTES/CB8398,13,14, the thio-diazo is near Y394 at the allosteric site.
Y394 could provide an aromatic ring for π stacking or –OH for hydrogen bond formation.
Since hydrogen bonds only form among N, O, and F atoms, Y394 residue is more likely to
interact with the sulfur of the thiodiazole ring through polar or non-polar interactions. In
addition, due to the increase in electro-donation from O and N to S, the electro-density
increased from furan and pyrrole to thiophene15. Therefore, replacing sulfur with selenium
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could increase the electro-density of the selenophene ring and might affect the interaction
between Y394 and a 1,3,4-selenadiazole ring. The thiadiazol group of CB839 is also involved
in a water-mediated interaction with ASP327, and replacing sulfur with bulky selenium might
increase this interaction. In addition, the allosteric inhibitor BPTES was reported to bind to
KGA through an induced fit mechanism8, and it is difficult to predict changes in inhibitory
activity based on structure docking.
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As a bioisostere for sulfur and oxygen atoms, selenium can produce unique biological
activity. For example, selenocysteine is an essential amino acid that is required for the
biological activity of many critical human enzymes, such as glutathione peroxidase (GSH-Px),
thioredoxin reductase (TrxR), and deiodinase16. Various selenium-containing molecules
showed good anticancer activity, including isoselenocyanates17, benzoselenadiazole18,
methylseleninic acid19, di-selenium ether20, selenoureas21, ethylselen22, and hexylselen23.
Therefore, it seems likely that replacing the thio-diazo group with a selenio-diazo group in
this series of compounds would generate improved anti-cancer or other interesting biological
activity. However, the mechanism still remains to be investigated.
Chemical synthesis of 1,3,4-selenadiazole was reported previously in harsh chemical
synthetic conditions24–30 that are not suitable for chemical synthesis of compounds containing
sensitive functional groups, such as amine, hydroxyl, ester, or amide. Therefore, we explored
chemical synthesis using amino selenourea and nitrile under mild conditions, successfully
incorporating the 1,3,4-selenadiazole moiety into BPTES and CB839 with high yield. As
shown in Figure 2, various 1,3,4-selenadiazole derivatives of BPTES and CB839 were
synthesized for structure activity relationship (SAR) analysis. The effects of selenium
substitution and different acyl groups were evaluated using a reliable KGA enzyme activity
assay, Biolayer interference (BLI)-based biophysical assay, and precise cell-based assay. Our
results demonstrated that some 1,3,4-selenadiazole derivatives feature enhanced inhibition of
KGA, inhibition of cancer cell growth, and tumor reduction in xenograft models.
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Figure 1. Examples of KGA inhibitors reported in the literature.
RESULTS AND DISCUSSION
Synthesis of 1,3,4-selenadiazole derivatives of BPTES and CB839
Prior studies synthesized 1,3,4,-selenadiazoles under very harsh conditions, such as
{PhP(Se)(l-Se)}24, Woollins’ reagent (WR)]29, or refluxing hydrazinecarboselenoamide with
carboxyl compounds in phosphorous oxychloride28. Since thiadiazole can be readily
synthesized from hydrazinecarbothioamide with a carboxylic acid or nitrile using P2O5 or
trifluoroacetic acid (TFA) as a catalyst8, we attempted to react hydrazinecarboselenoamide
derivatives31-32 with nitriles in the presence of P2O5 or TFA to synthesize various 1,3,4-
selenadizole derivatives, as shown in Figure 2.
Hydrazinecarboselenoamide (A1) was synthesized according to the method described in
the literature33. Phenyl- or alkyl-substituted hydrazinecarboselenoamides were synthesized
from an isoselencynate reaction with suitable amines31. Phenylamines containing a strong
electron-withdrawing group, such as NO2 or CF3, or a bulky group, like tri-phenyl
methylamine, yielded no reaction product, as shown in Figure 2 (compound A series).
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Figure 2. Synthesis of 1,3,4-selenadiazole analogs: (a) selenium urea derivatives; (b)
bis-cyanide derivatives; (c) bis-1,3,4-selenadiazole; (d) symmetric and asymmetric BPTES
derivatives; (e) mono-cyanide derivatives; and (f) CB839 derivatives.
The resulting amino seleniumurea compound (A1) reacted well with the cyano
compounds (B or E). In general, for bisnitriles, both cyano groups were derivatized to
1,3,4-selenadiazoles; when a large excess of bisnitrile was present, monosubstituted
1,3,4-selenazole could be obtained, providing more flexibility in chemical synthesis. In
addition, the reaction conditions are mild enough to yield selenadiazoles in the presence of
various functional groups, such as amine, amide, ether, and thioether. For example, the
1,3,4-selenadiazole analogs of BPTES and CB839 were synthesized with a good yield. The
BPTES analogs were synthesized using 3,3'-thiodipropanenitrile or adiponitrile as a starting
material, and then amide formation was performed using various acid derivatives9. CB839
analogs were synthesized from N-(6-(4-cyanobutyl)pyridazin-3-yl)-2-(3-(trifluoro-
methoxy)phenyl)acetamide according to the method described in the literature34 for SAR
analysis. The resulting 1,3,4-selenadiazole-containing BPTES and CB839 derivatives were
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subject to comparison of in vitro and in vivo activity with the corresponding 1,3,4-thiadiazole
compounds.
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SAR comparisons of selenadiazole analogs and their thiadiazole counterparts
Many previously reported KGA allosteric inhibitors were tested for activity in multiple
enzyme-coupled biochemical assays to assess SAR. These coupled assays include
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GluO-horseradish peroxidase measuring resorufin formation at ex530/em590 nm35-36
,
ammonia formation measured with Nessler’s reagent by absorbance at 450 nm36, glutamate
dehydrogenase (GDH)-diaphorase measuring resorufin formation at Ex544/Em590 nm36, and
[3H]-glutamate detection assay10. The complicated nature of these assays has resulted in great
variations in IC50 determination and, consequently, misinterpretation of SAR analysis. For
example, the IC50 values reported for BPTES in the literature range from 0.1–3.3 µM in
GluO-horseradish peroxidase-coupled assays. In addition, the solubility of BPTES was
determined to be 10 nM10,36; ebselen was mis-identified as a potent 9 nM KGA inhibitor23,36
and there were discrepancies in the optimal bridge length (4- or 6-carbon) in BPTES
analogs.10,23
;
Since GluO-horseradish peroxidase-coupled KGA assay36 is a three enzyme coupled
assay detecting H2O2 formation which is problematic in excluding false positives, we
performed our KGA assay using a stable
2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl)benzenesulfonate
sodium salt (EZMTT) dye for NAD(P)H detection. The coupled enzyme GDH was always
tested at the same time to serve as a blank control for identification of false positives. Because
KGA is activated by phosphate, the KGA enzyme was preincubated with the test compound
for 30 min at room temperature in a phosphate-free buffer, and then substrates and 200 mM
phosphate were added to initiate hydrolysis. After 4 h of incubation at room temperature, the
resulting glutamate was quantified by GDH-EZMTT detection reagents.
As shown in Table 1, the 1,3,4-selenadiazole compounds did not show inhibition of
GDH. In addition, the C series (CPD1-3) did not show KGA inhibition, perhaps due to the
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lack of an essential amide linkage, which is in agreement with the co-crystal structure of
KGA-BPTES, where the amide group forms via hydrogen bonding with multiple residues at
the allosteric site9.
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Table 1. Structure activity relationships of 1,3,4-selenadiazole and 1,3,4-thiadiazole
derivatives of BPTES and CB839
R2
X
X
N
R4
R3
N
N
N
A549
GDH
IC50
μM
KGA
IC50
μM
No.
R3
R4
R2
X
IC50
Max-INH
%
μM
H
N
CPD1
CPD2
CPD3
R3=R4
R3=R4
R3=R4
CH2CH2SCH2CH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
Se
Se
Se
>13
>13
>13
>13
>13
>13
>13
>13
>13
0±2%
0±3%
0±2%
H3CO
N
H
H
N
H
N
CPD4
CPD5
-NH2
CH2CH2CH2CH2
CH2CH2CH2CH2
S
S
>13
>13
>13a
>13a
>13
>13
0±3%
0±3%
O
O
H
N
R3=R4
O
N
H
CPD6
CPD7
CPD8
CPD9
CPD10
-NH2
-NH2
R3=R4
R3=R4
-NH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
Se
>13
>13
>13
>13
>13
1.1±0.03
1.4±0.05
0.027±0.003
0.28±0.05
2.1±0.3
>13
N
O
N
H
S
>13
N
O
N
H
Se
0.11±0.02
2.9±0.3
>13
81±4%
80±3%
N
N
O
N
H
S
O
HN
Se
N
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HN
CPD11
-NH2
CH2CH2SCH2CH2
S
>13
5.1±0.4
>13
N
N
O
HN
CPD12
CPD13
R3=R4
R3=R4
CH2CH2SCH2CH2
CH2CH2SCH2CH2
Se
>13
>13
0.06
1±0.3
82±4%
80±3%
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O
HN
S
0.13±0.02
2.2±0.4
N
H
N
CPD14
CPD15
CPD16
BPTES
-NH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
Se
>13
>13
>13
>13
2.8±0.2
>13
O
H
N
-NH2
S
5±0.5
>13
O
H
N
R3=R4
R3=R4
Se
S
0.06±0.01
0.13±0.03
0.23±0.06
0.42±0.05
82±3%
72±2%
O
O
H
N
H
N
O
CPD17
CPD18
CPD19
-NH2
R3=R4
-NH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
CH2CH2SCH2CH2
Se
Se
Se
>13
>13
>13
8±0.4
>13
0±2%
0±2%
60±3%
H
H
H
H
N
O
1.7±0.2
0.3±0.05
>13
H
H
H
O
N
H
5±0.3
H
H
H
N
O
N
N
NH
Se
CPD20
CB-839
>13
>13
0.001
0.017
82±2%
82±2%
N
O
N
F3CO
N
H
O
N
N
N
NH
0.002±0.0003
0.04±0.005
N
S
O
N
F3CO
N
H
N
O
O
N
F3CO
N
N
N
CPD21
>13
0.001±0.0003
0.017±0.004
80±3%
N
N
H
Se
H
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F
O
O
O
N
F3CO
CPD22
CPD23
NH
>13
>13
0.02±0.002
0.01±0.002
0.46
91±2%
90±3%
N
N
N
N
N
N
Se
O
H
H
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N
O
O
N
F3CO
N
N
N
0.01±0.002
N
N
Se
H
H
OCH3
a: The shape of the binding curve is abnormal, as shown in Figure 3.
Most D-series compounds (CPD4-19) showed KGA inhibition activity, except for CPD4
and CPD5. CPD5 was a BPTES derivative with a 4-carbon bridge between the two thioazoles
(R2)10 that was previously reported to be a better KGA inhibitor (IC50 = 1.6 µM) than BPTES
(IC50 = 3.3 µM; tested at the same time) in the GluO-horseradish peroxidase-coupled assay. In
our assay, CPD5 did not inhibit GDH and showed essentially no sigmoid dose response curve
with KGA (Table 1; Figure 3). However, we repeated the experiments several times with
different people. The KGA inhibition curves showed great variation in shape, and all lacked a
dose response. In a BLI biomolecular interaction assay (Figure 3), BPTES, CB839, CPD8,
and CPD20 prevented KGA from binding to BPTES immobilized on the chip, but CPD5 did
not. In addition, the solubility of CPD5 was very poor, possibly leading to non-specific
interaction with KGA and causing a noisy signal. Taken together, the optimal bridge for
bis-thiadiazole- or bis-selenadiazole-type KGA allosteric inhibitors is the 5-atom thioethyl
ether or the 6-carbon straight chain.
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Figure 3. Biochemical and biophysical analyses demonstrated that CPD8 and CPD20
have better KGA inhibitory activity than the corresponding BPTES and CB839. (a) GDH
inhibition assay; (b) KGA/GDH-coupled assay; (c) BLI subtraction assay to show compound
binding to KGA. Sensorgrams were plotted by subtracting two sensorgrams of KGA binding
to the immobilized BPTES in the presence or absence of a compound. One sample contained
only 100 nM KGA, and the other contained 100 nM KGA and a compound (BPTES, CB-839,
CPD8, CPD20, or CPD5). The subtracted sensorgram represents the amount of KGA bound
to the compound (BPTES, CB-839, CPD8, CPD20, or CPD5). The sensorgram showed no
binding of the compound (CPD5) to the KGA enzyme, because the enzyme could bind to
immobilized BPTES equally well in the presence or absence of the compound. When a
compound (10 µM BPTES, CB839, CPD8, or CPD20) can bind to the KGA enzyme, the
subtracted sensorgram will show the difference, shown as signals in pink or blue.
Even though researchers suggested that asymmetric BPTES analogs will have better
solubility and in vivo activity10, SAR analysis of CPD6-CPD17 demonstrated that, by
removing one of the corresponding phenylacetyl moiety (such as CPD6, CPD7, CPD10,
CPD11, CPD14, CPD15, or CPD17), the asymmetric analogs (CPD7 & CPD9; CPD8 &
CPD10) lost more than 10-fold activity in both the KGA assay and glutamine-dependent
A549 cancer cell proliferation assay. Also, the methylene next to the amide group is
important to activity; CPD17 and CPD19 showed a 10-fold difference in IC50 values. This is
supported by the co-crystal structure of BPTES and KGA8; since the KGA allosteric site
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exists at the interface of the tetramer, both thiodiazole and the amide bond extension of
BPTES showed important hydrogen interactions with tetrameric KGA, which may have led
the symmetric BPTES analogs to interact more than the asymmetric ones.
9
Interestingly, the BPTES selenadiazole analogs demonstrated more than two times better
potency than the corresponding 1,3,4-thiadiazole analogs in the KGA enzyme assay. SAR
analysis suggests that 1,3,4-selenadiazole fits better in the KGA allosteric site. In addition,
replacing the amide group with an amine lowered its KGA inhibitory activity, which is in
agreement with the co-crystal structure of KGA-BPTES, where the amide group forms via
hydrogen bonding with multiple residues at the allosteric site.
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To increase solubility, heteroaromatic groups were used to replace the phenyl group.
SAR analysis of seven pairs of 1,3,4-selenadiazoles and their 1,3,4-thiadiazole analogs
demonstrated that the 1,3,4-selenadiazoles compound showed better activity than the
corresponding 1,3,4-thiadiazoles compound in a KGA biochemical assay and cancer cell
growth inhibition assay. In particular, the indole-substituted 1,3,4-selenadiazole compound
showed five-fold improvement in potency and complete inhibition of KGA activity.
Additionally, the heteroaromatic group achieved complete inhibition of KGA enzyme
activity and increased compound solubility.
Selenadiazole analogs of CB839 were also synthesized (CPD20, CPD21, CPD22,
CPD23). CPD20 is selenium-substituted CB839, and it showed two times better potency
than CB839 in the KGA inhibition assay.
Comparison of the growth inhibitory activity of BPTES, CB839, and their
1,3,4-selenadiazole analogs in glutamine-dependent cell lines
Although BPTES and CB839 are potent KGA allosteric inhibitors, they achieved a
maximum of only approximately 50% reduction in tumor size in vivo. The poor solubility of
BPTES was blamed for its poor in vivo outcome. To select a suitable cell line for the assay,
we measured the glutamine dependence of several cell lines and their inhibition by KGA
inhibitors. Using non-toxic EZMTT reagents, we discovered that all the tested cell lines
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showed severe growth reduction in the absence of glutamine for five days, but they remained
alive. Interestingly, even though complete inhibition of KGA was achieved by CPD8, CB839,
and the selenium analogs in a biochemical assay, these compounds did not achieve complete
inhibition of glutamine-dependent cell lines such as A549 and Caki-1 (maximal inhibition:
70–90%) (Figure 4; Table 2). It is not likely that this is due to the solubility issue because the
water solubility was measured at 0.1 µg/ml for BPTES and 3 µg/ml for CB839. Thus, a
question remains: Why do potent and apparently efficacious KGA inhibitors (CB839 and
CPD20 compounds) show limited efficacy in a cell-based assay; is this a KGA-target-related
issue or a cellular penetration problem?
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Figure 4. KGA allosteric inhibitors (BPTES or CB839 type) only partially inhibit cancer cell
growth, whereas a KGA/GDH dual allosteric inhibitor (hexylselen) showed complete
inhibition.
Table 2. Inhibition of cancer cell growth by 1,3,4-selenadiazole and 1,3,4-thiadiazole
derivatives of BPTES and CB839
H22
IC50
(μM)
Caki-1
IC50
HCT116
IC50
Maximal
Maximal
Maximal
Inhibition %
(μM)
Inhibition %
(μM)
Inhibition %
BPTES
CB839
CPD9
CPD8
>10
29±3
44±3
42±2
71±3
1.16±0.12
0.04±0.009
3.92±0.35
0.4±0.05
73±3
88±2
85±3
87±4
0.54±0.08
0.028±0.005
2.2±0.13
69±4
90±3
93±2
94±2
>10
>10
1.38±0.12
0.32±0.09
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CPD20
6.78±0.21
>10
75±4
10±2
100±2
70±4
0.019±0.004
0.014±0.003
1.29±0.16
89±2
90±2
100±2
90±3
0.009±0.002
0.009±0.003
2.17±0.23
90±4
92±3
100±2
92±2
CPD21
Hexylselen
CPD23
0.84±0.09
4.05±0.25
0.02±0.003
0.02±0.003
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Comparison of cellular concentrations of 1,3,4-thiadiazole compounds and their
corresponding 1,3,4-selenadiazole analogs by Raman analysis37
As shown in Figures 3 and 4, KGA inhibitors showed complete inhibition of enzyme
activity, but only partial inhibition of cancer cell lines. However, the bis-selenadiazole
containing compounds showed improved efficacy in comparison with the corresponding
bis-thiadiazole compounds. We were interested in investigating whether the cellular uptake or
metabolism of the compounds played a role in cellular efficacy.
CPD6, CPD8, and CPD9 were subjected to a substrate uptake assay using Raman
analysis (Figure 5a), which was possible because their 1:1 mixtures with nanogold particles
showed strong SERS spectra. As shown in Figure 5, within wavelengths of 1100–1800 cm-1,
the 1:1 mixtures showed a dose-dependent increase in absorbance up to a compound
concentration of 2 µM, whereas in the absence of nanogold particles, no absorbance was
observed at these wavelengths. The intensity at 1100–1800 cm-1 wavelengths started to drop
completely at compound concentrations above 2 µM. Therefore, for our compound uptake
assay, we used premade 1:1 compound/nanogold mixtures up to a concentration of 2 µM.
In addition, because the peak at 1352 cm-1 wavelength does not exist in the control cells
in the absence of the compounds, we selected the 1352 cm-1 wavelength as the compound
marker for the cellular compound uptake assay. After treating the glutamine-dependent
Caki-1 cells with 1:1 compound/nanogold mixture particles for 3 h, 9 h, 16 h, and 24 h, SERS
spectra were collected to compare the cellular concentration of the compounds. Interestingly,
as shown in Figure 5c, the most potent selenadiazole compound (CPD8) showed the highest
cellular concentration; the intensity at 1100–1800 cm-1 was three to four times greater than its
corresponding thiadiazole analog (CPD9) or asymmetrical analog (CPD7). Overall, the
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bis-1,3,4-selenadiazole compounds accumulated to a much greater extent than the
corresponding bis-thiadiazole and mono-1,3,4-selenadiazole compounds.
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Figure 5. Raman analysis of the compounds (CPD6, CPD9, and CPD8) before and after their
cellular uptake. (a) SERS spectra show the dose response of the 1:1 mixtures of compound
with nanogold particles. (b) The dose response of the 1:1 mixtures at an absorbance of 1352
cm-1. (c) Cellular uptake and distribution of the 1:1 mixtures of 2 µM compound with
nanogold particles. (d) Light microscope pictures of the corresponding cell images in C.
1,3,4-selenadiazole analogs showed significantly enhanced ROS in A549 cancer cells
ROS is a key regulator of cancer cell growth. Many selenium-containing compounds
showed activation of ROS, although most reported selenium compounds are reactive16-23
Even though the mechanism of action remains to be investigated, as shown in Figure 6,
CPD20 and CPD22 generated more ROS than CB839 at a concentration of 10 µM.
.
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Figure 6. ROS activity. 10000/well A549 cells treated or not treated (Ctrl) with 100 µg/ml
ROSUP (positive control) or different concentration of CB839 or CPD20 or CPD23 (0, 3, or
10 µM) for 12 h. At a compound concentration of 10 µM, the selenium-containing analogs
showed improved ROS activity in comparison with CB839.
1,3,4-selenadiazole analog CPD20 showed improved efficacy in the glutamine-dependent
HCT116 tumor model
Because the growth of HCT116 cancer cells is inhibited by 1,3,4-selenadiazoles (CPD20)
more than the corresponding CB839 compound (Table 2 and Figure 4), we used the HCT116
cancer cell xenograft model to compare their in vivo efficacy. We subcutaneously injected 10
mg/kg of the compounds (CB839 and CPD20) into young nude mice (n=6). As shown in
Figure 7, CB839 and CPD20 reduced the size and weight of the HCT116 tumor, and
statistical analysis showed that the 40% reduction in tumor weight by CPD20 is statistically
significant.
In addition, H&E staining of the end-point tumor tissues was performed to assess the
tumor tissue damage. In the untreated control group (Figure 7e), the tumor cells were
arranged in sheets with large cell size, large nuclei, and a high nucleus/cytoplasm ratio, and
they were infiltrated with scattered lymphocytes. In the CPD20- or CB839-compound-treated
groups (Figure 7d), the tumor cells could not be clearly seen under a microscope, and the
CPD20-compound-treated tumors showed more necrosis and deterioration than the
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CB839-compound-treated ones. Overall, the in vivo efficacy is in good agreement with the
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cell growth inhibition results reported in Figures 4 and 6).
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Figure 7. Comparison of the in vivo efficacy of three different groups of HCT116 xenograft
models (n=6 mice per group). The selenium analog (CPD20) showed better in vivo activity
than CB839 after 15 injections of N.S. control, 10 mg/kg CB839, or 10 mg/kg CPD20
compound. (a) Comparison of the tumor weights of untreated and treated groups was
performed using an unpaired t test (*: P<0.05); (b) body weight changes; (c) tumor growth;
(d) H&E staining of untreated tumor controls; (e) H&E staining of CB839-treated tumor
slices; and (f) H&E staining of CPD20-treated tumor slices.
The 1,3,4-selenadiazole analog (CPD23) showed significantly improved efficacy for
reducing H22 tumor growth and prolonging survival
Because the growth of H22 cancer cells is inhibited by the 1,3,4-selenadiazole compound
(CPD23) more than the corresponding CB839 compound (Table 2 and Figure 4), we used the
H22 liver cancer cell xenograft model to compare their efficacy in vivo. The compounds
(CB839 and CPD23) were subcutaneously injected once a day in young ICR mice (n=10) at a
dose of 10 mg/kg. H22 is an aggressive cancer cell line that undergoes tumor metastasis
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quickly38. Therefore, we evaluated the compound activity based on not only the tumor’s
size/weight and tissue damage but also the animal’s survival rate.
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Figure 8 showed that CPD23 treatment resulted in a statistically significant reduction in
tumor size compared with the untreated control. Further quantification determined that the
H22 tumor weight was statistically significantly reduced by 40% (Figure 8), whereas at the
same dose, CB-839 showed only a minor reduction in comparison with the control group. In
addition, a simple viability assay showed that CPD20 could statistically significantly prolong
the survival of H22-bearing mice (Figure 8d), whereas in the control group, most animals
died within 7–12 days. Interestingly, HPLC analysis showed that no significant amounts of
CPD20 or CPD23 metabolites were found in tumors treated with the compounds. Figure 8e
showed that a higher level of selenadiazoles (CPD20 and CPD23) than CB839 was found in
the treated tumor, indicating higher uptake of selenium-containing compounds. This is
consistent with the results of Raman analysis. In addition, Figures 8f and 8g showed that
CPD20, CPD23, and CB839 are relatively stable in the blood and in mouse liver microsomes.
After incubation in blood for 2 h, essentially no changes in the compound concentration were
observed. After incubation in the mouse liver microsomes for 4 h, as quantified by HPLC
analysis based on UV absorbance at 254 nm, the level of CB83939, CPD20, CPD23, and the
control coumarin dropped to 46%, 50%, 30%, and 14%, respectively. Further, LC-MS
analysis revealed that CB839 (m/z 571; tR 7.97min) formed a metabolite, with a molecular ion
corresponding to the loss of the terminal pyridine and an oxygen (m/z = 475.11; tR 5.6 min).
However, for CPD20 or CPD23, LC-MS showed no difference in components between the 0
min and 4 h fractions, except that the amount of CPD20 (m/z 624.11; tR 8.2 min) or CPD23
(m/z 742.13; tR 10.7 min) was reduced. Perhaps it was difficult to extract the metabolites of
selenadiazoles. Because CPD20 and CB839 showed essentially the same metabolic rate as
liver microsomes, we consider selenadiazoles to be stable building blocks. Also, the
prolonged survival rate in mice treated with CPD20 indicates that the metabolites are less
likely to be toxic to the animals.
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Figure 8. The in vivo efficacy of CB-839, CPD20, and CPD23 (n=10 mice per group).
CPD23 significantly reduced tumor size and increased the necrotic area within tumors after
10 injections of N.S. control, 10 mg/kg CB839, and 10 mg/kg CPD23 (n= 10). (a) Picture of
the end point tumors; (b) tumor weight; (c) body weight changes; (d) survival analysis; (e)
compound accumulation in tumor; (f) compound stability in blood; (g) compound stability in
microsomes; (h) HPLC analysis of the liver microsomes treated with CPD20; (i) H&E
staining of the untreated tumor control; (j) H&E staining of CB839-treated tumor slices; (k)
H&E staining of CPD23-treated tumor slices. An unpaired t-test was used to compare the
treated group and the control and the CPD20- or CPD23-treated group and the CB839-treated
group. *: P<0.05; **: P<0.01; ***: P<0.001.
To evaluate CPD23-related tumor tissue damage, we H&E stained the end-point tumor
tissues. In the untreated control group (Fig. 8e), the tumor cells were arranged in sheets
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featuring large cell size, large nuclei, and a high nucleus/cytoplasm ratio that were infiltrated
with scattered lymphocytes. In the CPD23-treated group (Fig. 8g), tumor cells could not be
clearly seen under a microscope. The site of the original tumor was replaced by fibrous
hyperplasia, inflammatory cell infiltration, a foam-like histiocytic reaction, and scattered
deformed nuclei. This is evidence of tumor deterioration. Perhaps with longer treatment, a
more significant reduction in tumor size could be observed.
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Overall, selenium-substituted CB-839 analogs showed improved activity in inhibiting
aggressive liver tumor growth and prolonged survival. These results are in good agreement
with the cell growth inhibition results reported in Table 1.
DISCUSSION AND CONCLUSION
Targeting KGA has attracted many research groups to development of highly effective
cancer drugs with low toxicity that can prolong patients’ lives. So far, hundreds of
BPTES/CB839-type KGA allosteric inhibitors40,41 containing the thiadiazole moiety8-14 have
been reported. Major structural variations in the derivatives include the middle bridge chain
length8,10,42 or replacement of the bridge chain with a 5-6 membered heterocyclic ring12,42
,
replacement of the thiadiazole with another heteroaromatic group13,42,43, or replacement of the
terminal benzylester with other heterocycles or a substituted aromatic acetate group8-14. Due
to the limitations concerning quantitative precision of coupled-enzyme KGA assays, SAR
analysis of hundreds of KGA inhibitors is difficult. CB839 and UPGL00004 have the
potential to have significantly improved compound solubility, but their in vivo efficacies still
remained around 50% inhibition at up to 200 mg/kg doses. It has been suggested23 that
alternative bypass pathways might be the cause of the lack of efficacy, so we introduced
additional selenium to the KGA inhibitors to improve both their potency and anti-cancer
activity.
To evaluate the benefit of selenium in the molecule, we synthesized selenium
derivatives—BPTES and CB839—and then measured the biological activity of both the
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sulfur and corresponding selenium-containing compounds under the same conditions for SAR
analysis. In general, the SAR results are in good agreement with previous reports, except for
the compounds containing bis-thiadiazole with four carbons, which were reported to be potent
KGA inhibitors in the GluD-peroxidase-coupled assay10. However, in our well-controlled
KGA-GDH coupled assay, the compound (CPD5) did not show any dose-dependent
inhibition of either GDH or KGA enzymes.
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In addition, we observed that both BPTES and CB839 derivatives are potent and
efficacious KGA allosteric inhibitors that can achieve complete inhibition of KGA enzyme
activity. The selenadiazole scaffold significantly improved potency (by five times) in the
KGA biochemical assay. Also, selenium-containing derivatives showed better cellular uptake
and tumor accumulation than the corresponding sulfur-containing molecules. However, in
growth inhibition assays using glutamine-dependent cancer cells, these compounds (CPD8,
CPD9, CPD20, CB839, CPD21, and CPD23) only showed 60–90% inhibition, whereas the
KGA/GDH dual inhibitor hexylselen showed complete inhibition. Taken together, our results
support our proposal that 50% in vivo efficacy might be the limit for a reversible KGA
allosteric inhibitor.
In summary, most selenium building blocks are reactive, but, to our surprise,
selenadiazole is a stable chemical moiety that could be selectively taken up by cancer cells, as
shown by Raman analysis. Its accumulation in tumors was further demonstrated by HPLC
analysis. The selenadiazole CPD20 and CPD23 compounds achieved a statistically significant
weight reduction in HCT116 and H22 tumors and caused more severe tumor damage than
CB839. In addition, the selenium-containing CB839 analog showed an improved survival rate
in the H22 xenograft model. Even though the exact selenium-involved mechanism must still
be investigated, early results suggest that selenium substitution improved KGA inhibition,
compound tumor accumulation, and ROS-mediated activity, which further contributed to its
improved anti-cancer activity and prolonged animal survival.
Experimental section
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Materials and cell lines. Chemicals were purchased from Aladdin (California, USA) or other
commercial suppliers (Adamas Beta, Energy Chemical, J&K, Shanghai Chemical Reagent
Co., TCI) and used without further purification.1HNMR was performed with a Varian
Mercury 500 or 600 MHz NMR spectrometer. Low- and high-resolution mass spectra were
obtained in the ESI mode. EZMTT detection reagents were obtained from JNF Biotech Inc.
(Hangzhou, CN). A549, Caki-1, HCT116, H22, and other cell lines, cloning enzymes, and
assay kits were purchased from Labpal (Shanghai, China). Proteins (KGA, GDH, etc.) were
cloned and purified as reported previously44. UV-vis absorbance was measured using a
Flexstation 3 (Molecular Device, USA). Fluorescence images were taken using a
MiniMaxPro fluorescent microscope (Molecular Device). RPMI 1640 medium was purchased
from M&C Gene Technology Inc. (Beijing, China). Trypsin and EDTA were purchased from
Amresco (Solon, OH, USA). Fetal bovine serum (FBS) was purchased from Zhejiang
Tianhang Biological Technology Co., Ltd. (Zhejiang, China). ICR mice (SPF) were
purchased from the Zhejiang Institute of Medical Science (Hangzhou, CN). Nude mice (SPF)
were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China).
Animal experiments were carried out at the Zhejiang University of Technology Laboratory
Animal Center, and all experimental procedures were conducted in compliance with ethical
standards and institutional guidelines for the care and use of laboratory animals at the
Zhejiang University of Technology (Hangzhou, China) as well as the National Institutes of
Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1996).
The synthetic compounds (CPD1–CPD23) used for biological tests had a purity above 95%,
as indicated by HPLC analysis. ICR/CD1 mouse liver microsomes was purchased from the
Research Institute for Liver Diseases Co., Ltd. (Shanghai, China). Coumarin was purchased
from Meryer Chemical Technology Co., Ltd. (Shanghai, China). NADPNa2 was purchased
from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). G6P
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(glucose-6-phosphate) and G6PDH (glucose-6-phosphate dehydrogenase) were purchased
from Plantcell Bio-Technology Co., Ltd. (Beijing, China).
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Synthesis of selenadiazole containing amines (C series). As shown in Figure 2, the
compound A series (4.67 mmol) was mixed with a bis-cyano compound B series (2 mmol) in
trifluoroacetic acid (10 ml) and stirred for 4 h until the reaction was completed. The
selenadiazole-containing BPTES derivatives were purified by column chromatography,
leading the compound C series (CPD1-3) to have approximately 60% yield.
5,5'-(thiobis(ethane-2,1-diyl))bis(N-(4-methoxyphenyl)-1,3,4-selenadiazol-2-amine)
(CPD1)
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Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.7). Analytical HPLC method tR =11.9
min; 97% purity. 1H NMR (500 MHz, DMSO-d6) δ 10.05 (s, 1H), 7.51 (d, J = 9.0 Hz, 1H),
6.90 (d, 1H), 3.72 (s, 2H), 3.17 (t, J = 9.0, 5.0 Hz, 1H), 2.91 (t, J = 7.0 Hz, 1H). LRMS (ESI,
[M + H]+) m/z 597.4.
5,5'-(thiobis(ethane-2,1-diyl))bis(N-benzyl-1,3,4-selenadiazol-2-amine)(CPD2)
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.6). Analytical HPLC method tR =11.2
min; 99% purity. 1H NMR (500 MHz, DMSO-d6) δ 8.15 (t, J = 5.6 Hz, 1H), 7.40 – 7.30 (m,
5H), 4.45 (d, J = 5.5 Hz, 2H), 3.07 (t, J = 7.0 Hz, 2H), 2.83 (t, J = 7.0 Hz, 2H). LRMS (ESI,
[M + H]+) m/z 565.0. HRMS: 565.0156 (M + H+).
5,5'-(thiobis(ethane-2,1-diyl))bis(N-phenethyl-1,3,4-selenadiazol-2-amine)(CPD3)
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.6). Analytical HPLC method tR =12.2
min; 98% purity. 1H NMR (500 MHz, DMSO-d6) δ 7.75 (t, J = 5.5 Hz, 1H), 7.29 (t, 2H), 7.25
(d, J = 1.5 Hz, 1H), 7.24 – 7.18 (m, 2H), 3.47 (dd, J = 12.5, 7.0 Hz, 2H), 3.08 (t, J = 7.0 Hz,
2H), 2.87 (t, 2H), 2.84 (t, J = 6.0 Hz, 2H). LRMS (ESI, [M + H]+) m/z 593. HRMS: 593.0467
[M + H]+.
Synthesis of selenadiazole containing BPTES derivatives (D series; CPD4-19).
5,5'-(thiobis(ethane-2,1-diyl))bis(1,3,4-selenadiazol-2-amine) (1.04 mmol, 2.0 eq), acid (0.52
mmol, 1.0 eq), HBTU (0.62 mmol, 1.2 eq), DIPEA (0.62 mmol, 1.2 eq), DMF (8 ml) were
stirred overnight at room temperature. After the reaction completed, the reaction solution was
poured into water (50 ml) and the precipitated solid was collected, dried, and separated by
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column chromatography. The compound CPD6, CPD7, CPD10, CPD11, CPD14, CPD15,
CPD17, CPD19 is more polar (Rf =0.8) than their corresponding symmetrical compound Rf
=0.9.
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N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide
(CPD4)
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LRMS (ESI, [M + Na]+) m/z 397.3; Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.4).
Analytical HPLC method tR =5.4 min; 99% purity.
N,N'-(butane-1,4-diylbis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide) (CPD5)
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.6). Analytical HPLC method tR =11.6
min; 98% purity. 1H NMR (600 MHz, DMSO-d6) δ 11.49 (s, 1H), 7.36 (d, J = 7.8 Hz, 2H),
7.12 (t, J = 7.8, 2H), 6.98 (t, J = 7.2 Hz, 1H), 3.72 (s, 2H), 3.10 (t, J = 7.2 Hz, 2H), 2.83 (t, J =
7.2Hz, 2H). LRMS (ESI, [M + Na]+) m/z 515.0
N-(5-(2-((2-(5-amino-1,3,4-selenadiazol-2-yl)ethyl)thio)ethyl)-1,3,4-selenadiazol-2-yl)-2-(
1-methyl-1H-indol-3-yl)acetamide（CPD6）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.5). Analytical HPLC method tR =4.8 min;
98% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.73 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.40 (d,
J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.21 (s, 2H), 7.14 (t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H),
3.92 (s, 2H), 3.76 (s, 3H), 3.23 (t, J = 15.0, 8.0 Hz, 2H), 3.06 (t, J = 7.0 Hz, 2H), 2.90 (t, J =
7.0 Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H). LRMS (ESI, [M + H]+) m/z 556. HRMS: 555.9904 [M +
H]+.
N-(5-(2-((2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl)thio)ethyl)-1,3,4-thiadiazol-2-yl)-2-(1-me
thyl-1H-indol-3-yl)acetamide（CPD7）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.5). Analytical HPLC method tR =8.0 min
with 95% purity. 1H NMR (500 MHz, DMSO-d6) δ 7.57 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0
Hz, 1H), 7.26 (s, 1H), 7.17 – 7.12 (m, 1H), 7.06 – 7.00 (m, 3H), 3.88 (s, 2H), 3.76 (s, 3H),
3.23 (t, J = 7.0 Hz, 2H), 3.05 (t, J = 7.0 Hz, 2H), 2.91 (t, J = 7.0 Hz, 2H), 2.84 (t, J = 7.5 Hz,
2H). LRMS (ESI, [M + H]+) m/z 460 [M+H]+. HRMS: 460.1021 [M + H]+.
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-3-yl)acetamide)（CPD8）
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Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.7). Analytical HPLC method tR =12.9
min; 99% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.73 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H),
7.39 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.5Hz, 1H), 3.89 (s,
2H), 3.75 (s, 3H), 3.24 (t, J = 7.0 Hz, 2H), 2.91 (t, J = 7.0 Hz, 2H). LRMS (ESI, [M+H]+)
m/z=727 . HRMS: 727.0580 [M + H]+.
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N,N'-((thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1-methyl-1H-indol-3-
yl)acetamide)（CPD9）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.7). Analytical HPLC method tR =13.4
min; 95% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.62 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H),
7.39 (d, J = 8.0 Hz, 1H), 7.25 (s, 1H), 7.16 – 7.12 (m, 1H), 7.04 – 7.00 (m, 1H), 3.88 (s, 2H),
3.75 (s, 3H), 3.23 (t, J = 7.0 Hz, 2H), 2.91 (t, J = 7.0 Hz, 2H). LRMS (ESI, [M+H]+) m/z=631
N-(5-(2-((2-(5-amino-1,3,4-selenadiazol-2-yl)ethyl)thio)ethyl)-1,3,4-selenadiazol-2-yl)-2-(
1-methyl-1H-pyrrol-2-yl)acetamide（CPD10）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.5) and 1H NMR. Analytical HPLC
method tR =5.2 min with 99% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.69 (s, 1H), 7.18 (s,
1H), 6.67 (s, 1H), 5.89 (d, J = 9.0, 4.4 Hz, 2H), 3.83 (s, 2H), 3.53 (s, 3H), 3.25 (t, 2H), 3.07 (t,
J = 7.0 Hz, 1H), 2.92 (t, J = 7.0 Hz, 1H), 2.86 (t, J = 7.0, 4.0 Hz, 2H), 2.73 (t, J = 7.0 Hz, 1H),
2.34 (t, J = 7.0 Hz, 1H). LRMS (ESI, [M + H]+) m/z = 506. HRMS: 505.9753 [M + H]+.
N-(5-(2-((2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl)thio)ethyl)-1,3,4-thiadiazol-2-yl)-2-(1-me
thyl-1H-pyrrol-2-yl)acetamide（CPD11）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.4). Analytical HPLC method tR =6.9 min
with 98% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.61 (s, 1H), 7.03 (s, 2H), 6.67 – 6.65 (m,
1H), 5.91 (dd, J = 3.5, 2.0 Hz, 1H), 5.89 – 5.87 (m, 1H), 3.81 (s, 2H), 3.54 (s, 3H), 3.25 (t, J =
7.0 Hz, 2H), 3.07 (t, J = 9.5, 5.0 Hz, 2H), 2.92 (t, J = 7.0 Hz, 2H), 2.86 (t, 2H). LRMS (ESI,
[M + H]+) m/z =410. HRMS: 410.0866 [M + H]+.
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N,N'-((thiobis(ethane-2,1-diyl))bis(1,3,4-selenadiazole-5,2-diyl))bis(2-(1-methyl-1H-pyrr
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Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.6). Analytical HPLC method tR =9.7 min;
98% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.69 (s, 1H), 6.66 (t, 1H), 5.91 – 5.89 (m, 1H),
5.89 – 5.87 (m, 1H), 3.82 (s, 2H), 3.53 (s, 3H), 3.27 (t, J = 7.1 Hz, 2H), 2.93 (t, J = 7.1 Hz,
2H). LRMS (ESI, [M + H]+) m/z =627. HRMS: 627.0275 [M + H]+.
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N,N'-((thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(1-methyl-1H-pyrrol-
2-yl)acetamide)（CPD13）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.5) and 1H NMR. Analytical HPLC
method tR =9.8 min; 98% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.60 (s, 1H), 6.66 (t, 1H),
5.91 – 5.89 (m, 1H), 5.89 – 5.87 (m, 1H), 3.81 (s, 2H), 3.54 (d, J = 2.5 Hz, 3H), 3.25 (t, J =
7.2 Hz, 2H), 2.94 (t, 2H).
N-(5-(2-((2-(5-amino-1,3,4-selenadiazol-2-yl)ethyl)thio)ethyl)-1,3,4-selenadiazol-2-yl)-2-p
henylacetamide（CPD14）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.5). Analytical HPLC method tR =6.5 min;
99% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.79 (s, 1H), 8.53 (s, 2H), 7.42 – 7.17 (m, 5H),
3.82 (s, 2H), 3.26 (t, J = 7.0 Hz, 2H), 3.12 (t, J = 7.0 Hz, 2H), 2.93 (t, J = 7.0 Hz, 2H), 2.88 (t,
J = 7.0 Hz, 2H). LRMS, (ESI, [M + H]+) m/z=502 [M+H]+ . HRMS: 502.9651 [M + H]+.
N-(5-(2-((2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl)thio)ethyl)-1,3,4-thiadiazol-2-yl)-2-pheny
lacetamide（CPD15）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.4). Analytical HPLC method tR =6.1 min
with 99% purity. 1H NMR (600 MHz, DMSO-d6)) δ 12.68 (s, 1H), 7.35 – 7.24 (m, 5H), 7.03
(s, 2H), 3.80 (s, 2H), 3.25 (t, J = 7.2 Hz, 2H), 3.06 (t, J = 7.2 Hz, 2H), 2.92 (t, J = 7.2 Hz, 2H),
2.85 (t, J = 7.2 Hz, 2H).
N,N'-((thiobis(ethane-2,1-diyl))bis(1,3,4-selenadiazole-5,2-diyl))bis(2-phenylacetamide)
（CPD16）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.6). Analytical HPLC method tR =11.9
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min with 99% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.76 (s, 1H), 7.34 – 7.24 (m, 5H),
3.81 (s, 2H), 3.26 (t, J = 7.0 Hz, 2H), 2.92 (t, J = 7.0 Hz, 2H). LRMS, (ESI, [M + H]+) m/z
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Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.7). Analytical HPLC method tR =10.8
min; 98% purity. 1H NMR (500 MHz, DMSO-d6) δ 2.84 (t, J = 7.0 Hz, 4H), 3.06 (t, J = 7.0
Hz, 4H), 7.04 (s, 4H)
(3r,5r,7r)-N-(5-(2-((2-(5-amino-1,3,4-selenadiazol-2-yl)ethyl)thio)ethyl)-1,3,4-selenadiazo
l-2-yl)adamantane-1-carboxamide（CPD17）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.6). Analytical HPLC method tR =12.4
min with 99% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.14 (s, 1H), 7.17 (s, 2H), 3.25 (t, J
= 7.0 Hz, 2H), 3.08 (t, J = 7.0 Hz, 2H), 2.92 (t, J = 7.0 Hz, 2H), 2.86 (t, J = 7.0 Hz, 2H), 2.00
(m, J = 9.0 Hz, 3H), 1.92 (m, J = 6.2 Hz, 6H), 1.69 (m, 6H). LRMS, (ESI, [M + H]+) m/z
=547. HRMS: 547.0268 [M + H]+.
(3r,3'r,5r,5'r,7r,7'r)-N,N'-((thiobis(ethane-2,1-diyl))bis(1,3,4-selenadiazole-5,2-diyl))bis(a
damantane-1-carboxamide)（CPD18）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.7) and 1H NMR. Analytical HPLC
method tR =13.1 min; 96% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.14 (s, 1H), 3.27 (t, J
= 7.0 Hz, 3H), 2.94 (t, J = 7.0 Hz, 3H), 2.00 (s, 4H), 1.93 (d, J = 2.0 Hz, 8H), 1.69 (s, 8H).
LRMS, (ESI, [M + H]+) m/z 731.1
2-((3r,5r,7r)-adamantan-1-yl)-N-(5-(2-((2-(5-amino-1,3,4-selenadiazol-2-yl)ethyl)thio)eth
yl)-1,3,4-selenadiazol-2-yl)acetamide（CPD19）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.6). Analytical HPLC method tR =12.9
min with 99% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.40 (s, 1H), 7.17 (s, 2H), 3.26 (t, J
= 7.0 Hz, 2H), 3.08 (t, J = 7.0 Hz, 2H), 2.92 (t, J = 7.0 Hz, 2H), 2.87 (t, J = 7.0 Hz, 2H), 2.24
(s, 2H), 1.96 – 1.88 (m, 2H), 1.70 – 1.61 (m, 2H), 1.60 – 1.54 (m, 11H).
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Synthesis of selenadiazole containing CB839 derivatives (F series). As shown in Figure 2
and S2, compound E series (1 g, 7.14 mmol) and selenacarbazide (1.18 g, 8.56 mmol) were
mixed in TFA (10 ml) and refluxed at 85 ° C for 4 hours. The reaction mixture was poured
into ice water (20 ml), neutralized to precipitate pink solid containing the selenadiazole group
which was coupled with an acid (3.14 mmol) in the presence of DIPEA (3.9 mmol) and
HBTU (3.12 mmol) in DMF (10 ml) for overnight at room temperature to give corresponding
compound CPD20-23.
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2-(pyridin-2-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)but
yl)-1,3,4-selenadiazol-2-yl)acetamide（CPD20）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.6). Analytical HPLC method tR =11.5
min; 99% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.75 (s, 1H), 11.31 (s, 1H), 8.49 (dd, J =
5.0, 2.0, 1.0 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H), 7.77 (td, J = 8.0, 2.0Hz, 1H), 7.55 (d, J = 9.0
Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.41 – 7.34 (m, 3H), 7.31 – 7.22 (m, 2H), 4.01 (s, 2H), 3.85
(s, 2H), 3.02 (t, J = 6.5 Hz, 2H), 2.88 (t, J = 7.0 Hz, 2H), 1.82 – 1.68 (m, 4H). LRMS, (ESI,
[M + H]+) m/z =620 [M+H]+ . HRMS: 620.1115 [M + H]+.
2-(1-methyl-1H-indol-3-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyrida
zin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)acetamide (CPD21)
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.7). Analytical HPLC method tR =12.9
min; 97% purity. 1H NMR (500 MHz, DMSO-d6) δ 12.68 (s, 1H), 11.31 (s, 1H), 8.19 (t, J =
10.0 Hz,2H), 7.56 (dd, J = 10.5, 6.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 7.5 Hz, 2H),
7.29 (d, J = 9.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.21 (s, 1H), 7.10 (d, J = 2.5 Hz, 1H), 6.79
(dd, J = 9.0, 2.5 Hz, 1H), 3.84 (d, J = 8.0 Hz, 3H), 3.74 (s, 2H), 3.71 (s, 2H), 3.66 – 3.56 (m,
2H), 3.14-3.08 (m , 2H), 3.01-2.96 (m, 2H), 2.90 – 2.84 (m, 2H). LRMS, (ESI, [M + Na]+)
m/z 693.0
2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)p
henyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)acetamide (CPD22)
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5
6
7
8
9
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.3). Analytical HPLC method tR =9.5 min;
97% purity. 1H NMR (500 MHz, DMSO) δ 13.30 – 12.68 (m, 1H), 11.96 (s, 1H), 11.31 (s,
1H), 8.19 (d, J = 9.1 Hz, 1H), 8.08 (d, J = 6.7 Hz, 1H), 7.56 (d, J = 9.2 Hz, 1H), 7.47 (t, J =
7.9 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.1 Hz, 1H), 4.62 (s, 2H), 3.85 (s, 2H), 3.03
(t, 2H), 2.89 (t, 2H), 1.83 – 1.68 (m, 4H). LRMS, (ESI, [M + Na]+) m/z =671
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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54
55
56
57
58
59
60
2-(5-methoxy-1-methyl-1H-indol-3-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetam
ido)pyridazin-3-yl)butyl)-1,3,4-selenadiazol-2-yl)acetamide（CPD23）
Single spot on TLC (CH3OH: CH2Cl2 = 1:10; Rf = 0.7). Analytical HPLC method tR =12.9
min; 97% purity. 1H NMR (500 MHz, CDCl3) δ 10.58 (s, 1H), 8.44 (d, J = 9.4 Hz, 1H), 8.02
(s, 1H), 7.32 (s, 2H), 7.19 (d, J = 8.5 Hz, 2H), 7.13 (s, 1H), 7.08 (d, J = 10.0 Hz, 1H), 7.04 (s,
1H), 7.00 (s, 1H), 6.89 (d, J = 8.0 Hz, 1H), 3.86 (s, 3H), 3.78 (s, 3H), 3.73 (s, 2H), 3.70 (s,
2H), 2.95 – 2.8 (m, 8H). LRMS, (ESI, [M + Na]+) m/z =724; HRMS: 702.1549 [M + H]+.
GDH activity assay. The GDH assay was optimized to achieve linear enzyme dose response.
For the GDH inhibition assay, three-fold dilutions of the compounds (0–26 μM) were
preincubated with the GDH enzyme for 0.5 h, and a mixture of glutamate (10 mM final),
NADP+ (200 μM final), and EZMTT(4 nM) in 50 mM Tris-Cl (pH 8.2) detection reagent was
added. After 1 h of incubation, UV-vis absorbance changes at 450 nm were measured to
calculate the inhibition of GDH activity.
KGA/GDH coupled assay. The compound was premixed with human KGA protein (1 nM)
in Hepes buffer A (100 μl; 50 mM Hepes, 100 mM NaCl, 0.5 mM EDTA, 0.001 % Tween,
pH 7.5) for 15 min. Gln (20 mM) in Hepes buffer B (100 μl; 50 mM Hepes, 200 mM
KH2PO4, 100 mM NaCl, 0.5 mM EDTA, 0.01 % BSA, 0.001 % Tween 20, pH 7.5) was
added to the 96-well plate, and the reaction was shaken at 25 ℃ for 3 h. The glutamate
formation was then measured with a GDH-coupled assay by adding a mixture containing
GDH (8 nM), NADP+ (20 μM), and EZMTT detection reagent23. After the solution was
incubated at 25 ℃ for 5 min, the absorbance at 450 nm was measured against the reference
wavelength of 620 nm.
29
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