Design, Synthesis, and Evaluation of Thiazolidine-2,4-dione Derivatives as a Novel Class of Glutaminase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b00282

Humans have two glutaminase genes, GLS (GLS1) and GLS2, each of which has two alternative transcripts: the kidney isoform (KGA) and glutaminase C (GAC) for GLS, and the liver isoform (LGA) and glutaminase B (GAB) for GLS2. Initial hit compound (Z)-5-((1-(

Full text of DOI:10.1021/acs.jmedchem.7b00282

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Article
Design, synthesis, and evaluation of thiazolidine-2-4-
dione derivatives as a novel class of glutaminase inhibitors
Teng-Kuang Yeh, Ching-Chuan Kuo, Yue-Zhi Lee, Yi-Yu Ke, Kuang-Feng Chu, Hsing-Yu Hsu,
Hsin-Yu Chang, Yu-Wei Liu, Jen-Shin Song, Cheng-Wei Yang, Li-Mei Lin, Manwu Sun, Szu-
Huei Wu, Po-Chu Kuo, Chuan Shih, Chiung-Tong Chen, Lun Kelvin Tsou, and Shiow-Ju Lee
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 13 Jun 2017
Downloaded from http://pubs.acs.org on June 13, 2017
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Design, synthesis, and evaluation of thiazolidine-2-4-dione derivatives as a novel class of
glutaminase inhibitors
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Teng-Kuang Yeh , Ching-Chuan Kuo , Yue-Zhi Lee , Yi-Yu Ke
,
Kuang-Feng Chu , Hsing-Yu Hsu ,
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Hsin-Yu Chang , Yu-Wei Liu , Jen-Shin Song , Cheng-Wei Yang , Li-Mei Lin , Manwu Sun ,
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Szu-Huei Wu , Po-Chu Kuo , Chuan Shih , Chiung-Tong Chen , Lun Kelvin Tsou *, Shiow-Ju Lee *
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Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes,
Miaoli 35053
# Authors with equal contribution
Running title: Novel thiazolidine-2-4-dione derivatives as glutaminase inhibitors
Six key words
α-ketoglutarate ; Glutamine; Glutamate; Glutaminase; thiazolidine-2-4-dione; Warburg effect.
Abstract
Humans have two glutaminase genes, GLS (GLS1) and GLS2, each of which has two alternative
transcripts: the kidney isoform (KGA) and glutaminase C (GAC) for GLS; and the liver isoform (LGA)
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and
glutaminase
B
(GAB)
for
GLS2.
Initial
hit
compound
(
Z)-5-((1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)thiazolidine-2,4-dione (2),
a
thiazolidine-2-4-dione, was obtained from a high throughput screening of 40,000 compounds
against KGA. Subsequently, a series of thiazolidine-2-4-dione derivatives was synthesized. Most of
these were found to inhibit KGA and GAC with comparable activities; were less potent inhibitors of
GAB; and were moderately selective for GLS1 over GLS2. The relationships between chemical
structure, activity and selectivity were investigated. The lead compounds obtained were found to:
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1
) offer in vitro cellular activities for inhibiting cell growth, clonogenicity, and cellular glutamate
production; 2) exhibit high concentrations of exposure in plasma by a pharmacokinetic study; and
3) reduce the tumor size of xenografted human pancreatic AsPC-1 carcinoma cells in mice.
Introduction
Cancer cells produce most of their energy by glycolysis and glutaminolysis, rather than
pyruvate oxidation. Warburg effect is the observation that cancer cells reprogram the cellular
1
metabolism for cell growth mainly by the elevating glycolysis and glutaminolysis . Glutaminolysis
occurs through deamido-hydrolysis of glutamine by glutaminase (GA) to give glutamate, which is
further oxidatively deaminated by glutamate dehydrogenase into α-ketoglutarate. This
2
α-ketoglutarate enters the tricarboxylic acid cycle to replenish cellular nutrients . Mammalian cells
contain two GLS genes, GLS1 (or GLS) and GLS2, each of which encodes two alternatively spliced
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mRNAs that are translated into isozymes: kidney GA (KGA) and GA isoform C for GLS1; liver GA
3
(
LGA) and GA isoform B for GLS2 .
GLS1 plays a vital role in up-regulating cell metabolism for tumor cell growth in vitro and in
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vivo . Proteins or microRNAs that have been demonstrated to impact glutaminase expression or
activity levels are involved in a number of diverse pathways and mechanisms, e.g. cMyc, miRNA23,
5
EGFR. Treatments with GLS1 inhibitors for Burkitt lymphoma P493 or breast carcinoma
xenografted murine tumor models have validated GLS1 as a therapeutic target and demonstrated
4
, 8
in vivo efficacy, significantly reducing tumor progression . Elevated GLS2 enzymatic activity has
9
10
also been correlated with tumor cell growth in vitro and in vivo . N-Myc activates GLS2 to
promote conversion of glutamine to glutamate in MYCN-amplified neuroblastoma cells. Abrogation
of GLS2 function profoundly inhibits glutaminolysis and dramatically decreases cell proliferation
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, 10
10
and survival in vitro
and in vivo . However, there is controversy over the role of GLS2 as a
tumor suppressor. Enzymatic activity independent of GLS2 is up-regulated via p53 or p63, and plays
1
1-14
a role of tumor suppressor
. The p53 elevated GLS2 interacts with rac1 through its C-terminal
domain to interrupt the interaction of rac1 with its partner, the small GTP binding protein
exchanger, thereby inhibiting the rac1 functions of tumorigenesis. Expression levels and enzymatic
15-18
activity of GLS1 and GLS2 in different type of tumors are varied
GLS2 could both be targeted for anti-tumor cell growth.
Glutamine analogous antagonists 6-diazo-5-oxo-L-norleucin
, therefore the existing GLS1 or
5
, 19-21
22, 23
and acivicin
exert
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antitumoral efficacy in different animal models and were tested as chemotherapeutic agents in
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different clinical studies, but were never approved as therapeutic agents for toxicity reasons.
Naturally occurring ardisianones allosterically binding to the C-terminal end of each GAB
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monomer and physapubescin inhabiting in the substrate binding pocket were also reported to
inhibit GAB or KGA with IC50 values ranging from sub- to several ꢀM. The most recent progress in
the development of potent GA inhibitors has focused on allosteric small molecules, such as
5,
7,
25,
33
bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl
sulfide
(BPTES)
and
2
-(pyridin-2-yl)-N-(5-(4-(6-(2-(3-(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)-1,3,4-thi
8
, 26
adiazol-2-yl)acetamide (CB-839) (1) (Chart 1). BPTES and 1 bind to the gating loop, which affects
substrate binding and is located at the dimer-dimer interface that participates in the formation of
active tetrameric GA. Their binding causes a conformational change that deactivates the active
5
,
7,
25,
26
tetrameric
GA
.
In
addition,
5
-(3-Bromo-4-(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a]phenanthridin-4(1
5
, 6
H)-one (bromo-benzophenanthridinone 968) binds between the N and C termini of two GAC
monomers at the monomer-monomer interface and also exerts inhibition through modulating the
5, 27
conformation into inactive forms . These inhibitors all exhibit high selectivity for GLS1 over GLS2
and thus target GLS1 elevated cancer types.
Exploration of GA inhibitors with different inhibitory mechanisms should result in more
options for developing GA inhibitors into therapeutic agents. Herein, we report a novel class of
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thiazolidine-2-4-dione derived glutaminase inhibitors that preferentially inhibit the enzymatic
activity of GLS1 over GLS2 and show in vivo efficacy in the AsPC-1 xenograft tumor model.
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Results
Analysis of structure –activity relations
Compound (Z)-5-((1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)thiazolidine-2,4-dione
(
2), was identified by a high throughput screening of 40,000 compounds against KGA at the
3
National Health Research Institutes (NHRI) HTS core facility. Replacement of the –CH group with a
thiophene (3) or phenyl (4) group increased the enzymatic inhibitory potency (IC50) from 3097 nM
of 2 to 754 nM of 3 or moderately decreased to 4896 nM of 4 for KGA respectively (Scheme 1,
Table 1).
Hit compound 2 was further modified through an alternative route which started from
1
intermediate D shown in scheme 1 to give a symmetric structured compound 5. This modification
improved potency further, and compound 5 had an IC50 of 102 nM. Compounds 3 and 4 were
similarly modified to give compounds 6 and 7 respectively, and also exhibited improved potency
(
(
50 nM and 45 nM) by a ~16-98-fold increase (Scheme 1, Table 1). Substitutions at R1
3 2 3
with –OCH , –CN, or –OH) or at R2 (with –CH CH or –Cl) of compounds 5-7 gave compounds 8-17.
All of 8-17 retained the potency of 5-7 and each possessed an improved selectivity for KGA/GAC
over GAB from selectivity index of 2-12 to 8-74 (Scheme 1, Table 1).
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In general, most of compounds in this series inhibited KGA/GAC but exerted less potency for
GAB. Thus, these compounds are moderately selective for GLS1 over GLS2.
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In vitro activities of inhibiting cellular glutamate production, cell growth, and clonogenicity
This series of compounds was also found to 1) inhibit carcinoma cell growth, including cell
lines of triple negative breast cancer MDA-MB-231 and pancreatic cancer AsPC-1 (Table 2); 2)
inhibit colony formation (Table 2, Figure 1A); and diminish cellular glutamate production (Figure
1
B). Of the compounds tested, compound 17 was the most potent, with the highest selectivity for
KGA/GAC over GAB (Table 1). However, 17 was found to exert the least inhibitory potency for cell
growth and colony formation (Table 2), suggesting it to have poor properties, e.g. cell permeability,
and therefore disqualifying it from further in vivo efficacy testing. Accordingly, other compounds
with good inhibitory potency for KGA/GAC enzymatic activity (Table 1), cell growth and colony
formation (Table 2) were instead pursued.
In order to confirm the selectivity of these potent compounds for glutaminase isoforms, their
GLS-/-
inhibition of cellular glutamate production was examined with AsPC-1
cells, in which the GLS1
gene was knocked out and only GLS2 activity remained (Supporting Information Figure S1).
Compounds 6 and 7 inhibited the glutamate production in a dose dependent manner in both
WT
GLS-/-
AsPC-1 and AsPC-1
cells. However, BPTES treatments were only able to inhibit the glutamate
WT
GLS-/-
production in AsPC-1 by ~60-70 %, and did not inhibit glutamate production in AsPC-1
cells
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at all. The results clearly demonstrated that only the highly GLS1 selective compound BPTES could
GLS-/-
not inhibit GLS2 activity in AsPC-1
cells (Figure 1C). Since the thiazolidine-2-4-dione derived
glutaminase inhibitors 6 and 7 reported herein possessed a selectivity index of ~2 and ~6 for GLS1
over GLS2 respectively (Table 1), the results obtained are consistent in that both 6 and 7 inhibit
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cellular glutamate production in either AsPC-1 or AsPC-1
cells (Figure 1B, 1C).
Pharmacokinetics and in vivo efficacy tests
Pharmacokinetic studies entailed intravenous administration of the compounds to rats at 2
mg for BPTES or 0.1 mg for 5 and 6; and oral administration at 10 mg for BPTES or 1 mg for 5 and 6
per kg of body weight. Blood samples were taken and the plasma analyzed. BPTES, 5, and 6 all
exhibited very little bioavailability. However, 5 and 6 were well exposed in blood and their area
under the concentration time curve (AUC) values (via intravenous route) of 43580 and 3414
ng/mL·h respectively were obtained, much higher than that of BPTES (636 ng/mL·h; Table 3).
Due to the poor bioavailability and high blood exposure of compounds 5 and 6, in vivo
efficacy tests were carried out by intraperitoneal injection of the tested compounds in a human
pancreatic AsPC-1 xenograft tumor model. Nude mice bearing established AsPC-1 tumors were
treated with BPTES (positive control) (Figure 2A), 5 (Figure 2A), and 6 (Figure 2B), respectively. In
vivo antitumor efficacy was expressed as tumor growth inhibition, a treatment outcome indicator.
BPTES and compounds 5 and 6 significantly and effectively reduced tumor growth at a dose of 25
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mg/kg (BPTES and 5) or 50 mg/kg (5 and 6), resulting in 50-60% tumor growth-inhibition by the
end of the test (Figure 2, left panels). No overt signs of adverse effects or a body weight loss of
>
10% were observed during the experimental period (Figure 2, right panels). These results clearly
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demonstrate that 5 and 6 effectively inhibit tumor growth in a preclinical mouse model.
Molecular modeling, site-directed mutagenesis studies and binding site analysis
Based on the structure-activity relationships analyzed above, we sought to determine the
possible binding site(s) of thiazolidine-2-4-dione derived lead compounds in KGA. Molecular
modeling and docking studies were conducted using the human GLS1 crystal structure (PDB ID:
3UO9) and BIOVIA software (BIOVIA, Inc., San Diego, CA). Two different binding sites of
glutaminase inhibitors were simulated through Biovia based on molecular docking on the human
GLS1 crystal structure (3UO9). The first was for the allosteric inhibitors BPTES and 1 (data not
7, 25, 26
shown), which are known to bind KGA/GAC at the dimer-dimer interface
. The second was for
thiazolidine-2-4-dione derived glutaminase inhibitors in Table 1. As shown in Figure 3, compound 5
was predicted to bind to the substrate binding pocket of KGA (Figure 3A). R387 of KGA was
predicted to interact with thiazolidine-2-4-dione derived glutaminase inhibitors based on simulated
docking results (Figure 3A & 3B). Therefore, R387 was mutated into Ala to give KGA R387A, against
which compounds 5, 7, and 17 were also tested; a ~190 to >490 fold-decrease in IC50 values
compare to wild type KGA was observed (Table 4). These results suggested R387 interacts with
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compounds 5, 7, and 17 through hydrophobic bonding interactions.
Those residues, S384 and K507 of KGA, which did not show interaction in above simulated
docking model with the thiazolidine-2-4-dione derived glutaminase inhibitors (Figure 3A), were
also mutated into Ala to give KGA S384A and K507A, against which compounds 5, 7, and 17 were
also tested; only a ~2–10 fold-decrease in IC50 values compared to wild type KGA was observed
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Table 4). Finally, R387 was mutated into Leu to give KGA R387L, against which compounds 5, 7,
and 17 were also tested; only a ~12 to ~14 fold-decrease in IC50 values compared to wild type KGA
was observed (Table 4). These results further validate the hydrophobic bonding interaction
hypothesis of R387 with compounds 5, 7, and 17 (Figure 3A & 3B).
Kinetic studies on the KGA inhibition by thiazolidine-2-4-dione glutaminase inhibitors
Compound 5 was found to inhibit KGA in a mixed mode of a competitive and a partial
non-competitive inhibition (Figure 4); in a competitive mode when compound 5 concentration was
increased from 0 to 0.1 µM, the Vmax retained almost the same while the Km increased; in a
partial non-competitive mode when compound 5 concentration was increased from 0.1 µM to 0.4
µM the Vmax decreased while the Km increased (Figure 4C). This inhibition mode of compound 5
against KGA was in consistent with the above results from docking and site-directed mutagenesis
studies.
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Combined treatments of GA inhibitors at different binding sites or with doxorubicin afforded
synergistic effects in suppressing carcinoma cell proliferation
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, 25
Combined treatments of GA inhibitors 5, 6 or BPTES
with doxorubicin (a stabilizer of the
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2
8
topoisomerase-DNA complex) respectively resulted in a synergistic effect for suppressing AsPC-1
carcinoma cell proliferation (Figure 5A) with combination indexes from 0.45 to 0.68 at ED50
(
Supporting Information Table S1). The combined treatment of 6 and BPTES (both GA inhibitors but
with different binding sites) exerted a more profound synergistic effect for suppressing HCC1806
breast carcinoma cell proliferation (Figure 5B) with combination indexes from 0.13 to 0.14 at ED50
(
Supporting Information Table S1). These combined treatments should be considered when GA
allosteric inhibitors are developed into the therapeutic agents and treatment regimens are sought.
Discussion and conclusion
Compared to the highest selectivity of BPTES or 1 for GLS1, the novel class of
thiazolidine-2-4-dione glutaminase inhibitors presented herein can inhibit either cellular GLS1 or
GLS2 activity in both in vitro cell-based assays and in vivo tumor growth model as demonstrated in
WT
GLS1-/-
reduction of glutamate production in AsPC-1 and AsPC-1
(Figures 1 & 2). The solubility and
bioavailability of these compounds is inadequate for their immediate use as therapeutic
anti-cancer drugs, and further development is required. Replacement of the heterocyclic or phenyl
rings of these thiazolidine-2-4-dione glutaminase inhibitors with corresponding heterocyclic based
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isosteres or heteroaromatic rings, which potentially increase solubility, should improve the
pharmacokinetic properties.
The combination of thiazolidine-2-4-dione glutaminase inhibitors with the existing anti-cancer
agents was also tested. A synergistic effect was observed for BPTES, 5, or 6 with doxorubicin
0
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(
Figure 5A). A synergistic effect was also observed when compound 6 was administered in
combination with BPTES (Figure 5B). This result indicates that glutaminase inhibitors binding at
different sites of KGA can work cooperatively, in addition to be combination with doxorubicin – a
very interesting finding that merits further investigation.
Because this thiazolidine-2-4-dione compound class shares a key moiety with the
thiazolidinedione peroxisome proliferator-activated receptor agonists (PPARs) used in the
2
9
treatment of diabetes , off-target activities toward PPARs of these thiazolidine-2-4-dione
glutaminase inhibitors were also investigated. No significant transactivation of PPARγ, PPARα, or
PPARδ was found (Supporting Information Figure S2). Commercially available glitazone drugs were
also tested for KGA inhibitory activity; none was found (Supporting Information Table S2).
Accordingly, the lead thiazolidine-2-4-dione compounds reported herein are concluded to impart
glutaminase inhibitory activity without targeting cellular PPARs.
In summary, a novel class of thiazolidine-2-4-dione compounds capable of selectively
inhibiting glutaminase activity in vitro and in vivo has been discovered and developed. Tumor cells
such as AsPC-1, which expresses both GLS1 and GLS2 in significant amounts, are suitable for
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treatment with thiazolidine-2-4-dione glutaminase inhibitors, in order to reduce tumor size. This
finding opens another potential arena for GA allosteric inhibitors which bind KGA at the substrate
binding pocket. They are neither substrate analogues (e.g. 6-diazo-5-oxo-L-norleucin or acivicin)
nor inter monomer allosteric inhibitors such as BPTES or 1 for developing GA inhibitors for cancer
therapy.
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Experimental Section:
1
1
. Chemistry.
.1. General Remarks
According to the substructures listed in ‘Baell & Holloway, J. Med. Chem.2010, 53,
3
0
2719–2740.’ , the core structure of thiazolidine-2-4-dione moiety is in the category as a PAIN hit.
However,
compound
(Z)-5-((1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)-4-
thioxothiazolidin-2-one, with only one oxygen replaced by sulfur at the thiazolidine-2-4-dione
3
0
moiety of compound 2, is also a PAIN hit but exhibited no inhibitory activity against KGA (IC50
>
1
00 µM). Therefore the KGA inhibitory activity of compound 2, described herein is highly likely
not caused by pan assay interference. Moreover, we have provided firm experimental evidence
from in vitro and in vivo assays and demonstrated that the novel glutaminase inhibitors
thiazolidine-2-4-dione derivatives reported herein are specifically active and their apparent activity
is not an artifact.
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13
C H and C NMR spectra were measured with a Varian Mercury 300 or Varian Mercury 400
spectrometer and purity of the final compounds was determined with an Hitachi 2000 series HPLC
3
1
system using a C18 column as described . The purity of all tested compounds is >95% purity
except for compound 9 (>94%), 10 (>94%), and 17 (>92%). See Supporting Information Table S3 for
actual pictures of compounds 2-17 and intermediates with NMR data shown as follows.
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.2. General synthetic procedures for thiazolidine-2-4-dione derivatives.
1 3
Diketone A - (1.0 equiv,), amine B1-3 (1.2 equiv) and p-toluenesulfonic acid (TsOH; 0.1 equiv)
was dissolved in toluene (0.5M) and refluxed for 12 hours. The solution was cooled to room
temperature and concentrated in vacuo. The residue was purified by column chromatography over
silica gel (n-Hex:EA = 4:1) to give intermediate product C1-7
.
o
3
A solution of DMF containing 182 mg of POCl was stirred for 0.5 h at 0 C. The reaction
mixture was allowed to warm up to room temperature. Subsequently, 100 mg (1.0 equiv) of
starting material C1-7 was added and the mixture was stirred for one hour at room temperature.
o
The reaction was cooled to 0 C and quenched by dropwise addition of aqueous NaOH solution. The
resulting mixture was extracted with DCM and the organic layer was dried over magnesium sulfate
and concentrated in vacuo. The crude product obtained was purified by column chromatography
over silica gel to provide 76.3 mg of the aldehyde product D1-7
.
The corresponding arylboronic acid (1.5 equiv), sodium carbonate (2M, 4 equiv) and
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bromoarene D1-7 (1 equiv) were added into a degassed suspension with the catalyst Pd (PPh ) (0.1
2
equiv) in the mixture of aqueous EtOH/H O/DME (1:1:2) under argon atmosphere. This reaction
mixture was refluxed under argon for 3 hours. The reaction was quenched with 5% aqueous HCl,
extracted with ether, and the organic layer was dried by magnesium sulfate. The filtrate was
evaporated under reduced pressure and was purified by column chromatography over silica gel. A
solution mixture of aldehyde D1-7 or (1.0 equiv) (or E1-13), thiazolidine-2,4-dione (2.2 equiv),
piperidine (in a catalytic amount) and AcOH was then refluxed for 3 hours in toluene. The solution
was cooled to room temperature and triturated with 10 mL of methanol and filtered to afford
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yellow solids 2-4 or 5-17 as the corresponding products.
1
1.3. 1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (D
1
) : H-NMR (300 MHz, CDCl
3
)
δ 9.87 (s, 1H), 7.66 (dd, J = 8.6, 2.0 Hz, 2H), 7.09 (dd, J = 8.6, 2.0 Hz, 2H), 6.38 (d, J = 1.3 Hz, 1H),
2
.28 (s, 3H), 1.98 (s, 3H).
1
1
2
.4. 1-(4-bromo-3-methoxyphenyl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (D ) : H-NMR (400
3
MHz, CDCl ) δ 9.92 (s, 1H), 7.72 – 7.64 (m, 2H), 6.72 – 6.64 (m, 2H), 3.90 (s, 3H), 2.32 (s, 3H), 2.01
(
s, 3H).
1
1
4 3
.5. 1-(4-bromophenyl)-2,5-diphenyl-1H-pyrrole-3-carbaldehyde (D ): H-NMR (400 MHz, CDCl )
δ 9.70 (s, 1H), 7.39 – 7.13 (m, 10H), 7.11 – 7.03 (m, 2H), 6.96 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 8.8,
2
.5 Hz, 2H).
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1
5
1.6. 1-(4-bromo-3-methoxyphenyl)-2,5-diphenyl-1H-pyrrole-3-carbaldehyde (D ): H-NMR (400
3
MHz, CDCl ) δ 9.71 (s, 1H), 7.39-7.28 (m, 4H), 7.28-7.16 (m, 5H), 7.16-7.05 (m, 2H), 6.96 (d, J = 2.1
Hz, 1H), 6.47-6.34 (m, 2H), 3.49 (s, 3H).
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9
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1
6
.7. 1-(4-bromophenyl)-2,5-di(thiophen-2-yl)-1H-pyrrole-3-carbaldehyde (D ) : H-NMR(400 MHz,
3
CDCl ): 9.84 (s, 1H), 7.52-7.48 (m, 2H), 7.38 (dd, J = 1.2, 5.2 Hz, 1H), 7.18 (dd, J = 1.2, 5.2 Hz, 1H),
7
.10-7.07 (m, 2H), 7.03 (s, 1H), 7.01-6.99 (m, 1H), 6.97 (dd, J = 1.2, 3.6 Hz, 1H), 6.88 (dd, J = 3.6, 5.2
Hz, 1H), 6.64 (dd, 1.2, 3.6 Hz, 1H).
1
1
7
.8. 1-(4-bromo-3-methoxyphenyl)-2,5-di(thiophen-2-yl)-1H-pyrrole (D ): H NMR (400 MHz,
3
CDCl ) δ 7.57 (dq, J = 7.8, 0.8 Hz, 1H), 7.10 (dt, J = 5.1, 1.3 Hz, 2H), 6.85 (ddd, J = 5.1, 3.7, 1.3 Hz,
2H), 6.80 (t, J = 1.5 Hz, 2H), 6.59 (dt, J = 3.7, 1.3 Hz, 2H), 6.53 (d, J = 1.2 Hz, 2H), 3.75 (s, 3H).
3
1.9. 1-(3'-ethyl-4'-formyl-[1,1'-biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (E ):
1
3
H-NMR(400 MHz, CDCl ): 10.35 (s, 1H), 9.90 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.79-7.77 (m, 2H),
7
.63 (dd, J = 2.0, 8.0 Hz, 1H), 7.56 (d, J = 1.6 Hz, 1H), 7.34-7.30 (m, 2H), 6.42 (d, J = 1.2 Hz, 1H), 3.16
(
q, J = 7.6 Hz, 2H), 2.34 (s, 3H), 2.05 (s, 3H), 1.35 (t, J = 7.6 Hz, 3H).
1
4
.10. 1-(4'-formyl-2-methoxy-[1,1'-biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (E ):
1
3
H-NMR(400 MHz, CDCl ): 10.34 (s, 1H), 9.91 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.58 (dd, J = 1.6, 7.6
Hz, 1H), 7.49 (d, J = 1.2 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 6.91 (dd, J = 2.0, 8.0 Hz, 1H), 6.81 (d, J = 1.6
Hz, 1H), 6.42 (d, J = 0.8 Hz, 1H), 3.84 (s, 3H), 3.14 (q, J = 7.6 Hz, 2H), 2.37 (s, 3H), 2.08 (s, 3H), 1.33
(
t, J = 7.6 Hz, 3H).
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1.11.
3'-ethyl-4'-formyl-4-(3-formyl-2,5-dimethyl-1H-pyrrol-1-yl)-[1,1'-biphenyl]-2-carbonitrile
1
(
6 3
E ): H-NMR(400 MHz, CDCl ): 10.38 (s, 1H), 9.91 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz,
1
8
H), 7.68 (d, J = 2.0 Hz, 1H), 7.62 (dd, J = 2.0, 8.0 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H), 7.56 (dd, J = 2.0,
.4 Hz, 1H), 6.45 (d, J = 1.2 Hz, 1H), 3.19 (q, J = 7.6 Hz, 2H), 2.36 (s, 3H), 2.07 (s, 3H), 1.37 (t, J = 7.6
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Hz, 3H).
1
.12. 1-(4'-formyl-2-methoxy-[1,1'-biphenyl]-4-yl)-2,5-di(thiophen-2-yl)-1H-pyrrole-3-
1
carbaldehyde (E11): H-NMR(400 MHz, CDCl
3
): 10.06 (s, 1H), 9.86 (s, 1H), 7.93 (d, J = 10.8 Hz, 2H),
7
1
.72 (d, J = 11.2 Hz, 2H), 7.39 (dd, J = 2.0, 6.8 Hz, 1H), 7.34 (d, J = 10.4 Hz, 1H), 7.19 (d, J = 7.2 Hz,
H), 7.07 (s, 1H), 7.05-7.00 (m, 2H), 6.94-6.89 (m, 2H), 6.81 (d, J = 2.8 Hz, 1H), 6.75 (d, J = 3.6 Hz,
1H), 3.66 (s, 3H).
1.13. (Z)-5-((1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)thiazolidine-2,4-dione (2):
1
H-NMR(400 MHz, DMSO): 13.45 (s, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.51 (s, 1H), 7.35 (d, J = 8.0 Hz,
1
3
2
1
H), 6.20 (s, 1H), 2.14(s, 3H), 2.00 (s, 3H); C-NMR(400 MHz, DMSO): 168.29, 167.50, 136.16,
35.77, 132.59 (x2), 131.50, 130.12 (x2), 126.08, 121.99, 115.15, 115.00, 105.15, 12.49, 10.72.
+
ESI-MS C H BrN O S: 377.25 found: 377/379 [M ] . HPLC chromatograms: purity >96%.
1
6 13
2 2
1
2
.14. (Z)-5-((1-(4-bromophenyl)-2,5-di(thiophen-2-yl)-1H-pyrrol-3-yl)methylene)thiazolidine-
1
,4-dione (3): H-NMR(400 MHz, DMSO): 12.44 (s, 1H), 7.69 (dd, J = 1.2, 5.2 Hz, 1H), 7.64 (d, J = 8.8
Hz, 2H), 7.26 (s, 1H), 7.47 (dd, J = 1.2, 5,2 Hz, 1H), 7.31, (d, J = 8.8 Hz, 2H), 7.10 (dd, J = 3.6, 5.2 Hz,
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1H), 7.48 (dd, J = 1.2, 3.6 Hz, 1H), 6.99 (dd, J = 3.6, 5.2 Hz, 1H), 6.93, (dd, 1.2, 3.6 Hz, 1H), 6.77 (s,
1
3
1
1
1
H); C-NMR(400 MHz, DMSO): 167.81, 167.38, 136.04, 133.47, 132.24, 131.89, 131.66, 131.60,
31.26, 129.87, 129.03, 128.89, 128.20, 127.42 (x2), 126.95, 126.79, 125.31, 124.86, 122.87,
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+
+ +
19.55, 118.51. ESI-MS C H BrN O S : 513.44 found: 513/515 [M] , 536/538 [M+Na ] . HPLC
2 13 2 2 3
2
chromatograms: purity >98%.
1
.15. (Z)-5-((1-(4-bromophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)thiazolidine-2,4-dione (4):
1
6
H-NMR(300 MHz, DMSO-d ), δ (ppm): 7.46 (dd, J = 6.9, 1.8 Hz, 2H), 7.38-7.14 (m, 12H), 7.08 (dd, J
1
3
=
1
6.9, 1.8 Hz, 2H), 6.68 (s, 1H) ; C-NMR(176 MHz, DMSO): 140.95, 137.14, 132.28 (x2), 131.56 (x2),
31.51 (x2), 131.38, 129.99 (x2), 129.08, 129.03 (x3), 128.80, 128.77 (x3), 127.99 (x2), 121.72,
+
+
+ +
117.83, 107.86. ESI-MS C H BrN O S: 501.39 found: 502 [M+H ] , 524 [M+Na ] . HPLC
6 17 2 2
2
chromatograms: purity >98%.
1
.16. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-
1
[
1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (5): H-NMR(300 MHz, DMSO): 13.00-12.00
(
brs, 2H), 7.95 (dd, J = 3.3, 8.7 Hz, 4H), 7.86 (s, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.69 (s, 1H), 7.48 (d, J =
13
8
1
1
.4 Hz, 2H), 6.21 (s, 1H), 2.17 (s, 3H), 2.05 (s, 3H); C-NMR(400 MHz, DMSO): 168.26, 167.87,
67.55, 167.45, 140.37, 139.05, 136.73, 135.87, 132.59, 131.60, 131.00, 130.74 (x2), 128.50 (x2),
27.87 (x2), 127.57 (x2), 126.15, 123.86, 114.94, 114.92, 105.10, 12.59, 10.80. ESI-MS
+
+
C H N O S : 501.57 found: 502 [M+H ] . HPLC chromatograms: purity >95%.
6 19 3 4 2
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1.17. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-di(thiophen-2-yl)-1H-
1
pyrrol-1-yl)-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (6): H-NMR(400 MHz, DMSO):
1
7
2.53 (S, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.82 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H),
.67 (dd, J = 2.0, 4.8 Hz, 1H), 7.56 (s, 1H), 7.44 (m, 3H), 7.08 (m, 2H), 6.99(m, 2H), 6.79 (s, 1H);
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13
C-NMR(400 MHz, DMSO): 168.00, 167.86, 167.82, 167.44, 139.79, 139.56, 136.57, 133.61,
1
32.79, 132.14, 131.74, 131.14, 130.79, 130.70 (x2), 130.22, 129.79, 129.25, 127.46 (x3), 127.39
(
x3), 127.32, 126.82, 126.57, 125.00, 124.22, 119.43, 118.47, 107.07. ESI-MS C H N O S :
2 19 3 4 4
3
+
+
6
37.76 found: 638 [M+H ] . HPLC chromatograms: purity >96%.
1.18. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-diphenyl-1H-pyrrol-1-yl)-
1
[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (7): H-NMR(400 MHz, DMSO): 13.00-12.00
(
brs, 2H), 7.74 (d, J = 7.2 Hz, 2H), 7.66 (d, J = 7.2 Hz, 2H), 7.58 (d, J = 7.2 Hz, 2H), 7.41 (s, 1H),
1
3
7
1
1
1
.36-7.18 (m, 13H), 6.70 (s, 1H) ; C-NMR(75 MHz, DMSO): 167.98, 167.85, 167.49, 167.41, 140.93,
39.78, 138.03, 137.25, 137.00, 132.53, 131.25, 131.25 (x2), 131.08, 130.99, 130.67, 129.64 (x2),
29.45, 128.89 (x2), 128.59, 128.42 (x2), 128.32 (x2), 127.51 (x2), 127.27 (x2), 126.95, 125.88,
+
+
23.80, 118.11, 117.20, 107.44. ESI-MS C H N O S : 625.71 found: 626 [M+H] , 648 [M+Na] .
6 23 3 4 2
3
HPLC chromatograms: purity >97%.
1
.19. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-2'-
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
methoxy-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (8): H-NMR(300 MHz, DMSO):
1
7
3.00-12.00 (brs, 2H), 7.80 (s, 1H), 7.74-7.64 (m, 4H), 7.53 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 1.8 Hz, 1H),
1
3
.02 (dd, J = 7.8, 1.5 Hz, 1H), 6.20 (s, 1H), 3.82 (s, 3H), 2.21 (s, 3H), 2.08 (s, 3H); C-NMR(101 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
4
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7
8
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3
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7
8
9
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DMSO): 168.31, 167.50, 159.87, 156.81, 138.94, 137.83, 136.01, 132.39, 132.21, 132.08, 131.71,
1
1
30.96, 130.12, 129.84, 129.32, 128.81, 126.20, 124.09, 120.18, 119.10, 116.32, 114.89, 111.84,
+
04.98, 56.20, 12.66, 10.87. ESI-MS C H N O S : 531.60 found: 532 [M+H] . HPLC
2
7 21 3 5 2
chromatograms: purity >95%.
1
.20. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-di(thiophen-2-yl)-1H-pyrrol-
1
1-yl)-2'-methoxy-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (9): H-NMR(400 MHz,
DMSO): 7.68 (dd, J =2.4, 4.0 Hz, 1H), 7.60 (m, 5H), 7.52 (s, 1H), 7.50 (s, 1H), 7.47 (dd, J = 1.2, 4.8 Hz,
1
H), 7.40 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 1.6 Hz, 1H), 7.11 (d, J = 1.6 Hz, 1H), 7.11 (s, 1H), 7.00-6.95
1
3
(
m, 4H), 6.77 (s, 1H), 3.66 (s, 3H) ; C-NMR(400 MHz, DMSO): 167.97, 167.88, 167.57, 167.43,
1
1
1
56.44, 138.66, 137.49, 133.59, 132.17, 132.04, 131.70, 131.28, 131.23, 130.57, 130.13 (x2),
29.87 (x2), 129.80, 129.41, 129.30, 127.40 (x2), 126.86, 126.69, 125.06, 123.70, 121.94, 119.39,
+
18.49, 113.87, 107.04, 56.23. ESI-MS C H N O S : 667.78 found: 668 [M+H] . HPLC
3
3 21 3 5 4
chromatograms: purity >94%.
1
.21. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-diphenyl-1H-pyrrol-1-yl)-
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2'-methoxy-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (10): H-NMR(400 MHz, DMSO):
1
3
1
1
3
3.00-12.00 (brs, 2H), 7.76 (d, J = 2.0 Hz, 1H), 7.58 (s, 4H), 7.41-7.23 (m, 14H), 6.91 (s, 1H), 3.46 (s,
1
3
H) ; C-NMR(101 MHz, DMSO): 168.07, 168.01, 167.82, 167.44, 156.00, 140.75, 138.58, 138.02,
36.86, 131.90, 131.37, 131.02, 130.16, 129.94, 129.83, 129.76, 128.89, 128.62, 128.29, 128.19,
27.85, 127.52, 125.84, 123.81, 121.03, 118.20, 117.24, 113.10, 107.41, 55.79, 40.12, 39.91, 39.70,
10
11
12
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18
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25
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38
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48
49
50
51
52
53
54
55
56
57
58
59
60
+
+
9.49, 39.29, 39.08, 38.87. ESI-MS C H N O S : 655.74 found: 656 [M+H] , 678 [M+Na] . HPLC
7 25 3 5 2
3
chromatograms: purity >94%.
1
.22. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-3-
1
ethyl-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (11): H-NMR(400 MHz, DMSO):
13.00-12.00 (brs, 2H), 7.95 (s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.77-7.76 (m, 2H), 7.68 (s, 1H), 7.54 (d, J
=
7
1
1
8.4 Hz, 1H), 7.46 (d, 2H), 6.20 (s, 1H), 2.84 (q, J = 7.6 Hz, 2H), 2.29 (s, 3H), 2.04 (s, 3H), 1.22 (t, J =
1
3
.6 Hz, 3H); C-NMR(400 MHz, DMSO): 168.28, 168.08, 167.46, 167.28, 145.49, 140.57, 139.33,
36.63, 135.89, 131.61, 130.89, 128.54, 128.44 (x2), 128.13, 127.95, 127.87 (x2), 126.17, 125.44,
24.99, 114.95, 114.92, 105.10, 26.10, 15.64, 12.59, 10.81. ESI-MS C H N O S : 529.62 found:
8 23 3 4 2
2
+ +
5
30 [M+H ] . HPLC chromatograms: purity >95%.
1
.23. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl
1
)
-3-ethyl-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (12): H-NMR(400 MHz, DMSO):
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13.00-12.00 (brs, 2H), 7.91 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.72-7.75 (m, 2H), 7.67 (dd, J = 2.0, 4.8
Hz, 1H), 7.56 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.45-7.43 (m, 3H), 7.10-7.07 (m, 2H), 6.98-6.93 (m, 2H),
1
3
6
1
1
1
.79 (s, 1H), 2.82 (q, J = 7.6 Hz, 2H), 1.91 (t, J = 7.6 Hz, 3H); C-NMR(400 MHz, DMSO): 168.54,
68.17, 167.94, 167.56, 145.46, 139.82, 139.80, 136.45, 133.60, 132.19, 131.75, 131.21, 131.12,
30.15, 129.74, 129.30, 128.91, 128.22, 128.14, 127.94, 127.76 (x2), 127.40, 127.29, 126.78,
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26.53, 126.29, 124.97, 124.90, 119.53, 118.49, 107.07, 26.14, 15.72. ESI-MS C H N O S :
4 23 3 4 4
3
+ +
6
65.81 found: 666 [M+H ] . HPLC chromatograms: purity >98%.
1
.24. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-diphenyl-1H-pyrrol-1-yl)-3-
1
ethyl-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (13): H-NMR(400 MHz, DMSO): 7.90
(s, 1H), 7.71-7.63 (m, 4H), 7.46 (d, J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.37-7.19 (m, 11H), 6.70 (s, 1H), 3.46
1
3
(
(
s, 3H), 2.63 (q, J = 8.0 Hz, 2H), 1.16 (t, J = 8.0 Hz, 3H) ; C-NMR(75 MHz, DMSO): 168.14, 168.00
x2), 167.43, 145.48, 139.87, 138.24, 137.15, 137.02, 131.28, 131.10 (x2), 130.87 (x2), 129.66 (x2),
1
1
29.40 (x2), 128.91 (x2), 128.60 (x2), 128.59 (x2), 128.33 (x2), 128.08 (x2), 127.60, 127.52, 126.92,
25.90, 124.73, 118.10, 117.19, 107.42, 26.09, 15.72. ESI-MS C H N O S : 653.77 found: 654
8 27 3 4 2
3
+
+
[
M+H] , 676 [M+Na] . HPLC chromatograms: purity >95%.
1
.25. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-3-
1
ethyl-2'-methoxy-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (14): H-NMR(400 MHz,
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DMSO): 13.00-12.00 (brs, 2H), 7.94 (s, 1H), 7.69 (s, 1H), 7.57-7.49 (m, 4H), 7.12 (d, J = 2.0 Hz, 1H),
7
3
1
1
.02 (dd, J = 2.0, 8.0 Hz, 1H), 6.20 (s, 1H), 3.82 (s, 3H), 2.81 (q, J = 7.6 Hz, 2H), 2.21 (s, 3H), 2.09 (s,
13
H), 1.19 (t, J = 7.6 Hz, 3H); C-NMR(400 MHz, DMSO): 168.58, 168.34, 168.13, 167.53, 156.84,
44.43, 139.03, 137.71, 136.03, 131.73, 130.96, 130.44, 130.34, 129.10, 128.32, 127.58, 127.16,
26.23, 125.86, 120.14, 114.91, 114.89, 111.79, 104.99, 56.19, 26.02, 15.65, 12.66, 10.87. ESI-MS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+ +
+ +
C H N O S : 559.65 found: 560 [M+H ] , 582 [M+Na ] . HPLC chromatograms: purity >96%.
9 25 3 5 2
2
1
.26. 4'-((E)-(2,4-dioxothiazolidin-5-ylidene)methyl)-4-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)
1
methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-3'-ethyl-[1,1'-biphenyl]-2-carbonitrile (15): H-NMR(400
MHz, DMSO): 13.00-12.00 (brs, 2H), 8.13 (J = 2.0 Hz, 1H), 7.96 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.83
(dd, J = 2.0, 8.4 Hz, 1H), 7.69-7.67 (m, 3H), 7.61 (d, J = 8.0 Hz, 1H), , 6.23 (s, 1H), 2.87 (q, J = 7.6 Hz,
1
3
2
1
1
1
H), 2.20 (s, 3H), 2.08 (s, 3H), 1.23 (t, J = 7.6 Hz, 3H) ; C-NMR(400 MHz, DMSO): 168.26, 168.10,
67.47, 167.33, 144.96, 143.73, 138.37, 136.86, 135.90, 133.24, 133.12, 132.23, 131.69, 131.40,
29.98, 128.26, 127.77, 127.03, 126.59, 125.92, 117.62, 115.55, 115.29, 111.35, 105.47, 25.94,
+ +
+ +
5.29, 12.56, 10.83. ESI-MS C H N O S : 554.63 found: 555 [M+H ] , 577 [M+Na ] . HPLC
9 22 4 4 2
2
chromatograms: purity >98%.
1
.27. (E)-5-((4'-(3-((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-2'-
1
hydroxy-[1,1'-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (16): H-NMR(400 MHz, DMSO):
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2
2
2
2
2
2
2
2
3
3
3
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3
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13.00-12.00 (brs, 2H), 10.10 (s, 1H), 7.72 (d, J = 8.4 Hz, 3H), 7.65 (s, 1H), 7.60 (d, J = 8.4 Hz, 2H),
7
3
1
1
.49 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 6.86 (dd, J = 1.6, 8.4 Hz, 1H), 6.82 (d, J = 1.6 Hz, 1H), 2.18 (s,
1
3
H), 2.06 (s, 3H); C-NMR(400 MHz, DMSO): 169.20, 168.81, 168.36, 167.56, 155.22, 139.19,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
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4
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37.25, 135.82, 131.98, 131.52, 131.15, 130.46, 129.89 (x2), 129.81(x2), 127.07, 126.22, 124.70,
18.92, 115.32, 114.92, 114.87, 105.06, 12.63, 10.84. ESI-MS C H N O S : 517.57 found: 518
6 19 3 5 2
2
+
[
M+H] . HPLC chromatograms: purity >95%.
1
.28. (Z)-5-((1-(3'-chloro-4'-((E)-(2,4-dioxothiazolidin-5-ylidene)methyl)-[1,1'-biphenyl]-4-yl)-2,5-
1
dimethyl-1H-pyrrol-3-yl)methylene)thiazolidine-2,4-dione (17): H-NMR(300 MHz, DMSO): 12.80
(s, 1H), 12.23 (s, 1H), 8.08 (s, 1H), 8.00-7.87 (m, 3H), 7.69 (m, 2H), 7.51-7.43 (m, 3H), 6.21 (s, 1H),
1
3
2.17 (s, 3H), 2.04 (s, 3H); C-NMR(400 MHz, DMSO): 168.29, 167.55, 167.46, 167.16, 141.89,
1
1
37.63, 137.23, 135.80, 135.53, 131.54, 130.14, 129.31, 128.54 (x2), 128.26, 128.06 (x2), 127.06,
26.21, 126.10, 125.98, 115.05, 115.03, 105.21, 12.61, 10.80. ESI-MS C H ClN O S : 536.01
6 18 3 4 2
2
+
found: 536/538 [M] . HPLC chromatograms: purity >92%.
2
.
Cell culture and reagents. The human carcinoma cell lines breast MDA-MB-231 [BCRC 60425],
pancreatic AsPC-1 [BCRC 60494], breast HCC1806 [(ATCC® CRL-2335™] and breast MCF7 [BCRC
6
0436] were used in this study. MCF7 cells were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM, Hyclone Laboratory Inc.) and supplemented with 10% fetal bovine serum (FBS,
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Hyclone Laboratory Inc.). AsPC-1, MDA-MB-231 and HCC1806 cells were cultured in RPMI 1640
medium (GIBCO-Life Technologies) supplemented with 10% FBS.
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3
.
Cloning, expression, and purification of glutaminases. The human KGA and GAB were
9
prepared as described . The human GAC gene was amplified from a cDNA library derived from
MCF7 cells. Briefly, total RNAs were isolated from MCF7 cells using TRIzol reagent (Invitrogen, Inc.)
and converted into cDNAs using the SuperScript III Transcriptor First-Strand cDNA Synthesis System
for RT-PCR (Invitrogen, Inc.) according to the manufacturer. A cDNA fragment of 1428bp, which
encodes a human GAC polypeptide of residues Leu123 to Ser598, was obtained by PCR against the
obtained MCF7 cDNAs library with primers of 5’- ATACGCGGATCCCTGGTGGCCTCAGGTGAAAA-3’
(containing
a
BamHI
restriction
site)
and
5
’-GCAAGGAAAAAAGCGGCCGCAGCGTTAGCTTTTCTCTCCCAGACTTTCCA -3’ (containing a Notl
restriction site). The GAC gene sequence included in this cDNA fragment of 1428bp was verified by
sequence analysis and found the same as that of accession no. AF158555 except three different
nucleotides (underlined as follows) that resulted into the changes of a.a. 172 Asp(GAT)ꢀGly(GGT),
a.a. 268 Ala(GCA)ꢀVal(GTA), and a.a. 376 Ala(GCA)ꢀThr(ACA). This PCR amplified cDNA fragment
and the protein expression vector pET28a(+) (Novagen) were separately digested with BamHI and
NotI. The desired DNA fragments were cleaned and purified prior to being ligated into a His-tagged
GAC protein expression plasmid.
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The resultant His-tagged GAC-expression plasmid, pET28a(+)-GAC, was transformed into
Escherichia coli strain C41(DE3)pLysS (Yeastern Biotech Co., Taiwan) and then cultured in Terrific
Broth medium in the presence of kanamycin. Once the Escherichia coli culture reached an
absorbance at 600nm of 0.6 - 0.8, isopropyl β-D-1-thiogalactopyranoside (1 mM) was added to
induce the His-tagged GAC protein expression for 6 h at 25 °C. The resultant bacteria were
0
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2 4 2
collected by rapid centrifugation and then re-suspended in a lysis buffer (50 mM NaH PO .H O, 300
mM NaCl, 5 mM Tris-HCl pH 8.5, 5% glycerol , 0.1% Triton X-100, 10 mM β-ME and 50 mM
Imidazole) prior to breaking bacteria by sonication. The supernatants from sonication treatment
2
+
were collected for further protein purification at cold room with a Ni charged resin (GE
Healthcare) column and the His-tagged GAC bound resin column was washed with the lysis buffer
containing 50 mM and 75 mM Imidazole. His-tagged GAC was eluted with the lysis buffer
2 4 2
containing 500 mM imidazole, which buffer was then exchanged to 50 mM NaH PO .H O, 50 mM
Tris-HCl pH 8.0, 2 mM dithiothreitol, and 50% glycerol by using PD-10 Desalting column (GE
9
Healthcare). The concentrations of the purified protein were determined as described.
4
.
Glutaminase activity assays and kinetic studies. Human glutaminase activity was measured
9, 32, 33
9
using a two-step assay as described previously
. Kinetic studies were measured as described .
5
.
In vitro carcinoma cell growth inhibition and colony formation assays. MDA-MB-231 breast
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carcinoma, AsPC-1 pancreatic carcinoma, HCC1806 breast carcinoma cells were seeded at 8500,
4
000, and 3000 cells/well respectively in 96-well plates, as previously described for cell growth
9
inhibition assay . To assess anchorage-dependent colony formation effect, the cells (1000 cells/well)
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9
were seeded in a six-well plate, as previously described .
6
.
Determination of intracellular glutamate levels. AsPC-1 cells were seeded at 30,000
2
cells/well in 96-well plates and incubated in a CO incubator at 37°C for 24 h. The cells were treated
with either the vehicle control (1% DMSO) or 3.13 to 100 μM glutaminase inhibitors as indicated
for an additional 4 h. After 4 h treatment, the medium was removed and cells per well were
washed twice with 1x PBS, and 50 μL of nonidet-P40 lysis buffer was added per well at 4°C for
20min. Aliquots (each 10 μL) of the resultant cell lysates were subjected to Amplex red glutamic
acid assay (Invitrogen) followed by manufacturer’s recommendations.
7
.
GLS gene knockout. Knockout of GLS was accomplished by ZEN cleavage using zinc-finger
nuclease mRNA (GLS-KO ZFN mRNA) designed to target the exon 1 site of GLS glutaminase gene in
the CompoZr® Knockout ZFN Kit (CKOZFN9361, Sigma) by manufacturer’s recommendations.
AsPC-1 cells were transfected with GLS-KO ZFN mRNA using TransIT®-mRNA Transfection Reagent
(
MIR2225, Mirus) and incubated for 5 days. Then, a portion of the transfected cells were harvested
to isolate the genomic DNA which was used as the template for further PCR amplification. The
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sequences of PCR primers used for amplification of the GLS target locus were as follows: forward
primer 5’-AGCCCTTGGTCACCCTGT-3’ and reverse primer 5’-GGATAGGAGCCGAGGGTCTA-3’. The
obtained CR product was denatured and re-annealed to allow the heteroduplex formation
between wild type (402-bp) and modified (401-bp) amplicons. Subsequently, the CEL-I mismatched
nuclease assay was performed to identify the heteroduplex formation and ZEN cleavage. Once
heteroduplex was found from the CEL-I assay, the correspondent GLS-KO ZFN mRNA transfected
cells were subject for single-cell sorting and each single cell was cultured in RPMI 1640 medium
without glutamine (Catalog number 21870-076, GIBCO Life Technologies) supplemented with 10%
FBS. Identification of GLS knockout cell line was analyzed by directly sequencing GLS alleles that
were isolated by PCR amplification as described above. The verified sequencing data and western
analysis for verification of GLS gene knocked out were provided in Supporting Information Figure
S1.
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Animal study protocols. Animal study protocols were reviewed and approved for the in vivo
experiments herein by the Institutional Animal Care and Use Committee (IACUC) of National Health
Research Institutes, Taiwan.
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Pharmacokinetic analysis. Sprague-Dawley rats used for pharmacokinetic studies were
obtained from BioLASCO Taiwan Co. (Ilan, Taiwan) and housed in the animal facility at the NHRI,
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Journal of Medicinal Chemistry
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Taiwan. Male rats (330-380 g, 9-10 weeks old) were quarantined for one week before use. The rats
were surgically implanted with a jugular-vein cannula one day before treatment. Compounds
BPTES, 5, and 6 were given to rats (n = 3) by intravenous or oral administration as prepared in a
mixture of DMA/PEG400 (20%/80%, v/v) and 1% CMC/0.5% Tween80 for intravenous and oral
administration respectively. At 0 (immediately before dosing), 2 min, 5 min (intravenous only), 15
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min and 30 min and 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after compound administration, a blood sample
o
(
100 µL) was taken from each animal via the jugular-vein cannula and stored in ice (0-4 C). The
processing of the plasma and subsequent analysis by high performance liquid
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1
chromatography-tandem mass spectrometry were performed as described . The pharmacokinetic
parameters were obtained by a standard non-compartmental method.
nu
10. In vivo anti-tumor activity. Male nude mice (BALB/cAnN.Cg-Foxn1 /CrlNarl,National
Laboratory Animal Center, Taipei, Taiwan) were 7-8 weeks old, and had a body weight range of
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1
8-22 g. Mice were inoculated subcutaneously at the right flank region with AsPC-1 cells (4.5×10
cells/mouse) in 0.1 mL of Hank's Balanced Salt Solution (ThermoFisher Scientific) mixing with equal
volume Matrigel (BD Biosciences) for tumor development. Tumors were monitored twice weekly.
The mice were sorted into different groups randomly and the treatments were started when the
3
mean tumor size reached ~100 mm (this day was thus designated as Day 1). The tumor volume
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was calculated using the equation V (mm ) = a × b /2, where a is the largest diameter and b is the
smallest diameter. %TGI was determined for anti-tumor effects which are expressed as
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