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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* Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli 35053, Taiwan S Supporting Information *

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) 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 (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.

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EGFR. 5 Treatments with GLS1 inhibitors for Burkitt lymphoma P493 or breast carcinoma xenografted murine tumor models have validated GLS1 as a therapeutic target and demonstrated in vivo efficacy, significantly reducing tumor progression.4,8 Elevated GLS2 enzymatic activity has also been correlated with tumor cell growth in vitro9 and in vivo.10 NMyc 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 and survival in vitro9,10 and in vivo.10 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 a role of tumor suppressor.11−14 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

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 metabolism for cell growth mainly by the elevating glycolysis and glutaminolysis.1 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 α-ketoglutarate enters the tricarboxylic acid cycle to replenish cellular nutrients.2 Mammalian cells contain two GLS genes, GLS1 (or GLS) and GLS2, each of which encodes two alternatively spliced mRNAs that are translated into isozymes: kidney GA (KGA) and GA isoform C for GLS1; liver GA (LGA) and GA isoform B for GLS2.3 GLS1 plays a vital role in up-regulating cell metabolism for tumor cell growth in vitro and in vivo.4−8 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, © 2017 American Chemical Society

Received: February 23, 2017 Published: June 13, 2017 5599

DOI: 10.1021/acs.jmedchem.7b00282 J. Med. Chem. 2017, 60, 5599−5612

Journal of Medicinal Chemistry

Article

decreased to 4896 nM of 4 for KGA respectively (Scheme 1, Table 1). Hit compound 2 was further modified through an alternative route that started from intermediate D1 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- to 98-fold increase (Scheme 1, Table 1). Substitutions at R1 (with −OCH3, −CN, or −OH) or at R2 (with −CH2CH3 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). 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. 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 (3) diminish cellular glutamate production (Figure 1B). 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 inhibition of cellular glutamate production was examined with AsPC-1GLS−/− 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 AsPC-1WT and AsPC-1GLS−/− cells. However, BPTES treatments were only able to inhibit the glutamate production in AsPC-1WT by ∼60−70% and did not inhibit glutamate production in AsPC-1GLS−/− cells at all. The results clearly demonstrated that only the highly GLS1 selective compound BPTES could not inhibit GLS2 activity in AsPC1GLS−/− 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 cellular glutamate production in either AsPC-1WT or AsPC-1GLS−/− cells (Figure 1B and Figure 1C). Pharmacokinetics and in Vivo Efficacy Tests. Pharmacokinetic studies entailed intravenous administration of the compounds to rats at 2 mg for BPTES and 6 or 0.1 mg for 5 and oral administration at 10 mg for BPTES and 6 or 1 mg for 5 per kg of body weight. Blood samples were taken, and the plasma was 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 43 580 and 3414 (ng/mL)·h,

tumorigenesis. Expression levels and enzymatic activity of GLS1 and GLS2 in different types of tumor are varied;15−18 therefore the existing GLS1 or GLS2 could both be targeted for antitumor cell growth. Glutamine analogous antagonists 6-diazo-5-oxo-L-norleucin5,19−21 and acivicin22,23 exert antitumoral efficacy in different animal models and were tested as chemotherapeutic agents in different clinical studies but were never approved as therapeutic agents for toxicity reasons.21,23 Naturally occurring ardisianones allosterically binding to the C-terminal end of each GAB monomer9 and physapubescin inhabiting in the substrate binding pocket24 were also reported to inhibit GAB or KGA with IC50 values ranging from sub-μM to several μM. The most recent progress in the development of potent GA inhibitors has focused on allosteric small molecules, such as bis-2-(5phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) 5,7,25,33 and 2-(pyridin-2-yl)-N-(5-(4-(6-(2-(3(trifluoromethoxy)phenyl)acetamido)pyridazin-3-yl)butyl)1,3,4-thiadiazol-2-yl)acetamide (CB-839)8,26(1) (Chart 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 tetrameric GA.5,7,25,26 In addition, 5-(3-bromo-4(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a]phenanthridin-4(1H)-one (bromobenzophenanthridinone 968)5,6 binds between the N and C termini of two GAC monomers at the monomer−monomer interface and also exerts inhibition through modulating the conformation into inactive forms.5,27 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 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 National Health Research Institutes (NHRI) HTS core facility. Replacement of the −CH3 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 5600



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Scheme 1. Synthesis of a Series of Thiazolidine-2,4-dione Derivativesa

a Reagents and conditions: (a) TsOH(cat.), toluene, reflux; (b) POCl3, DMF, rt; (c) thiazolidine-2,4-dione, piperidine(cat.), AcOH(cat.), toluene, reflux; (d) (4-formylphenyl)boronic acid or (3-ethyl-4-formylphenyl)boronic acid or (3-chloro-4-formylphenyl)boronic acid, Pd(PPh3)4 (cat.), Na2CO3(aq), DME, EtOH, reflux; (e) BBr3, DCM, 0 °C. The percentage (%) following each set of R groups is the percent yield for that reaction.

on molecular docking on the human GLS1 crystal structure (3UO9). The first was for the allosteric inhibitors BPTES and 1 (data not shown), which are known to bind KGA/GAC at the dimer−dimer interface.7,25,26 The second was for thiazolidine2,4-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 and Figure 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 compared to wild type KGA was observed (Table 4). These results suggested R387 interacts with 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- to 10-fold decrease in IC50 values compared to wild type KGA was observed (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

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 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 demonstrate that 5 and 6 effectively inhibit tumor growth in a preclinical mouse model. Molecular Modeling, Site-Directed Mutagenesis Studies, and Binding Site Analysis. On the basis of 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 code 3UO9) and BIOVIA software (BIOVIA, Inc., San Diego, CA). Two different binding sites of glutaminase inhibitors were simulated through Biovia based 5601



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Table 1. Thiazolidine-2,4-dione Derivatives That Inhibit the Enzymatic Activity of Human Glutaminase Isoforms KGA, GAC, and GABa

Chemical core structures are shown. Measurement of the IC50 values is described in Experimental Section. Values shown are the mean ± SD from at least three independent experiments, each in duplicate.

a

with combination indexes from 0.45 to 0.68 at ED 50 (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.

interaction hypothesis of R387 with compounds 5, 7, and 17 (Figure 3A and Figure 3B). Kinetic Studies on the KGA Inhibition by Thiazolidine2,4-dione Glutaminase Inhibitors. Compound 5 was found to inhibit KGA in a mixed mode of a competitive and a partial noncompetitive 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 value while the Km increased; in a partial noncompetitive 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. Combined Treatments of GA Inhibitors at Different Binding Sites or with Doxorubicin Afforded Synergistic Effects in Suppressing Carcinoma Cell Proliferation. Combined treatments of GA inhibitor 5, 6, or BPTES7,25 with doxorubicin (a stabilizer of the topoisomerase−DNA complex)28 respectively resulted in a synergistic effect for suppressing AsPC-1 carcinoma cell proliferation (Figure 5A)

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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 reduction of glutamate production in AsPC-1WT and AsPC-1GLS1−/− (Figures 1 and 2). The solubility and bioavailability of these compounds are inadequate for their immediate use as therapeutic anticancer drugs, and further development is required. Replacement of the heterocyclic or phenyl rings of these thiazolidine-2,4-dione 5602



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thiazolidine-2,4-dione moiety is in the category as a PAIN hit. However, compound (Z)-5-((1-(4-bromophenyl)-2,5-dimethyl-1Hpyrrol-3-yl)methylene)-4-thioxothiazolidin-2-one, with only one oxygen replaced by sulfur at the thiazolidine-2,4-dione moiety of compound 2, is also a PAIN hit30 but exhibited no inhibitory activity against KGA (IC50 > 100 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. 1 H and 13C NMR spectra were measured with a Varian Mercury 300 or Varian Mercury 400 spectrometer, and purity of the final compounds was determined with a Hitachi 2000 series HPLC system using a C18 column as described.31 The purity of all tested compounds is >95% except for compounds 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. 1.2. General Synthetic Procedures for Thiazolidine-2,4dione Derivatives. Diketone A1−3 (1.0 equiv), amine B1−3 (1.2 equiv), and p-toluenesulfonic acid (TsOH, 0.1 equiv) were dissolved in toluene (0.5 M) and refluxed for 12 h. 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. A solution of DMF containing 182 mg of POCl3 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 1 h at room temperature. 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 (2 M, 4 equiv), and bromoarene D1−7 (1 equiv) were added to a degassed suspension with the catalyst Pd(PPh3)4 (0.1 equiv) in the mixture of aqueous EtOH/H2O/DME (1:1:2) under argon atmosphere. This reaction mixture was refluxed under argon for 3 h. 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 (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 h in toluene. The solution was cooled to room temperature and triturated with 10 mL of methanol and filtered to afford yellow solids 2−4 or 5− 17 as the corresponding products. 1.3. 1-(4-Bromophenyl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (D1). 1H NMR (300 MHz, CDCl3) 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). 1.4. 1-(4-Bromo-3-methoxyphenyl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (D2). 1H NMR (400 MHz, CDCl3) 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). 1.5. 1-(4-Bromophenyl)-2,5-diphenyl-1H-pyrrole-3-carbaldehyde (D4). 1H NMR (400 MHz, CDCl3) 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). 1.6. 1-(4-Bromo-3-methoxyphenyl)-2,5-diphenyl-1H-pyrrole-3-carbaldehyde (D5). 1H NMR (400 MHz, CDCl3) 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). 1.7. 1-(4-Bromophenyl)-2,5-di(thiophen-2-yl)-1H-pyrrole-3carbaldehyde (D6). 1H NMR (400 MHz, CDCl3): 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,

Table 2. Growth Inhibition and Anticlonogenic Activity of Glutaminase Inhibitors, Thiazolidine-2,4-dione Derivatives, against Pancreatic Carcinoma Cellsa clonogenicity inhibition (%)

growth inhibition

AsPC-1 compd BPTES 5 6 7 9 10 11 12 13 17

2 μM 41 13 64 79 89 90 28 44 51 18

± ± ± ± ± ± ± ± ± ±

6 11 11 8 0 2 9 4 2 12

IC50 (μM) 10 μM

AsPC-1

MDA-MB-231

± ± ± ± ± ± ± ± ± ±

10.2 ± 1.3 34.5 ± 1.4 14.8 ± 2.0 10 ± 1.5 11.9 ± 1.1 12 ± 1.3 12.5 ± 2.4 24.8 ± 3.9 8.8 ± 1.7 >50

6.8 ± 0.3 42 ± 1.9 28.7 ± 1.0 13.4 ± 1.5 10.6 ± 5.1 8.9 ± 1.0 15.3 ± 3.1 26.3 ± 2.1 8.2 ± 2.0 >50

98 59 87 94 95 95 59 89 91 51

1 8 3 3 2 2 11 5 6 25

Shown are the mean ± SD from at least three independent experiments, each in duplicate. The clonogenicity inhibition treated with indicated compounds was presented as percentage relative to the vehicle treatment. a

glutaminase inhibitors with corresponding heterocyclic based 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 anticancer agents was also tested. A synergistic effect was observed for BPTES, 5, or 6 with doxorubicin (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 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 proliferatoractivated receptor agonists (PPARs) used in the treatment of diabetes,29 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-2,4dione 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 treatment with thiazolidine2,4-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-Lnorleucin or acivicin) nor intermonomer allosteric inhibitors such as BPTES or 1 for developing GA inhibitors for cancer therapy.

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EXPERIMENTAL SECTION

1. Chemistry. 1.1. General Remarks. According to the substructures listed by Baell and Holloway,30 the core structure of 5603



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Figure 1. Effects of glutaminase inhibitors, thiazolidine-2,4-dione derivatives, in inhibiting AsPC-1 cellular glutamate production and clonogenicity. Thiazolidine-2,4-dione derivatives inhibited colony formation of AsPC-1 (A) and diminished the cellular glutamate levels in both of AsPC-1WT (B, C) and AsPC-1GLS−/− cells (C). The highly selective compound BPTES, which completely lost glutaminase inhibition in AsPC-1GLS−/− cells, was used as reference compound control. The cells treated with thiazolidine-2,4-dione derivatives or the reference compound BPTES were subjected to assays for glutamates levels or clonogenicity as described in Experimental Section. Shown are the mean ± SD from three independent experiments, each in duplicate for (B) and (C): (∗∗) P < 0.01, (∗∗∗) P < 0.001 versus vehicle treatment. 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). 1.11. 3′-Ethyl-4′-formyl-4-(3-formyl-2,5-dimethyl-1H-pyrrol1-yl)-[1,1′-biphenyl]-2-carbonitrile (E6). 1H NMR (400 MHz, CDCl3): 10.38 (s, 1H), 9.91 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 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, 8.4 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 Hz, 3H). 1.12. 1-(4′-Formyl-2-methoxy-[1,1′-biphenyl]-4-yl)-2,5-di(thiophen-2-yl)-1H-pyrrole-3-carbaldehyde (E11). 1H NMR (400 MHz, CDCl3): 10.06 (s, 1H), 9.86 (s, 1H), 7.93 (d, J = 10.8 Hz, 2H), 7.72 (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, 1H), 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).

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). 1.8. 1-(4-Bromo-3-methoxyphenyl)-2,5-di(thiophen-2-yl)1H-pyrrole (D7). 1H NMR (400 MHz, CDCl3) 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). 1.9. 1-(3′-Ethyl-4′-formyl-[1,1′-biphenyl]-4-yl)-2,5-dimethyl1H-pyrrole-3-carbaldehyde (E3). 1H NMR (400 MHz, CDCl3): 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). 1.10. 1-(4′-Formyl-2-methoxy-[1,1′-biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrole-3-carbaldehyde (E4). 1H NMR (400 MHz, CDCl3): 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, 5604


2 1 NAc

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1.13. (Z)-5-((1-(4-Bromophenyl)-2,5-dimethyl-1H-pyrrol-3yl)methylene)thiazolidine-2,4-dione (2). 1H 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, 2H), 6.20 (s, 1H), 2.14(s, 3H), 2.00 (s, 3H). 13C NMR (400 MHz, DMSO): 168.29, 167.50, 136.16, 135.77, 132.59 (×2), 131.50, 130.12 (×2), 126.08, 121.99, 115.15, 115.00, 105.15, 12.49, 10.72. ESI-MS C16H13BrN2O2S, 377.25; found, 377/379 [M ]+. HPLC chromatograms: purity >96%. 1.14. (Z)-5-((1-(4-Bromophenyl)-2,5-di(thiophen-2-yl)-1Hpyrrol-3-yl)methylene)thiazolidine-2,4-dione (3). 1H 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, 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, 1H). 13C NMR (400 MHz, DMSO): 167.81, 167.38, 136.04, 133.47, 132.24, 131.89, 131.66, 131.60, 131.26, 129.87, 129.03, 128.89, 128.20, 127.42 (×2), 126.95, 126.79, 125.31, 124.86, 122.87, 119.55, 118.51. ESI-MS C22H13BrN2O2S3, 513.44; found, 513/515 [M]+, 536/538 [M + Na+]+. HPLC chromatograms: purity >98%. 1.15. (Z)-5-((1-(4-Bromophenyl)-2,5-diphenyl-1H-pyrrol-3yl)methylene)thiazolidine-2,4-dione (4). 1H NMR (300 MHz, DMSO-d6), δ (ppm): 7.46 (dd, J = 6.9, 1.8 Hz, 2H), 7.38−7.14 (m, 12H), 7.08 (dd, J = 6.9, 1.8 Hz, 2H), 6.68 (s, 1H). 13C NMR (176 MHz, DMSO): 140.95, 137.14, 132.28 (×2), 131.56 (×2), 131.51 (×2), 131.38, 129.99 (×2), 129.08, 129.03 (×3), 128.80, 128.77 (×3), 127.99 (×2), 121.72, 117.83, 107.86. ESI-MS C26H17BrN2O2S, 501.39; found, 502 [M + H+]+, 524 [M + Na+]+. HPLC 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′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (5). 1H 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 = 8.4 Hz, 2H), 6.21 (s, 1H), 2.17 (s, 3H), 2.05 (s, 3H). 13C NMR (400 MHz, DMSO): 168.26, 167.87, 167.55, 167.45, 140.37, 139.05, 136.73, 135.87, 132.59, 131.60, 131.00, 130.74 (×2), 128.50 (×2), 127.87 (×2), 127.57 (×2), 126.15, 123.86, 114.94, 114.92, 105.10, 12.59, 10.80. ESI-MS C26H19N3O4S2, 501.57; found, 502 [M + H+]+. HPLC chromatograms: purity >95%. 1.17. (E)-5-((4′-(3-((Z)-(2,4-Dioxothiazolidin-5-ylidene)methyl)-2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)-[1,1′-biphenyl]4-yl)methylene)thiazolidine-2,4-dione (6). 1H NMR (400 MHz, DMSO): 12.53 (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), 7.67 (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). 13C NMR (400 MHz, DMSO): 168.00, 167.86, 167.82, 167.44, 139.79, 139.56, 136.57, 133.61, 132.79, 132.14, 131.74, 131.14, 130.79, 130.70 (×2), 130.22, 129.79, 129.25, 127.46 (×3), 127.39 (×3), 127.32, 126.82, 126.57, 125.00, 124.22, 119.43, 118.47, 107.07. ESIMS C32H19N3O4S4, 637.76; 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′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (7). 1H 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), 7.36−7.18 (m, 13H), 6.70 (s, 1H). 13C NMR (75 MHz, DMSO): 167.98, 167.85, 167.49, 167.41, 140.93, 139.78, 138.03, 137.25, 137.00, 132.53, 131.25, 131.25 (×2), 131.08, 130.99, 130.67, 129.64 (×2), 129.45, 128.89 (×2), 128.59, 128.42 (×2), 128.32 (×2), 127.51 (×2), 127.27 (×2), 126.95, 125.88, 123.80, 118.11, 117.20, 107.44. ESI-MS C36H23N3O4S2, 625.71; found, 626 [M + H]+, 648 [M + Na]+. 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′-methoxy-[1,1′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (8). 1H NMR (300 MHz, DMSO): 13.00−12.00 (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), 7.02 (dd, J = 7.8, 1.5 Hz, 1H), 6.20 (s, 1H), 3.82 (s, 3H), 2.21 (s, 3H), 2.08 (s, 3H). 13C NMR (101 MHz, DMSO): 168.31, 167.50, 159.87, 156.81, 138.94, 137.83, 136.01, 132.39, 132.21, 132.08, 131.71, 130.96,

a iv, intravenous administration; po, oral administration; T1/2, terminal half-life; CL, total body clearance; Vss, volume of distribution at steady state; AUC, area under the concentration time curve; Cmax, the highest peak concentration; Tmax, time to reach the highest peak concentration; F, % oral bioavailability. bND: not determined. cNA: not available.

AUC(0−inf) ((ng/mL)·h) Tmax (h)

73 ± 2 222 ± 89 NDb 40 ± 8.2 16.6 ± 4.6 NDb

Cmax (ng/mL) T1/2 (h)

2.0 ± 0.1 NAc NDb 10 1.0 10

dose (mg/kg) AUC(0−inf) ((ng/mL)·h)

636 ± 90 3414 ± 1184 43580 ± 8136 8.8 ± 1.6 0.4 ± 0.1 0.1 ± 0

Vss (L/kg) kg ) CL (mL min

2.0 0.1 2.0

T1/2 (h) dose (mg/kg) compd

BPTES 5 6

5.3 ± 1.4 NAc 5.0 ± 1.4

53 ± 7.6 0.4 ± 0.1 0.9 ± 0.2

−1

iv

−1

Table 3. Pharmacokinetic Properties of Glutaminase Inhibitors, BPTES, and Thiazolidine-2,4-dione Derivatives 5 and 6 in Ratsa

po

0.7 ± 0.3 4.0 NDb

F (%)


5605



Article

Figure 2. In vivo antitumoral efficacy of GA inhibitors in AsPC-1 xenograft tumor model. (A) BPTES and compound 5: AsPC-1 bearing mice were sorted into four groups. Group 1 mice received vehicle (2% DMSO/PBS), group 2 mice received BPTES at 25 mg/kg, groups 3 and 4 mice received compound 5 at 25 and 50 mg/kg through intraperitoneal (ip) injection, respectively, once daily on 5 on/2 off (5 days on and then 2 days off) schedule for the entire period of observation. (B) Compound 6: AsPC-1 bearing mice were sorted into 3 groups. Group 1 mice received vehicle (2% DMSO/PBS), and groups 2 and 3 received compound 6 at 25 and 50 mg/kg through ip injection, respectively, once daily on 5 on/2 off schedule for the entire period of observation. % TGI was determined for antitumor effects which are expressed as [1 − ((Tt − T0)/(Ct − C0))] × 100 where Tt = median tumor volume of treated at time t, T0 = median tumor volume of treated at time 0, Ct = median tumor volume of control at time t, and C0 = median tumor volume of control at time 0. “t” is indicated as day 43 for (A) and day 68 for (B) in this study. Statistical significance was established at p < 0.05. found, 656 [M + H]+, 678 [M + Na]+. HPLC chromatograms: purity >94%. 1.22. (E)-5-((4′-(3-((Z)-(2,4-Dioxothiazolidin-5-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-3-ethyl-[1,1′-biphenyl]4-yl)methylene)thiazolidine-2,4-dione (11). 1H 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 = 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 = 7.6 Hz, 3H). 13C NMR (400 MHz, DMSO): 168.28, 168.08, 167.46, 167.28, 145.49, 140.57, 139.33, 136.63, 135.89, 131.61, 130.89, 128.54, 128.44 (×2), 128.13, 127.95, 127.87 (×2), 126.17, 125.44, 124.99, 114.95, 114.92, 105.10, 26.10, 15.64, 12.59, 10.81. ESIMS C28H23N3O4S2, 529.62; found, 530 [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)-3-ethyl-[1,1′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (12). 1H NMR (400 MHz, DMSO): 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), 6.79 (s, 1H), 2.82 (q, J = 7.6 Hz, 2H), 1.91 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz, DMSO): 168.54, 168.17, 167.94, 167.56, 145.46, 139.82, 139.80, 136.45, 133.60, 132.19, 131.75, 131.21, 131.12, 130.15, 129.74, 129.30, 128.91, 128.22, 128.14, 127.94, 127.76 (×2), 127.40, 127.29, 126.78, 126.53, 126.29, 124.97, 124.90,

130.12, 129.84, 129.32, 128.81, 126.20, 124.09, 120.18, 119.10, 116.32, 114.89, 111.84, 104.98, 56.20, 12.66, 10.87. ESI-MS C27H21N3O5S2, 531.60; found, 532 [M + H]+. HPLC 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-yl)-2′-methoxy[1,1′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (9). 1H 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, 1H), 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 (m, 4H), 6.77 (s, 1H), 3.66 (s, 3H). 13C NMR (400 MHz, DMSO): 167.97, 167.88, 167.57, 167.43, 156.44, 138.66, 137.49, 133.59, 132.17, 132.04, 131.70, 131.28, 131.23, 130.57, 130.13 (×2), 129.87 (×2), 129.80, 129.41, 129.30, 127.40 (×2), 126.86, 126.69, 125.06, 123.70, 121.94, 119.39, 118.49, 113.87, 107.04, 56.23. ESI-MS C33H21N3O5S4, 667.78; found, 668 [M + H]+. HPLC chromatograms: purity >94%. 1.21. (E)-5-((4′-(3-((Z)-(2,4-Dioxothiazolidin-5-ylidene)methyl)-2,5-diphenyl-1H-pyrrol-1-yl)-2′-methoxy-[1,1′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (10). 1H NMR (400 MHz, DMSO): 13.00−12.00 (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, 3H). 13C NMR (101 MHz, DMSO): 168.07, 168.01, 167.82, 167.44, 156.00, 140.75, 138.58, 138.02, 136.86, 131.90, 131.37, 131.02, 130.16, 129.94, 129.83, 129.76, 128.89, 128.62, 128.29, 128.19, 127.85, 127.52, 125.84, 123.81, 121.03, 118.20, 117.24, 113.10, 107.41, 55.79, 40.12, 39.91, 39.70, 39.49, 39.29, 39.08, 38.87. ESI-MS C37H25N3O5S2, 655.74; 5606



Article

Figure 3. Assignment of the KGA binding site of thiazolidine-2,4-dione derived glutaminase inhibitors. (A) Docking simulation of the compound 5 binding site in KGA. Shown were the alkyl to alkyl and alkyl to π hydrophobic bonding interactions (in blue dashed lines) of compound 5 (highlighted in cyan) with the residue Arg 387, which is located in the substrate binding pocket and highlighted in orange, of KGA in the best docked pose. The best docked pose was determined based on the results from site-directed mutagenesis (Table 4) and the molecular modeling and docking studies, which were conducted using the human GLS1 crystal structure (PDB code 3UO9) and the BIOVIA/LigandFit program (BIOVIA, Inc., San Diego, CA) with the CHARMM force field.34 The residues (Ser 286, Glu 381, Asn 335, and Tyr 414) involved in substrate glutamine binding site of KGA25 were highlighted in yellow. Those residues, Ser 384 and Lys 507, which did not show interaction with the thiazolidine-2,4-dione derived glutaminase inhibitors as demonstrated by site-directed mutagenesis (Table 4), were highlighted in magenta and green, respectively. (B) Close look of the hydrophobic interactions between the side chain CH2 of compound 5 and Arg 387. (C) Overview of the proposed association of compound 5 in a GA tetramer. DMSO): 168.14, 168.00 (×2), 167.43, 145.48, 139.87, 138.24, 137.15, 137.02, 131.28, 131.10 (×2), 130.87 (×2), 129.66 (×2), 129.40 (×2), 128.91 (×2), 128.60 (×2), 128.59 (×2), 128.33 (×2), 128.08 (×2), 127.60, 127.52, 126.92, 125.90, 124.73, 118.10, 117.19, 107.42, 26.09, 15.72. ESI-MS C38H27N3O4S2, 653.77; found, 654 [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-ethyl-2′-methoxy-

119.53, 118.49, 107.07, 26.14, 15.72. ESI-MS C34H23N3O4S4, 665.81; 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-ethyl-[1,1′-biphenyl]4-yl)methylene)thiazolidine-2,4-dione (13). 1H 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 (s, 3H), 2.63 (q, J = 8.0 Hz, 2H), 1.16 (t, J = 8.0 Hz, 3H). 13C NMR (75 MHz, 5607



Article

Table 4. Inhibitory Activities (IC50) of Thiazolidine-2,4-dione Derivatives and BPTES against Human KGA Mutantsa compound BPTES

a

5

7

17

KGA

IC50 (nM)

V.S. KGA (fold)

IC50 (nM)

V.S. KGA (fold)

IC50 (nM)

V.S. KGA (fold)

IC50 (nM)

V.S. KGA (fold)

WT S384A R387A R387L K507A

81 ± 15 65 ± 11 63 ± 10 345 ± 36 66 ± 5

1 0.8 0.8 4.3 0.8

102 ± 20 361 ± 3 >50000 1270 ± 40 1045 ± 94

1 3.5 >490 12.5 10.2

45 ± 4 78 ± 17 8883 ± 136 603 ± 32 187 ± 46

1 1.7 197 13.4 4.2

57 ± 15 87 ± 24 10901 ± 759 770 ± 57 269 ± 79

1 1.5 191 13.5 4.7

Shown are the mean ± SD from three independent experiments, each in duplicate.

Figure 4. Inhibition mode of thiazolidine-2,4-dione derived glutaminase inhibitors against KGA. (A) Glutamine saturation profiles for KGA at 0−0.4 μM of compound 5. (B) Lineweaver−Burk double-reciprocal for KGA and compound 5. (C) Data of Vmax and Km at different concentrations of compound 5. [1,1′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (14). 1H NMR (400 MHz, 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), 7.02 (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, 3H), 1.19 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz, DMSO): 168.58, 168.34, 168.13, 167.53, 156.84, 144.43, 139.03, 137.71, 136.03, 131.73, 130.96, 130.44, 130.34, 129.10, 128.32, 127.58, 127.16, 126.23, 125.86, 120.14, 114.91, 114.89, 111.79, 104.99, 56.19, 26.02, 15.65, 12.66, 10.87. ESI-MS C29H25N3O5S2, 559.65; found, 560 [M + H+]+, 582 [M + Na+]+. HPLC chromatograms: purity >96%. 1.26. 4′-((E)-(2,4-Dioxothiazolidin-5-ylidene)methyl)-4-(3((Z)-(2,4-dioxothiazolidin-5-ylidene)methyl)-2,5-dimethyl-1Hpyrrol-1-yl)-3′-ethyl-[1,1′-biphenyl]-2-carbonitrile (15). 1H 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, 2H), 2.20 (s, 3H), 2.08 (s, 3H), 1.23 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz, DMSO): 168.26, 168.10, 167.47, 167.33, 144.96, 143.73, 138.37, 136.86, 135.90, 133.24, 133.12, 132.23, 131.69, 131.40, 129.98, 128.26, 127.77, 127.03, 126.59, 125.92, 117.62, 115.55, 115.29, 111.35, 105.47, 25.94, 15.29, 12.56, 10.83. ESI-MS

C29H22N4O4S2, 554.63; found, 555 [M + H+]+, 577 [M + Na+]+. HPLC chromatograms: purity >98%. 1.27. (E)-5-((4′-(3-((Z)-(2,4-Dioxothiazolidin-5-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-2′-hydroxy-[1,1′-biphenyl]-4-yl)methylene)thiazolidine-2,4-dione (16). 1H NMR (400 MHz, DMSO): 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), 7.49 (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, 3H), 2.06 (s, 3H). 13C NMR (400 MHz, DMSO): 169.20, 168.81, 168.36, 167.56, 155.22, 139.19, 137.25, 135.82, 131.98, 131.52, 131.15, 130.46, 129.89 (×2), 129.81(×2), 127.07, 126.22, 124.70, 118.92, 115.32, 114.92, 114.87, 105.06, 12.63, 10.84. ESI-MS C26H19N3O5S2, 517.57; found, 518 [M + H]+. HPLC chromatograms: purity >95%. 1.28. (Z)-5-((1-(3′-Chloro-4′-((E)-(2,4-dioxothiazolidin-5ylidene)methyl)-[1,1′-biphenyl]-4-yl)-2,5-dimethyl-1H-pyrrol3-yl)methylene)thiazolidine-2,4-dione (17). 1H 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), 2.17 (s, 3H), 2.04 (s, 3H). 13C NMR (400 MHz, DMSO): 168.29, 167.55, 167.46, 167.16, 141.89, 137.63, 137.23, 135.80, 135.53, 131.54, 130.14, 129.31, 5608



Article

Laboratory Inc.). AsPC-1, MDA-MB-231, and HCC1806 cells were cultured in RPMI 1640 medium (GIBCO-Life Technologies) supplemented with 10% FBS. 3. Cloning, Expression, and Purification of Glutaminases. The human KGA and GAB were prepared as described.9 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 RTPCR (Invitrogen, Inc.) according to the manufacturer. A cDNA fragment of 1428 bp, 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 1428 bp was verified by sequence analysis and found to be the same as that of accession no. AF158555 except three different nucleotides (underlined as follows) that resulted into the changes of aa 172 Asp(GAT) → Gly(GGT), aa 268 Ala(GCA) → Val(GTA), and aa 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. 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 600 nm of 0.6−0.8, isopropyl β-D-1thiogalactopyranoside (1 mM) was added to induce the His-tagged GAC protein expression for 6 h at 25 °C. The resultant bacteria were collected by rapid centrifugation and then resuspended in a lysis buffer (50 mM NaH2PO4·H2O, 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 were collected for further protein purification at cold room with a Ni2+ 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 containing 500 mM imidazole, and the buffer was then exchanged to 50 mM NaH2PO4·H2O, 50 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, and 50% glycerol by using PD-10 desalting column (GE Healthcare). The concentrations of the purified protein were determined as described.9 4. Glutaminase Activity Assays and Kinetic Studies. Human glutaminase activity was measured using a two-step assay as described previously.9,32,33 Kinetic studies were measured as described.9 5. In Vitro Carcinoma Cell Growth Inhibition and Colony Formation Assays. MDA-MB-231 breast carcinoma, AsPC-1 pancreatic carcinoma, HCC1806 breast carcinoma cells were seeded at 8500, 4000, and 3000 cells/well, respectively, in 96-well plates, as previously described for cell growth inhibition assay.9 To assess anchorage-dependent colony formation effect, the cells (1000 cells/ well) were seeded in a six-well plate, as previously described.9 6. Determination of Intracellular Glutamate Levels. AsPC-1 cells were seeded at 30 000 cells/well in 96-well plates and incubated in a CO2 incubator at 37 °C for 24 h. The cells were treated with either the vehicle control (1% DMSO) or 3.13−100 μM glutaminase inhibitors as indicated for an additional 4 h. After a 4 h treatment, the medium was removed and cells per well were washed twice with 1× PBS, and 50 μL of nonidet-P40 lysis buffer was added per well at 4 °C for 20 min. 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

Figure 5. Synergistic effects of combined treatments of GA inhibitors with doxorubicin or GA inhibitors at different binding sites in inhibition of carcinoma cell growth. AsPC-1 (A) and HCC1806 (B) cells were grown and treated with indicated compounds. Cell viabilities were assayed by MTS as described in Experimental Section. Combination index (CI) values at ED50, ED75, and ED90 were calculated using Calcusyn software based on the Chou−Talalay method for drug combination35 and summarized in Supporting Information Table S1. Combination index (CI) = 1 refers to additive effect, CI < 1 refers to synergism, and CI > 1 means antagonism. ED50, ED75, and ED90 represent effective doses of 50%, 75%, and 90% inhibition, respectively. Results shown are representative of three independent experiments. The dashed line, isobologram of ED50, is drawn for clarity to define area for the additive effect, synergism, or antagonism. When the combination data point falls on the dashed line (CI = 1), an additive effect is suggested. When data point falls on the lower left area of the dashed line (CI < 1), a synergism is suggested, and if it falls on the upper right area (CI > 1), an antagonism is suggested. Data of two combinations for each experiment all fell on the lower left area in the isobologram indicating a synergism. 128.54 (×2), 128.26, 128.06 (×2), 127.06, 126.21, 126.10, 125.98, 115.05, 115.03, 105.21, 12.61, 10.80. ESI-MS C26H18ClN3O4S2, 536.01; 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 60436] were used in this study. MCF7 cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone Laboratory Inc.) and supplemented with 10% fetal bovine serum (FBS, Hyclone 5609



Article

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 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 PCR product was denatured and reannealed 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 corresponding GLS-KO ZFN mRNA transfected cells were subject to 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. 8. 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. 9. 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, Taiwan. Male rats (330−380 g, 9−10 weeks old) were quarantined for 1 week before use. The rats were surgically implanted with a jugular-vein cannula 1 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% Tween-80 for intravenous and oral administration, respectively. At 0 (immediately before dosing), 2 min, 5 min (intravenous only), 15 min, and 30 min and 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after compound administration, a blood sample (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 chromatography−tandem mass spectrometry was performed as described.31 The pharmacokinetic parameters were obtained by a standard noncompartmental method. 10. In Vivo Antitumor Activity. Male nude mice (BALB/ cAnN.Cg-Foxn1nu/CrlNarl, National Laboratory Animal Center, Taipei, Taiwan) were 7−8 weeks old and had a body weight range of 18−22 g. Mice were inoculated subcutaneously at the right flank region with AsPC-1 cells (4.5 × 106 cells/mouse) in 0.1 mL of Hank’s balanced salt solution (ThermoFisher Scientific) mixing with equal volume of 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 mean tumor size reached ∼100 mm3 (this day was thus designated as day 1). The tumor volume was calculated using the equation V (mm3) = ab2/ 2, where a is the largest diameter and b is the smallest diameter. % TGI was determined for antitumor effects which are expressed as [1 − ((Tt − T0)/(Ct − C0))] × 100 where Tt = median tumor volume of treated at time t, T0 = median tumor volume of treated at time 0, Ct = median tumor volume of control at time t, and C0 = median tumor volume of control at time 0. Data were presented as the mean ± SD and analyzed using IBM SPSS Statistics version 20.0 software (IBM Corp., Armonk, NY, USA). The Student’s t test was carried out to calculate the percent of tumor growth inhibition (% TGI) in the treatment group versus the vehicle control at the end point. Statistical significance was established at p < 0.05. 11. Site-Directed Mutagenesis and Docking. Site-directed mutagenesis was performed on the pET28a(+)-KGA9 plasmids using PfuUltra II fusion HS DNA polymerase (Agilent Technologies) to construct the KGA mutants as described.9 The mutated codons in mutants KGA S384A, KGA R387A, KGA S387L, and KGA K507A were verified by sequencing. The GLS1 crystal structure (PDB code

3UO9) was applied to BIOVIA 2017 for the docking analysis which was conducted using the BIOVIA/LigandFit program (BIOVIA, Inc., San Diego, CA) with the CHARMm force field.34 The population size of docked poses was set as 100 with default parameters. The best pose was determined according to the result of site-directed mutagenesis. 12. Statistical Analysis. The statistical significance between two groups was evaluated by two-tailed unpaired Student’s t test and ∗, ∗∗, and ∗∗∗ were used to denote the statistical significance for p < 0.05, p < 0.01, and p < 0.001, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00282. Glutaminase inhibitory activity of commercial available glitazones, summary of synergistic effects of combined treatments of GA inhibitors, pictures/structures of compounds 2−17 and intermediates, verification of GLS gene knocked out in AsPC-1GLS−/− cells, and effects of PPAR agonists or thiazolidine-2,4-dione compounds on the transactivation activities of PPARα, PPARγ, and PPARδ (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*L.K.T.: telephone, +886-37-246-166, extension 35769; fax, +886-37-586-456; e-mail, [email protected]. *S.-J.L.: telephone, +886-37-246-166, extension 35715; fax, +886-37-586-456; e-mail, [email protected]. ORCID

Yue-Zhi Lee: 0000-0001-8640-9525 Yu-Wei Liu: 0000-0001-8495-4941 Cheng-Wei Yang: 0000-0002-3994-475X Lun Kelvin Tsou: 0000-0002-1593-5226 Shiow-Ju Lee: 0000-0003-4012-0149 Author Contributions †

T.-K.Y., C.-C.K., Y.-Z.L., Y.-Y.K., and K.-F.C. are authors with equal contribution. L.K.T. and S.-J.L. are cocorresponding authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grants 101-EC-17-A-02-04-1099, 102-EC-17-A-02-04-1099, 103-EC-17-A-22-1099, 104-EC-17A-22-1099, and 105-EC-17-A-22-1099 from Ministry of Economic Affairs, Taiwan R.O.C.

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ABBREVIATIONS USED AsPC-1, human pancreatic carcinoma cells; CMC, carboxymethylcellulose; cMyc, v-myc avian myelocytomatosis viral oncogene homolog; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3phosphate dehydrogenase; GA, glutaminase; GLS, glutaminase gene; GAB, glutaminase isoform B; KGA, glutaminase kidney isoform; LGA, glutaminase liver isoform; miRNA, microRNA; MDA-MB-231, human breast adenocarcinoma epithelial cells; p53, tumor protein p53; p63, tumor protein p63; PPAR, peroxisome proliferator-activated receptor; rac1, Ras-related C3 5610



Article

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botulinum toxin substrate 1; SD, standard deviation; SI, selectivity index; WT, wild type

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