Glucose transporter 1/GLUT1 (IC50 = 2 nM); GLUT2/3/4 (IC50 = 10~ 0.3 μM)

Growth inhibitory BAY-876 (25–75 nM; 24 and 72 hours) reduces the number of SKOV-3 and OVCAR-3 cells in a dose-dependent manner [2].

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To understand the biological outcomes of GLUT1 dysregulation in oncogenesis, we asked if ovarian cancer cells rely on GLUT1 to support glycolytic metabolism. As we and others have demonstrated previously, ovarian cancer cells exhibited high basal glycolytic activity in regular culture conditions (Figure 1A). Although BAY-876 has been shown to inhibit glucose uptake, its effect on the downstream glycolytic metabolism is not, known. We thus examined the effects of BAY-876 on glycolysis and lactate production in ovarian cancer cell lines including A2780 known to lack a functional GLUT1 as a negative control. As shown in Figure 1A, incubation with BAY-876 dose-dependently decreased glycolytic rates in SKOV-3, OVCAR-3, and HEY cells. Similarly, BAY-876 reduced lactate levels present in culture supernatants of these cells (Figure 1B). Although this anti-glycolytic effect of BAY-876 was detectable at single-digital nanomolar concentrations, half-maximum suppression was achieved with 25–50 nM of the compound. We also observed similar anti-glycolytic activity of BAY-876 in other commonly used ovarian cancer cell lines such as OVCR-429 and OVCA-432.[2]

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In the A2780 ovarian cancer cell line, GLUT1 was reported to co-localize with mutant PTEN at the nucleus instead of the plasma membrane. Consistent with BAY-876 suppressing glycolysis via specific inhibition of GLUT1 rather than other GLUT members or off-target proteins, BAY-876 did not affect glycolysis or lactate production in A2780 cells (Figure 1A,B). The specific effect of BAY-876 on GLUT1-mediated glycolysis was further confirmed by siRNA silencing of GLUT1. The molecular approach decreased glycolysis rate and lactate production in SKOV-3 and OVCAR-3 cells (Figure 1C). [2]

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GLUT1 Mediates Upregulation of Glycolysis in Stress Conditions [2]
The basal aerobic glycolysis is generally high in ovarian cancer and other malignant cells compared to their normal counterparts. The glycolytic activity of cancer cells also fluctuates in response to tumor microenvironmental cues to meet the bioenergetic and biosynthetic needs of rapidly growing tumor cells. In particular, glycolysis is heightened in response to hypoxic conditions. GLUT1 is known to be one of hypoxia target genes, upregulated by HIF. We used cobalt chloride (CoCl2) as a means of stabilizing HIF to examine GLUT1 inducibility in ovarian cancer cells. As shown in Figure 2A, GLUT1 was highly induced by CoCl2 in all ovarian cancer cell lines. The induction of GLUT1 was associated with a rise in cellular glycolysis (Figure 2A). BAY-876 reduced both basal and CoCl2-stimulated glycolysis in SKOV-3, OVCAR-3 and HEY cells. Although CoCl2 increased GLUT1 expression and glycolysis in A2780 cells, BAY-876 had no effects on either basal or CoCl2-driven glycolysis, suggesting that GLUT1-independent mechanisms are involved in HIF-driven glycolysis in A2780 cells.

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Inhibition of GLUT1 Causes Bioenergetic Loss and Activation of AMP-Activated Protein Kinase (AMPK) [2]
Unlike normal cells that use oxidative phosphorylation as a major bioenergetic source, cancer cells rely heavily on glycolysis for substrate-based phosphorylation of ADP to form ATP [44]. We next determined whether GLUT1 inhibition is sufficient to reduce ATP production in ovarian cancer cells. As shown in Figure 3A, cellular ATP levels were significantly decreased by treatment of SKOV-3 and OVCAR-3 with BAY-876. In agreement with decreased ATP abundance, the treatment led to activation of 5′ adenosine monophosphate-activated protein kinase α (AMPKα) as reflected by increased phosphorylation of AMPKα at Thr-172 (Figure 3B). Once again, BAY-876 had no effect on ATP level or AMPKα phosphorylation in A2780 cells. Together, the results support the conclusion that BAY-876 impairs glycolysis-associated ATP generation via specific inhibition of GLUT1.

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To further understand the metabolic outcomes of GLUT1 inhibition, we measured oxidative phosphorylation by following oxygen consumption rate (OCR) in SKOV-3, OVCAR-3 and A2780 cells treated with or without BAY-876. In contrast to the effect on glycolysis, BAY-876 increased OCR in these cells as determined using the Seahorse XF24 Analyzer (Figure 3C). Significant increases in basal mitochondrial respiration (basal OCR), ATP-linked OCR, and maximal respiratory capacity were observed in SKOV-3 cells. In OVCAR-3, increases in basal and ATP-linked OCR were detected. The mechanism leading to increased respiration following BAY-876 treatment was not fully understood. Since it was absent in A2780 cells, this effect of BAY-876 on OCR in SKOV-3 and OVCAR-3 cells was also GLUT1 dependent, likely as a compensatory response to inhibition of the GLUT1-glycolysis-ATP cascade. [2]

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Inhibition of GLUT1 Suppresses Proliferation, Viability, and Anchorage-Independent Growth of Ovarian Cancer Cells [2]
Given the importance of hyperactive glycolysis in support of the malignant features of cancer, we next assessed the effects of BAY-876 on growth and viability of ovarian cancer cells. First, we treated ovarian cancer cell lines with <100 nM concentrations of BAY-876 that significantly decreased glycolysis with little cytotoxicity as shown in Figure 1. Treatment with these concentrations of BAY-876 for a single day led to a dose-dependent decrease in numbers of SKOV-3 and OVCAR-3 cells (Figure 4A). Three-day growth curves in the presence of 75 nM BAY-876 further confirmed the growth-inhibitory effect in SKOV-3, OVCAR-3 and HEY, but not in A2780 cells (Figure 4B). To evaluate the combined effects of BAY-876 on cell growth and cytotoxicity, these cell lines were treated with a greater dose range of BAY-876 up to 10 µM for 3 days. The mitochondrial activity of viable cells was measured with the MTT assay. The results in Figure 4C revealed that OVCAR-3 was most sensitive to BAY-876 with IC50 value of approximately 60 nM. The IC50 values of SKOV-3 and HEY were 188 and 1002 nM, respectively. In agreement with the lack of functional GLUT1 and anti-glycolytic effect, A2780 cells were refractory to treatment with BAY-876 even at as high as 2 µM concentration (Figure 4C).

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Starting from moderately potent and metabolically labile HTS hit 1 carrying a furanyl moiety at position 2 of the quinoline and four methyl groups in total, we quickly improved both, potency and metabolic stability by substituting the furan and taking out the two quinoline methyl groups. Using the quinoline core of compound 3 we found an unsubstituted methylene to be the optimal spacer between the phenyl and pyrazole ring. After successful replacement of one of the pyrazole methyl groups for the metabolically more stable CF3 group (12), further SAR exploration of the quinoline core demonstrated an unsubstituted amide at position 2 and a fluorine at position 7 (19, BAY-876) to be very beneficial regarding GLUT1 potency and selectivity against the other GLUTs. At the pyrazole core a double substitution at positions 3 and 5 was found to be crucial for the excellent potency/selectivity profile especially against GLUT3. Regarding a substituent at the benzyl ring, the para position yielded more promising compounds than the ortho or meta position. With a para‐cyano group and an additional ring nitrogen, compound 54 demonstrated also an interesting GLUT profile. The synthesis of the N‐(1H‐pyrazol‐4‐yl)quinoline‐4‐carboxamides was straightforward as exemplified for BAY-876 (19). In vitro PK data showed that both BAY‐876 (19) and 54 were very stable in liver microsomes and hepatocytes, although 54 had a strong efflux ratio of around 16 [1].

Mice treated orally with BAY-876 (1.5–4.5 mg/kg/day for 28 days) showed a notable dose-dependent reduction in carcinogenicity [2].

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BAY-876 Inhibits Tumorigenicity of Ovarian Cancer Cell Lines and Ovarian Cancer PDXs [2]
The roles of GLUT1 in regulation of glycolysis, energy metabolism, and anchorage-dependent and -independent growth of ovarian cancer cells suggest that GLUT1 is a promising anti-cancer target. Since BAY-876 has not been thoroughly evaluated as an anti-cancer agent in vivo, we assessed its anti-tumor potential and safety in female NOD-scid IL2rgnull (NSG) mice carrying SKOV-3 subcutaneous (s.c.) xenografts. All animal experiments in this study were conducted following the policies and regulations of VCU IACUC. Four cohorts of mice with a similar range of tumor sizes (~100 mm3) were orally fed 0, 1.5, 3.0 and 4.5 mg/kg/day for 4 weeks. The tumor growth curves and changes in body weights were monitored and presented in Figure 5. There was a clear dose-dependent inhibition of tumorigenicity by BAY-876. The maximal effect was observed in the group treated with 4.5 mg/kg/day. The tumors shrunk significantly after 2 weeks of treatment. At endpoint, the final average tumor volumes and tumor weights decreased by 68% and 66%, respectively, compared to these parameters in the vehicle control group. However, the 4.5 mg/kg/day dose was somewhat toxic in NSG mice. The mouse body weights started to drop during the last week of treatment. At the end of 4 weeks, the weight loss in this group reached an average of 18% compared to the control group or groups of other two lower doses. Except for the weight loss, these mice, however, had no other noticeable health conditions.

We have developed PDXs from patients with high-grade serous ovarian carcinomas. H&E staining of these PDXs confirmed histological appearances of papillary adenocarcinomas, resembling the original patient tumor tissues (Supplementary Figure S2). We examined the effects of BAY-876 in two PDXs OVC-PDX2 and OVC-PDX3 that expressed GLUT1 protein (Figure 6D,H). Female NSG mice bearing PDXs were treated with 4.0 mg/kg/day, a dose determined on the basis of the earlier SKOV-3 xenograft experiment. Treatment with this dose of BAY-876 over 30 days dramatically decreased tumor growth with no significant loss of body weights (Figure 6B,F). The average endpoint tumor volumes reduced by more than 60% in both PDXs (Figure 6A,E). The final tumor weights in OVC-PDX2 and OVC-PDX3 were decreased by 50% and 71%, respectively (Figure 6C,G).

These results indicate that BAY-876 is an effective and safe anti-cancer agent when orally administrated at 4.0 mg/kg/day or less. We next asked whether acute treatment with a higher dose over a shorter period could achieve a better therapeutic benefit. Accordingly, female NSG mice carrying OVC-PDX2 were treated with 7.5 mg/kg/day and closely monitored for tumor growth and health conditions. Unfortunately, the mice did not tolerate this dose and all succumbed by 18 days of treatment. Shown in Supplementary Figure S3 were the effects of BAY-876 at 7.5 mg/kg/day on tumor growth, body weights, and Kaplan Meier survival of control and treated groups.

Ultra‐high‐throughput screen (uHTS) with human GLUT1: [1]
It is well known that a combination of small‐molecule inhibitors of mitochondrial electron transport chain and glucose catabolism synergistically suppress ATP production. For uHTS, CHO‐K1 cells were stable transfected with human GLUT1 and a constitutively expressing luciferase as described previously. Cells were seeded in 1536 microtiter plates with a density of 1000 cells per well and starved for 24 h in glucose free DMEM in the presence of 1 % FCS. Prior to measurements cells were incubated for 30 min at 37 °C in the presence of 10 μm rotenone to fully block oxidative phosphorylation. Test compounds and caged luciferin were loaded simultaneously. Before application of 0.5 mm glucose and corresponding activation of GLUT1, basal ATP was indirectly measured by luciferase activity in order to identify effects on cellular ATP levels independent of glucose; 10 min kinetic luciferase recordings after application of 500 μm glucose allowed the investigation of compound induced inhibition of GLUT1.

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GLUT isoform specificity testing: [1]
For specificity testing between GLUT1, GLUT2, GLUT3 and GLUT4 we used DLD1 (for GLUT1), DLD1GLUT1−/− (for GLUT3), CHO‐hGLUT2 and CHO‐hGLUT4 (GLUT2 and 4) cells in combination with an oxidative phosphorylation inhibitor (rotenone 1 μm). Cell lines were maintained in DMEM medium supplemented with 10 % FCS and 1 % penicillin‐streptomycin solution and 2 % Glutamax under standard conditions. The cells were treated with trypsin and seeded into 384 plates at a density of 4000 cells per well. The cells were then cultured overnight in glucose free media containing 1 % FCS to reduce intracellular ATP levels. For GLUT1/2/3, after 16 h the cells were incubated with appropriate glucose concentration or in case of GLUT2 fructose concentration (0.1 m for GLUT1, 0.3 m for GLUT3 and 30 mm fructose for GLUT2, respectively) with or without compounds and 1 μm rotenone for 15 min. The CellTiter‐Glo® Luminescent Cell Viability Assay was then used to measure ATP levels. Assay was normalized to the control cytochalasin B (IC50 GLUT1: 0.1 μm GLUT2: 2.8 μm, GLUT3: 0.12 μm, GLUT4: 0.28 μm), assay variance: 9 %, IC50 calculation R 2>0.9. For GLUT4, after 16 h the glucose free medium was removed and cells were adapted to KCl free tyrode buffer for 3 h. Compounds and rotenone were added and after 20 min cells were incubated with glucose (0.1 m final concentration) for 15 min. The CellTiter‐Glo® Luminescent Cell Viability Assay from Promega was then used to measure ATP levels.

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Glucose competition: [1]
For the glucose competition DLD1 cells were treated with trypsin and seeded into 384 plates at a density of 4000 cells per well. The cells were then cultured overnight in glucose free media containing 1 % FCS to reduce intracellular ATP levels. After 16 h the cells were incubated with different glucose concentration (0.1; 1 and 10 mm, respectively) together with compound (30 μm to 1 nm) and 1 μm rotenone for 15 min. The CellTiter‐Glo® Luminescent Cell Viability Assay from Promega was then used to measure ATP levels.

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GLUT1 Knockdown [2]
GLUT1 siRNA (Thermo Fisher Scientific, siRNA ID: s12925) or non-targeting control siRNA were transfected into SKOV-3 or OVCAR-3 cells using Amaxa™ Nucleofector™ Kit V according to the manufacturer. One million cells were electroporated with 100 pmol siRNA. Twenty-four hours after plating, transfected cells were fed fresh medium and incubated for another 72 h before analysis of glycolysis and immunoblotting.

Cell Proliferation Assay[2]
Cell Types: SKOV-3 and OVCAR-3 cells
Tested Concentrations: 25, 50, 75 nM
Incubation Duration: 24 and 72 hrs (hours)
Experimental Results: Led to a dose-dependent decrease in numbers of SKOV-3 and OVCAR- 3 cells.

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OCR Measurement[2]
OCR (pMoles/min) of cultured cells treated with or without BAY-876 was measured using the Seahorse XF24 Extracellular Flux Analyzer as we described previously. Ovarian cancer cell lines were seeded in XF24 microplates and cultured for 18–30 h before switching to the seahorse assay medium supplemented with 10 mM glucose and 2 mM glutamine. Basal mitochondrial respiration, ATP-linked respiration, maximal respiratory capacity and reserve capacity were calculated as described previously.

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Cell Growth Assays[2]
To assess the effect of BAY-876 (0, 25, 50, 75 nM) on proliferation, cells in 12-well plates were counted with Coulter counter after trypsinization. To determine the cytotoxic effect of BAY-876, cells were treated with a wider range of indicated concentrations for 3 days followed by MTT staining and measuring the absorbance at 570 nm. The IC50 values were calculated using SigmaPlot 13.0. Anchorage-independent growth was performed to assess the effect of BAY-876 on the ability of cells to grow in semi-solid soft agar. Six-well plates were pre-coated with 1.5 mL of 0.6% soft agar in complete medium. Cells suspended in 1.5 mL growth medium containing 0.3% soft agar were overlayered onto the pre-coated wells. Fresh complete medium containing 0.3% agar (1.5 mL) was added to the top every 3–5 days. The colonies larger than 100 μm (SKOV-3, HEY and A2780) or 50 μm (OVCAR-3) in diameter were counted after 3 weeks.

Animal/Disease Models: Female NOD-scid IL2rgnull (NSG) mice carrying SKOV-3 subcutaneous (sc) xenografts[2]
Doses: 1.5, 3, 4.5 mg/kg
Route of Administration: Oral administration; daily; for 28 days
Experimental Results: Caused a clear dose -dependent inhibition of tumorigenicity.

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Analysis of Anti-Cancer Activity of BAY-876 in Mice [2]
The above-described PDXs and cell-line-derived xenografts were utilized to assess the anti-tumor effect of BAY-876. The cell lines in exponential growth phase were trypsinized, washed twice with PBS and resuspended in the serum-free medium. SKOV-3 cells (4 × 106) were injected s.c. on the right flank of 6–7 weeks old female NSG mice. The formation of s.c. tumors was monitored and measured with a digital caliper. The tumor volumes were calculated based on the formula lw2/2 where l is the length and w is the shortest width of the tumor. When PDXs or cell line-derived tumors reached an average volume of 100 mm3, the mice were divided into control and experimental groups (5 mice/group) with a similar range of tumor volumes. The mice were treated by gavage feeding with the indicated doses of BAY-876.

Regarding further characterization we first examined BAY-876 (19) and other candidates in metabolic stability assays using liver microsomes and hepatocytes of different species. Furthermore, the most promising compounds were checked in the Caco‐2 permeability assay. Starting from compounds 2, 3, and 12 with moderate to high metabolic clearance in rat hepatocytes (Figures 1 and 2), installation of a 2‐carboxamide at the quinoline and a CF3 at the pyrazole, like in compound 13, already resulted in compounds with low metabolic clearance in human, dog and mouse liver microsomes as well as dog hepatocytes. However, rat hepatocyte clearance was higher giving only average maximal bioavailability according to the well‐stirred model. Comparing the Caco‐2 data of 2, 3, 12, and 13, the permeability from low (3.5 nm s−1) to high (200 nm s−1) (apical to basolateral) showed the high variation possible within the N‐(1H‐pyrazol‐4‐yl)quinoline compound class. [1]

BAY-876 (19) showed low metabolic in vitro clearance in all tested species except for monkey hepatocytes displaying moderate clearance (Table 6). The Caco‐2 permeability was high and the efflux ratio not considered critical. Compared with the moderate stability of triazole 33 in mouse liver microsomes and in rat hepatocytes, dimethylpyrazole 39 revealed good bioavailability of 88 % in mouse liver microsomes and 75 % in rat hepatocytes. Its stability in human microsomes was slightly lower with 66 %. In comparison with 39 the corresponding isopropylpyrazole 43 showed double the clearance in human microsomes. This metabolic liability was not only observed for 43 but also for other isopropylpyrazole compounds within the project. Due to this finding no isopropylpyrazole was selected for advanced in vivo studies. [1]

Despite the sub‐nanomolar potency of compound 52 its metabolic liability discouraged further in vivo characterization. Cyanopyridine 54 did not only show promising potency and selectivity in the GLUT assays but also very good metabolic stability in liver microsomes and hepatocytes across all tested species. However, the strong efflux ratio in the Caco‐2 assay appeared to be a major drawback of this compound relative to BAY-876 (19). The pyrazine 57 and the pyrimidine 58 showed a two‐ to threefold higher clearance in rat hepatocytes than BAY‐876 (19). [1]

Taking into account GLUT inhibition, metabolic stability, and Caco‐2 performance we selected BAY-876 (19) as candidate for in vivo pharmacokinetic studies that were conducted in two different species (Table 7). In good agreement with the in vitro hepatocyte data BAY‐876 displayed low clearance also in vivo in rat and in dog. The volume of distribution in steady state (V ss) was moderate in both species. Terminal half life was intermediate in rat and long in dog due to the very low clearance. As would be expected from the low blood clearance oral bioavailability was high at the given doses and formulations. Overall, the preliminary data of BAY‐876 demonstrate a favorable in vivo PK profile.

[1]. Identification and Optimization of the First Highly Selective GLUT1 Inhibitor BAY-876. ChemMedChem. 2016 Aug 23.

[2]. Ovarian Cancer Relies on Glucose Transporter 1 to Fuel Glycolysis and Growth: Anti-Tumor Activity of BAY-876. Cancers (Basel). 2018 Dec 31;11(1).

Despite the long-known fact that the facilitative glucose transporter GLUT1 is one of the key players safeguarding the increase in glucose consumption of many tumor entities even under conditions of normal oxygen supply (known as the Warburg effect), only few endeavors have been undertaken to find a GLUT1-selective small-molecule inhibitor. Because other transporters of the GLUT1 family are involved in crucial processes, these transporters should not be addressed by such an inhibitor. A high-throughput screen against a library of ∼3 million compounds was performed to find a small molecule with this challenging potency and selectivity profile. The N-(1H-pyrazol-4-yl)quinoline-4-carboxamides were identified as an excellent starting point for further compound optimization. After extensive structure-activity relationship explorations, single-digit nanomolar inhibitors with a selectivity factor of >100 against GLUT2, GLUT3, and GLUT4 were obtained. The most promising compound, BAY-876 [N4 -[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide], showed good metabolic stability in vitro and high oral bioavailability in vivo. [1]

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Starting from moderately potent and metabolically labile HTS hit 1 carrying a furanyl moiety at position 2 of the quinoline and four methyl groups in total, we quickly improved both, potency and metabolic stability by substituting the furan and taking out the two quinoline methyl groups. Using the quinoline core of compound 3 we found an unsubstituted methylene to be the optimal spacer between the phenyl and pyrazole ring. After successful replacement of one of the pyrazole methyl groups for the metabolically more stable CF3 group (12), further SAR exploration of the quinoline core demonstrated an unsubstituted amide at position 2 and a fluorine at position 7 (19, BAY-876) to be very beneficial regarding GLUT1 potency and selectivity against the other GLUTs. At the pyrazole core a double substitution at positions 3 and 5 was found to be crucial for the excellent potency/selectivity profile especially against GLUT3. Regarding a substituent at the benzyl ring, the para position yielded more promising compounds than the ortho or meta position. With a para‐cyano group and an additional ring nitrogen, compound 54 demonstrated also an interesting GLUT profile. The synthesis of the N‐(1H‐pyrazol‐4‐yl)quinoline‐4‐carboxamides was straightforward as exemplified for BAY-876 (19). In vitro PK data showed that both BAY-876 (19) and 54 were very stable in liver microsomes and hepatocytes, although 54 had a strong efflux ratio of around 16. Preliminary in vivo PK studies of BAY‐876 (19) demonstrated that a good oral bioavailability and long terminal half‐life is attainable making it an excellent chemical probe to further evaluate the hypothesis of cancer treatment with a very selective GLUT1 inhibitor. [1]

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The recent progresses in understanding of cancer glycolytic phenotype have offered new strategies to manage ovarian cancer and other malignancies. However, therapeutic targeting of glycolysis to treat cancer remains unsuccessful due to complex mechanisms of tumor glycolysis and the lack of selective, potent and safe glycolytic inhibitors. Recently, BAY-876 was identified as a new-generation inhibitor of glucose transporter 1 (GLUT1), a GLUT isoform commonly overexpressed but functionally poorly defined in ovarian cancer. Notably, BAY-876 has not been evaluated in any cell or preclinical animal models since its discovery. We herein took advantage of BAY-876 and molecular approaches to study GLUT1 regulation, targetability, and functional relevance to cancer glycolysis. The anti-tumor activity of BAY-876 was evaluated with ovarian cancer cell line- and patient-derived xenograft (PDX) models. Our results show that inhibition of GLUT1 is sufficient to block basal and stress-regulated glycolysis, and anchorage-dependent and independent growth of ovarian cancer cells. BAY-876 dramatically inhibits tumorigenicity of both cell line-derived xenografts and PDXs. These studies provide direct evidence that GLUT1 is causally linked to the glycolytic phenotype in ovarian cancer. BAY-876 is a potent blocker of GLUT1 activity, glycolytic metabolism and ovarian cancer growth, holding promise as a novel glycolysis-targeted anti-cancer agent.
In conclusion, our findings provide direct evidence that GLUT1 is causally linked to the glycolytic phenotype in ovarian cancer. Selective targeting of GLUT1 with the newly developed candidate inhibitor BAY-876 is sufficient to suppress glycolytic metabolism and in vitro and in vivo growth of ovarian cancer. Therefore BAY-876 is an ideal glycolysis-targeted anti-cancer agent.[2]

C24H16F4N6O2

496.4165

496.127

C, 58.07; H, 3.25; F, 15.31; N, 16.93; O, 6.45

1799753-84-6

1799753-84-6(BAY-876)

118191391

White to off-white solid powder

1.5±0.1 g/cm3

632.3±55.0 °C at 760 mmHg

336.2±31.5 °C

0.0±1.9 mmHg at 25°C

1.649

3.89

2

9

5

36

870

0

FC(C1C(=C(C)N(CC2C=CC(C#N)=CC=2)N=1)NC(C1=CC(C(N)=O)=NC2C=C(C=CC1=2)F)=O)(F)F

BKLJDIJJOOQUFG-UHFFFAOYSA-N

InChI=1S/C24H16F4N6O2/c1-12-20(21(24(26,27)28)33-34(12)11-14-4-2-13(10-29)3-5-14)32-23(36)17-9-19(22(30)35)31-18-8-15(25)6-7-16(17)18/h2-9H,11H2,1H3,(H2,30,35)(H,32,36)

4-N-[1-[(4-cyanophenyl)methyl]-5-methyl-3-(trifluoromethyl)pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.04 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 5 mg/mL (10.07 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)