LDHA (IC50 = 3 nM); LDHB (IC50 = 5 nM)
At a dose of 5 μM, (R)-GNE-140 demonstrated cell proliferation in 37 out of 347 pancreatic lineages that were evaluated. With an IC50 of 0.8 μM, (R)-GNE-140 inhibits two chondroma (bone) hexane lines expressing IDH1 dimming [1].
(R)-GNE-140 proved to possess the optimal combination of strong enzymatic and MiaPaca2 cellular potency, moderate permeability, and reduced plasma protein binding compared to other potent compounds in this series.

At a dose of 5 μM, (R)-GNE-140 demonstrated cell proliferation in 37 out of 347 pancreatic lineages that were evaluated. With an IC50 of 0.8 μM, (R)-GNE-140 inhibits two chondroma (bone) hexane lines expressing IDH1 dimming [1].
(R)-GNE-140 proved to possess the optimal combination of strong enzymatic and MiaPaca2 cellular potency, moderate permeability, and reduced plasma protein binding compared to other potent compounds in this series.

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R-GNE-140 demonstrated potent cellular activity in the MiaPaca2 pancreatic cancer cell line, inhibiting lactate production with an IC₅₀ of 0.67 µM. [1]
In a broad panel of 347 cancer cell lines, R-GNE-140 inhibited proliferation in 37 cell lines (11%) at a potency cut-off of 5 µM. Two IDH1-mutant chondrosarcoma cell lines were particularly sensitive, with an IC₅₀ of 0.8 µM. [1]

In mice, (R)-GNE-140 (5 mg/kg) exhibits a high bioavailability. In the prior gun simulation, (R)-GNE-140 shown increased exposure at 50 to 200 mg/kg.

In vitro drug treatment experiments.[1]
All cell lines were obtained from our in-house tissue culture cell bank (original source was ATCC). Lines were authenticated by short tandem repeat (STR) and genotyped upon re-expansion. Cells were maintained in RPMI 1640 media supplemented with 10% FBS. Cells were plated using optimal seeding densities in 384-well plates using RPMI, 5% FBS, 100 ug/ml penicillin, 100 units/ml streptomycin. Optimal seeding densities were established for each cell line in order to reach 75-80% confluence at the end of the assay. The following day, cells were treated with compound 29 using a 6 pt dose titration scheme. After 72 hours, cell viability was assessed using the CellTiter-Glo® Luminescence Cell Viability assay. Absolute inhibitory concentration (IC) values were calculated using four-parameter logistic curve fitting.

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The enzymatic inhibitory activity of compounds against LDHA and LDHB was determined using a biochemical assay. Recombinant human LDHA or LDHB protein was incubated with the test compound, along with the substrates NADH and pyruvate (for the forward reaction). The reaction progress was monitored by measuring the decrease in absorbance at 340 nm, which corresponds to the oxidation of NADH to NAD⁺. IC₅₀ values were calculated from dose-response curves. The assay was performed in at least two separate runs, and values are reported as geometric means. [1]

Treatment with GNE-140 phenocopies LDHA/B double genetic disruption in both the LS174T and B16 cell lines[2]
Recently, Boudreau et al. demonstrated the ability of GNE-140, a specific LDHA and LDHB inhibitor, to cause growth arrest in highly glycolytic pancreatic cancer cell lines such as MiaPaca2. Hence, we were curious to see whether this inhibitor could reactivate OXPHOS without delay and maintain the viability and growth of the WT LS174T and B16 cell lines. We treated WT and LDHA/B-DKO cells with different concentrations of GNE-140 and showed that a concentration of 10 μm, known to collapse LDHA and B activity, reduced the growth of the WT but not of the two LDHA/B-DKO cell lines reported here. This long-term experiment (9 to 12 days) proved the lack of off-target effects of this compound at the concentration used. Furthermore, we analyzed the metabolic consequences of the short-term GNE-140 treatment of the WT cells by Seahorse bioanalyzer. As shown in Fig. 8, E–H, 1-h treatment with 10 μm GNE-140 was sufficient to phenocopy the effect of the LDHA/B-DKO cells in terms of suppression of glycolysis and reactivation of OXPHOS. Hence, the growth phenotype of DLHA/B-DKO cells does not result from long-term growth selection during the two steps of genetic disruption. This finding, based on genetics and specific pharmacological disruption of LDHA and LDHB, firmly attests that, under normoxia, the Warburg effect is dispensable for in vitro tumor growth.

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Cellular activity was assessed using a lactate production assay in the MiaPaca2 cell line. Cells were cultured and treated with serial dilutions of the test compound. After an incubation period, lactate levels in the culture supernatant were quantified, typically using a colorimetric or enzymatic detection method. The concentration of compound that inhibited lactate production by 50% (IC₅₀) was determined. The assay was performed in at least two separate runs, and values are reported as geometric means. [1]
Anti-proliferative activity was evaluated across a panel of cancer cell lines. Cells were treated with R-GNE-140 (e.g., at 5 µM), and cell viability/proliferation was measured after a defined period (e.g., 72-96 hours) using methods like CellTiter-Glo. IC₅₀ values were calculated for sensitive lines. [1]

Mouse Pharmacokinetics Study [1]
The pharmacokinetics of compound 29 ((R)-GNE-140) was evaluated following a single intravenous bolus (IV) dose of 1.0 mg/kg and oral administration (PO) of solutiomorphous suspension at a dose of 5 mg/kg in female CD-1 mice (N=3). The vehicle used for IV administration was 10/50/40 EtOH/PEG400/50mM citrate pH3 (v/v, 10/50/40), and for PO, 0.5% methycellulose:0.2% Tween in water (MCT). Blood samples for the IV dose group were collected at 0.033, 0.25, 1, 2, 4, 6 hours post dose. Blood samples for PO dose groups were collected at 0.25, 0.5, 1, 2, 4, and 6 hours post dose. For the high dose oral PK study at 50, 100, and 200 mg/kg, blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, and 8 hours post dose. Blood samples were centrifuged within 29 minutes of collection, and plasma was harvested. Plasma samples were stored at approximately –70°C until the analysis of the compound concentration by a liquid chromatography/tandem mass spectrometry (LCMS/MS) method. PK parameters were determined by non-compartmental methods using WinNonlin

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In vivo pharmacokinetic studies were conducted in mice. R-GNE-140 was administered via intravenous (IV) injection at a dose of 1 mg/kg and via oral gavage (PO) at doses of 5, 50, 100, and 200 mg/kg. The specific formulation or vehicle used for dosing is not described in the manuscript. Blood samples were collected at various time points post-dose for plasma concentration analysis. [1]

(R)-GNE-140 is stable in liver microsomes, with predicted hepatic clearance (Clp) of 3.3, 8.1, and 14 mL/min/kg based on human, rat, and mouse microsomes, respectively. We performed in vivo pharmacokinetic studies on (R)-GNE-140 in mice and were pleased to find that this compound has a low Clp, consistent with the predicted hepatic clearance, and high bioavailability at an oral dose of 5 mg/kg (Table 5; concentration-time curves are shown in Figure S2). At higher oral doses (50 to 200 mg/kg), (R)-GNE-140 showed even higher exposures (Table 5 and Figure S3), confirming that (R)-GNE-140 can be used as a tool compound for in vivo pharmacodynamic studies in mice. [1] In a mouse pharmacokinetic study, after intravenous injection of 1 mg/kg, the AUC of (R)-GNE-140 was 1.5 µM·h, the Cₘₐₓ was 2.5 µM, and the plasma clearance (Cₗ) was consistent with the predicted liver clearance. [1]
After oral administration, the exposure was dose-dependent: 5 mg/kg oral: AUC = 5.1 µM·h, Cₘₐₓ = 1.7 µM; 50 mg/kg oral: AUC = 125 µM·h, Cₘₐₓ = 60.7 µM; 100 mg/kg oral: AUC = 250 µM·h, Cₘₐₓ = 224 µM; 200 mg/kg oral: AUC = 403 µM·h, Cₘₐₓ = 440 µM. [1]
R-GNE-140 showed high oral bioavailability in mice (the exposure was significant after oral administration, but the specific F% was not calculated in the text). [1]
Its predicted hepatic clearance in microsomes was low: human (3.3 mL/min/kg), rat (8.1 mL/min/kg), mouse (14 mL/min/kg). [1]
The compound showed moderate permeability in MDCK cell permeability assays. [1]
The plasma protein binding rate in mice was 99.1%. [1]
LogD₇.₄ value was 0.6. [1]

[1]. Cell Active Hydroxylactam Inhibitors of Human Lactate Dehydrogenase with Oral Bioavailability in Mice. ACS Med Chem Lett. 2016 Aug 26;7(10):896-901.

series of trisubstituted hydroxylactams have been identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Several lactate IC50 < 10 μM inhibitors were discovered in the MiaPaca2 cell line using structure-based design and physical property optimization. After optimization of this series of compounds, compound 29 was obtained, which is a potent cellularly active molecule (MiaPaca2 IC50 = 0.67 μM) and also showed good exposure after oral administration to mice. [1]

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R-GNE-140 (compound 29) is a trisubstituted hydroxylactam discovered through structure-based design. It has a weaker acidic hydroxylactam core (pKₐ ~4.1) compared to the previous diketone/dihydropyranone skeleton, which was designed to improve its physicochemical properties. [1]
It is a pan-LDH inhibitor with low nanomolar inhibitory activity against both LDHA and LDHB isoforms. [1]
It is the first reported LDHA inhibitor with submicromolar cellular activity (MiaPaca2 IC₅₀ = 0.67 µM) and good pharmacokinetic properties (low clearance in mice and high oral exposure), making it a suitable compound for in vivo research. [1]
Its mechanism of action is to inhibit the glycolytic enzyme LDHA, thereby blocking the conversion of pyruvate to lactate, which is a key step in the Wahlberg effect observed in many cancers. [1]
It exhibits selective antiproliferative activity in some cancer cell lines, especially showing significant sensitivity in an IDH1 mutant chondrosarcoma model, suggesting its potential application value in the treatment of metabolically vulnerable tumors. [1]

C25H23CLN2O3S2

499.0447

498.083

C, 60.17; H, 4.65; Cl, 7.10; N, 5.61; O, 9.62; S, 12.85

2003234-63-5

GNE-140 racemate;1802977-61-2;(S)-GNE-140;2003234-64-6; 1809794-70-4 (racemate)

121225870

Light yellow to yellow solid

4.8

2

6

5

33

739

1

C1COCCN1C2=CC=C(C=C2)[C@]3(CC(=C(C(=O)N3)SC4=CC=CC=C4Cl)O)C5=CSC=C5

SUFXXEIVBZJOAP-RUZDIDTESA-N

InChI=1S/C25H23ClN2O3S2/c26-20-3-1-2-4-22(20)33-23-21(29)15-25(27-24(23)30,18-9-14-32-16-18)17-5-7-19(8-6-17)28-10-12-31-13-11-28/h1-9,14,16,29H,10-13,15H2,(H,27,30)/t25-/m1/s1

(R)-3-((2-Chlorophenyl)thio)-4-hydroxy-6-(4-morpholinophenyl)-6-(thiophen-3-yl)-5,6-dihydropyridin-2(1H)-one

R-GNE-140; (R)-GNE-140; (R)-GNE-140; 2003234-63-5; (R)-3-((2-chlorophenyl)thio)-4-hydroxy-6-(4-morpholinophenyl)-6-(thiophen-3-yl)-5,6-dihydropyridin-2(1H)-one; (2R)-5-(2-chlorophenyl)sulfanyl-4-hydroxy-2-(4-morpholin-4-ylphenyl)-2-thiophen-3-yl-1,3-dihydropyridin-6-one; R-GNE-140; (2~{r})-5-(2-Chlorophenyl)sulfanyl-2-(4-Morpholin-4-Ylphenyl)-4-Oxidanyl-2-Thiophen-3-Yl-1,3-Dihydropyridin-6-One; inhibitor GNE-140; GNE 140; GNE140.

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)

DMSO : ≥ 50 mg/mL (~100.19 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.01 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.01 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.01 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 4: 2.5 mg/mL (5.01 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 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.)