Pyrazole-Based Lactate Dehydrogenase Inhibitors with Optimized Cell Activity and Pharmacokinetic Properties

Abstract

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug-target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.

Figure 1.

Representative previously described LDH inhibitors and comparison with new leads 43 = NCATS-SM1440 and 52 = NCATS-SM1441. ^aNamed as NCI-006 in refs and . ^bNamed as NCI-737 in ref .

Figure 2.

Crystal structure of LDHA bound to inhibitor 23 (A, pdb code 6Q0D) and 52 (B, pdb code 6Q13). LDHA is shown in ribbon (blue) and key residues in the active site are shown in green. Small-molecule inhibitors are shown in sticks with salmon- and magenta-colored carbons.

Figure 3.

LDH inhibitor-dependent suppression of glycolytic flux in A673 cells. The GST was performed in A673 cells to measure the ECAR over time; cellular basal ECAR was measured and then compound 43 (panel A) or 52 (panel B) was injected at increasing concentrations. After 48 min, subsequent injections of glucose (10 mM; glycolysis), oligomycin (1 μg/mL) (O; reaching maximal glycolytic capacity), and 2-deoxyglucose (50 mM) (2-DG; inhibition of glycolysis) were made. (C) Quantification of the glycolysis (ECAR after glucose injection minus basal ECAR) and (D) quantification of the glycolytic capacity of the LDHA inhibitors, 43 and 52, in a dose–response manner in comparison with compounds 1 and 23. Data represent the mean ± standard error of the mean, n = 4–6 per group. All LDH inhibitors completely suppressed both basal and maximal glycolysis between 1 and 3 μM, with 43 and 52 being the most potent and 23 the least.

Figure 4.

In vivo target engagement and tumor LDH activity upon administration of compounds in mice bearing A673 flank xenograft tumors. Mice received a single IV injection of LDHA inhibitors 14, 43, 47, 52, 56, 69, and 71 at 50 mg/kg. At the time of sacrifice, the samples of tumor and plasma were collected and flash-frozen in liquid nitrogen. Compound levels in plasma and tumor were determined by LC–MS/MS, and LDHA activity was measured in tumor lysates.

Figure 5.

SAR summary and essential structural moieties contributing to the cellular potency, binding affinity, and PK properties of analogue 52 (NCATS-SM1441) as an example.

Scheme 1.

Syntheses of Intermediates VIa–r and Analogues 1–9^{a a}Reagents and conditions: (a) SOCl₂, CH₂Cl₂, 4 h, and 91–100%; (b) MgBr₂·OEt₂, iPr₂NEt, CH₂Cl₂, 12 h, and 60–69%; (c) Cs₂CO₃, DMSO, 1 h, and 55–83%; (d) (i) pyrrolidine (0.5 equiv), TsOH (0.5 equiv), EtOH, reflux, and 1–2 h and (ii) ethyl 2-hydrazinylthiazole-4-carboxylate·HBr and reflux overnight; and (e) LiOH, THF/MeOH/H₂O, and 1 h.

Scheme 2.

Syntheses of Analogues 10–32 and 36–89^{a a}Reagents and conditions: (a) [P(t-Bu)₃]Pd(crotyl)Cl, DABCO, dioxane, RT, and 1–12 h; (b) [DTBN_pP]Pd(crotyl)Cl, DABCO, dioxane, 60 °C, and 12 h; (c) SPhosPd(crotyl)Cl, K₃PO₄, dioxane/H₂O, 100 °C, and 0.5 h; (d) LiOH, THF/MeOH/H₂O, and 1 h; and (e) CsF, THF–EtOH, RT, and 2 h.

Scheme 3.

Syntheses of Analogues 33–35^{a a}Reagents and conditions: (a) Dess–Martin periodinane, DCM, RT, and 2 h; (b) NaN₃, CF₃SO₃H, ACN, RT, and 12 h; (c) (CH₃)₃Sn–OH, dichloroethane, MW, 110 °C, and 1 h; (d) Deoxo-Fluor, DCM, RT, and 12 h; (e) P(t-Bu)_3.HBF₄, ([PdCl(allyl)]₂, dioxane, 80 °C, and 4 h; and (f) LiOH, THF/MeOH/H₂O, and 1 h.