Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH)

Abstract

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.

Figure 1

Representative previously described LDH inhibitors and qHTS “hit” 5.

Figure 2

(A) Crystal structure of inhibitor 27 bound to LDHA in complex with zinc. The inhibitor is shown in sticks with salmon-colored carbons. The protein is shown in ribbon representation and the metal zinc is shown as a magenta sphere. A water molecule (red sphere) and protein residues R168, H192 and T247 (yellow-colored carbons) are coordinated with Zn or form H-bonding interactions with the inhibitor. PDB: 5W8I (B) Inhibitor 33 docked in the binding pocket of LDHA and overlaid with 4 (purple-colored carbons). The benzyl sulfonamide moiety shown as magenta-colored carbons.

Figure 3

(A) Crystal structure of LDHA bound with 33. The inhibitor is shown in sticks and the protein is shown in surface representation. Key protein residues Asp140, Glu191 and lle141 form H-bonding interactions with the sulfonamide moiety as shown by dotted red lines. PDB: 5W8J (B) Overlay of inhibitor 33 (salmon) and 4 shown in purple (Figure 1) bound in the binding pocket of LDHA.

Figure 4

Decrease in glycolytic flux caused by LDH inhibitors in A673 cells. The Glycolysis Stress Test was performed in A673 cells, The Extracellular acidification rate (ECAR) over time; cellular basal ECAR was measured, then compounds (A) GNE140, (B) 61 or (C) 63 were injected in a dose-response manner, after 40 min, subsequent injections of glucose (10 mM), oligomycin (O at 1μg/mL; reaching maximal glycolytic capacity), and 2-deoxyglucose (2-DG at 50 mM; inhibition of glycolysis) were made. (D) Quantification of the maximal ECAR (% from control of maximal ECAR minus ECAR with 2-DG) of increasing concentrations of the five LDH inhibitors.

Figure 5

Incucyte long-term cellular growth data. (A) Cellular confluency of MiaPaCa-2 cells treated with LDHA inhibitors at 2.22 μM over 120 hours. (B) Dose-response relative growth of MiaPaCa-2 cells treated with 63 for 120 hours. Relative growth of (C) MiaPaCa-2 and (D) A673 cells treated with LDHA inhibitors for 120 hours.

Scheme 1. Synthesis of 5, 9 and 12–15 ^a

^aReagents and conditions: (a) NH₂CSNHNH_2, EtOH, reflux, 12 h, 55–75% (b) i. BrCH₂COCO₂Et, EtOH, reflux, 1 h; ii. EtOH, H₂SO₄, reflux, 12 h, 24–35% (c) i. Reversed-phase chromatography separation of regioisomers ii. HCl, AcOH, 120 °C, 1 h.

Scheme 2. Synthesis of precursors (I, II, and III) and analogs 7 and 10 ^a

^aReagents and conditions: (a) BF₃·OEt₂, CH₂Cl₂‒THF, 12 h, 88% (b) N₂H₄, EtOH, reflux, 2 h, 82% (c) EtOH, reflux, 5 h (d) EtOH, AcOH, reflux, 12 h, 77% (e) TFA, CH₂Cl₂ (f) Ac₂O, Pyridine, 100 °C.

Scheme 3. Synthesis of 11, 16–32 and 33–41 ^a

^aReagents and conditions: (a) CH₃CO₂Et, LDA, THF, −78 °C, 2–6 h, 15–90% (b) II, AcOH, EtOH, reflux, 12 h, 12–70% (c) TFA, CH₂Cl₂, 1 h (d) NaH, Dioxane, 1 h, 0 °C, 60–75% (e) III, EtOH, TsOH, MW, 15 min, 46–86% f) LiOH, THF/MeOH/H₂O, 1 h. ^bCommercially available.

Scheme 4. Synthesis of 43–45^a

^aReagents and conditions: (a) ethyl 2-bromothiazole-4-carboxylate, K₂CO₃, 120 °C, 3–4 h, 27–78% (b) bis(pinacolato)diboron, PdCl₂(dppf), AcOK, 1,4-dioxane, 95 °C, overnight 49 % (c) 4-(bromomethyl)benzenesulfonamide, 2N Na₂CO₃ (aq), Pd(Ph₃P)₄, tolune/EtOH (3/1), 80 °C, 2 h 64–95% (d) 1.5N LiOH (aq), THF, 2h (e) 4-hydroxybenzenesulfonamide, K₂CO₃, acetone, 20 h, 92% (f) 1,1-dimethoxy-N,N-dimethylmethanamine, 90 °C, overnight, then N₂H₄, EtOH, 60 °C, 4 h, 27%.

Scheme 5. Synthesis of analog 46 and 47 ^a

^aReagents and conditions: (a) iPr₂NEt, CH₂Cl₂, 0 °C, 1 h, 96% (b) Fe, NH₄Cl, EtOH, reflux, 94% (c) t-BuBrettPhos, t-BuBrettPhos-palladacycle, LHMDS, THF, 80 °C, 14 h, 62–83% (d) K₂CO₃, 125 °C, 14 h, 60% e) TFA, CH₂Cl₂, 100 °C, MW, 15 min.

Scheme 6. Synthesis of analogs 48–50 ^a

^aReagents and conditions: (a) K₂CO₃, DMSO, 120 °C, 24 h, 25% (b) HATU, iPr₂NEt, DMF, 60 °C, 4 h (c) NaBH₃CN, AcOH, MeOH, (d) TFA, CH₂Cl₂, 1 h (e) K₂CO₃, DMSO, 125 °C, 3 h, 52%.

Scheme 7. Synthesis of analogs 51–57 ^a

^aReagents and conditions: (a) MeCN, LDA, −78 °C, 4 h, 78–97% (b) Hantzsch ester, L-Pro, EtOH, 60 °C, 0.5 h, 86–98% (c) III, TsOH, EtOH, 150 °C, MW, 15 min, 60–77 % (d) TsOH, NaNO₂, KI, MeCN, 12 h, 36–47% (e) 52: CuCN, DMSO, 160 °C, 0.5 h, 78% (f) 51, 53–55: requisite boronic acid, SiliCat-DPP-Pd, Na₂CO₃, DME, MW, 130 °C, 0.5h (g) 57: (1,10-Phenanthroline)(trifluoromethyl)copper(I), DMF, 55 °C, 1 h, 97% (i) LiOH, THF-MeOH, 1 h.

Scheme 8. Syntheses of analogs 58–63 ^a

^aReagents and conditions: (a) 1,2,3-Benzotriazole, SOCl₂, CH₂Cl₂, 4 h, 91–100% (b) MgBr₂·OEt, iPr₂NEt, CH₂Cl₂, 12 h, 60–69% (c) 4-(bromomethyl)benzenesulfonamide, Cs₂CO₃, DMSO, 1 h, 55–83% (d) TMS-Cl, NCS, CH₂Cl₂, 0 °C-rt, 3 h, 73–94 % (e) 4-hydroxybenzenesulfonamide, K₂CO₃, DMSO, 50 °C, 1 h, 31–49% (f) TsOH, EtOH, reflux, 12 h, 77–83% (mixture) (g) LiOH, THF-MeOH, 1 h.

Scheme 9. Syntheses of analogs 64–68 ^a

^aReagents and conditions: (a) NH₃, EtOH, 60 °C, 18 h, 90% (b) TFAA, iPr₂NEt, CH₂Cl₂ (c) NaN₃, NH₄Cl, DMF, 125 °C, 2 h (d) LiAlH₄, THF, 1 h (e) MnO₂, CHCl₃, 2 h (f) TMS-CF₃, TBAF, THF, 4 h.