Evaluation of substituted N,N'-diarylsulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase

Abstract

The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel antiproliferation strategy. In this manuscript, we detail the discovery of a series of substituted N,N'-diarylsulfonamides as activators of PKM2. The synthesis of numerous analogues and the evaluation of structure-activity relationships are presented as well as assessments of mechanism and selectivity. Several agents are found that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55 (AC(50) = 43 nM, maximum response = 84%; solubility = 7.3 microg/mL), 56 (AC(50) = 99 nM, maximum response = 84%; solubility = 5.7 microg/mL), and 58 (AC(50) = 38 nM, maximum response = 82%; solubility = 51.2 microg/mL). The small molecules described here represent first-in-class activators of PKM2.

Figure 1

A. The fate of glucose metabolism to pyruvate in anaerobic and aerobic environments in differentiated, healthy tissue. B. The fate of glucose metabolism to pyruvate in rapidly proliferating tissue and tumors. PKM2 is activated by FBP and inhibited by tyrosine-phosphorylated proteins derived from growth factor signaling.

Figure 2

A. Summary of actives from PKM2 qHTS of 276,309 small molecules. B. Concentration response curve (CRC) representation of the full qHTS dataset. C. CRC’s for actives from the primary screen associated with curve classes 1a, 1b and 2a showing potency and maximum response. D. Chemical structures of the two lead activators of PKM2 substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone NCGC00031955 (1) and substituted N,N′-diarylsulfonamide NCGC00030335 (2).

Figure 3

A. Initial velocity as a function of PEP and ADP concentration in the presence (open triangles) or absence (filled circles) of 2 (10 μM). B. Initial velocity as a function PEP and ADP concentration in the presence (open circles) or absence (filled circles) of FBP (10 μM). V_o, initial rate in pmol/min as determined in the PK-LDH coupled assay (kinetic assays were carried out at approximately 22 °C with 10 nM PKM2 using [KCl] = 200 mM, [MgCl₂] = 15 mM, and with either [ADP] or [PEP] = 4.0 mM; see supporting information).

Figure 4

Selectivity assessment for 2 versus PKM2 (open circles), PKM1 (filled squares), PKL (open squares), and PKR (filled circles).

Scheme 1

Conditions and reagents: (i) TEA, CH₂Cl₂, 0 °C; (ii) TFA, CH₂Cl₂, 0 °C; (iii) TEA, CH₂Cl₂, 0 °C.

Scheme 2

*Conditions and reagents: (i) K₂CO₃, DMF; (ii) mCPBA, CH₂Cl₂, 0 °C; (iii) TFA, CH₂Cl₂, 0 °C; (iv) TEA, CH₂Cl₂, 0 °C;

Scheme 3

Conditions and reagents: (i) TEA, CH₂Cl₂, 0 °C; (ii) LHMDS, THF, −78 °C - then Ar₂SO₂Cl.