Evaluation of thieno[3,2-b]pyrrole[3,2-d]pyridazinones as activators of the tumor cell specific M2 isoform of pyruvate kinase

Abstract

Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure-activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.

Figure 1

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 2

A. Initial velocity of PKM2 as a function of PEP and ADP concentration in the presence (open squares) or absence (filled circles) of NCGC00031955 (1) (10 μM). B. Initial velocity as a function of 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 [KCl] = 200 mM, [MgCl₂] = 15 mM, and with either [ADP] or [PEP] = 4.0 mM; see supporting information).

Figure 3

Selectivity assessment for NCGC00031955 (1) versus PKM2 (open circles), PKM1 (filled squares), PKL (open squares), and PKR (filled circles).

Scheme 1

Conditions and reagents: (i) Na, EtOH, 0 °C; (ii) o-Xylene, reflux; (iii) POCI₃, DMF, 60 °C; (iv) R₂I, K₂C0₃, DMF, r.t.; (v) 2-Ethoxyethanol, hydrazine, reflux; (vi) Benzyl bromide or alkyl bromide, KOtert-Bu, DMF, r.t.; (vii) iodobenzene, Cul, trans-cyclohexane-1,2-diamine, 1,4-dioxane, reflux.

Scheme 2

Conditions and reagents: (i) Na, MeOH, Cul, 1,4-dioxane, reflux; (ii) Acetamide, Cul, trans-cyclohexane-1,2-diamine, dioxane, reflux; (iii) CuCN, DMF, 140 °C; (iv) CO (1 atm), Pd(OAc)₂,1,3-bis(diphenylphosphino)propane, Et₃N, MeOH, DMSO, 65 °C. (v) vinyl or isopropenyboronic acid pinacol ester, Pd(PPh₃)₂CI₂,1M Na₂CO₃/CH₃CN, 120 °C, microwave; (vi) Pd/C, H₂ (1 atm), MeOH, r.t.; (vii) ⁱPrMgBr, tetramethylethylenediamine, THF, 15 °C, 20 min, then starting material, r.t., 25 min; (viii) B(OMe)₃, 0 °C, then 0.1 N HCl; (ix) CH₃CHO, 0 °C; (x) procedure ix followed by IBX, DMSO, r.t.; (xi) NaSMe, CuBr, DMF, 140 °C; (xii) mCPBA (1.5 eq.), CH₂CI₂, r.t.;

Scheme 3

Conditions and reagents: (i) POCI₃, DMF, 60 °C; (ii) Cu(NO₃)₂, Ac₂O, 0 °C to r.t.; (iii) Mel, K₂CO₃, DMF; (iv) hydrazine, EtOH, r.t.; (v) 2-fluororobenzyl bromide, K₂CO₃, DMF, r.t..

Scheme 4

Conditions and reagents: (i) POCI₃, DMF, CICH₂CH₂CI, reflux; (ii) NaBH₄, MeOH.

Scheme 5

Conditions and reagents: (i) MeMgCI, THF, −78 °C; (ii) IBX, DMSO, r.t.. (iii) steps iv through vi (scheme 1).

Scheme 6

Conditions and reagents: (i) Cu(NO₃)₂, Ac₂O, 0 °C to r.t.; (ii) Mel, K₂CO₃, DMF; (iii) SnCI₂, HCl, EtOH/H₂O, 35 °C; (iv) NH₂CHO, ammonium formate, 120°C; (v) 2-fluorobenzyl bromide, K₂CO₃, EtOH, reflux.