Hemoglobin (Hb) modifiers that stereospecifically inhibit sickle hemoglobin polymer formation and/or allosterically increase Hb affinity for oxygen have been shown to prevent the primary pathophysiology of sickle cell disease (SCD), specifically, Hb polymerization and red blood cell sickling. Several such compounds are currently being clinically studied for the treatment of SCD. Based on the previously reported non-covalent Hb binding characteristics of substituted aryloxyalkanoic acids that exhibited antisickling properties, we designed, synthesized and evaluated 18 new compounds (KAUS II series) for enhanced antisickling activities. Surprisingly, select test compounds showed no antisickling effects or promoted erythrocyte sickling. Additionally, the compounds showed no significant effect on Hb oxygen affinity (or in some cases, even decreased the affinity for oxygen). The X-ray structure of deoxygenated Hb in complex with a prototype compound, KAUS-23, revealed that the effector bound in the central water cavity of the protein, providing atomic level explanations for the observed functional and biological activities. Although the structural modification did not lead to the anticipated biological effects, the findings provide important direction for designing candidate antisickling agents, as well as a framework for novel Hb allosteric effectors that conversely, decrease the protein affinity for oxygen for potential therapeutic use for hypoxic- and/or ischemic-related diseases.

Curcumin and trans-cinnamaldehyde are acrolein-based Michael acceptor compounds that are commonly found in domestic condiments, and known to cause cancer cell death via redox mechanisms. Based on the structural features of these compounds we designed and synthesized several 2-cinnamamido-N-substituted-cinnamamide (bis-cinnamamide) compounds. One of the derivatives, (Z)-2-[(E)-cinnamamido]-3-phenyl-N-propylacrylamide 1512 showed a moderate antiproliferative potency (HCT-116 cell line inhibition of 32.0 µM), good selectivity profile (no inhibition of normal cell lines), and proven cellular activities leading to apoptosis. SAR studies led to more than 10-fold increase in activity. Our most promising compound, [(Z)-3-(1H-indol-3-yl)-N-propyl-2-[(E)-3-(thien-2-yl)propenamido)propenamide] 4112 killed colon cancer cells at IC50 = 0.89 µM (Caco-2), 2.85 µM (HCT-116) and 1.65 µM (HT-29), while exhibiting much weaker potency on C-166 and BHK normal cell lines (IC50 = 71 µM and 77.6 µM, respectively). Cellular studies towards identifying the compounds mechanism of cytotoxic activities revealed that apoptotic induction occurs in part as a result of oxidative stress. Importantly, the compounds showed inhibition of cancer stem cells that are critical for maintaining the potential for self-renewal and stemness. The results presented here show discovery of covalently-acting Michael addition compounds that potently kill cancer cells by a defined mechanism, with minimal effect on normal noncancerous cell.

Curcumin and trans- cinnamaldehyde are acrolein-based Michael acceptor compounds that are commonly found in domestic condiments, and known to cause cancer cell death via redox mechanisms. Based on the structural features of these compounds we designed and synthesized several 2-cinnamamido-N-substituted-cinnamamide (bis-cinnamamide) compounds. One of the derivatives, ( Z )-2-[( E )-cinnamamido]-3-phenyl-N-propylacrylamide ( 1512) showed a moderate antiproliferative potency (HT116 cell line inhibition of 32.0 µM ± 2.6) with proven cellular activities leading to apoptosis. Importantly, 1512 exhibited good selectivity toxicity on cancer cells over noncancerous cells (IC ₅₀ of C-166 cell lines >100 µM), and low cancer cell resistance at 100 µM dose (growth rate 10.1±1.1%). We subsequently carried out structure activity relationship studies with 1512 . Derivatives with electron rich moiety at the aryl ring of the 2-aminocinnamaide moiety exhibited strong antiproliferative action while electron withdrawing groups caused loss of activity. Our most promising compound, 4112 [( Z )-3-(1 H -indol-3-yl)- N -propyl-2-[( E )-3-(thien-2-yl)propenamido)propenamide] killed cancer cells at IC ₅₀ = 0.89 ± 0.04 µM (Caco-2), 2.85 ± 1.5 (HCT-116) and 1.65 ± 0.07 (HT-29), while exhibiting much weaker potency on C-166 and BHK normal cell lines (IC ₅₀ = 71 ± 5.12 and 77.6 ± 6.2 µM, respectively). Cellular studies towards identifying the compounds mechanism of cytotoxic activities revealed that apoptotic induction occurs in part due to oxidative stress. Importantly, the compounds showed inhibition of cancer stem cells that are critical for maintaining the potential for self-renewal and stemness. The results presented here show discovery of Michael addition compounds that potently kill cancer cells by a defined mechanism, with minimal effect on normal noncancerous cell.