Design, Synthesis, and Investigation of Novel Nitric Oxide (NO)-Releasing Prodrugs as Drug Candidates for the Treatment of Ischemic Disorders: Insights into NO-Releasing Prodrug Biotransformation and Hemoglobin–NO Biochemistry

Abstract

We have developed novel NO-releasing prodrugs of efaproxiral (RSR13) for their potential therapeutic applications in a variety of diseases with underlying ischemia. RSR13 is an allosteric effector of hemoglobin (Hb) that decreases the protein’s affinity for oxygen, thereby increasing tissue oxygenation. NO in the form of ester prodrugs has been found useful in managing several cardiovascular diseases by increasing blood flow and oxygenation in ischemic tissues, due to its vasodilatory property. We synthesized three NO-donor ester derivatives of RSR13 (DD-1, DD-2 and DD-3) by attaching the NO-releasing moieties, nitrooxyethyl, nitrooxypropyl, and 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate, respectively, to the carboxylate of RSR13. In-vitro studies demonstrated that the compounds released NO in a time-dependent manner when incubated with L-cysteine (1.8 – 9.3%) or human serum (2.3 – 52.5%); and also reduced Hb affinity for oxygen in whole blood (ΔP50 of 4.9 – 21.7 mmHg vs 25.4 – 32.1 mmHg for RSR13). Crystallographic studies showed RSR13, the hydrolysis product of the reaction between DD-1 and deoxygenated Hb, bound to the central water cavity of Hb. Also, the hydrolysis product, NO was observed exclusively bound to the two α-hemes; the first such HbNO structure to be reported, capturing the previously proposed physiological bis-ligated nitrosylHb species. Finally, nitrate was observed bound to βHis97. UPLC-MS analysis of the compounds incubated with matrices used for the various studies demonstrated the presence of the predicted reaction products. Our findings, beyond the potential therapeutic application, provide valuable insights into the biotransformation of NO-releasing prodrugs and their mechanism of action, as well as hemoglobin-NO biochemistry at the molecular level.

Hemoglobin: Structure, Function and Allostery

“…Crystallographic studies with the prodrugs showed RSR-13, the hydrolysis product of the prodrugs bound to the central water cavity of deoxygenated Hb (as described above) that explained these compounds ability to decrease Hb affinity for oxygen (Xu et al 2015). Moreover, the released NO, was observed exclusively bound to the two α hemes, which was suggested to be due to RSR-13 decreasing the protein's affinity for the ligand at the β hemes (Xu et al 2015).…”

“…Safo's group recently developed novel nitric oxide (NO)-releasing prodrugs of RSR-13 derivatives by attaching the NO-releasing moieties nitrooxyethyl, nitrooxypropyl, and 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate, respectively, to the carboxylate of RSR-13 (Xu et al 2015). Crystallographic studies with the prodrugs showed RSR-13, the hydrolysis product of the prodrugs bound to the central water cavity of deoxygenated Hb (as described above) that explained these compounds ability to decrease Hb affinity for oxygen (Xu et al 2015). Moreover, the released NO, was observed exclusively bound to the two α hemes, which was suggested to be due to RSR-13 decreasing the protein's affinity for the ligand at the β hemes (Xu et al 2015).…”

π-Hole Interactions Involving Nitro Compounds: Directionality of Nitrate Esters

“…The Brookhaven protein databank (PDB) was also consulted for ligands bearing a nitrate ester. While merely four such structures were found (3k2f, 3ni5, 4fr8, and 5e29), in two of these the nitrate ester’s N atom is in close contact with an electron rich atom. In 4fr8 (Figure a) the tris-nitrate ligand TNG-601 is buried within an aromatic-rich pocket and one of the nitrate N atoms is in close contact (2.89 Å) with a S atom from the disordered cysteine 302.…”

Design, Synthesis, and Investigation of Novel Nitric Oxide (NO)-Releasing Aromatic Aldehydes as Drug Candidates for the Treatment of Sickle Cell Disease

“…It is notable that these compounds are stable in DMSO at room temperature for extended periods of time, suggesting that the NO release is facilitated in the whole blood. The mechanism of biotransformation of the nitrate esters to release NO has been proposed to involve both an enzymatic action and a chemical hydrolysis [ 64 , 65 , 66 ].…”