Formation of Methemoglobin by3- (Substituted) Phenylsydnones: an Evidence for their Nitric Oxide Donor Property

INTRODUCTION

Nitric oxide (NO), the simplest highly reactive tiny free radical biosynthesised in the body from L-arginine by nitric oxide synthases (NOS), is found to play an important role virtually in every biological activity ranging from a critical endogenous regulator of blood flow and thrombosis, to a principal neurotransmitter mediating penile erection, to a major pathophysiological mediator of inflammation and host defence.^1,2 Thisversatile nature of NO spurred hectic research activities in biosciences over last three decades making NO an important therapeutic target. The diverse and important physiological roles of NO also suggested that, modulation of its levels in physiological milieu could be a useful strategy for the treatment of some diseases. This paved the way for the development of NO modulators such as NO donors and NOS inhibitors.

NO is highly unstableand is rapidly converted in vivo and in vitro to nitrite and nitrate.³ Nitrite is a potent methemoglobin (MHb) forming agent.⁴ MHb is the form of haemoglobin in which the heme iron is in oxidised Fe³⁺ state (in contrast to Fe²⁺ in haemoglobin) and therefore, cannot bind and carry molecular oxygen.⁵ Thus, the formation of MHb by any NO donor could be an evidence for its NO donor activity. Measurement of the formed MHb can be correlated quantitatively with the release of NO from an NO donor.

Sydnones are the mesoionic heteroaromatic compounds having peculiar chemical structure and exciting physicochemical and biological properties.^6,7 They are structurally very much similar to sydnonimines, a known class of NO donors.⁸ Both sydnones and sydnonimines contain a proactive NO releasing N-nitroso moiety disguised in the ring. Different substituted sydnones for their in vitro NO donor activities were studied spectrophotometrically by measuring nitrite formed with Griess reagent and found them to be weak and slow donors of NO in presence of thiol.⁹ This work was undertaken to further substantiate the claimed NO donor activities of sydnones by their potential to form MHb.

MATERIALS AND METHODS

All the chemicals used were of analytical reagent grade and used as received. The 3-(phenyl/ substituted phenyl) sydnonesand their alkaline hydrolytic products N-nitroso phenyl/substituted phenyl glycines were synthesised according to the protocol reported.10 Human blood sample was collected from Bagalkot blood bank, Bagalkote. Centrifugation was done in R-24 research centrifuge (Remi, India). Absorbances were taken on a double beam UV-Vis spectrophotometer (UV-240, Shimadzu, Japan).

Preparation of Haemolysate¹¹

Human blood sample was centrifuged at 2000 rpm for 15 min to remove the plasma and buffy coat of white blood cells. Erythrocytes thus obtained were suspended in phosphate buffer saline for 5 min and centrifuged again to remove the supernatant liquid containing white blood cells. The same procedure was repeated twice. The packed cells thus obtained were suspended in 20 volume of 0.7% sodium chloride solution for 30 min at room temperature and centrifuged at 2000 rpm for 15 min to remove membrane and cell debris. The resulting solution (hemolysate, brilliant red color) was diluted with 0.7% sodium chloride solution to yield a final concentration of oxyhemoglobin suitable for spectroscopic analysis.

Determination of MHb

The test compounds at 8.0µM concentration in DMF were incubated with 2mL hemolysate (having absorbance 0.5 at 577nm) at 37o C of for 15 min. The absorbance of formed MHb was measured against same reaction mixture without test compounds at 631 nm.

RESULTS AND DISCUSSION

MHb formation by 3-(phenyl/substituted phenyl) sydnones was measured as per the reported method11 where in, the formed MHb was determined spectrophotometrically at 631 nm. The chemical structures of 3-(phenyl/ substituted phenyl) sydnones and their alkaline hydrolytic products N-nitroso (phenyl/ substituted phenyl) glycines are shown in Fig. 1 and2 . It is hypothesized that; sydnones might release NO via their alkaline hydrolytic products N-nitrosophenylglycines in slightly alkaline pH of plasma. Since many nitrosamines are known to be NO donors,12 therefore, N-nitrosophenylglycines were also screened for MHb formation activity. The human hemolysate has the λmax at 577 nm and an absorbance of 0.5 for 2 mLwhile, λmax of MHb is 631 nm. After the method standardization, bothsydnones and N-nitrosophenylglycines were tested at 8.0 μM by incubating them with human blood hemolysate for 15 min. The results computed are mean of triplicate readings. 3-phenylsydnone (IV.1) formed the highest MHb amongst the tested sydnones, followed by 3-(4-chlorophenyl) sydnone (IV.3) and 3-(4-toluyl) sydnone (IV.2). Presence of a substituent on the phenyl ring decreasedthe MHb formation. Electron releasing substituent like methyl group drastically decreased the MHb formation than electron withdrawing substituent such as chloro group, suggesting the favorable effect of electron withdrawal on MHb formation and thus on NO donor activity. Among the N-nitrosamines, N-nitrosophenylglycine (III.1) showed the higher MHb formation than its chloro counterpart (III.3), and N-nitroso (4-toluyl) glycine (III.2) displayed the least MHb formation. The effect of substituent on MHb formation remained the same as seen with sydnones. The results are illustrated in Fig.3. In general, the N-nitrosophenylglycines exhibited better MHb formation and thus better NO donors than their cyclic counterparts the sydnones, suggestingsydnones may probably act after hydrolysis. The results obtained were in close agreement with earlier study9 and formed a strong evidence for it. However, the extent of MHb formation by these compounds was too weak to bebiologically significant.

Reaction mixtures containing sydnones (IV.1- IV.3) or N-nitroso phenylglycines (III.1-III.3) in DMF at 8.0 µM and 2 mL human blood hemolysate was incubated at 37 o C for 15 m. The absorbance was measured at 631 nm against blank which did not contain test samples.

CONCLUSION

This study was undertaken to further substantiate the NO donor activity of 3-(phenyl/ substituted phenyl) sydnones and their alkaline hydrolytic products N-nitroso (phenyl/ substituted phenyl) glycines by in vitro MHb formation with human blood hemolysate. Though, the MHb formation by these compounds was found to be weak but formed a strong evidence for their NO donor activity. Thus, the present study substantiated the NO donor property of 3-(phenyl/substituted phenyl) sydnones and N-nitroso (phenyl/substituted phenyl) glycines.

CONFLICTS OF INTEREST

The authors declare no conflicts of interests.

ACKNOWLEDGEMENTS

The authors are grateful to The Medical Officer, Bagalkot Blood Bank, Bagalkote for providing human blood sample.