Protonation of Nitrofurantoin and Furazidine Molecules in Acidic Media-Molecular Modelling Studies

The molecular modeling studies on protonation sites of Nitrofurantoin and Furazidine as well as on the stability of particular protonated forms were performed using quantum chemical MP2 method. Performed calculations show that Furazidine oxygen and nitrogen atoms are better proton acceptors than in Nitrofurantoin, therefore the acidity of the media may differentiate Nitrofurantoin and Furazidine antibacterial activity.


Introduction
According to literature data in nitrofuran derivatives the major role in antimicrobial activity plays the acidity of physiological medium in urinary tract [1]. It was pointed out [2] that dissociation of Furazidine is hampered in the presence of ascorbic acid (vitamin C). The role of any acidifying agent like ascorbic acid is to prevent alkalization of infected urine and preserve pH close to 5.5 in which Furazidine molecule stays in non-dissociated form what enhances its antibacterial activity [1]. More acidic medium can probably cause better protonation of the Furazidine molecule than of Nitrofurantoin, what can further enhance Furazidine therapeutic efficacy. Therefore studies of possible protonation sites of Nitrofurantoin and Furazidine, along with stability of particular protonated form, can provide valuable estimate for insight into activity determinates of Furazidine moiety, and also for further modification of its structure. Selection of Nitrofurantoin and Furazidine is a wise model because those two compounds differ in two carbon atoms in the rings bridging part of molecules. Moreover there is distinct difference between their antimicrobial activities. In Escherichia coli test they display MIC ≤ 32 μg/ml and 1 μg/ml for Nitrofurantoin and Furazidine, respectively [3], as reported in information leaflets of medicine present on the market.

Results and Discussion
Calculation on neutral Nitrofurantoin tautomer clearly indicates that N 1 -H tautomer is the most stable form both in water medium. The same holds for Furazidine molecule.
The neutral Nitrofurantoin and Furazidine molecules can potentially exist in various tautomeric forms. As shown in Tables 1 and 2 the N 1 -H tautomer of Nitrofurantoin is the most stable.
The second most stable tautomer is the one bearing the proton on the O 2 nitrogen atom at the MP2/6-31G*//MP2/6-31G* level with hydration energy included

Journal of In Silico & In Vitro Pharmacology ISSN 2469-6692
When proton is placed on O 3 oxygen atom then it relocates to one of the atoms of the nitro group. The relative stability of Nitrofurantoin tautomers in water medium is as: N1-H>O 2 -H>O 1 -H>O 4 -H form. The same trend is observed when the Δ (ΔGo) values are considered.
In the case of Furazidine molecule also the N 1 -H tautomer appeared to be the most stable. Here however the N 2 -H and N 3 -H tautomers exist. The N 3 -H tautomer is stabilized through C-H interaction of more flexible bridging chain with O 2 atom of five membered rings. As in Nitrofurantoin, when proton is placed on O 3 oxygen atom then it relocates to one of the atoms of the nitro group.
In Nitrofurantoin and Furazidine molecules there is 8 potential protonation centers, 3 oxygen atoms and 3 nitrogen atoms and two oxygen atoms of the nitro group (Figure 1). Nevertheless the nitro group, in each of two equivalent resonance structures, can potentially interact via the hydrogen bonding.  Table 2.
The dication of Nitrofurantoin with the highest stability is the one formed from the most stable N 1 -H; N 3 -H cation by protonation of O 1 or O 2 oxygen atom. The same holds for Furazidine molecule ( Table 1). The nitro group can be protonated at each of the oxygen atoms yielding resonance structure -NOOH+ similar to -COOH [4]. Further protonation of -NO 2 could lead to reduction of -NO 2 yielding the -NH 2 derivative. This is one of the mechanisms that activate Furazidine active substance in the living organism [5,6].

Formation of the most stable monocation N
If additional proton is placed on O 3 oxygen of five membered ring of neutral Furazidine, then it relocates to O 2 oxygen atom. It means that formation of cation III is very unlikely. Cation II gets additional stabilization due to intramolecular bonding with one of the oxygen atoms of -NO 2 group. The most stable cation is the N-1 H, N 3 -H. The total energy and thermodynamics (ΔG) analysis leads to the same conclusions.
Similar conclusions regarding possible protonation sites appear from analysis of electron charges on the atoms that are eager to accept proton (Table 3).  Table 3: Electrostatic, Mulliken and natural charges calculated for nitrofurantoin and furazidine at MP 2 /631G*//MP 2 /6-31G* level.