Chloramine - T - induced oxidation of Rizatriptan Benzoate: An integral chemical and spectroscopic study of products, mechanisms and kinetics

: Oxidation is a prominent degradation route of biological molecules that produces a wide variety of degradation products through complex mechanisms and hence quali ﬁ es to be a vital pharmaceutical process. This article presents the kinetic and spectral study of the oxida - tion of an antimigraine drug rizatriptan benzoate ( RTB ) in an acid medium with the aid of a mild biocidal oxidant N - chloro - p - toluenesulfonamide, referred to as chloramine - T ( CAT ) . The kinetic experimental studies reported here, such as fractional order dependency on RTB, pseudo - ﬁ rst - order dependency on CAT, negative fractional order dependency on the acid medium, independent of the rate on the ionic concentration and increasing rate with increasing dielectric constant, have led to the evalua - tion of stoichiometry, thermodynamic properties, and derivation of a rate equation. E ﬀ ective interpretation of UV – Vis, IR, 1 H and 13 C NMR investigation was per - formed to identify and con ﬁ rm the identity of the oxida - tion products and discuss the involved plausible mechanism. This study provides an extended insight into the products of oxidation formed during the metabolism of RTB.


Introduction
The study of oxidative degradation behavior helps to establish the metabolic pathway of drugs and therefore qualifies to be essential data.Oxidative metabolism, being a crucial route of degradation, decides the ability to uptake, modify or eliminate the drug in most organisms and therefore deserves a quantitative exploration.In vitro oxidative conversions of polycyclic phenols [1], lipids [2], flavonoids [3] and various other organic moieties [4] are always a concern of pharmaceutical scientists as they indicate stability and also resemble the in vivo oxidative degradation of ingredients in a drug formulation [5] and its effects on biological systems [6].The most popularly used novel reagents for degradation studies include enzymes [7], N-aryl halo sulfonamides [8], nanometallic oxide composites [9], nanocomposites with heterojunction [10], vitreous fibers [11], and glass surfaces acting as bases or nucleophiles [12].Among all these oxidants, chloramines like chloramine-T (CAT) are familiar for their versatile oxidative behavior, affecting a wide range of functional groups to induce molecular modifications.Chattaway [13] was the first to prepare CAT by reacting toluene with chlorosulfonic acid and subsequently with aqueous sodium hypochlorite; the colorless compound in its stable form has been successfully used since years in bringing effective oxidative transformations in medicinal compounds such as kojic acid [14], imidazoles [15], D-mannitol [16] and L-alanine [17].The component-level details on such oxidation degradation products of organic compounds are often revealed by many analytical tools, such as density functional theory [18], non-isothermal thermogravimetric analysis [19], liquid chromatographymass spectrometry [20], EC/Q-TOF/MS [21] and 1 H and 13 C NMR [22].
N,N-Dimethyl-5-(1H-1,2,4-triazol-1-yl methyl)-1H-indole-3-ethanamine monobenzoate, commercially familiar by the name rizatriptan benzoate (RTB), is the most widely used drug known for its clinically useful antimigraine action.The central actions exerted as serotonin 5-HT 1 receptor agonist followed by constriction of intracranial nerves and reversal of neurogenic vasodilation contributes to the pain-free state.Despite producing an excellent doserelated pain-relieving effect of RTB in migraine attacks, its action is often associated with the incidences of liver toxicity [23] and renal infarction [24].This has aroused the interest of researchers to investigate the mechanistic mode of oxidative degradation by stress [25], photocatalysis [26] and forced degradation [27] along with its quantification using multiwall carbon nanotube-supported Fe 3 O 4 [28], nickel-ferrite-modified carbon paste electrode [29] and Gr/αFe 2 O 3 /carbon paste [30].
The mechanism of degradation of RTB under the influence of chloramine-B was explored by us earlier [31] but the molecular structures of these products of degradation remain ambiguous.Here, the methodology is modified by employing N-chloro-p-toluenesulfonamide, often referred to as CAT, to cause oxidative degradation of RTB in an HClO 4 medium.We used 1 H and 13 C NMR spectroscopy data in conjunction with UV and IR analysis as complementary and confirmatory techniques to characterize the degradation products.The detailed study presented here through spectral structural assignment provides an extended insight into the metabolic activity of RTB.The unambiguous spectral evidence for the transformation of N,N-dimethyl-5-(1H-1,2,4-triazol-1-yl methyl)-1H-indole-3-ethanamine monobenzoate to N,N-dimethyl-5-(1H-1,2,4triazol-3,5-dione-1-yl methyl)-1H-indole-3-ethanamine significantly enhances the understanding of biological mechanism and also serves as a reference for any further 1 H and 13 C NMR investigation of antimigraine agents belonging to the class of triptan drugs.
Oxidative studies were conducted in aqueous media using double-distilled water.KBr employed in generating pellets for IR spectral scanning was kept in an oven at 120°C and brought to room temperature in a desiccator.Highly pure TMS served as an internal standard for calibration and investigation of delta values in generating 1 H and 13 C spectra. Figure 1 depicts the procedural steps involved in the oxidation of the RTB.

Kinetic procedures
The kinetic procedures were carried out in an experimental system designed for pseudo-first-order conditions.UV analysis of oxidation was initiated in thermostatted black color-coated Pyrex boiling tubes with glass stoppers to avoid some degree of photochemical degradation.RTB was constantly stirred in the acid medium, and a known quantity of CAT was introduced to trigger the reaction under thermostatic conditions.About 4 ml of the reaction mixture was immediately pipetted into a cuvette placed in a UV-Visible spectrophotometer and the absorbance was measured.The progress of the reaction was monitored by the measurement of absorbance at a chosen wavelength for two half-lives.The slope k gives the pseudo-first-order rate constant from the plot of log (A 0 /A t ) versus time, where A 0 and A t are absorbance readings at time t = 0 and t, respectively.

Stoichiometry by Job's method
Job's method of continuous variation was employed where a series of solutions with varying mole fractions of the substrate and oxidant were prepared; the absorbance of each was noted, keeping the total molar concentration constant.The mole fraction of the oxidant or the substrate was plotted against the absorbance readings.The maximum of the plots indicated the stoichiometry of the oxidant and the substrate.

Product isolation
RTB and CAT were allowed to react at a stoichiometric ratio with constant stirring for 48 h in the acid medium.The reaction was allowed to progress under stirred conditions for 48 h and at the end of the process two products, water-insoluble (Product I) and water-soluble by-product (product II), were isolated.
The water-insoluble product (Product I) was filtered using Whatman filter paper 40, dried and the purity was checked using the thin layer chromatography (TLC) technique with 1:1 benzene/ethanol as an eluting solvent.The product was identified by the data obtained by UV-Visible, IR, and NMR spectrophotometry techniques.
The water-soluble product (Product II) was extracted by evaporation of water using a vacuum rotatory evaporator.The product was subjected to TLC, where a blend of petroleum-ether, chloroform and n-butanol (2:2:1, v/v) served as an eluting mixture, and gas chromatographymass spectroscopy (GC-MS).

Instrumentation
A Shimadzu UV-1700 Pharma Spc double beam UV-Vis spectrophotometer was used to investigate the oxidation reaction.The Fourier transformed spectrum of IR was outlined using a Shimadzu FT-IR-8400S instrument.The oxidized product weighing 200-300 mg was crushed to a fine powder, and 1 H and 13 C NMR spectra were scanned using a 600 MHz NMR spectrometer (model BRUKER DSX).A spin speed of 10-12 kHz was generated, and a strong and consistent magnetic field of 7.04 T at room temperature was maintained to measure the chemical shift (in ppm) and coupling constants (J, in Hz) on the Linux & X-Winnmr Platform.
GC-MS spectra were recorded using an electron impact technique at 280°C on Hewlett Packard-5890 gas chromatograph, equipped with an automatic quartz sampler (model 7673A) and a mass selective detector (model 5989A) operated at 2,200 V.

UV analysis of the reactivity of RTB
The reactivity of RTB is reflected by the modifications in the UV spectral traces during the oxidation process and was monitored kinetically at 298 K, with the addition of CAT in an acid medium.A pseudo-first-order oxidation reaction conditions were achieved with RTB at higher concentrations than CAT.The electronic spectrum of RTB scanning between 200 and 350 nm displays maximum absorptions at 227 and 280 nm, concurrent with the earlier report by Altinoz et al. [32].The time-resolved kinetic curves, shown in Figure 2, with a scanning interval of 60 s, resulting from the oxidation process were evaluated.According to Watanabe et al. [33], during oxidation, the attack should occur at the triazole ring, with the highest frontier electron density and preferentially involving highly reactive nitrogen atoms.In the current study, this is indicated by a notable absorption band at 225 nm, which is generally attributed to π−π*, which decreases in intensity that can be attributed to the weakening of the conjugated C]N bond of the triazole ring.Similarly, a relatively weak absorbance at 267 nm undergoing an increase in the intensity is attributed to the formation of an imide with a carbonyl group possessing forbidden n−π* [34].These changes in the UV absorbance are clearly assignable to the transition of amine to imide [35], which reveals excellent stability of the oxidized product and reinforces the data in Table 1.
In addition, Figure 3(a) displays a negative slope of unity with regression coefficient of 0.993, indicating a firstordered reaction with regard to the oxidant by a decrease in the intensity of the absorbance peak at 227.This observation matches with the equation where A t , A 0 and A ∞ denote the absorbance at time t, 0 and infinity, respectively, plotted in Figure 3(b) with a regression coefficient of 0.994, marking an increased intensity due to the product formation.However, these conversions can also be confirmed by high-resolution crystal studies that are in progress in our laboratory.

Description of kinetic parameters
The pseudo-first-order rate constants (k l ) in Table 1 were determined by the graphical plots of log (A 0 /A t ) vs time,

Calculation of activation parameters
The variance of temperature allows us to quantify the activation parameters relevant for oxidative degradation.A graphical plot of log k vs 1/T with R 2 = 0.9973 (Figure 4(d)) leads to an Arrhenius plot in the temperature range 293.5-313.5K, resulting in the determination of the energy of activation, enthalpy, and Gibbs free energy as listed in Table 2.

Description of other secondary parameters
An inert electrolyte NaClO 4 was added during the kinetic runs to examine the impact of the ionic strength on the reaction rate, which is highly valuable to gain insight into active components in the rate-determining step [36].NaClO 4 , in the concentration range of 1.0 × 10 −4 to 1.0 × 10 −3 M, when added to the reaction mixture, had no significant effect on the rate of the reaction and hence no measures were attempted to keep a fixed ionic concentration during the degradation study.In addition, one of the reaction products, p-toluenesulfonamide, when increased from 0.2 × 10 −4 to 2.0 × 10 −4 M (Table 3) also had an insignificant influence on the rate of the reaction.
The oxidation reaction was conducted at 298 K in a mixture of methanol and water at varying concentrations (% v/v), with the concentrations of RTB and acid at 1.0 × 10 −4 M and CAT at 1.0 × 10 −3 M, respectively.The reaction rate increased with the increase in the concentration of methanol in the medium (Table 3), which is indicated by the linearity and a positive slope in the plot of log k against 1/D with R 2 = 0.9865 (Figure 4e).

Elucidation of oxidation products 3.5.1 Stoichiometry
The stoichiometric ratio of RTB oxidized by CAT is determined by the Job plot [37] based on the absorbance changes for different mole fraction combinations of reactants.The absorbance computation of the mixtures at regular intervals of 10 min with reactants in ratios 0.2:1.8,0.3:1.7,0.4:1.6,… in a 2 ml calibrated flask against the reagent blank leads to a maximum absorbance for the ratio 2.00:1.00,as shown in Figure 5.This indicates the formation of the oxidized product in the ratio of 2:1 (CAT/RTB) and suggests the following stoichiometric equation:     where Ts = p-CH 3 C 6 H 4 SO 2 .

Comparative analysis of RTB, Product I and
Product II by FTIR measurements (ν max , in cm −1 ) RTB and the oxidized Product I are compared, and reproducible changes are observed, especially with the triazole ring.Pure RTB, in Figure 6(a), shows a peak at 3,124 cm −1 , including a shoulder 3,014 cm −1 because of an aromatic N-H stretch.A couple of weak spikes at 2,926 and 2,868 cm −1 are identified to be of the C-H stretching vibration and a sharp peak at 1,371 cm −1 is attributed to the C-N amine stretch.On oxidation, Product I in Figure 6(b) shows a moderately weak peak at 1,274 cm −1 , including a prominent peak at 3,398 cm −1 with a shoulder at 3,122 cm −1 , which was a weak peak in RTB at 3,437 cm −1 attributed to N-H and the aromatic secondary N-H stretch, respectively.A detailed study by Ghaemy et al. [38] provided the IR spectral details of triazole-derived imide rings according to which the carbonyl group of the imide ring showed a signal at 1,735 cm −1 , which matches well with the current study where a weak peak at 1,734 cm −1 and a strong sharp peak of 1,616 cm −1 is seen and represents an imide inphase together with imide out-of-phase, respectively.Similarly, the peak pertaining to C-N-C stretching at 1,371 cm −1 in RTB is observed to undergo severe modification on the formation of the imide product.However, the stretching vibrations of the methylene group, typically appearing at 2926.11 and 2868.24cm −1 , have not undergone a significant change.
Product III in Figure 6(c) depicts an asymmetric stretch sharply visible at 1,040 cm −1 , and a doublet at 1,190 cm −1 along with 1,130 cm −1 is attributed to O]S]O.The peaks pertaining to C-S and N-H are located at 680 and 3,630 cm −1 in addition to 3,420 cm −1 .The signals at 1,700 and 810 cm −1 are related to the carbonyl group and the substituted benzene ring.

Analysis of Product I by 1 H NMR measurements: δ
(in ppm) (600 MHz, DMSO, TMS) Based on the spectra displayed in Figure 7(a)-(e), predictions are made regarding Product I correlating with 1 H NMR peaks, and the characteristic features are listed in Table 4.The electron-rich linkage of N-H in indole leads to a small singlet at 8.8 ppm, as shown in Figure 7(b).The signal at 4.49 ppm is related to cyclic imide hydrogens of the N-H linkage, which are highly deshielded flanked by two carbonyl groups that induce a magnetic field but remains as a singlet due to no neighboring interactions.A set of signals between 8.2 and 8.5 ppm is assigned to the aromatic protons where circulating п electrons exert a deshielding effect [39], shifting them to higher frequency.A high-intensity singlet, representing the terminal methyl hydrogens of the tertiary amine unaffected by neighboring moieties, typically expected in the range of 2.9-3.3 ppm, is shifted downfield due to the attachment of methyl groups to nitrogen and is observed at 4.81 ppm [40].The additional peaks at 3.30 ppm and the small set of peaks close to 2.2 ppm are assigned to 4 and 2 hydrogens in the methylene groups.

Analysis of Product I by 13 C NMR
All the 13 C NMR chemical shifts for Product I are well correlated, as shown in Figure 8(a)-(c), suggesting the formation of a highly pure oxidized product, and the details are listed in Table 5.The high-frequency peak corresponding to the imide appears at 152.481 ppm, which is a downfield carbonyl shift typical for five-membered ring imides as compared to six-membered ring, which generally appears in the range 170-180 ppm [41].The downfield  shift of 3-4 ppm is often due to the reduced effect of resonance contribution from the lone pair of electrons on nitrogen toward the carbonyl carbon.Signals at 145.041 ppm may be assigned to the two deshielded terminal methyl groups directly attached to the electronegative nitrogen.The olefin carbons in the pyrrole component of the hetero aromatic indole can be correlated with 134.048 and 130.002 ppm, respectively [42].
The presence of the heteroatom adds to the aromatic effect and results in a significant up-field shift compared to other olefin analogues.A peak of moderate intensity at 53.763 ppm along with a set of peaks at 49.843 ppm represent the methylene carbon and a set of overlapping peaks with a high intensity accounting for six carbons representing the aromatic phenyl group in indole, respectively.

Analysis of Product I and Product II by MS
The water-insoluble Product I is shown in Figure 10 Product II recognized as CAT, which is a well-known metabolite para-toluenesulfonamide, was detected in the present case by unambiguous TLC detection, where Product II was eluted using a combination of 2:2:1 v/v petroleum-ether, CHCl 3 and ethanol, respectively, followed by iodine spray similar to the that reported elsewhere [43].
The same was confirmed by MS with many peaks corresponding to cationic fragments matching with the pioneering work reported by Idowu et al. [44].The peaks reported by the authors at m/z 155.05, 91.31 and 65.08 represented by the structures in Figure 9 is similar to that obtained in the current study.
Further, an intense signal at 155.05 corresponds to the detachment of the phenyl group connected to the sulfonyl group via a homolytic breakup of bond violating the "evenelectron rule" [45].Interestingly, the m/z 91.31 ion is formed due to the elimination of the SO 2 molecule from the ion m/z 156.41 generated by the initial benzyne loss [46].
In addition, a molecular ion peak at 171.02 amu displayed by the GC-MS peaks, shown in Figure 10(b), confirms the formation of p-toluenesulfonamide [47], similar to the degradation of Donepezil, an Alzheimer's drug reported previously by the authors.

Discussion
The most common cause for idiosyncratic reactions is the metabolites of drugs formed during oxidation.Nitrogencontaining compounds are likely to generate reactive metabolites as they are more susceptible to oxidation Figure 9: Representative structures corresponding to some fragments in the mass spectrum.
[48] and hence deserve greater attention.Table 6 lists some of the significant oxidation reactions of drugs by several advanced techniques whose degradation has revolutionized bioremediation.The variation of various reactants, CAT, RTB and pH, reveals the first-order rate dependency with regard to the oxidant: less than unit order toward [RTB] and inverse fractional order toward [H + ] ions.Bishop and Jennings [55], Morris et al. [56] and Kumar et al. [57] have concluded that the oxidizing species produced by CAT are TsNHCl, TsNCl 2 and HOCl, among which TsNCl 2 and HOCl are excluded as second-order reaction rate with respect to the oxidant, and first-order retardation of rate on adding p-toluenesulfonamide was not observed.Further, a fractional order dependency of the reaction rate on [RTB] points out to the involvement of [RTB] and specifies that a RTB-CAT complex is formed during the fast pre-equilibrium state preceding the rate-determining step, which resembles the Michaelis-Menten type of kinetics [58].
TsNHCl has a great affinity to get protonated at pH < 2: Since the present observations are carried out at pH > 3, it may be concluded that TsNHCl is the oxidizing agent.

Rate constants and the ratedetermining step
A range of data obtained in Table 1 for varying concentrations provides the best-fit curves that forms the basis for deriving a rate equation.In the present study, all the above observations are consistent with Scheme 1, described as follows.
TsNHCl reacts with RTB to give the intermediate complex X, undergoing hydrolysis to give X I which on further oxidation induces the generation of the products.The rate law can be expressed as Assuming [CAT]t to symbolize the active component of CAT, The above rate expression is in concurrence with the empirical results such as positive fractional order dependency of the rate on [RTB], first-order dependency over [CAT] and inverse fractional order dependency over [H + ].

Mechanistic elucidation
A recently reported article by Khana et al. [59] describes the oxidation of RTB and the details of a mechanism using a disintegrated complex of silver(III), which proves the vital role of pH that matches with the current scenario.In addition, a variation in the ionic strength of the medium did not cause any change in the rate of the reaction, which implied that no ions of opposite signs are involved in the slow step.However, increase in the percentage composition of methanol in the reaction medium to vary the dielectric permittivity led to the increase in the reaction rate.This positive dielectric effect in the present study seems to agree with the expected interaction between the cationic form of the substrate (X) and dipolar water in the slow step of the presented mechanism.In a restricted case where both dipoles or ions and dipoles are drawn toward each other at zero angle, Amis [60] proposed that a graph of log K vs 1/D yields a linearity with a decreasing slope for any two dipoles reacting.Similarly, a positive slope for the reaction is observed when a positive ion reacts with a dipole.This explanation is well applicable to the present reaction under study.The mechanism supports an apt energy of activation, negative entropy of activation, which generally is well suited for the generation of a compact activated complex and also confirms the fairly positive value of enthalpy assuring the presence of a highly solvated transition state.In view of the above results, the following mechanistic pathway (Scheme 2) may be outlined to explain the oxidation process of RTB.
The reaction is proposed to be initiated by the leaving group chlorine that triggers the nitrogen in the triazole ring to form an adduct complex X, which subsequently reacts with a molecule of water to form X I whose positive charge is stabilized by deprotonation leading to X II .Thus, the larger adduct X II undergoes deprotonation and subsequent hydrolysis to generate the imide oxidized Product I and CAT as the by-product.
The by-product, namely CAT, being a stable watersoluble compound will not undergo further oxidation and hence can be finally isolated easily.The mechanism outlined is in accordance with all the experimental data obtained.

Conclusions
The present kinetic modeling provides an insight into the oxidative susceptibility and oxidation pathway of RTB, an antimigraine drug utilizing a mild biocidal oxidant N-chloro-p-toluenesulfonamide.Kinetic observations revealed fractional order dependency on RTB, pseudofirst-order dependency on CAT, negative fractional order dependency on the acid medium, independency of the rate on the ionic concentration and increasing rate with increasing dielectric constant.The stoichiometric kinetic study of the drug is based on time-resolved UV spectra displaying maxima at 227 and 280 nm.Product isolation followed by extremely informative spectroscopic analysis through IR peaks, which are concurrent to 1 H, 13 C NMR spectral studies and MS led to a reliable identification and structure determination.
The results emerging by varying the concentration of the substrate, oxidant and ionic strength of the medium is complementary with spectroscopic results and hence has assisted in the assessment of critical parameters and thermodynamic properties of the oxidation reaction.Further, a system of reaction rate equations based on the kinetic model is derived for the reaction that contributes for the first time in proposing a mechanism of action of CAT on RTB.The development of this model would benefit many studies related to oxidation reactions in pharmacokinetic conditions.

Figure 1 :
Figure 1: Steps involved in the oxidation study of RTB: (a) oxidation workup, (b) kinetic procedures and (c) product isolation followed by characterization.

Figure 2 :
Figure 2: UV spectra of (a) RTB and (b) time-resolved UV spectra of RTB reacting with CAT.

Figure 3 :
Figure 3: Absorbance intensities: (a) decreasing intensities at 225 nm and (b) increasing intensities at 267 nm in accordance with a single exponential equation.
where A 0 and A t represent the absorbance at t = 0 and t, respectively, for the oxidation of RTB in an HClO 4 medium with varying concentrations of RTB, CAT and HClO 4 .The linear regression graph obtained by the range of data by varying the concentrations of RTB, CAT and HClO 4 yields a best-fit curve, which is highly valuable in solving the rate equation.Varying the concentration of RTB across 0.2 × 10 −4 and 2.0 × 10 −4 M with a combination of CAT and HClO 4 to be sustained at 1.0 × 10 −3 M and 1.0 × 10 −4 M individually leads to linear plot (log k vs log [RTB]), as shown in Figure4(a), with R 2 = 0.9799 and a slope of 0.6, indicating the reaction to be fractional order

Figure 5 :
Figure 5: Job's plot of continuous variance for the RTB/CAT reaction product.

Figure 6 :
Figure 6: IR spectra of (a) RTB, (b) oxidized Product I and (c) oxidized Product II.

Figure 10 :
Figure 10: GC-mass spectra of (a) Product I and (b) Product II.

Table 3 :
Effect of different concentrations of p-toluenesulfonamide and the solvent on the reaction rate at 298 K

Table 4 :
1H NMR evidence of the oxidized Product I of RTB

Table 5 :
13C NMR specifications of oxidized Product I

Table 6 :
Comparison of significant oxidation reactions of various drugs Substrate Scheme 2: Proposed scheme with structures for the oxidation of RTB on reacting with CAT.Rizatriptan Benzoate oxidation: chemical and spectroscopic study  13