key: cord-0810835-9knla8sw authors: Tuwar, Suresh M; Hanabaratti, Rohini M title: Kinetics and mechanistic investigations on antiviral drug-valacyclovir hydrochloride by heptavalent alkaline permanganate date: 2021-11-03 journal: J Chem Sci (Bangalore) DOI: 10.1007/s12039-021-01969-4 sha: ae7b0e7635aa3ad3f88f12ec644173a2e9c35d22 doc_id: 810835 cord_uid: 9knla8sw ABSTRACT: Kinetics of Permanganate (MnO(4)(−)) oxidation of antiviral drug, valacyclovir hydrochloride (VCH) has been studied spectrophotometrically at a constant ionic strength of 0.1 mol dm(−3). The reaction exhibiting a 2:1 stoichiometry (MnO(4)(−):VCH) has been studied over a wide range of experimental conditions. It was found that the rate enhancement was associated with an increase in concentrations of alkali, reductant and temperature. A plausible mechanism involving an intermediate Mn(VII)-VCH complex (C) was expected and rate law is derived accordingly. Calculated activation parameters also supported the anticipated mechanism. GRAPHIC ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12039-021-01969-4. Valacyclovir (VCH), a valine ester contains a guanine acyclic nucleoside. The two moieties are linked by a couple of alkyl oxygen bonds. Chemical name of VCH is L-valine-2-[(2-amino-1, 6-dihydro-6-oxo-9-hipurin-9-yl) methoxy] ethyl ester, also named as Valtrex. It is an L-valyl ester prodrug of the antiviral drug acyclovir that exhibits activity against herpes simplex virus types, (HSV-1), (HSV-2) and Varicella-zoster virus (VZV), 1 Scheme 1 reveals the structure of VCH. In the mechanism of its action on herpes, acyclovir involves a highly selective inhibition of DNA replication virus, via enhanced uptake in herpes virus-infected cells and phosphorylation by viral thymidine kinase. VCH is rapidly converted to acyclovir and further phosphorylated to acyclovir triphosphate (ATP). The incorporation of ATP into the growing chain of viral DNA results in chain termination. 2, 3 The substrate specificity of ATP for viral, rather than cellular, DNA polymerase contributes to the specificity of the drug 4,5 but VCH has side effects, like skin rash central nervous system effects with symptoms such as dizziness, confusion, headache numbness etc. These side effects may be due to the oxidative product of VCH. Currently, COVID-19 (Coronavirus disease-2019) is treated with Remdesivir which is one of the expensive drugs. Recently, clinical trials are going on 6 to treat the COVID-19 pandemic particularly SARS-CoV-2 infection with acyclovir which is formed in vivo by administrating valacyclovir. Oxidation by permanganate had earned more attention in green chemistry due to its versatile applications in several organic 7, 8 and inorganic 9,10 redox reactions. The permanganate occurs in a few oxo-compounds 11 and has tetrahedral geometry with extensive p-bonding. The mechanistic pathways of MnO 4 oxidation of organic substances like alcohols, aldehydes, alkenes and alkynes are depending upon the active species involved and its sensitivity to solvent, pH and other variables. Literature survey revealed that the dissolution studies, 12,13 pharmacological data 14, 15 and a few methods are recommended for its analysis in pharmaceutical dosage forms by spectrophotometry, 16 HPLC 17 and RP-HPLC 18 methods. It is pertinent to mention that VCH hydrochloride undergoes acyl-oxygen bond cleavage in hydrolysis and biological systems to generate acyclovir 19, 20 and valine. Its electrochemical oxidation has led to the formation of imidazolone 21 moiety without affecting the side chain (Scheme 2). To the best of our knowledge and literature survey, there are no kinetic studies reported for oxidation of VCH using KMnO 4 in alkaline medium. Hence, the present investigation has been taken up to understand the mechanistic pathway of KMnO 4 oxidation and to identify the product obtained in this reaction. All chemicals used were of AR grade and double distilled water was used throughout the study. VCH was procured from SD Fine Chemicals (India) with 98.0% purity. Further, its purity was checked by its melting point (198°C) and GC-MS. A stock solution of VCH was prepared by dissolving an appropriate quantity of sample in double-distilled water. The permanganate solution was prepared by dissolving the required amount of KMnO 4 crystals in distilled water and standardized against sodium oxalate. 22, 23 In addition, it is well-preserved in an amber glass bottle to avoid degradation due to exposure to sunlight and is characterized by a spectrophotometer. Potassium manganate (K 2 MnO 4 ) solution was prepared as follows; an aqueous solution of KMnO 4 was heated to boiling [ 100°C in alkali. A green solution of K 2 MnO 4 formed and was characterized by its visible spectrum at 608 nm (e = 1530 ± 100 dm 3 mol -1 cm -1 ) ( Figure 1 ). Further, it was used to verify the product effect on the rate of reaction. at its k max , 526 nm with a 1 cm quartz cell in Specord-200 plus spectrophotometer set up with a Peltier accessory as a function of time at 298 K unless otherwise stated. Prior to the reaction, it was confirmed that there is no interference from the other reagents at this wavelength. The reaction was initiated by mixing previously thermostated MnO 4 and VCH solutions, which also contained necessary concentrations of KOH and KNO 3 to maintain constant alkali and ionic strength respectively in the reaction. Obedience to Beer's law for permanganate at 526 nm had been previously confirmed, giving e = 2241 ± 30 dm 3 mol -1 cm -1 (Lit. value = 2200 dm 3 mol -1 cm -1 ). Since, the first-order plots, log 10 (Abs.) versus Time were found to be linear up to 80% of the reaction, the rate constants, k obs were calculated from the slopes of such plots for various experimental conditions. The experimental results were reproducible within ± 5%. (1). MnO 4 2as a reduction product was identified by measuring its optical density at 608 nm. The oxidation products of VCH were evident for formylmethyl 2-amino-3-methylbutanoate and 2-amino-9-(hydroxymethyl)-1H-purin-6(9H)-one. After completion of the reaction, the solution was subjected to TLC for separation of components. It gave two spots with reference to VCH, which confirmed the established products. Further, the formation of aldehyde was identified by spot test. 24 In addition, the solution was analyzed by LC-ESI-MS for mass evidence of the expected products. After completion of the reaction, it was treated with 50% methanol followed by acidification with HCl and 3% acetonitrile and 1% formic acid to make the solution in a positive ion mode. The solution was subjected at the rate of 5 lL/min with retention time 0.51-0.98 s in the applied voltage of 30 kV with a glass microsyringe. The nitrogen gas was used as a nebulizer. The LC-ESI-MS spectra exhibited, m/z peak at 159 and 181 which are expected for formylmethyl 2-amino-3-methylbutanoate (a) and 2-amino-9-(hydroxymethyl)-1H-purin-6(9H)-one (b) respectively ( Figure 2 ). The reaction orders were calculated from the slopes of plot, log 10 k obs versus log 10 (concentration) for varying [VCH] and [OH -] in turn keeping all other reactant conditions constant. Since the first-order plots were linear up to 80% completion of the reaction, the basic rate methods were used for determining the order of reactive species. Effect of [MnO 4 ]on the rate of reaction was studied by varying the [MnO 4 -] from 4.0 9 10 -5 to 4.0 9 10 -4 mol dm -3 at constant ionic strength by keeping all other conditions constant (Table 1 ). It was found that k obs values were constant for different [MnO 4 -] and also found to be linear and parallel in pseudo-first-order plots. The order in [MnO 4 -] was well-thought-out to be unity. Effect of VCH on rate was studied by varying its concentration between 8.0 9 10 -4 and 8.0 9 10 -3 mol dm -3 by keeping other conditions constant (Table 1) . It had been noticed that k obs values increased with increasing [VCH] . The order in [VCH] was calculated from the plot of log 10 k obs versus log 10 [VCH] and was found to be a positive fraction (0.4). Effect of alkali on rate was studied by varying the [OH -] between 1.0 9 10 -2 and 1.0 9 10 -1 mol dm -3 by keeping other conditions constant at 298 K. It was observed that k obs values were increased with an increase in [OH -] ( Table 1 ). The order in [OH -] was calculated from the plots of log 10 k obs versus log 10 [OH -] and was found to be a positive fraction (0.4). The effect of ionic strength on rate was carried out by varying the [KNO 3 ] between 0.05 and 0.6 mol dm -3 and keeping all other conditions constant ( Table 2 ). It was observed that the added salt had no effect on the rate. The effect of change in the dielectric constant of the medium on the reaction rate was studied by using different compositions (v/v) of t-butanol and water. 25 As 'D' decreases k obs values decreased ( Table 2 ). The dielectric constants of their different compositions were calculated by considering their D in pure form using the equation: where V 1 and V 2 are volume fractions and D 1 and D 2 are dielectric constants of water and t-butanol as 78.5 and 10.5, respectively at 298 K. Prior to the reaction, it was confirmed the inertness of the solvent with oxidant and other components of the reaction mixture. The results indicate that the added product did not affect the rate. In the present study MnO 4 is one equivalent oxidant in alkali. Hence, the reaction may proceed via free radical formation. In view of this, acrylonitrile was used as a free radical scavenger and tested in the reaction mixture as follows; the reaction mixture was mixed with acrylonitrile monomer and kept for 24 h in O 2 and CO 2 free atmosphere. A copious precipitation was formed on diluting the reaction mixture with methanol, indicating the intervention of free radicals in the reaction. The experiment of either MnO 4 or VCH with acrylonitrile alone did not induce the polymerization under similar condition as those induced with reaction mixture. Initially added acrylonitrile also decreased the rate, indicating a free radical intervention. By keeping constant conditions of the reaction, the temperature was raised to 298, 303, 308, 313 and 318 K. The rise in temperature shows an increase in the rate of reaction and calculated k obs values are presented in Table 3 . The activation parameters for the reaction are calculated by using linear regression analysis (also known as the method of least square). In generalized notation, the formula for the straight line is y = ax ? b. The most tractable form of linear regression analysis assumes that values of the independent variables 'x' are known without error and that experimental error is manifested only in values of the kcal were calculated using k = 3.18 9 10 -3 s -1 , K 1 = 4.42 dm 3 mol -1 , and K 2 = 3.64 9 10 3 dm 3 mol -1 at 298 K in rate eqn. (9). where 'n' is the number of data points and summation are for all data points in the set. These data were subjected to least square analysis and verified with experimental values. From the Arrhenius plot, log k obs versus 1/T, activation parameters were figured out (Table 4) . The other activation parameters were calculated as follows. The Arrhenius factor 'A' was calculated by, log A = log k obs þ E a 2:303RT The entropy of activation was calculated by using the equation, The k obs should be in s -1 , and temperature in Kelvin, then the E a results in J mol -1 and DS = in J K -1 mol -1 . The enthalpy of activation was calculated by, DH = = E a -RT and free energy of activation from DG = = DH = -TDS = . The rate constants (k) were obtained from intercept of 1/k obs versus 1/[VCH] for slow step (Scheme 3). Other equilibrium constants, K 1 and K 2 were obtained from the slope and intercept of the plots, 1/k obs versus 1/[VCH] and 1/[OH -] (Figure 3 ). In the present study, added [OH -] has a positive effect and thus combines with permanganate ion in Table 3 . Effect of temperature on the oxidation of VCH by alkaline permanganate. ¼3:0 Â 10 À3 mol dm À3 I ¼ 0:1 mol dm À3 T (K) 10 3 9 k obs (s -1 ) Table 4 . Activation parameters for the oxidation of VCH by alkaline permanganate at 298 K. alkaline medium to form alkaline permanganate ion in pre-equilibrium step as shown below. This is in accordance with the earlier work 26, 27 . The proposed structure of MnO 4 complex (Scheme 3) is based on the MnO 4 oxidation of heteroaryl formamidines 28 in an alkaline medium. Since the progress of the reaction was monitored for change in color of oxidant, it exhibited changeover in coloration from violet to blue and then to green. Spectral changes during the oxidation as shown in Figure 4 is evidence for the formation of MnO 4 2complex by the appearance of two new bands at 432 and 608 nm followed by the disappearance of permanganate bond at 526 nm. OH À ½ ¼ 0:05 mol dm À3 VCH ½ ¼3:0 Â 10 À3 mol dm À3 ] and positive fractional order in both alkali and substrate concentrations. The permanganate species acts as a one-electron oxidant and affords via free radical intermediate and it is evidenced by the free radical test. The evidence for such free radical in a slow step is also reported in earlier work. 31, 32 In the first step (1) (4) to yield the products 2-amino-9-(hydroxymethyl)-1H-purin-6(9H)-one and formylmethyl 2-amino-3-methyl butanoate. This was ascertained from their LC-ESI-Mass spectra, m/z peak at 159 and 181, expected for formylmethyl2-amino-3-methylbutanoate and 2-amino-9-(hydroxymethyl)-1H-purin-6(9H)-one respectively ( Figure 2 ). This proposed mechanism leading to the formation of aldehyde is supported by earlier studies and to quote a few, amino acid, ester, etc. 33 In the proposed mechanism (Scheme 3), complex (C) decomposes to give Mn 6? by abstracting an electron leading to a CH(methylene) free radical. In the next step, cleavage of alkyl oxygen (AL 2 ) bond rather than acyl-oxygen bond leads to an aldehyde and hydroxyl methyl purine-one. The formation of such aldehyde has been observed in the earlier reports of oxidation of amino acid ester. 33 Further, cleavage of AL 2 bond is found, leading the N-CH 2 -OH group on imidazole and is stabilized by an intramolecular hydrogen bonding. The other possibility of direct '2' electron reduction was i.e., hypomanganate (MnO 4 3-) to yield a final product. Such single step oxidation was rejected as the development of Mn V O 4 3ion was not noticed in the progress of the reaction, which was expected for the absorbance at 667 nm. Hence, it is concluded that the oxidative mechanism of VCH by alkaline permanganate follows as per Scheme 3. Table 1 ). (1) (9) (9) (1) (1) The rate law (eqn. 9) has been proved by plotting of 1/k obs versus 1/[VCH] and 1/[OH -] which gave linear plots (Figure 3 ). From the slopes and intercepts of these plots, the values, k = 3.18 9 10 -3 s -1 , K 1 = 4.42 dm 3 mol -1 , and K 2 = 3.64 9 10 3 dm 3 mol -1 for 298 K were calculated. The K 1 value obtained is in good agreement with the literature value of (6.6 dm 3 mol -1 ). 29, 34 Further, equilibrium constants K 1 , K 2 along with k were used to regenerate k obs values for the different experimental conditions. It is found that the regenerated results are in good agreement with experimental results (Table 1 ). This strengthens the proposed mechanism (Scheme 3) and rate law (eqn. 9). In the proposed mechanism (Scheme 3), the reaction takes place via complex formation (step 2). The value of DS = (-165) strengthens a relatively rigid complex formation and hence its stability. 35 The higher negative value of DS = proves that the complex is more ordered than other species present in the reaction. It is noticed in the reaction that as the dielectric constant of the media increases rate increases. This indicates that the reaction is more favorable in aqueous media. Oxidation of VCH by alkaline permanganate proceeds through the intervention of free radicals generated from VCH (methylene moiety). The active species of permanganate is found to be [MnO 4 (OH)] 2which was formed in a prior equilibrium step of the mechanism. The mechanism occurs through a complex formed between MnO 4 and VCH. The relatively large value of k obs and small value of log A supports that the reaction was led through the inner-sphere mechanism. The overall mechanistic sequence described here is consistent with product studies and kinetic studies. The spectrum of alkaline permanganate at 298 K, Order plot of [VCH] and [OH -], Effect of dielectric constant (D) (log k vs. 1/D), Effect of initially added product, [MnO 4 2-] and Arrhenius plot for the oxidation of VCH by alkaline permanganate (Figure S1-S5 and Table S1 ) are available at www.ias.ac.in/chemsci. Valacyclovir: A review of its long term utility in the management of genital herpes simplex virus and cytomegalovirus infections Valacyclovir: A review of its antiviral activity, pharmacokinetic properties, and clinical efficacy Electrooxidation of the antiviral drug valacyclovir and its square-wave and differential pulse voltammetric determination in pharmaceuticals and human biological fluids Acyclovir an updated review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy Gene expression in the human intestine and correlation with oral valacyclovir pharmacokinetic parameters Acyclovir for SARS-CoV-2: An Old Drug with a New Purpose Clin Kinetics and mechanism of uncatalysed and ruthenium(III)-catalysed oxidation of d-panthenol by alkaline permanganate Transit Spectrophotometric evidence for the formation of short-lived hypomanganate(V) and manganate(VI) transient species during the oxidation of K-carrageenan by alkaline permanganate Short-Lived manganate(VI) and manganate(V) intermediates in the permanganate oxidation of sulfite ion Mechanism of oxidation of L-histidine by heptavalent manganese in alkaline medium E The Pharmacological Basis of Therapeutics 7 th edn Comparative activity of various compounds against clinical strains of herpes simplex virus Eur Comparative activity of selected antiviral compounds against clinical isolates of varicella-zoster virus Eur Novel spectrophotometric determination of valacyclovir and cefotaxime using 1, 2-napthaquinone-4-sulfonic acid sodium in bulk and pharmaceutical dosage form Arch Rapid determination of valaciclovir and acyclovir in human biological fluids by high-performance liquid chromatography using isocratic elution Development and validation of an RP-HPLC method for the determination of valacyclovir in tablets and human serum and its application to drug dissolution studies Stability of valacyclovir: Implications for its oral bioavailability Int Stability evaluation and sensitive determination of antiviral drug, valacyclovir and its metabolite acyclovir in human plasma by a rapid liquid chromatography-tandem mass spectrometry method J. Chromatogr Electrochemical oxidation and thermodynamic parameters for an anti-viral drug valacyclovir Anal Vogel's Text Book of Quantitative Chemical Analysis 5th edn A Text Book of Quantitative Inorganic Analysis Spot Tests in Organic Analysis Translated by Oxidation of mandelic acid by alkaline potassium permanganate-A kinetic study Kinetics of oxidation of free and coordinated dimethylsulfoxide with permanganate in aqueous solution Inorg Kinetic and mechanistic investigations on the oxidation of N'-heteroaryl unsymmetrical formamidines by The authors are obliged to the Principal, Karnataka Science College, Dharwad, Karnataka, India for offering the essential laboratories to execute the present work. According to Scheme 3,However,-] f is neglected compared to 1 in the denominator as low concentration of MnO 4 used. Therefore,On substituting eqns. (5), (6) , and (7) in eqn. (4), eqn. (8) resultsFor verification of rate law, the subscripts 'T' and 'f' are omitted and hence eqn. (8) becomes,Equation (9) is rearranged into eqn. (10) , which is suitable for verification.