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.
Scheme 1
Structure of valacyclovir
Structure of valacyclovirIn 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 MnO4− 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).
Scheme 2
General mechanisms of oxidation of VCH
General mechanisms of oxidation of VCHTo the best of our knowledge and literature survey, there are no kinetic studies reported for oxidation of VCH using KMnO4 in alkaline medium. Hence, the present investigation has been taken up to understand the mechanistic pathway of KMnO4 oxidation and to identify the product obtained in this reaction.
Experimental
Materials and reagents
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 KMnO4 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 (K2MnO4) solution was prepared as follows; an aqueous solution of KMnO4 was heated to boiling > 100 °C in alkali. A green solution of K2MnO4 formed and was characterized by its visible spectrum at 608 nm (ε = 1530 ± 100 dm3 mol−1 cm−1) (Figure 1). Further, it was used to verify the product effect on the rate of reaction.
Figure 1
The spectrum of Potassium manganate (K2MnO4) in an aqueous alkaline medium at 298 K [K2MnO4] = 1.0 × 10−4 mol dm−3 [OH−] = 0.05 mol dm−3
The spectrum of Potassium manganate (K2MnO4) in an aqueous alkaline medium at 298 K [K2MnO4] = 1.0 × 10−4 mol dm−3 [OH−] = 0.05 mol dm−3
Kinetic analysis
Kinetic analyses were carried out with [VCH] > [MnO4−], supporting pseudo-first-order condition by following absorption of MnO4− at its λmax, 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 MnO4− and VCH solutions, which also contained necessary concentrations of KOH and KNO3 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 ε = 2241 ± 30 dm3 mol−1 cm−1 (Lit. value = 2200 dm3 mol−1 cm−1). Since, the first-order plots, log10 (Abs.) versus Time were found to be linear up to 80% of the reaction, the rate constants, kobs were calculated from the slopes of such plots for various experimental conditions. The experimental results were reproducible within ± 5%.
Results and Discussion
Stoichiometry and product analysis
Different sets of reaction mixtures containing varying ratios of [VCH] to [MnO4−] at constant ionic strength, 0.1 mol dm−3 in presence of constant [OH−] and [NO3−] were kept for 24 h. in an inert atmosphere. The unreacted [MnO4−] in every case was determined spectrophotometrically at 526 nm. The results indicate a 1:2 (VCH: MnO4−) stoichiometry as shown in Eqn. (1). MnO42− as 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 μL/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).
Figure 2
LC-ESI-MS (+) spectra of VCH products
LC-ESI-MS (+) spectra of VCH products
Reaction orders
The reaction orders were calculated from the slopes of plot, log10kobs
versus log10 (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.
Influence of [permanganate]
Effect of [MnO4]− on the rate of reaction was studied by varying the [MnO4−] from 4.0 × 10−5 to 4.0 × 10−4 mol dm−3 at constant ionic strength by keeping all other conditions constant (Table 1). It was found that kobs values were constant for different [MnO4−] and also found to be linear and parallel in pseudo-first-order plots. The order in [MnO4−] was well-thought-out to be unity.
Table 1
Effect of variations of [MnO4−], [VCH] and [OH−] in the oxidation of VCH by alkaline permanganate at 298 K.
I = 0.1 mol dm−3
104 × [MnO4−]mol dm−3
103 × [VCH]mol dm−3
[OH−]Mol dm−3
103 × kobs (s−1)
103 × kcal (s−1)
0.4
3.0
0.05
2.63
2.12
0.6
3.0
0.05
2.23
2.12
1.0
3.0
0.05
2.72
2.12
2.0
3.0
0.05
2.72
2.12
3.0
3.0
0.05
2.16
2.12
4.0
3.0
0.05
2.30
2.12
2.0
0.8
0.05
1.18
1.11
2.0
1.5
0.05
1.63
1.59
2.0
2.0
0.05
1.89
1.82
2.0
3.0
0.05
2.24
2.12
2.0
6.0
0.05
2.62
2.55
2.0
8.0
0.05
2.89
2.70
2.0
3.0
0.01
1.04
1.01
2.0
3.0
0.03
1.87
1.79
2.0
3.0
0.05
2.10
2.12
2.0
3.0
0.07
2.35
2.30
2.0
3.0
0.09
2.45
2.41
2.0
3.0
0.10
2.50
2.45
kcal were calculated using k = 3.18 × 10−3 s−1, K1 = 4.42 dm3 mol−1, and K2 = 3.64 × 103 dm3 mol−1 at 298 K in rate eqn. (9).
Effect of variations of [MnO4−], [VCH] and [OH−] in the oxidation of VCH by alkaline permanganate at 298 K.kcal were calculated using k = 3.18 × 10−3 s−1, K1 = 4.42 dm3 mol−1, and K2 = 3.64 × 103 dm3 mol−1 at 298 K in rate eqn. (9).
Influence of [VCH]
Effect of VCH on rate was studied by varying its concentration between 8.0 × 10−4 and 8.0 × 10−3 mol dm−3 by keeping other conditions constant (Table 1). It had been noticed that kobs values increased with increasing [VCH]. The order in [VCH] was calculated from the plot of log10
kobs
versus log10 [VCH] and was found to be a positive fraction (0.4).
Influence of [OH−]
Effect of alkali on rate was studied by varying the [OH−] between 1.0 × 10−2 and 1.0 × 10−1 mol dm−3 by keeping other conditions constant at 298 K. It was observed that kobs values were increased with an increase in [OH−] (Table 1). The order in [OH−] was calculated from the plots of log10
kobs versus log10 [OH−] and was found to be a positive fraction (0.4).
Influence of ionic strength (I) and dielectric constant of the medium (D)
The effect of ionic strength on rate was carried out by varying the [KNO3] 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.
Table 2
Effect of ionic strength (I) and dielectric constant (D) on the oxidation of VCH by alkaline permanganate at 298 K.
Effect of ionic strength (I) and dielectric constant (D) on the oxidation of VCH by alkaline permanganate at 298 K.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 kobs values decreased (Table 2). The dielectric constants of their different compositions were calculated by considering their D in pure form using the equation:where V1 and V2 are volume fractions and D1 and D2 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.
Influence of initially added product
Effect of initially added product MnO42− on the rate of reaction was verified between the series of 1.0 × 10−5 and 1.0 × 10−4 mol dm−3 at 298 K by keeping all other conditions constant. The results indicate that the added product did not affect the rate.
Polymerization study
In the present study MnO4− 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 O2 and CO2 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 MnO4− 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.
Influence of temperature
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 kobs values are presented in Table 3.
Table 3
Effect of temperature on the oxidation of VCH by alkaline permanganate.
Effect of temperature on the oxidation of VCH by alkaline permanganate.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 dependent variable ‘y’. Most sets of kinetic data approximate this situation, in as much as times of observation are more accurately measurable than the chemical or physical quantities related to reactant concentrations. The straight-line selected by the common linear regression analysis is that which minimizes the sum of the squares of the derivations of the ‘y’ variable from the line. The slope ‘a’ and intercept ‘b’ parameters for the above equation can be calculated by linear regression analysis by any of several mathematically equivalent but different looking experiments.Most familiar arewhere ‘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 kobs
versus 1/T, activation parameters were figured out (Table 4).
Table 4
Activation parameters for the oxidation of VCH by alkaline permanganate at 298 K.
Activation parameters
Values
Ea (k J mol−1)
39 ± 2
∆H≠ (k J mol−1)
36 ± 1.5
∆S≠ (J K-1 mol−1)
− 165 ± 8
∆G≠ (k J mol−1)
86 ± 4
log A
4.0 ± 0.2
Activation parameters for the oxidation of VCH by alkaline permanganate at 298 K.The other activation parameters were calculated as follows.The Arrhenius factor ‘A’ was calculated by,The entropy of activation was calculated by using the equation,On substituting the universal gas constant ‘R’ as 8.314 J K−1 mol−1, the Boltzmann constant (k) = 1.3807 × 10−23 J K−1 and the Plank’s constant (h) = 6.630 × 10−34 J s.The kobs should be in s−1, and temperature in Kelvin, then the Ea results in J mol−1 and ΔS≠ in J K−1 mol−1. The enthalpy of activation was calculated by, ΔH≠ = Ea − RT and free energy of activation from ΔG≠ = ΔH≠ − TΔS≠.The rate constants (k) were obtained from intercept of 1/kobs versus 1/[VCH] for slow step (Scheme 3). Other equilibrium constants, K1 and K2 were obtained from the slope and intercept of the plots, 1/kobs versus 1/[VCH] and 1/[OH−] (Figure 3).
Scheme 3
Mechanism of oxidation of VCH by Permanganate in aqueous alkali.
Figure 3
Verification of the rate law (eqn. 9) for oxidation of VCH by alkaline permanganate at 298 K. Plot of (A) 1/kobs versus 1/[VCH] and (B) 1/kobs versus 1/[OH−] (Conditions as in Table 1).
Mechanism of oxidation of VCH by Permanganate in aqueous alkali.Verification of the rate law (eqn. 9) for oxidation of VCH by alkaline permanganate at 298 K. Plot of (A) 1/kobs versus 1/[VCH] and (B) 1/kobs versus 1/[OH−] (Conditions as in Table 1).In the present study, added [OH−] has a positive effect and thus combines with permanganate ion in 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 MnO4− complex (Scheme 3) is based on the MnO4− 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 MnO42− complex by the appearance of two new bands at 432 and 608 nm followed by the disappearance of permanganate bond at 526 nm.
Figure 4
Spectral changes during the oxidation of VCH by alkaline permanganate with scanning time interval of: (1) 1.0, (2) 2.0, (3) 3.0, (4) 4.0, (5) 5.0, (6) 6.0, (7) 7.0, (8) 8.0, (9) 9.0 min
Spectral changes during the oxidation of VCH by alkaline permanganate with scanning time interval of: (1) 1.0, (2) 2.0, (3) 3.0, (4) 4.0, (5) 5.0, (6) 6.0, (7) 7.0, (8) 8.0, (9) 9.0 minFormation of Mn5+ was rejected based on the absence of absorbance at 667 nm, expected for MnO43−. Further, the reduction of MnO4- is stopped[29,30] at MnO42− and become stable in alkali concentration maintained in this study.The reaction with 1:2 of [VCH]:[MnO4−] stoichiometry proceeded with pseudo-first-order dependence on [MnO4−] 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) of the proposed mechanism (Scheme 3), potassium permanganate combines with alkali to form alkali-permanganate ion [MnO4(OH)]2− and in succeeding step (2) the alkali-permanganate ion combines with the VCH molecule to form a complex (C). Formation of such complex (C) is confirmed kinetically by Michaelis–Menten plot (1/kobs
versus 1/[VCH] (Figure 3). The unstable complex (C) decomposes in a slow step to give a free radical (3) with the formation of MnO42−. The unstable free radical as an intermediate reacts with another molecule of [MnO4(OH)]2− in consequent fast step (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 Mn6+ by abstracting an electron leading to a CH(methylene) free radical. In the next step, cleavage of alkyl oxygen (AL2) 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 AL2 bond is found, leading the N–CH2–OH group on imidazole and is stabilized by an intramolecular hydrogen bonding.The other possibility of direct ‘2’ electron reduction was i.e., hypomanganate (MnO43−) to yield a final product. Such single step oxidation was rejected as the development of MnVO43− ion 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.According to Scheme 3,However,At low concentration ofThe term K1
K [OH−]f [MnO4−]f is neglected compared to 1 in the denominator as low concentration of MnO4− 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.The rate law (eqn. 9) has been proved by plotting of 1/kobs
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 × 10−3 s−1, K1 = 4.42 dm3 mol−1, and K2 = 3.64 × 103 dm3 mol−1 for 298 K were calculated. The K1 value obtained is in good agreement with the literature value of (6.6 dm3 mol−1).[29,34]Further, equilibrium constants K1, K2 along with k were used to regenerate kobs 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 ΔS≠ (− 165) strengthens a relatively rigid complex formation and hence its stability.[35] The higher negative value of ΔS≠ 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.
Conclusions
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 [MnO4(OH)]2− which was formed in a prior equilibrium step of the mechanism. The mechanism occurs through a complex formed between MnO4− and VCH. The relatively large value of kobs 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.
Supplementary Information (SI)
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, [MnO42−] 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.Below is the link to the electronic supplementary material.Supplementary file1 (PDF 24 kb)
Authors: G Andrei; R Snoeck; D Reymen; C Liesnard; P Goubau; J Desmyter; E De Clercq Journal: Eur J Clin Microbiol Infect Dis Date: 1995-04 Impact factor: 3.267
Authors: Christopher P Landowski; Duxin Sun; David R Foster; Sujatha S Menon; Jeffrey L Barnett; Lynda S Welage; Chandrasekharan Ramachandran; Gordon L Amidon Journal: J Pharmacol Exp Ther Date: 2003-05-15 Impact factor: 4.030
Scheme 1
Scheme 2
Figure 1
Figure 2
Scheme 3
Figure 3
Figure 4