Studies on the oxidation of levofloxacin by N-bromosuccinimide in acidic medium and their mechanistic pathway

Journal of Molecular Liquids 218 (2016) 604–610

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Studies on the oxidation of levofloxacin by N-bromosuccinimide in acidic medium and their mechanistic pathway Aftab Aslam Parwaz Khan a,b,⁎, Anish Khan a,b, Abdullah M. Asiri a,b, Salman Ahmad Khan a a b

Chemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia Center of Excellence for Advanced Materials Research, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 5 January 2016 Accepted 19 February 2016 Available online xxxx Keywords: Levofloxacin Oxidation N-bromosuccinimide Rate laws Mechanism Activation parameters

a b s t r a c t Kinetics and mechanism of oxidation of levofloxacin(LF) by N-bromosuccinimide (NBS) have been studied in acid medium with pH and ionic strength at 25 °C. It is a first order reaction with respect to both LF and NBS, but inverse first order in succinimide (reduction product of NBS). The stoichiometry of the reaction is 1:2 in this investigation. The effects of added products, halide ion and ionic strength have also been investigated. Polymerization was not observed with acrylonitrile. The main oxidation products as, (3S)-9-fluoro-3-methyl-10-(4methylpiperazin-1-yl)-5,7-dioxo-2,3,6,7-tetrahydro-5H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid and succinimide were identified by Fourier transform infrared spectroscopy (FTIR), Liquid chromatography–mass spectrometry (LC–MS), Nuclear magnetic resonance (NMR) and Thin-layer chromatography (TLC) data. The rate of reaction was examined with reference to variation of ionic strength, solvent polarity, free radicals, halides ions, mercuric acetate for fix up the Br − in reaction mixture and also addition of the product succinamide (RNH).·On the basis of the experimental results, the mechanism of the reaction has been proposed and supported by kinetic orders, spectrophotometric evidence and negative entropy of activation parameters. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Levofloxacin [S-isomer of racemic OFL] and 3D views shows in Fig. 1 is one of the commonly used fluoroquinolones class of antibiotics. They are synthetic broad spectrum antibacterial drugs that exhibit significant activity against gram-positive and gram-negative bacteria. [1]. LF is valued for their broad spectrum of activity, excellent tissue penetration, and for their availability in both oral and intravenous formulations [2].It has been used for the treatment of infectious diseases, including pneumonia and abdominal infections [3,4]. Several techniques and procedures were reported in the scientific literature for complex formation, determination fluorescence quenching and also the evidenced results of oxidation of levofloxacin using the oxidants such as permanganate and hexacyanoferrate(III) [5–15]. We have also published of well-defined mechanistic approach for the oxidative decarboxylation by chloroamine-T in acidic medium [16]. In this work focuses on the another oxidizing agent N bromosuccinamide for the oxidative studies. Among many N-halo compounds available, N-bromosuccinimide was chosen as an oxidant for the oxidation of LF, since NBS is a source of positive halogen, is well understood. In particular, it is known for its capacity to oxidize a variety of substrates in both acidic and alkaline solutions [17–21].We have also published Micro concentrations of Ru(III) ⁎ Corresponding author at: Chemistry Department & Center of Excellence for Advanced Materials Research (CEAMR), Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia. E-mail address: [email protected] (A.A.P. Khan).

http://dx.doi.org/10.1016/j.molliq.2016.02.051 0167-7322/© 2016 Elsevier B.V. All rights reserved.

used as homogenous catalyst in the oxidation of levothyroxine by NBS for the mechanistic pathway [22]. To our knowledge, our efforts described in this work constitute the report of a novel, efficient procedure of oxidative studies of LF by NBS and elucidation of the mechanistic pathway of oxidation. A literature survey revealed no mention of the mechanistic details of the oxidation of LF with N-bromosuccinimide. The objectives of the present work are to elucidate plausible reaction mechanisms, synthesize a complex that decomposes at the slow step in the mechanism, obtain and identify the reaction products and compare the reaction behavior at different temperatures to determine the activation energy. 2. Experimental section 2.1. Materials and methods LF was obtained from Sigma. All other reagents were of Merck analytical grade or Fluka. Solutions were prepared by using double distilled water in all kinetic runs and preparations, commercial sample of NBS was used as such. Standard solution of NBS was prepared in water and its purity was checked iodometrically. 2.2. Instrumentation The absorbance spectra were measurement and recording of absorption spectra of reactants and product with double beam UV–vis

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605

Fig. 1. Chemical structure of LF with 3D view.

spectrophotometer (UVD-2960). FT-IR spectrometer (Perkin-Elmer, spectrum 100, Waltham, MA, USA) was recorded in the range of 600 to 4000 cm−1.All pH-metric measurements were done with EL20 Education Line pH Meter from Mettle Toledo Inc., Quartz cuvette and all Glasswares were cleaned by immersion in nitric acid and subsequently rinsed thrice with deionized water to remove traces of any impurities deposited on it. 3. Experimental 3.1. Kinetic preparation and studies The oxidation of LF by NBS was followed under pseudo-first order conditions where its concentration was maintained by keeping [LF]NNN[NBS]. The reaction was carried out in glass stopper pyrex boiling tubes, whose outer surface was coated black to eliminate photochemical effects. Requisite amounts of NBS, HCl and water were taken in the tube and it was placed in an electrically operated thermostat for thermal equilibrium. The first order rate constant Kobs was evaluated from a plot of log [NBS] versus time (Fig. 2B). The reaction was followed spectrophotometrically at 440 nm. The spectral changes during the reaction under standard conditions at room temperature are given in Fig. 2A.The results

were reproducible within ±4% in repeat kinetic runs. Regression analysis of experimental data to obtain regression coefficient ‘r’ and the standard deviation ‘s’, of points from the regression line, was performed with the Microsoft Office Excel-2010 and OrginPro 6.1 Program. 3.2. Stoichiometry reaction between LF and NBS The stoichiometry of the reaction was studied adopting the limiting logarithmic method [23,24]. The ratio of the reaction between (log Abs versus log [LF], log [NBS] were calculated by dividing the slope of NBS over the slope of the drug curve. It was found that, the ratio was 1:2 (LF to NBS) and accordingly, stoichiometric equation for the oxidation of LF by NBS may be written as Eq. (1).

ð1Þ

Fig. 2. Uv–Vis spectra (A) and first order plot (B) of product, (3S)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-5,7-dioxo-2,3,6,7-tetrahydro-5H- [1,4]oxazino[2,3,4-ij]quinoline-6carboxylic obtained during the oxidation of LF by NBS in acidic medium solutions recorded at room temperature.

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3.3. Characterization of the product of LF The main product in the form of stoichiometric proportion under stirred condition was reasonable to run for one day in an inert atmosphere at 35 °C. After completion of 24 h the reaction products checked by spot tests, and extracted with ether. The products obtained were (3S)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-5,7-dioxo2,3,6,7-tetrahydro-5H- [1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid with 3D view shown in Fig. 3 and succinimide. The Fourier transforms infrared spectra of the main reaction product was recorded in wavelength range 4000 to 650 cm−1 as shown in Fig. 4 where, the appears of absorption band at 1647 cm−1 and 1623 cm−1 which is characteristic band of acidic and ketonic C_O stretching respectively; 2971 cm− 1 is due to CH3 stretching of the piperazine moiety; and a broad peak at 3361 cm−1 is due to ν (OH) stretching (Fig.IR). The product was also confirmed by 1H NMR (DMSO) spectra where, the acidic OH protons appeared at 7.92 δppm and NH of methyl piperzine moiety singlet appears at of 2.30 δppm and the another singlet of NH of benzoxazine moiety at 3.82 δppm,and also the singlet of phenolic OH at 4.23 δppm ppm, which disappears on D2O exchange and confirms the formation of product (3S)-9-fluoro-3-methyl-10-(4-methylpiperazin1-yl)-5,7-dioxo-2,3,6,7-tetrahydro-5H- [1,4]oxazino[2,3,4-ij]quinoline6-carboxylic acid (Scheme 1) and The reduction product of NBS, succinimide (RNH) was detected by spot test [25]. The product was also further confirmed by using an Agilent 1260 liquid chromatograph (Agilent Technologies, Wilmington, DE), coupled within Agilent 6400 triple quadruple mass spectrometer (Agilent Technologies, Wilmington, DE, USA). The mass spectrum showing an M/Z parent ion peak at 330 amu clearly confirms (3S)-9-fluoro-3-methyl-10-(4methylpiperazin-1-yl)-5,7-dioxo-2,3,6,7-tetrahydro-5H- [1,4]oxazino [2,3,4-ij]quinoline-6-carboxylic (Fig. 5). Other peaks observed in the spectrum can be interpreted in accordance with the observed structure. 4. Results and discussion Kinetics of oxidation of NBS by (NBS) in Sodium acetate/acetic acid buffered medium was investigated under pseudo-first-order kinetics, that is, [LF]. [NBS]. The rate was measured at the start of the slow reaction by measuring the absorbance of the oxidation products at λ = 440 nm for a definite period of time at fixed ionic strength, pH, and temperature and for a range of NBS and LF concentrations. 4.1. Effect of acid concentration The reaction between LF and NBS was performed in different acid media. Better results were obtained in hydrochloric acid medium. The effect of acid concentration on the reaction between LF and NBS was studied by varying the concentration of HCl keeping the concentrations of NBS, LF, ionic strength, pH and temperature fixed. The reaction was found to be rapid yielding a constant absorbance with maximum

sensitivity and stability when the HCl concentration was maintained in the range of 0.10–1.00 mol dm−3. An increase in acid concentration was found to accelerate the rates of the present oxidation reaction. The reaction order with respect to [H+] was found to be of fractional order. 4.2. Effect of [LF] The dependence of the rate constant on LF concentration was determined at different [LF] from 5.0 × 10−4 to 5.0 × 10−3 mol dm−3 at constant values of other reactants at 25 °C. The kobs increased with increase in the concentration of [LF] but it was found to be less than unity. The results are summarized in Table 1. 4.3. Effect of [NBS] The [NBS] was varied from 2.0 × 10−4 mol dm− 3 to 2.0 × 10−3 mol dm−3 at constant concentration of other reactants at 25 °C (Table 1). The pseudo-first order plot of log [NBS] versus time was linear and slope was found to be constant for all the varied [NBS] (Table 1.). Thus, order in [NBS] is considered as a unity. 4.4. Effects of varying ionic strength and relative permittivity of the medium The effect of ionic strength was studied by the NaClO4 concentration (0.1–0.8 mol dm−3) had no significant effect on the rate. The effect of change in dielectric constant (D) of the medium on the reaction rate was studied by increasing the percentage of acetic acidH2O content (20–50% (v/v)) of the reaction medium when all other reactants concentration and other conditions being constant. The plot of log kobs versus 1/D was linear and had a positive slope. The values of D for various CH3CN–H2O mixtures were calculated from the following equation: The values of D for CH3CN–H2O v/v was calculated from the following equation. D ¼ DW V W þ DA V A where DW and DA are the dielectric permittivities of H2O and CH3CN and VW and VA are the volume fractions of H2O and CH3CN. The blank experiments of reacting with NBS and LVT with alone CH3CN did not affect on the rate of the reaction. 4.5. Effect of halides ions The effect of halides ions on the reaction rate was studied at a constant HCl concentration of (1.5 × 10−3 mol/dm3). The addition of NaCl had no effect on the kobs, confirming that the observed effects of HCl were due solely to changes in [H +]. Similarly, the addition of NaBr

Fig. 3. Chemical structure of the product of LF with balls & sticks 3D image.

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Fig. 4. FT-IR spectrum of product, (3S)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-5,7-dioxo-2,3,6,7-tetrahydro-5H- [1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic obtained during the oxidation of LF by NBS in acidic medium.

did not show any effect on the rate. These results indicated that halide ions play no role in the reaction mechanism. 4.6. Effect of [Hg(OAc)2] The effect of [Hg(OAc)2] observed in the range 1 × 10−3to5 × 10 moldm−3 was found to be negligible effect on the rate of reaction. The function of added Hg(OAc)2 is therefore only to fix up Br- formed in the course of reaction as HgBr2 or HgBr2− 4 . −3

4.7. Effect of succinimide on the rate It is noteworthy that even when the succinimide concentration was varied in the range of 1.0 × 10−4 to 1.0 × 10−5 mol dm−3 and all other parameters were held constant, had retards the reduction product of the oxidant on the rate of reaction.

4.10. Effect of temperature The effect of temperature on the reaction rate was studied for all the kinetic runs at four different temperatures (20–37 °C), keeping other experimental conditions constant. The activation parameters (Ea, ΔH# and Δ S#) have been evaluated using Arrhenius and Eyring equations from the linear Arrhenius plot of log kobs versus 1/T. 4.10.1. Mechanism and derivation of rate law A probable mechanism for the oxidation of LF by NBS has been proposed based on the experimental. In general, reactions of NBS is a two equivalent oxidant which involve changes many substrate in the form of Br+, N+ HBr and hypobromite anion. The oxidation potential of NBS fluctuate with the pH of the medium [26]. Depending on the pH of the medium, NBS hand over different types of reactive species in solutions as shown in equations

4.8. Effect of pH

RNBrþ Hþ →RNþ HBr

ð2Þ

The effect of pH on the absorbance of the produced color was studied at lmax over the pH range 1.0–8.0. Different buffer solutions (acetate, monochloroacetate, and phosphate) were tested. Sodium acetate/acetic acid buffer solution gave the best results. Highest absorbance values were achieved at pH 3.6, which was used in all experiments.

RNBrþ H2O→RNHþ HOBr

ð3Þ

HOBrþ OH− →OBrþ H2 O

ð4Þ

RNBrþ Hþ →RNHþ Brþ

ð5Þ

4.9. Test for free radical intermediates

Brþ þ H2 O→H2 OBrþ

ð6Þ

RNBrþ OH− →RNHþ OBr−

ð7Þ

To get more information about the reaction mechanism, acrylonitrile was added to the reaction mixture. No polymerization found, so the oxidation of LF does not occur via free radical formation mechanism.

Scheme 1. A general Scheme for oxidation of LF by NBS in acidic medium.

(where, R represents (CH2CO)2 group). In acidic solutions, the probable reactive species of NBS are NBS itself or Br+ or protonated NBS viz., RN+ HBr, and the reactive species in alkaline solutions are NBS, HOBr or OBr−. Furthermore, if Br+ or H2OBr+ are chosen as possible oxidizing species, then it forms to retardation of the rate of succinimide avoid us to take H2OBr+ as the active oxidizing species. As such, there is no clear choice of reactive oxidizing species of NBS in acidic medium. As prescribed in the literature, all measurements have been performed in the presence of mercuric (II) acetate to avoid any possible oxidation of bromine. Mercuric (II) acetate acts as a capture or unionized agent for Br formed in the reaction and exists as HgBr2− 4

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Fig. 5. LC–MS spectra of the product, 3S)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-5,7-dioxo-2,3,6,7-tetrahydro-5H- [1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic showing a molecular ion peak at 330 amu.

HgBr2. Its presence ensures that oxidation takes place purely through NBS [27]. The results of oxidation of LF with NBS in acidic medium indicated the first-order dependence each on the concentration of NBS, LF and H+. Acceleration of the rate by increasing concentration of H+ assumes that, protonated species of NBS, i.e., (CH2CO)2N+ HBr is the most likely oxidizing species. It is known that NBS serves as a source of bromonium ion (Br+) and undergoes a simple two-electron reduction leading to the formation of bromide ion, succinimide and the products of reaction [28]. The protonated NBS reacts with LF to form a complex which further undergo hydrolysis and intramolecular rearrangement to form products. In Scheme 1, X and X′ are the intermediate species whose structures are shown in Scheme 2, where a detailed mechanistic interpretation of LF oxidation with NBS in acid medium is proposed. Herein, we report kinetics of oxidation of antibacterial agent LF by NBS. The characteristic band for the investigated complex in the UV/visible spectra lies at λmax = 340–600 nm. The absorption spectrum of the Table 1 Calculated values of rate constants for the variations of [NBS], [LF] and [H+] at 35 °C (Scheme 2). [NBS] (×104mol dm−3) Variation of [NBS] 2.0 4.0 6.0 8.0 10.0 12.0 Variation of [LF] 6.0 6.0 6.0 6.0 6.0 6.0 Variation of [HCl] 6.0 6.0 6.0 6.0 6.0 6.0

[LF] (×103 mol dm−3)

[H+] (×102 mol dm−3)

Kobs (×104 s−1)

4.0 4.0 4.0 4.0 4.0 4.0

20.0 20.0 20.0 20.0 20.0 20.0

3.25 3.22 3.26 3.28 3.27 3.28

1.0 2.0 4.0 6.0 8.0 10.0

20.0 20.0 20.0 20.0 20.0 20.0

2.43 2.96 3.26 3.38 3.93 4.16

4.0 4.0 4.0 4.0 4.0 4.0

10.0 15.0 20.0 25.0 75.0 100.0

2.92 3.16 3.26 3.92 4.26 5.11

reaction product has maximum absorption at 440 nm as shown in Fig. 2A. Repeated spectral scans show first-order kinetics of the oxidation of LF by NBS in acidic medium. These results were supported by the naked eye observation that change occurs in the yellow color of the complex fades slowly until the solution turned colorless and optically clear at the end of each run. These spectrophotometrically and visually observable facts would suggest the following mechanism for the oxidation of LF (Scheme 2). The reaction between NBS and LF in the presence of H+ ion has a stoichiometry of 1:2 with a first order dependence on the [NBS] and less than a unit-order dependence on both the [H+] and [LF]. No effect of added products such as RNH and halides ions was observed. It appears that the first acid combines with RNBr to form an acid succinimide species RN+ HBr. In the second step, RN+ HBr reacts with LF to form a intermediate complex. Further formed complex reacts with another 1 mol of H2O in a fast step to give the products, 2[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]acetaldehyde and succinimide identified by FTIR,NMR and TLC. To determine the overall rate from Scheme 1: RNBr Rate ¼ − ¼ k3 ½X dt

ð8Þ

If [RNBr]t represents the total effective concentration of NBS in solution can be written as.   ½RNBrt ¼ ½RNBr þ RNþ HBr þ ½X

ð9Þ

which leads to the following rate law: ½RNBr ¼

 þ  RN HBr  þ K1 H

 þ  RN HBr ¼

X K 2 ½LF

ð10Þ

ð11Þ

Substituting Eqs. (10) and (11) in Eq. (9): ½X ¼

  K 1 K 2 ½RNBrt ½LF Hþ  þ   1 þ K 1 H þ K 1 K 2 ½LF Hþ

ð12Þ

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Scheme 2. Detailed mechanistic pathway for the oxidation of LF by NBS in acidic medium.

By substituting [X] from Eq. (12) in Eq. (8): Rate ¼

  K 1 K 2 k3 ½RNBrt ½LF Hþ  þ   1 þ K 1 H þ K 1 K 2 ½LF Hþ

ð13Þ

Further rate law Eq. (13) is in agreement with the experimental data. Since. rate ¼ kobs ¼ ½RNBrt

ð14Þ

temperatures to obtain various values of activation parameters. (Table 2) From the Arrhenius plots of log Kobs versus 1/T, activation energy, and other thermodynamic parameters for the oxidation of LF by NBS have been calculated. The effect of dielectric constant (D) of the reaction medium on the reaction rate was investigated by varying the solvent composition of CH3CN content in CH3CN–H2O solvent mixtures. In the present investigation a plot of logkobs vs. 1/D was linear with a positive slope supports

Rate law Eq. (13) can be substituted in Eq. (14):   K 1 K 2 k3 ½LF Hþ   Rate ¼ 1 þ K 1 Hþ ½1 þ K 1 ½LF

ð15Þ

1 1 1 1  þ þ ¼ kobs K 1 K 2 K 3 ½LF Hþ K2 K3 ½LF K3

ð16Þ

According to Eq. (16), the values of K1, K2 and K3 are calculated from the slope and intercept of such plot of 1/kobs vs. 1/[LF] and 1/kobs versus 1/[H+] are linear (Fig. 6).These slopes and intercepts of such plots yield K1, K2 and K3 and the constants value were used to calculate the rate constants at different experimental conditions and compared with experimental kobs values. They are found to be in reasonably agreeing with each other which fortifies the mechanism as in Scheme 2. The thermodynamic quantities for the first and second equilibrium steps of Scheme 2 can be evaluated. The reactions were studied at different

Fig. 6. Double reciprocal plots of 1/kobs versus 1/[LF] and 1/kobs versus 1/[H+]. Experimental conditions are as in Table 1.

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Table 2 Effect of different temperature on the reaction rat and activation parameters for the oxidation of LF by NBS in acidic medium at fixed ionic strength and pH. Temperature (K)

`1/T × 103

Kobs (×104 s−1)

293 298 303 308

3.413 3.356 3.300 3.246

2.92 3.26 4.50 5.12

fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-5,7-dioxo-2,3,6,7tetrahydro-5H- [1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid by FTIR, LC-MS, and NMR. The active oxidizing species present RNHBr in acidic medium. Initially added of RNH in the reaction system did not induced any effect on the rate of reaction. Activation parameters and the reaction constants were calculated. The observed results have been explained from proposed mechanism and the rate law has been investigated.

Activation parameters Ea (kJ mol−1) ΔH± (kJ mol−1) ΔS±(J K−1 mol−1) ΔG± (kJ mol−1)

88.55 85.22 −76.12 90.14

the proposed mechanism. The dependence of the rate constant on the dielectric constant of the medium is given by the following equation: logkobs ¼ logk0 −

2

ZA ZB N 1  2:303ð4πε0 ÞdAB RT D

where ko is the rate constant in a medium of infinite dielectric constant, ZA and ZB are the charges of reacting ion, dAB refers to the size of activated complex, T is absolute temperature and D is dielectric constant of the medium. This equation shows that if a plot is made between log kobs versus 1/D straight line having slope equal to − ZAZB and e2N/ 2.303(4πεo)dABRT will be obtained. The effect of solvent on the reaction kinetics has been described [29–31]. Eq. (17) predicts a linear relation between log kobs versus 1/D. The slope of the line should be negative for a reaction between a negative ion and a dipole or between two dipoles, while a positive slope is obtained for positive ion–dipole reactions. In the present investigations, plots were linear with positive slopes, supporting the observation indicates the positive ion-dipole nature of the rate determining step in the reaction sequence and also points to extending of charge to the transition state (Scheme 1). The negligible influence of added succinimide and halide ions on the rate are in agreement with the proposed mechanism. The proposed reaction mechanisms are supported by the observed values of the enthalpy of reaction Δ H# and free energy of reaction Δ G# were calculated obtain a high positive values of the free energy of activation and of the enthalpy of activation support the formation of highly solvated transition state. The ΔS# value is surrounded by the expected range for radical reactions and has been attributed to the nature of pairing and unpairing electron processes and, the loss of degrees of freedom in the past presented to the reactants upon the formation of a rigid transition state [32–33]. The negative value of ΔS# indicates that complex is more ordered than the reactants [32–37]. Further, The experimental results indicated that, there is no significant effect of the ionic strength on the oxidation rates and supported that the reaction took place between charged and non charged species. To avoid any possible bromine oxidation, all kinetics studies were made with [mercuric acetate] greater than [NBS] which simply means that Br2 oxidation was completely suppressed. Mercuric acetate acted as a scavenger for any bromide (Br) formed in the reaction thus ensuring that oxidation took place purely through NBS itself.23,24. 5. Conclusion In view of the experimental results and the kinetic interpretations, it is obvious that oxidation of LF by NBS in acidic medium leads to the formation of the product. The oxidation products were identified (3S)-9-

Acknowledgments This project was funded by the Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah, under grant no. CEAMR-SG-9-437.

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