Indi an Journal of Chemi stry Vol. 4 1 A, October 2002, PP. 2025 -203 1

Voltammetric studies of an oxazolone derivative of cefaclor in aqueous buffers

Shim Bali Prasad*, Sandeep Gupta & ShoLII'i Saneljee

Analy tical Di vis ion , Department of C hemi stry, Faculty of Science. Banaras Hindu Univers ity.

Varan as i 221005, India

Received 24 Novell/bel' 2000; rC I!i.l'ed 5 lilly 2002

El ectrochemi ca l propertie s of cdaclor at a mercury e lectrode in aqueous so luti on of varying p H have been stud ied through tast, pul se (normal and differenti a l). and cyc lic vo lt ammetri c techniques. For thi s, the e lec tro inacti ve cc fa clor is tran sformed into e lec troactive peni c illenate analogue 'oxazolo ne de ri va ti ve' under strong ac idi c hydro lysis conditi on. T he de ri ved oxazo lone system underwent two-steps irrevers ible reduct io ns in volving proton-participations (2e·. H+; 4e-, 4H+) when voltammograms scanned cathodi cally. However, in cyclic vo ltammetry, a typi ca l wave due to sul phhydryl g ro up ox idati on is noticed on scan reversa l in anodic direction . The results have been di sc ussed by taking into account the implications of reactant and product adsorpti ons o nto e lec trode surface during the course of reducti ons.

Earlier recognition of ~-I actam antibiotics as a pharmaceutical class of compounds led wide attention for their analysesl.2 , Cephalospori ns hav ing a reducible substituted methyl group at 3-posi ti on have been analysed vo ltammetrically'·6. However, other

penicillins and cephalosporins with ~-amino­phenylacetamido group at 6- and 7-positions , respect ively had to be converted into their respective electroactive species viz. monoketopyrazine7

.8 and

diketopi prazine') analogues for quantitative analyses.

Some workers have made electro-inert ~-lactam s active after intensive hydroly sis in aqueous acidic and basic media

,o-' 2. There are a few reports where

pol arog raphic studi es of in tact benzylpenicillins are I 'bl ' h f h' I' 1'1- 15 on y POSSI e el t er a ter t elr comp exatI on . or

nitrosation 16.

The present work in volves the parti c ipatio n of side chain amido group 111 the conversion of cephalosporins and penicillins into respective electroactive ' penicillenate ' derivative with oxazolone structure l7 in the same way as benzylpenicillin undergoes spontaneous degradation into benzy lpenicillenic acid under strong acidi c condition ' 8. ' 9. Earlier studies20 on benzylpenicillenic acid were typically centered on the electrochemical reactivity of sulfhydryl group. However, no study has been reported on the voltammetric behaviour of penicillenate analogues implicating the reduction of oxazolone moiety. Although a preliminary study on conventional polarography of simple oxazolone is reported2 1

.22

, implicatio ns caused by the adsorption of

CI 1~ SH o H I

O~ cH-g-~nls HOOC N-CH = C-C -= O - I I I I

NH2 0 N h CI H N",C/ O

COOH + I -o~ H3N-CH

(Al (B)-

Structure> -1

the reactant or product onto electrode surface have not been taken into account. In order to establish an estimation technique for such antibiotics at phys io logical trace level, the adsorption charac­teri stics of depolarizer, whi ch have significant impact over sensiti vity, warrants a comprehensive study. As a

model representative of ~-Iactam systems, cefaclor (structure lA) is selected for the present study.

Materials and Methods Cefaclor is found to be e lectrochemically inert at

all pH values. Its electroactive peni ci llenate analogue,

i. e., oxazolone, [4-alkylidene-2-(a-aminobenzy l)-2-oxazo l in-S-one deri vati ve of cefaclorl (structure IB ) was obtained through drast ic acid hydrolysis (0. I M HCI , 80 ± SoC, 2h) following the reported procedure '7.

18. In support of oxazolo ne structure (lB ),

a separate experiment was carried out in which the acid hydro lysis of benzylpenicillin instantaneously resulted in benzy lpenicill enic acid product. The voltammetric reduction of this product revealed similar peaks as those for oxazolone deri vati ve

2026 INDIAN J CHEM, SEC. A, OCTOBER 2002

obtained from cefactor. This supports exclusive attack of acetamido group to the ~-lactam in strong acid medi um.

All voltammograms [sample DC polarography (DCP-Tast), normal pulse polarography (NPP), differential pulse polarography (DPP), and cyclic

voltammetry (CY)] were recorded at - 25°C with the help of a PAR 264A voltammetric analyzer in conjunction with PAR 303A stand consisting of a mercury electrode (DME, HMDE, and SMDE modes), a platinum wire auxiliary electrode, and an Ag/ AgCl reference electrode with vycor frit. Typical parameters employed were drop time 1 s, scan rate (v) 10 mY S·I, modulation amplitude 25 mY, and drop surface area 0.0092 cm2

. The PAR model RE0089 X-Y recorder was used to record voltammograms.

For controlled potential electrochemical (CPE) reduction, we have used a cell in which the electrolysis was performed for 10 mL of oxazolone derivative solution in 40% (v/v) ethanol -0.01 N HCI at a mercury pool cathode (area 3.5 cm2

) maintained at the potential -1.0 Y vs. SCE. The exponential decay of current measured with time in this experiment responded the quantity of electricity and thereby an estimate of total number of electrons involved in the electroreduction.

Procedure Stock solution (1 mM) was prepared by dissolving

the weighed amount of oxazolone derivative of cefaclor in ethanol. The supporting electrolytes used consisted of a HCI-KCI mixture and citrate and phosphate buffers of desired pH prepared in 40% (v/v) ethanol; water in excess volume (> 60%) causes instant hydrolysis of the oxazolone. The ionic

strengths (~) of buffers were adjusted to 0.1 M with the addition of requisite amount of KCI. The appropriate amount of stock solution was transfelTed into IO mL volume of ethanolic buffer in a voltammetric cell and purged with pure nitorgen gas for 4 min. All voltammograms were subsequently run under the blanket of nitrogen in the usual manner. Voltammograms obtained were quite reproducible even after prolonged contact (- 12 h) with the acidic medium in the cell; this rules out the formation of related degradation products of oxazolone derivative (lB) in the present experimental conditions2J. However, in the basic media, voltammograms were found to be somewhat perturbed, owing to the alkaline hydrolysis'8 in the cell, if recorded after couple of hours.

Results and Discussion

For all solutions studied, two well-defined DCP­Tast cathodic waves (I and II) with their wave-heights in 1:2 ratio were observed. Interestingly, no wave was obtained for the electrochemical reactivity of sulfhydryl group present in the compound. The reason is quite obvious as the reactivity of -SH group with mercury at dropping mercury electrode (DME) is negligible during the shorter life (t = 1 s) of the drop in DCP-Tast than that (t = 3 s) with conventional polarograph/. The heights of waves I and 11 are observed to be constant upto pH 8 and pH 5, respectively. Beyond these pH val ues, both waves observe proton dissociation curve"" giving decreasing limiting currents . Unlike conventional DC polarograph/, oxazolone revealed its adsorptive characteristics in the present study by showing a pre­wave to wave I and a post-wave to wave 11 at all pH values in DCP-Tast mode. This confirms the adsorption of first reduction product at the electrode surface. It should be noted that reactant itself has shown adsorption characteristics by revealing a peak­like perturbation at the start of the first limi ting plateau. However, no distinct post-wave to wave I is observed.

Despite the fact that the composition of medium (40% v/v ethanol) may favour alkaline hydrolysis of oxazolone and not alcoholysis22

, the pertinent equilibrium (Structure 2) in alkaline medium (pH> 8.0) leading the cyclization of -SH group to thiozolidine intermediate (thiozolidenyl oxazolone isomer)23 does not approve further reduction owing to the absence of exocyclic C=C double bond in the isomer in contrast to the earlier work2I.22 . The slope (dEII2/dpH) values (Fig. 1) for both waves indicate that H+ ions are involved in the electrode reaction. The relative ease in reduction is dependent on the restricted electron donor capacity of cx-aminobenzyl group as compared to the phenyl substituent attached at 2-position22

•

The total number of electrons (Il) consumed in the complete reduction of oxazolone derivative was obtained as 6.0 by CPE experiment. This was apportioned between both waves on the basis of their wave-height ratio (l :2) . The electrode processes can be suggested by accounting the adsorption-desorption equilibrium OCCUlTing at the electrode surface prior to the cathodic reductions (Structure 2). After complete CUlTent decay in CPE experiment, the ether extract of resulting solution yielded on evaporation a solid compound. The IR (KBr, Vmax , cm" ) spectrum of this

PRASAD et 01.: VOLTAMMETRIC STUDIES OF OXAZOLONE DERIVATIVE OF CEFACLOR 2027

-}

SH CI ,

N-CH=C - C=O HOO C H N b

~I ~' + 'Y CH- NH3

~SH

CI N-CH2-CH- C~OH

HOOC H I

Structure - 2

NH2 (A) (8 )

product revealed the absence of any peak at 1600 cm-I

cOITesponding to -N=C-O- of the parent compound. However, the presence of an -OH peak at 3380 cm- I

,

overlapping with the required peak of -NHJ + group indicates the reduction of -N=C-O- ring into the corresponding -CH20H group of aminopropanol derivative (A). Other peaks were identical with those of the parent compound. The ethereal layer apparently contained am inopropanol derivative (A) with impurity of sparingly soluble aminophenyl acetic acid (B) (el Structure 2)21.22.

Typical NPP and OPP runs of oxazolone derivative (lB) in different buffers are shown in Fig. 2. The peak potentials of all NPP and OPP waves are almost consistent with their cOITesponding values in OCP­Tast and are found to be negatively shifting with pH. Since t.E is small pulse amplitude (25 mY), the summit potential (Es) obtained in the case of OPP lies close to the OCP-Tast half-wave potential and the

relationship, E I/2 = Es + t.EI2 holds true within the limit of error for all waves. The basic difference observed between OCP-Tast and pulse

U '" « '-'" « ,;, >

~ S

w

-l4

-1 ·2

-10

-08

-06

--0"4

-::r 0

I I , I

8 pH-

10 12

Fig. I-Effect of pH on the half wave potential (El l" ) for oxazolone derivative of cefaclor in DCP-Tast polarography: -. wave I and 0, wave II.

voltammograms is in their magnitude of reduction current. While the trend of wave-height variation for both waves in OCP-Tast is in 1:2 ratios, this is not true with the NPP and OPP runs. This may be attributed to the strong adsorption of the reactant and first reduction product at OME at any concentration , which did not desorb from electrode surface readil y

within the smaller time of pulse (T-T1 = 17 ms) and

pulse amplitude (25 mY) in NPP and OPP modes, respectively. Thus, the normal diffusion currents for both waves were drastically restricted at the coated electrode surface.

In NP.P, the first wave assumed an unusual peak­shape at all pH values, whereas the shape of second wave was observed as peak-like at lower pH (upto pH = 5.83) turning to be a drawn-out wave form at higher pH. The appearance of peak nature of NPP waves supports the coating of reactant and the first reduction onto electrode surface. This was evident from the well-resolved pre-peak to wave I at - -0.35 V vs. Ag/AgCI at all pH values. This peak was found to be merged with wave I at pH 2.13. The pre-peak is due to the adsorption of first reduction product. The post-peak to wave I is missing in both types of pulse voltammograms, despite the strong adsorption of the starting material. Owing to the adsorption of first reduction product, which is also a successive reactant for the second wave, the post-peak is observed to be either drawn-out at the foot of the wave II or appeared to be merging as a consequence of excessive adsorption complications during pre-electrolysis time.

2028 INDIAN J CHEM , SEC. A, OCTOBER 2002

-0·2 - 0 ·3 -0,4 -0'5 - 0 ·6 - 0 ·7 - 0 ' 8 - 0 ·9 -1 '0 - H -1 '2 - 1'3 -1'~ -1·5 -1-6 - 1'7 -1'8 Pot~ntiol (V V5 . Ag / Agel)

Fi g. 2-NPP and DPP runs of 0.087 111M oxazolo ne deri va ti ve o f cefac lor at difrerent pH va lues: (NPP): (a) 2.1J , (b) 4.98, (c) 7.97, and (d ) 10.06; (DPP) : (A) 2. 13, (8) 4.98, (C) 7.97, and (D) 10.06 . Dotted runs are pul se polarogral11 s of benzy lpeni ciilen ic ac id at p H 2.00.

The determination of half-peak width (W Il2) from the ex press ion, W l /2 = 3,25 RT/a n F, is not feas ible for unsymmetri cal peaks. The asymmetricity is due to the massive adsorption in OPP mode despite the small time el apsed between current sampling at the beginning and the end of the pulse signal, over the end of lives of many successive mercury drops. However, in those cases where the pre-and post­adsorption peaks are well separated from normal reduction peaks, the W 1/2 values are obtained to be greater than 60 m V for rhe first 2-electrons reduction peak and 70 m V for the second 4-electrons reduction peak. Furthermore, the corresponding electron­transfer coefficients (an) for both waves are found to be fractional values. Thus both electrode reactions in OPP mode are irreversible in nature.

Typical cycl ic voltammograms of oxazolone deri vative (lB) recorded with a triangulm potential sweep starting from -0.20 vs. Ag/ AgCl in aqueous solutions of di fferent pH values are shown in Fig. 3. All results consisting the effect of pH, scan rate, and concentration obtained from the treatment of various voltammetric runs (including those are not shown in representative figure) are given in Table 1.

All voltammograms revealed two major cathodic peaks (ipe l and ipez ) and one anodic peak (ipa) at all pH values. The height of first cathodic peak is higher than second peak. The unprecedented ri se in ipe l at pH 8.00 is quite surpri si ng; however, the fall in ipel and ipc2

beyond pH 8 and 7, respectively (Table 1) is apparently due to dissociation of protonated

oxazolone derivative making the reactant less available for the electron-transfer reactions. The fi rst peak (ipe l) is accompanied with an irreversible pre­peak in cathodic scan. No post-peaks to peak 1 and peak 2 are observed.

At all concentrations and pH values, the reactant

was adsorbed over the electrode surface. Though the post-peak to peak 1 is mi ssing, the cathodi c shi ft of first peak potential (Epl ) with equilibrium time (0 -

120 s) and diminishing trend of current (ipel ) afte r repetitive recording of voltammograms (Fig. 4) confirm the strong adsorption of the product which

partially blocked the electrode surface. The effect of cathodic increase in switching potential (E,.) at pH

2.13 resulted in decreasing anodic current (ipa) (Fig. 3, dotted runs) . Thi s shows that the adsorbing first reduction species had sufficient time to block the electrode surface for the anodi c oxidation.

Cyclic voltammograms have shown a negative shift in peak potential with pH variation fo r both cathodic and anodic branches of voltammogram revealing the

direct involvement of proton-participation in electrode processes. The appearance of an anodic peak in the reverse scan of cyclic voltammogram is interesti ng (no anodic oxidation is observed in OCP-Tast and pulse voltammetry as these are recorded by potential

. scanning in the cathodic direction) . Since the fi rst cathodic reduction is totally irreversible, the peak il'a

cannot be considered as an anodic component of ipe l '

PRASAD et al.: VOLTAMMETRIC STUDIES OF OXAZOLONE DERIVATIVE OF CEFACLOR 2029

~ ~==============:===::~ __ ~~ __ -=====~~IP=C2===========d========1 I

It--__ o I I-_--_-=_-=_:-::_o-_--_-_-::::,~ IPo

- 0 -2 -0-3 -0-1; -0-5 -0-6 -0,7 -0·8 -0-9 -1,0 -1 -1 -1 '2 -1'3 -1·1; -1 -5 -1- 6 -1-7

Potential ( V VS_ Ag/AgCI)

Fig. 3--Cyclic voltammograms of 0.087 mM oxazolone derivative of cefaclor at scan rate 0.200 V S· I and different pH values: (a) 2. 13, (b) 4.98, (c) 7.97, and (d) 10.06. Dotted run shows the change in EA'

TO.025~ 0·1 V

.1t

Fig. 4-Repetitive cyclic voltammograms of 6.5 x 10.5 M oxazolone derivative of cefaclor at pH 7.00 after 120 s accumulation at -0.5 V.

This peak is attributed to the oxidation of sulfhydryl

group present in the compound as depicted in Structure 3.

Though the oxidation peak (ipa) is ill-defined and the corresponding peak height could be much affected due to adsorption perturbation at the electrode surface, this peak appeared explicitly when E"A was kept less cathodic. The negative shift of anodic peak potential with pH (Table 1) reflects the ease of oxidation in solutions of lower acidities. The anodic peak appears to be reversible as peak potential remained practically constant with variation of scan rate (Table 1).

The total non-reversible character of cathodic waves is evident by a shift of Ep towards negative potential by increasing scan rate and the lack of corresponding anodic peak at all scan rates (Table 1).

The non-linearity of ipCI and i pc2 with scan rate variation (Table 1) reveals the adsorption complications due to both reactant and product over

SH ~SH9

CI~N+ +Hg ~ CI }~+ + H+ + fl-

HOOC H HOOC

Structure - 3

electrode surface. The accumulation of different amount of the product at electrode is dependent upon the availability of time and concentration of adsorptive species. The adsorbed species on the electrode surface may attain the equilibrium, Reactant(strongly adsorbed) Reactant(dissol ved), at the electrode-electrolyte interface within the time span of voltammetry. Here the difference in energy for the reduction of adsorbed and dissolved species is small and thus the separate post-peak are not observed to both peaks. Under such circumstances, both adsorbed and diffused reactants contribute to the current resulting in enhanced peak current compared with that, in the absence of adsorption. With the increase of scan rate, the relative contribution of the effect of adsorption is virtually lessened and this makes a larger surface area available for diffusion-controlled cathodic reduction resulting in an increase in ipJv' 12

with log V for both reductions . The strong adsorption of the first reduction product is evident by the appearance of a pre-peak to peak 1. Both reactant as well as first reduction product are adsorbed at identical value of free energy of adsorption (t-,CO). The adsorption of both species is probably due to intramolecular conjugation in the structure. However,

203U INDIAN J CHEM, SEC. A, OCTOBER 2002

Table I---Cyclic voltammetric results of oxazolone derivative of cefaclor (0.087 mM) at 25°C"

pH Scan ra te Peak Ep i pc i p,lv 112

2.13 0 .100 -0.475 1.20 3.80 2 -0 .825 0.45 1.42

(-0.420)

0.200 1 -0.480 1.90 4 .25 2 -0.830 0.75 1. 68

( -0.420)

0.500 -0.485 3.80 5.37 2 -0 .835 1.45 2.05

(-0 .420)

3 .55 0 . 100 1 -0 .550 0.70 2 .2 1 2 -0.895 0.10 0.32

(-0.480)

0.200 -0 .560 0.85 1. 90 2 -0.910 0.40 0.89

(-0.480)

0.500 1 -0.570 1.40 I. 98 2 -0 .955 0.65 0.92

( -0.480)

5.83 0 . 100 -0.665 0.50 1.58 2 -1.025 0 . 10 0.32

(-0.600)

0 .200 -0.670 0 .85 I. 90 2 -1.040 0.25 0.56

(-0.600)

0.500 -0 .685 1.25 1. 77 2 -1.065 0.60 0.85

(-0.600)

9.11 0.200 -0 .785 1. 90 4.25 2 -1.95 0.25 0.56

(-0. 795)

0.500 1 -0.795 3.65 5.16 2 -1.205 0.45 0.63

(-0. 720)

"Unit s: if! = Cathodic pea k curren t ( IlA): Ep = Ca thodic p eak p o tenti a l (V V.I'. Ag/A gC I): Values given in parenth eses denote po te ntial of anodic peak (V vs. Ag /AgC I) ; v = Scan rate (V S· I). "On ly mid d le sc an. rate s data in rep resen tati ve bu ffers (fl = 0.1 M) are g iv e n .

the second reduction product as obtained at higher negative potential, is not adsorbable due to the lack of conjugation in its structural moiety .

The first CV peak is found to be facilitated as it is preceded by a protonation equilibrium and followed

by the strong adsorption of reduction product onto hanging mercury drop electrode (H MDE) surface (Structure 2). Under such condition, the electrode kinetics may be considered to be dependent on protonation constant and electrode coverage (8). The

PRASAD et af. : VOLT AMMETRIC STUDIES OF OXAZOLONE DERIV ATIV E OF CEFACLOR 2031

surface-attached first reduction product in the present instance imparts less uncovered fraction (1 - 8). Also the occupied film after the first reduction might restrict the rate of electron-transfer to some extent by partially blocking the electrode surface. This is why the cathodic cun-ent in CV, contrary to the DCP-Tast, for the second reduction is found to be smaller than that for the first reduction .

Acknowledgement The ass istance rendered by UGC under DSA

programme is greatly acknowledged .

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