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LSER AND TLSER ANALYSIS OF SOLVENT EFFECT ON THE SENSITIZED PHOTOOXYGENATION OF LIDOCAINE.

 

A. L. Zanocco,* E. Lemp M., N. Pizarro U.,

J. R. de la Fuente and G. Günther S.

Universidad de Chile, Facultad de Ciencias Químicas y Farmacéuticas Departamento de Química Orgánica y Fisicoquímica

Casilla 233, Santiago - 1, Santiago, Chile, FAX: 56 - 2 - 6782878, e-mail: azanocco@ll.ciq.uchile.cl

INTRODUCTION

Lidocaine, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide, is a local anaesthetic that reduces ventricular arrhythmia associated with myocardial infarction, myocardial infarct size and ischemic myocardial injury (1). The protective effects of lidocaine have been attributed to its membrane stabilizing properties, by acting as a short-lived free radical scavenger. Since lidocaine plays a protective role in ischemic myocardial damage (2) and has been used in cardioplegic solutions its antioxidant properties have been investigated. Das and Misra (3) using EPR, found that lidocaine is a powerful hydroxyl radical scavenger. Furthermore, these authors propose that lidocaine is a powerful scavenger of singlet oxygen. Also, they found that lidocaine was more effective than b -carotene, sodium azide and histidine in quenching singlet oxygen. However, there are no kinetic data accounting for its reactivity towards singlet oxygen in the different microenvironments present in biological systems, and no data regarding reaction mechanism.

In this work we report kinetic results obtained on the sensitized photo-oxidation of lidocaine using both steady state and time-resolved methods. Additionally, LSER and TLSER correlations were employed to analyze kinetic solvent effects and explain differences between lidocaine reactivity and that of a typical aliphatic amine. The acetamido group present in lidocaine (Fig. 1) is strongly electron withdrawing, which will affect the reactivity of the diethylamino group towards singlet oxygen, and these groups may interact intramoleculary, e.g., by hydrogen bonding, which will be solvent dependent.

Figure 1. Structure of lidocaine

EXPERIMENTAL

Chemical reaction rate constants were determined by using a 10 ml double wall cell, light-protected by black paint. A centred window allows irradiation with light of a certain wavelength by using Schott cut-off filters. Circulating water maintained the cell temperature at 22 ± 0.5 °C. Rose Bengal (l max = 557 nm) was the sensitizer. Illumination was with a visible, 200 W, Par lamp. In these experiments lidocaine consumption was monitored by using a Hewlett Packard 5890 gas chromatograph equipped with a NPD detector.

Steady state competitive experiments were performed in a cell allowing irradiation with light of a selected wavelength by using a Schott cut-off filter. The temperature was 22 ± 0.5 °C. In apolar solvents, Rubrene (l max = 520 nm) was the sensitizer. In polar solvents or in buffered solutions (pH = 5 and 10) Rose Bengal (l max = 557 nm) was the sensitizer. Illumination was with a visible, 150 W, Par lamp.

Time resolved phosphorescence measurements were carried out in 1 cm path fluorescence cuvettes. Phenazine excitation was by absorption of the third harmonic (355 nm, ca. 15 mJ per pulse) of the 6-ns light pulse of a Quantel Brilliant Q-Switched Nd:YAG laser. When TPP or Rose Bengal was the sensitizer, excitation was by absorption of the 500-ps light pulse of a PTI model PL-202 dye laser (419 nm or 556 nm, ca. 200 m J per pulse). A PTI model PL-2300 nitrogen laser was used to pump the dye laser. A liquid N2 cooled North Coast model EO-817P germanium photodiode detector with a built-in preamplifier was used to detect infrared radiation from the cuvette. The detector was coupled to the cuvette at a right-angle. The only elements between the cuvette face and the diode cover plate were an interference filter (1270 nm, Spectrogon US, Inc.) and a cut-off filter (995 nm, Andover Corp.). The preamplifier output was fed into the 1 MW input of a digitizing oscilloscope Hewlett Packard model 54540 A. Computerized experiment control, data acquisition and analysis were performed by LabView based software developed in our laboratory.

RESULTS AND DISCUSSION

Chemical reaction of lidocaine with singlet oxygen. Rate constants for the chemical reaction between lidocaine and O2(1D g) were determined in methanol, acetonitrile and N,N-dimethylformamide. Lidocaine consumption was followed during the reaction. Rate constants for the chemical reaction, kRLID, are (1.05 ± 0.061) x 105 M-1 s-1, (1.42 ± 0.073) x 105 M-1 s-1 and (0.61 ± 0.046) x 105 M-1 s-1 in acetonitrile, methanol and N,N-dimethylformamide, respectively.

By using the Mair method (4) for hydroperoxide determination, a concentration equivalent to 0.0153 M of hydroperoxide was found when 0.03 M lidocaine in acetonitrile was irradiated for 12 h in the presence of Rose Bengal. The amount of hydroperoxide produced agrees with the consumption of lidocaine. Although we cannot isolate reaction products in quantities adequate for spectroscopic characterization, a rough idea of the product distribution was obtained by GC-MS analysis of the main lidocaine derivatives produced in the photooxidations. When 0.03 M lidocaine was irradiated for 12 h in the presence of Rose Bengal the results shown in Fig. 2 a) are obtained with the mass spectrometer in the positive chemical ionization (CI+) mode. Only four peaks appear in the chromatogram, the main one, with a retention time of 15.59 min, is that of unreacted lidocaine. Fig. 2 b) shows that the mass spectrum is that of lidocaine. The CI+ and EI (not included) mass spectra corresponding to peaks at retention times of 14.57, 13.22 and 7.77 min, indicate that 2-(ethylvinylamino)-N-(2,6-dimethylphenyl)-acetamide, 2-(1-azapropily-den)-N-(2,6-dimethyl-phenyl)-acetamide and 2,6-dimethylaniline are the probable main products of photooxidation of lidocaine. Figs. 2 c), 2 d) and 2 e), show the CI+ mass spectra and corresponding structures.

 

Figure 2.a) GC-MS chromatogram of 30 mM lidocaine in acetonitrile after 12 h of irradiation in the presence of Rose Bengal. b) CI+ mass spectrum of compound with retention time 15.58 m. c) CI+ mass spectrum of compound with retention time 14.67 m. d) CI+ mass spectrum of compound with retention time 13.22 m. e) CI+ mass spectrum of compound with retention time 7.76 m.

Physical quenching of singlet oxygen by lidocaine. In most of the studied solvents, total rate constants, kTLID, for the reaction of lidocaine with O2(1D g), were determined by using time-resolved phosphorescence. Figure 3, shows a typical Stern-Volmer plot for quenching of singlet oxygen by lidocaine. Values of kTLID, in different solvents, were obtained from slopes of these plots. The insert shows the luminescence decay of singlet oxygen at 1270 nm in acetone as solvent and Rose Bengal as sensitizer. Decays obtained over several concentrations of lidocaine are included in this figure. The lifetime of O2(1D g) was obtained from single exponential decays in the absence or presence of variable concentrations of lidocaine.

Due to the low water solubility of lidocain, values of kTLID were determined by competitive methods with Rose Bengal as sensitizer and 9-anthrylmethanol whose reactivity is known. In heptane, values of kTLID were determined from inhibition of the Rubrene autooxidation rate. Table 1 includes values of kTLID in different solvents from the various methods.

 

  • Figure 2. Stern-Volmer plot for deactivation of singlet oxygen by lidocaine in acetone as the solvent. Insert (a) Singlet oxygen phosphorescence decay at 1270 nm, following dye laser excitation at 414 nm, with Rose Bengal as sensitizer in acetone. (b) and (c) as a), but with 1.6 mM and 9.6 mM of lidocaine, respectively.

  • Table 1. Total rate constants for reactions of O2(1D g) with lidocaine, kTLID, and triethylamine, kTTEA, in different solvents.

    •

     

    Solvent

    kTLID/106 M-1 s-1

    kTTEA/106 M-1 s-1

    1

    n-Heptane

    1.10 ± 0.08c

    68.8 ± 3.4

    2

    n-Hexane

    1.24 ± 0.05ª

    66.2 ± 2.6
       

    1.29 ± 0.06b

    		  

    3

    Diethylether

    3.30 ± 0.16b

    91.5 ± 3.2

    4

    Dioxane

    2.53 ± 0.13b

    275.2 ± 9.1

    5

    Ethyl Acetate

    5.26 ± 0.17b

    190.4 ± 7.6

    6

    Tetrahydrofurane

    7.02 ± 0.32b

    221.1 ± 6.6

    7

    Benzene

    2.00 ± 0.11c

    198.5 ± 7.1
       

    2.69 ± 0.09a

    		  

    8

    Tributylphosphate

    39.4 ± 0.16b

    71.4 ± 3.5

    9

    Anisole

    3.67 ± 0.19b

    314.8 ± 9.4

    10

    Propylene Carbonate

    8.15 ± 0.36b

    258.1 ± 8.9

    11

    N,N-dimethylformamide

    31.70 ± 1.27b

    347.6 ± 10

    12

    N,N-dimethylacetamide

    45.8 ± 2.40a

    422.2 ± 12

    13

    Benzonitrile

    4.28 ± 0.21b

    254.2 ± 9.1

    14

    Acetone

    6.14 ± 0.28b

    216.7 ± 7.6

    15

    Methylene chloride

    1.42 ± 0.06b

    128.4 ± 5.1

    16

    Acetonitrile

    2.36 ± 0.09d

    165.1 ± 6.6
       

    3.17 ± 0.15a

    		  
       

    4.10 ± 0.19b

    		  

    17

    Chloroform

    0.98 ± 0.05a

    45.3 ± 2.3
       

    0.90 ± 0.04b

    		  

    18

    Benzylic alcohol

    2.48 ± 0.11b

    15.2 ± 0.8

    19

    Formamide

    2.81 ± 0.12a

    21.6 ± 1.1

    20

    i-Propanol

    4.30 ± 0.22b

    27.8 ± 1.4

    21

    n-Octanol

    3.40 ± 0.16b

    30.4 ± 1.6

    22

    n-Hexanol

    -

    22.2 ± 1.0

    23

    i-Pentanol

    3.04 ± 0.13b

    19.7 ± 1.2

    24

    n-Pentanol

    3.52 ± 0.16b

    26.2 ± 1.3

    25

    n-Butanol

    3.54 ± 0.14b

    22.3 ± 1.1

    26

    n-Propanol

    2.96 ± 0.12a

    14.8 ± 0.7

    27

    Ethanol

    2.72 ± 0.13d

    23.6 ± 1.4
       

    2.93 ± 0.15a

    		  

    28

    Methanol

    2.08 ± 0.09a

    12.7 ± 0.5

    29

    Trifluoroethanol

    0,20 ± 0.09a

    -

    a sensitizer : Phenazine or TPP, Nd-YAG Laser

    b sensitizer : TPP or Rose Bengal, Dye Laser

    c sensitizer : Rubrene, Steady State Method

    d sensitizer : Rose Bengal, Steady State Method

    A comparison of kRLID and kTLID values shows that kTLID is greater than kRLID by between one and two orders of magnitude in the same solvent, indicating that the main path for interaction of lidocaine with singlet oxygen corresponds to physical quenching of excited oxygen by the anaesthetic, thus the rate constant for this process, kQ, approximately equals kTLID.

    Quenching of singlet oxygen by aliphatic amines is well studied (5,6), and is explained in terms of reversible formation of an exciplex, via charge-transfer interactions, due to electrophilic attack of excited oxygen on the amino group. The exciplex yields products by chemical reaction or undergoes intersystem crossing to regenerate amine and triplet oxygen.

    This behavior, expected for lidocaine, fits our steady state and time resolved experiments. Photooxidation product distribution, hydroperoxide detection, dependence of kTLID on pH for reaction in aqueous solution and the increase of lidocaine reactivity towards singlet oxygen in more polar aprotic media, indicate that reaction between singlet oxygen and lidocaine occurs via formation of a charge transfer exciplex followed by physical quenching or chemical reaction. Scheme 1 shows a mechanism compatible with these observations.

    Scheme 1

    However, although these results are consistent with a common mechanism for reactions of lidocaine or aliphatic amines with singlet oxygen, several differences require additional explanations. Mainly, values of kTLID are between one and two orders of magnitude lower than those previously reported for typical tertiary amines (7). This lower reactivity of lidocaine relative to tertiary amines is not easily understood in terms of steric effects and we note that dependence of kTLID on solvent has a different pattern from that for amines (7). For instance, kT values in acetonitrile are (2.36 ± 0.09) x106 M-1 s-1 and 33.3 x 107 M-1 s-1 (21) while in methanol, values of kT are (2.08 ± 0.09) x 106 M-1 s-1 and 1.3 x 107 M-1 s-1 for lidocaine and triethylamine, respectively. Tertiary aliphatic amines dramatically decrease their reactivities towards singlet oxygen when the media changes from aprotic polar solvents, such as acetonitrile or methylene chloride, to protic polar solvents such as aliphatic alcohols. In contrast to this well-established behavior, lidocaine reactivity in aprotic polar solvents is very close to that in aliphatic alcohols.

    In order to understand the solvent effect on kTLID and explain the lower reactivity of the drug we measured the rate constant for reaction between triethylamine (a typical tertiary amine) and singlet oxygen, kTTEA, in the solvents employed to determine kTLID. These results are included in Table 1.

    LSER and TLSER analysis of solvent effect on kTLID and kTTEA. With the aim of rationalizing differences in lidocaine reactivity relative to that typical of aliphatic amines and solvent effects on kTLID, we analyzed the kT dependence on microscopic solvent characteristics by using the semiempirical solvatochromic equation (LSER) of Taft, Kamlet et al. (Eqn. 1) (8).

    Log k = log ko + s (p * + d d ) + a a + b b + h r 2H (1)

    Also, we analyze the dependence of kTLID on the solvent by using a theoretical set of correlation parameters determined solely from computational methods. The theoretical linear solvation relationship (TLSER) descriptors have been developed to give optimal correlation with the LSER descriptors. The generalized TLSER equation proposed by Famini et al. (9,10), (Eqn. 2) can be used to analyze chemical reactivity.

    Log k = log ko + a r 2H + b p 1 + c e b + d q- + e e a + f q+ (2)

    The correlation equations obtained for the dependence of kTLID and kTTEA on the solvent parameters are included in Table 2. The results show that not all the descriptors are significant. Descriptor coefficients accepted in the correlation equation were those that have a significance level ³ 0.95.

    Table 2. LSER and TLSER correlation equations for the reactions of singlet oxygen with lidocaine and triethylamine.

    Log k = Log ko + a p * + b a + c b

    TEA

    Log ko

    a

    b

    c

    Coeff.

    7.924

    0.321

    -1.305

    0.362

    ±

    0.111

    0.151

    0.134

    0.184

    t-stat.

    71.126

    2.121

    -9.705

    1.967

    P(2-tail)

    < 0.0001

    0.0444

    < 0.0001

    0.0609

    VIF

    		  

    1.068

    1.721

    1.869

    N = 28 R = 0.927 SD = 0.200 F = 48.995

    LIDOCAINE

    Log ko

    a

    b

    c

    Coeff.

    5.946

    0.425

    -0.813

    1.318

    ±

    0.096

    0.128

    0.081

    0.121

    t-stat.

    62.106

    3.318

    -10.074

    10.872

    P(2-tail)

    < 0.0001

    0.0029

    < 0.0001

    < 0.0001