Ester Hydrolysis and Nitrosative Deamination of Novocaine in

594

Chem. Res. Toxicol. 2006, 19, 594-600

Ester Hydrolysis and Nitrosative Deamination of Novocaine in Aqueous Solutions Emilia Iglesias-Martinez,* Isabel Brandariz, and Francisco Penedo Departamento de Quı´mica Fı´sica e Enxenerıá Quı´mica I, Facultad de Ciencias, UniVersidad de La Corun˜ a, 15071-La Corun˜ a, Spain ReceiVed January 19, 2006

In aqueous solutions, the kinetic features of both the hydrolysis reaction of the ester function of novocaine in alkaline medium and the nitrosation reaction of the primary amine group of novocaine in mild acid medium were investigated by UV/vis spectroscopy. The ester hydrolysis shows first-order kinetics with respect to both the drug and the nucleophile, OH-, concentrations, thus following a typical SN2 (Ac) mechanism. The rate of the reaction decreases strongly with the polarity of the reaction media, analyzed for both dioxane-water and Me2SO-water mixtures. The effect of the presence of cationic micelles of tetradecyltrimethylammonium bromide, TTABr, was abnormal in that it inhibits the rate of the reaction throughout the analyzed concentration range of the surfactant. The same pattern of behavior is observed in the presence of anionic micelles of sodium dodecyl sulfate (SDS); however, the effect is more pronounced. The rate equation obtained in the kinetic study of the nitrosation reaction of novocaine in mild acid medium contains first- and second-order terms with respect to [nitrite], which correspond with the two parallel reaction paths due to nitrosation via both NO+ and N2O3, respectively; the rate of the reaction also increases with both the [H+] and the total acetic acid-acetate buffer concentration. In contrast to the ester hydrolysis, the nitrosation reaction is accelerated in aqueous micellar solutions of both cationic and anionic surfactants of TTABr and SDS, respectively. Introduction Anesthesia was discovered more than a century ago, but the mechanism of action of anesthetics is still unknown and is the subject of intense experimental and theoretical work (1-4). According to some models, anesthesia can be regarded as an interfacial phenomenon involving molecular processes that occur at the interface between biomembranes and their aqueous environment (5, 6). Chemical compounds used as local anesthetics are capable of blocking the initiation or conduction of nerve impulses following three main steps: (i) diffusion of the uncharged form of the local anesthetic to the membrane interface, (ii) reequilibration between the uncharged and the cationic forms in the aqueous environment, and (iii) penetration into and attachment to a receptor at a site within the sodium channels. However, not all substances displaying such properties have been accepted for clinical use as local anesthetics. Most of the local anesthetics now in use are amphiphilic molecules, i.e., molecules containing both a hydrophilic group (usually an amino moiety) and a hydrophobic group (generally containing an aromatic ring). Among the compounds displaying such chemical structure, the tertiary amine type molecules, e.g., novocaine and its derivatives, make up an important class of synthetic drugs of this therapeutic family (7). Tertiary amines induced some structural changes in plasma membrane reorganization that affects in transport properties of the membranes (8-10). However, local anesthetics often show a short duration of action and adverse side effects, such as cardiac and neurological toxicity, accompanied sometimes by allergic reactions. The origin of these secondary effects is often attributed to modifications of biomembranes molecular structure and permeability * To whom correspondence should be addressed. E-mail: [email protected].

properties, change of enzyme activities, immune response, etc. Therefore, it is important that the anesthetic drugs are studied with regard to three main aspects: their molecular structure, which is closely related with the third step of the mechanism of action; their dissociation equilibrium, which specifies the acidity conditions of the application point according to the first step of the mechanism; and their reactions under different conditions, which are of crucial importance in their duration during the application process. Therefore, this work reports the acid-base equilibrium and reactivity study of aqueous solutions of the local anesthetic novocaine, a tertiary amine that also contains a primary amine group linked to an aromatic ring; then, depending on the acidity of the medium, novocaine may exist as a neutral molecule (No),1 a monocation (NoH+), or a dication (NoH2+2). We studied the acid-base equilibrium of No, as well as its stability in both basic and acidic media. For that, we performed the kinetic study of the ester hydrolysis reaction in alkaline medium and the nitrosation of the primary amine group in acid medium. This latter reaction is widely used in the volumetric titration of this drug from the coupling of the diazo compound formed in the nitrosation reaction and the Bratton-Marshall reagent (Scheme 1).

Experimental Procedures Novocaine hydrochloride (NoHCl) was purchased from Sigma with a purity higher than 99% and was used without further purification. All other reagents were commercially available reagent grade and were used as received. All of the solutions were freshly prepared with double-distilled water over potassium permanganate. 1 Abbreviations: TTABr, tetradecyltrimethylammonium bromide; SDS, sodium dodecyl sulfate; No, neutral novocaine; NoH+, monoprotonated novocaine; NoH2+2, diprotonated novocaine.

10.1021/tx060013b CCC: $33.50 © 2006 American Chemical Society Published on Web 03/22/2006

Ester Hydrolysis and NitrosatiVe Deamination of NoVocaine

Chem. Res. Toxicol., Vol. 19, No. 4, 2006 595

Scheme 1. Reactions that Occur in the Titration of Novocaine

The pH was controlled using buffer solutions of acetic acidacetate and its chloride derivatives and was measured with a Crison 2001 pH meter equipped with a GK2401B combined glass electrode and calibrated using commercial buffers of pH 4.01, 7.02, and 9.26 (Crison). The reported pH values refer to the pH of the same sample used to record the spectra or to measure the kinetic rate constant. The reported [buffer] refers to the total buffer concentration. The UV/vis spectra and kinetic experiments were recorded with a Kontron-Uvikon double beam spectrophotometer fitted with thermostated cell holders at 25 °C and following the decrease in absorbance at 285 nm. Data acquisition and analysis of both UV/ vis spectra and kinetics were performed with software supported by the manufacturer and converted to ASCII format for their analysis with common packet programs. The kinetic measurements of absorbance vs time always fit the first-order integrated rate equation giving quite satisfactory correlation coefficients (r > 0.999).

Results and Discussion The UV/vis spectra of 6.5 × 10-5 M novocaine in aqueous solution were collected at different acidity conditions. Figure 1a shows that in strong acid medium (HCl at [H+] ) 0.05 M) the highest intense peak is centered around 228 nm, and a weaker broad band is extended between 260 and 310 nm; by contrast, in neutral medium, the weaker peak is centered at 221 nm, whereas the strongest absorption appears at approximately 290 nm, which, in alkaline medium, shifts with time to shorter wavelengths until it reachessat an infinite timesthe maximum absorption at 265 nm (Figure 1b); two well-defined isosbestic points are drawn that indicate that the absorption is due to two species, the initial novocaine and p-aminobenzoate anion generated in the ester hydrolysis. Ionization Equilibrium. In pharmacology, the pKa controls many aspects of the drug metabolism, including transport through membranes that are frequently permeable only to a particular species. In a similar manner, the effects of a drug of known pKa can be considered in light of the properties of the various species existing under the appropriate pH conditions. The novocaine molecule possesses two basic centers: a primary amine group attached to a phenyl ring and a tertiary amine group, whose pKa values are largely different since no interaction effects are expected between both centers. The analysis of the UV/vis spectrum of novocaine reveals that this analytical method is adequate to measure the ratio of dication/monocation (NoH2+2/NoH+) as a function of pH. This first ionization constant, pKa1, was then determined by dissolving the same amount of No (6.5 × 10-5 M) in aqueous solutions of acetic acid-acetate buffer or its chloro and dichloro derivatives at a 0.30 M concentration of known pH values. The absorbance of the samples was read at λ ) 289 nm as a function of pH. The results are displayed in Figure 2, where the curve was drawn in the fit of eq 1 to the experimental points with the

Figure 1. (a) UV/vis spectra of aqueous solutions of novocaine (6.5 × 10-5 M) in water (1) added as a hydrochloric salt, (2) in 0.30 M NaOH, and (3) in 0.050 M HCl. (b) Repeat scans every 3 min showing the reaction spectrum of alkaline hydrolysis of No at [OH-] ) 0.30 M; (-O-O-) at infinite time.

following values of the optimized parameters: Aa ) 0.03 ( 0.01 (the absorbance of the full diprotonated form), Ab ) 1.207 ( 0.008 (the absorbance of the monoprotonated form measured at pH 4.9), and pKa1 ) 2.31 ( 0.05 (r ) 0.9993).

A289 )

Aa 10pKa1 + Ab 10pH 10pKa1 + 10pH

(1)

The second ionization constant, pKa2, that corresponds to the acidity of the tertiary amine group appears referenced in the literature as 9.05 (11), 9.30 (12), 9.24 (13), or even 8.1 (14) all at 25 °C. Ester Hydrolysis. In aqueous alkaline medium, novocaine undergoes hydrolysis of the ester function at moderate reaction rates. The kinetic study of the reaction was conducted by conventional UV/vis spectroscopy noting the decrease in

596 Chem. Res. Toxicol., Vol. 19, No. 4, 2006

Figure 2. Absorbance readings of the novocaine aqueous solutions (6.5 × 10-5 M) as a function of the pH of the solution. The sigmoide curve represents the simulated results from eq 1 and the results reported in the text.

Figure 3. Variation of the pseudo-first-order rate constant, ko, for the alkaline hydrolysis of novocaine as a function of [OH-].

absorbance at 285 nm. Under pseudo-first-order conditions with the novocaine as the limiting reagent ([No] ) 6.5 × 10-5 M) and by applying the integration method, the absorbance-time (A-t) data were fit to the first-order integrated rate equation At ) A∞ + (Ao - A∞)e-kot to obtain the pseudo-first-order rate constant, ko, with At, Ao, and A∞ being the absorbance at t, zero, and infinite time. Figure 3 shows the influence of OHconcentration on reaction rate; ko increases with [OH-] being negligible in the reaction in the absence of OH- (eq 2); in fact, in aqueous buffered solutions of carbonate-bicarbonate buffer of pH 10.5, novocaine is quite stable and no appreciable hydrolysis was observed during days. The alkaline hydrolysis, due to the attack of the nucleophile OH- (a hard nucleophile) to the C atom of the carbonyl group (a hard electrophile because of the large electronegativity difference between C and O atoms), is a charge-controlled reaction (15).

ko ) kw[OH-]; kw ) (4.10 ( 0.04) × 10-3 M-1 s-1 (2) At [OH-] ) 0.17 M, the rate of the reaction was measured in aqueous mixtures of both dioxane and Me2SO solvents. Lowering the polarity of the reaction medium causes a strong reduction of the observed rate constant, with ko vs % v/v solvent profiles fitting first-degree exponential decays (Figure 4). It can be seen that under 40% v/v the reduction factor of approximately 2.5 in Me2SO [µ ) 4.05 D, ∈ ) 46.5 (16)] increases up to approximately 7 in dioxane [µ ) 0.45 D, ∈ ) 2.2 (16)].

Iglesias-Martinez et al.

Figure 4. Plot of ko obtained in the alkaline hydrolysis of novocaine at [OH-] ) 0.17 M as a function of the percentage of the solvent dimethyl sulfoxide (DMSO) (2) and dioxane (b) in the aqueous reaction sample.

Figure 5. Influence of TTABr concentration on the observed rate constant, ko, for the alkaline hydrolysis of novocaine at [OH-]0.13 (b) and 0.30 M (2).

Nucleophiles such as OH- react with esters by substitution rather than addition-elimination. The carboxylic acid group and its derivatives possess thermodynamic stabilization; therefore, reactions on these functional groups tend to retain this stabilization. The tetrahedral intermediate formed in the first step of the reaction of the ester function with OH- needs stabilization by solvation. Solvents with low polarity parameters (dipole moment, µ, and/or dielectric constant, ∈) show low solvating power of either reagents or transition state, and consequently, the activation energy increases in reaction media of low polarity. The ester hydrolysis reaction was also studied in aqueous micellar solutions of both cationic and anionic surfactants of tetradecyltrimethylammonium bromide (TTABr) and sodium dodecyl sulfate (SDS), respectively. In the presence of either cationic or anionic micelles, the reaction is strongly retarded; nevertheless, the effect of anionic micelles is approximately 100fold higher, a fact that makes the reaction extremely slow to follow it by continuous recording methods (ko is of the order of 10-6 s-1). Figure 5 displays values of ko measured as a function of TTABr concentration corresponding to two OH- concentrations of 0.13 (circles) and 0.30 M (triangles). Under these experimental conditions, the substrate (novocaine) is a neutral molecule of high hydrophobicity. Then, a strong association of the drug molecules to either TTABr or SDS micelles is expected. In the presence of cationic micelles, there is also a substantial



Table 1. Parameters Obtained in the Alkaline Hydrolysis of No in the Presence of Cationic Micelles of TTABr [OH-] (M)

kow (10-4 s-1)

Ks (M-1)

cmc (mM)

r

0.13 0.30

5.23 9.85

149 ( 8 152 ( 9

1.8 1.5

0.9997 0.9998

Scheme 2. Sketched 2D Structure of No Molecules into TTABr Micelles

amount of OH- in the micellar interface due to the ionic exchange process stated in eq 3 with KI ) 0.05 (17). Consequently, a catalysis of the hydrolysis reaction due to concentration effects of both reagents into the small volume of the micelle was expected. Typical results of the alkaline hydrolysis in the presence of cationic micelles go to maxima (18, 19). KI

OHw- + Brm- y\z OHm- + Brw-

(3)

By contrast, the experimental data show a continuous decrease of ko against [TTABr], whose profiles fit quite well in eq 4, where kow () kw[OH-]w) is the rate constant measured in water and Ks refers to the association constant of No molecules to TTABr micelles, being [TTABr]m ) [TTABr]t-cmc (cmc, critical micelle concentration). As [OH-]t () [OH-]w + [OH-]m) is kept constant throughout the surfactant concentration range, the kow could not be a real constant if [OH-]w decreases significantly. Nevertheless, because of the small value of KI, the highest [OH-]m was estimated lower than 10% of [OH-]t; hence, the corrected ko values no longer differentiate from the experimental ones. Therefore, the optimized values of Ks by applying eq 4, which correspond to the solid lines in Figure 5, are reported in Table 1. These results indicate that at approximately [TTABr] ) 0.1 M, 95% of total novocaine molecules are bound to micelles, where the rate of the hydrolysis is nil or very slow. To explain the absence of reaction of the substrate in the micelle, we have to consider first that the polarity of the micellar interface is lower than that of the bulk water phase (Figure 4 illustrates the strong effect of this parameter) and second that the orientation adopted by the novocaine molecules inside the micellar interface must protect the ester group from the attack of the OH- that exists in the micellar interface. As sketched in Scheme 2, the ester group is deep inside the micelle where the dielectric constant of the microenvironment surrounding the drug could be lower than 35 [the estimated value of the TTABr interface (20)]. In conclusion, the comparison of Figures 4 and 5 indicates that the micellar effect can be considered as a solvent effect similar to that of water-solvent mixtures.

ko )

kow 1 + Ks[TTABr]m

(4)

Amine Nitrosation. The ingestion or inhalation of nitrosating agents has been known to cause in vivo nitrosation, whose

Figure 6. (a) Plot of the observed rate constant, ko, against [nitrite] obtained in the kinetic study of the nitrosation of novocaine in aqueous buffered solutions of acetic acid-acetate ([buffer] e 0.20 M) of pH 4.30 (1), 4.50 (b), 4.75 (2), and 4.95 ([). (b) Linear plot of the previous results according to the linear form of eq 5.

significance greatly increases with the discovery of endogenous NO synthesis and the emergence of NO-releasing drugs. NO has a key role in the regulation of many biological processes including vasorelaxation, blood clotting, neuronal plasticity, and cytotoxic activity; nevertheless, NO is unable to react with nucleophiles under oxygen free conditions, suggesting that its higher oxides, such as N2O3, were actually nitrosylating agents and that the oxidation of NO to N2O3 is facilitated by micellar catalysis. Therefore, endogenous nitrosation involves mainly N2O3, which leads to DNA base deamination and interstrand cross-linking, causing a variety of disorders and diseases due to the alteration of both the activity and the function of a protein. Hence, a more detailed and quantitative understanding of nitrosation is required (21-26). In this work, we analyzed the kinetic characteristics of the nitrosation reaction of the primary amine group of novocaine to yield the diazonium ion, performed in mild acid conditions of acetic acid-acetate buffers. The progress of the reaction was followed by UV/vis spectroscopy by registering the absorbance decrease at 289 nm with the nitrite concentration in high excess over the drug concentration. Thus, the decay kinetics are wellbehaved and are easily fitted to first-order integrated rate equation by nonlinear least-squares regression. At constant pH and total buffer concentration, the effect of nitrite concentration on ko was studied. Representative results are shown in Figure 6a. The ko vs [nitrite] profiles indicate an increase of the reaction order with increasing [nitrite]. This kinetic feature evidences the existence of two parallel pathways

598 Chem. Res. Toxicol., Vol. 19, No. 4, 2006

Iglesias-Martinez et al.

Table 2. Rate Constants Obtained in the Nitrosation of No under Acetic Acid-Acetate-Buffered Solutions in the Analysis of the Effect of Nitrite Concentration (Eq 5)

Table 3. Rate Constants Obtained in the Nitrosation of No at [Nitrite] ) 3.3 × 10-3 M in the Analysis of the Influence of [Buffer] at Fixed pH (Eq 7)

[buffer] (M)

pH

k1 (M-1 s-1)

k2 (M-2 s-1)

(k1/k2) (10-3 M)

pH

koH (10-3 s-1)

kB (10-3 M-1 s-1)

(kB/koH) (M-1)

0.15 0.15 0.20 0.21

4.30 4.52 4.70 4.95

0.42 ( 0.02 0.252 ( 0.003 0.106 ( 0.006 0.080 ( 0.004

60 ( 2 38.8 ( 0.3 15.65 ( 0.6 8.5 ( 0.4

7.0 6.5 6.8 9.3

4.30 4.52 4.70 4.95

0.87 ( 0.02 0.53 ( 0.01 0.189 ( 0.009 0.121 ( 0.005

7.19 ( 0.07 4.95 ( 0.05 1.75 ( 0.03 1.14 ( 0.02

8.26 9.34 9.26 9.42

of rate constants k1 and k2, in agreement with the linear plots of Figure 6b, in accordance with the linear form of eq 5.

the [H+] of the buffer used and kB is the added counterpart by the presence of the buffer.

ko ) k1 [nitrite] + k2 [nitritre]2

ko ) koH + kB[buffer]

(5)

A rate equation consisting of a sum of two terms indicates the existence of two parallel reaction paths involving transition states of different composition generated in the nitrosation of the primary amine group of novocaine by the two major nitrosating agents present in the reaction medium under the experimental conditions: NO+, responsible for the first-order in [nitrite], and N2O3, responsible for the second-order in [nitrite]. Both nitrosating agents are generated in aqueous acid solutions of nitrite according to the equibrium reactions of eq 6 with K1 ) 3.5 × 10-7 M-1 and K2 ) 3 × 10-3 M-1 at 25 °C (27-29). K1

K2

HNO2 + H+ y\z NO+; 2HNO2 y\z N2O3

(6)

Values of the rate constants k1 and k2, obtained from each set of experiments performed at fixed pH and similar total buffer concentration, are collected in Table 2. Both rate constants increase with the medium acidity as a consequence of the enhancement of the nitrosating agent concentrations as stated in eq 6. Entry 5 in Table 2 reports the ratio of k1/k2, which as can be seen, is independent of pH within the experimental errors. Taking into account that the pKa of HNO2 is 3.15 (30, 31), the total nitrite concentration at pH > 4 is practically in the form of NO2-. Then, the equilibrium reactions stated in eq 6 lead to [NO+] ) [nitrite]K1[H+]2/Ka and [N2O3] ) [nitrite]2K2[H+]2/ Ka and, thus, the quotient of these expressions, which determines the acidity dependence of k1/k2, is independent of the acidity. The influence of the total buffer concentration on the nitrosation reaction has been analyzed at both fixed pH and [nitrite] ()3.3 mM). The variation of ko as a function of [buffer] is displayed in Figure 7. The ko vs [buffer] profiles fit eq 7, where koH represents the observed rate constant measured at

Figure 7. Plot of the pseudo-first-order rate constant, ko, as a function of the total buffer concentration measured in the nitrosation of novocaine at [nitrite] ) 3.3 mM and pH 4.30 (1), 4.50 (b),4.75 (2), and 4.95 ([).

(7)

Both rate constants are influenced by the pH of the reaction medium by increasing their values when the pH diminishes; see Table 3. The nitrosating agents concentration increases parallel to [H+] according to eq 6 with the concomitant increase of koH; in the same sense, at high pH (and high [buffer]), more important is the concentration of the acid form of the buffer, AcH, which would imply a higher HNO2 concentration (NO2+ HAc h HNO2 + Ac-) and, thus, a higher nitrosating agents concentration. At low [buffer] ()0.048 M) of pH 4.33 and low [nitrite] ()3.3 mM), we studied the nitrosation reaction in aqueous micellar solutions of both cationic and anionic surfactants. The presence of the cationic surfactant TTABr increases ko even at concentrations lower than the cmc (∼3.5 mM) (32); see Figure 8. Below the cmc, ko increases proportional to [TTABr], i.e., ko ) a + b[TTABr] with a ) (1.20 ( 0.05) × 10-3 s-1 and b ) (0.15 ( 0.02) M-1 s-1. Above the cmc, the ko vs [TTABr]

Figure 8. (a) Variation of ko as a function of the concentration of the cationic surfactant TTABr for the nitrosation of novocaine at [nitrite] ) 3.3 mM and [buffer]t ) 0.042 M of pH 4.30. (b) Expanded plot showing the points below the cmc.



profiles fit eq 8 with ko* ) (1.53 ( 0.02) × 10-3 s-1, δ ) (0.042 ( 0.002) M-1 s-1, and Ks ) (10.1 ( 0.6) M-1.

ko )

ko* + δ[TTABr]m 1 + Ks[TTABr]m

(8)

To explain this behavior, we have to remain that with the addition of TTABr, we are also adding Br- ions, which generate a new nitrosating agent, BrNO [Br- + HNO2 + H+ h BrNO + H2O; the corresponding equilibrium constant, K3 ) 0.051 M-2 at 25 °C (33), is higher than K1 or K2], whose concentration is higher than that of NO+ or N2O3. Therefore, the catalysis by TTABr is due to the formation of BrNO that introduces a new reaction path. Below the cmc, the [Br-] ) [TTABr], whereas above the cmc, the concentration of Br- ions in the bulk water phase is [Br-]w ) R[TTABr]m + cmc, with R being the ionization degree of the micelles, ∼25% (34). As a consequence, ko* should be equal to ko when [TTABr] ) cmc and δ ) b × R. The expected results are in good agreement with that obtained in the fit of eq 8 to the experimental data. On the other hand, Ks refers to the association constant of protonated novocaine molecules (NoH+) to cationic micelles. Obviously, this value is much lower than that obtained in alkaline hydrolysis, under which the conditions of the substrate are neutral and neither repulsions between equal charges occur nor the deprotonation of the substrate, previously to the binding process, is required (NoH+ + TTABrm h Nom + H+); finally, the conformation of novocaine molecules into the micellar interface would prefer the opposite orientation of Scheme 2, i.e., with the H2N group of novocaine entry first into the micelle, in which case, the nitrosatable group is located deeper inside the micelle, far from the attack of XNO. This conformation remembers that adopted in the action of this drug as an anesthetic is its action of blocking the Na+ channels through biomembranes. Previous studies on the influence of cationic micelles on nitrosation reactions have been explained in a similar manner, even though the resulting micellar effects were quite different, because of the distinct nature of the substrate (35, 36). The influence of SDS on the nitrosation of No was studied at 0.043 M buffer concentration (pH 4.30) and [nitrite] ) 3.3 mM. Figure 9a shows the plot of the observed rate constant, ko, against [SDS]. Under these experimental conditions, the expected cmc of SDS should be around 6.5 × 10-3 M (37). As can be seen in Figure 9b, we observed a strong catalysis by the presence of SDS below the cmc, whereas above this value, ko decreases to achieve at approximately 0.2 M SDS the measured value in the absence of SDS. This unusual behavior could be explained in the assumption of the formation of premicellar aggregates. This process is facilitated by both the cationic nature of No and its high hydrophobicity; both factors would induce the formation of premicellar aggregates with SDS monomers, given a kind of mixed micelles (38). Below the cmc, the novocaine concentration into the mixed micelles should be extremely high, and these nanosize structures would act as reaction centers. Above the cmc, the formation of SDS micelles provokes dilution of No molecules into the SDS micelles, and the rate of nitrosation decreases. Fitting the descending part of the curve in Figure 9a to eq 8, which takes into account the concentration effects in micellar catalysis, gives the single line shown in the plot for the values of ko* ) (2.09 ( 0.03) × 10-3 s-1, δ ) 0.0135 ( 0.0028 M-1 s-1, and Ks ) 19 ( 2 M-1 (r ) 0.999). However, further investigations are required for clear and quantitative interpretation of this unexpected behavior.

Figure 9. Influence of SDS on the observed rate constant, ko, for the nitrosation of novocaine at [nitrite] ) 3.3 mM and [buffer]t ) 0.043 M of pH 4.30; [SDS] < cmc (O) and [SDS] > cmc (b). (b) Expanded plot showing the points around the cmc.

Conclusions The local anesthetic novocaine hydrolyzes in aqueous alkaline medium at moderate reaction rates that increase with the concentration of OH- and are markedly reduced in water mixtures of aprotic solvents, such as dimethyl sulfoxide or dioxane. Despite the concentration effects of cationic micelles of TTABr, the ester hydrolysis of novocaine is strongly inhibited in aqueous micellar solutions of both cationic and anionic surfactants. By contrast, either cationic or anionic micellar solutions enhance the nitrosation reaction rate in mild acid aqueous medium. The existence of two amine groups in the novocaine molecule susceptible to protonation changes the nature of the substrate, which determines the orientation inside the micellar interface. Acknowledgment. Financial support from the Direccioń General de Investigacioń (Ministerio de Educacioń y Ciencia) of Spain and FEDER (Project CTQ2005-07428/BQU) is gratefully acknowledged.

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Ester Hydrolysis and Nitrosative Deamination of Novocaine in

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