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11 Borg, D. C., and G. C. Cotzias, these PROCEEDINGS, 48, 623 (1962).12Ibid., 643 (1962).13The effect of alkalinization was not to facilitate the autoxidation of manganese on the inter-

face provided by precipitated phenothiazine. For example, with dibasic trifluoperazine plusmanganous salts, aerobic alkalinization produced color as the first positive charge was neu-tralized, corresponding to the initial step of the titration curve of Figure 3. Precipitation, per se,occurred only after the second proton was titrated (Fig. 3).

14Pauling, L. C., General Chemistry (San Francisco: W. H. Freeman and Company, 1953).16 Cotzias, G. C., and D. C. Borg, Association for Research in Nervous and Mental Disease,

Proceedings, vol. 40 (Baltimore: Williams and Wilkens (in press), 1962).16Yamamoto et al. (Jap. J. Pharm., 10, 38 (1960)) also noted a pink color with maximum ab-

sorption at 525 mg (sic) upon mixing chlorpromazine with FeCl3, but FeCl2 and CuC12 did notreact.

17 The apparent inactivity of these ions is in concordance with the observation of Yamamotoet al. that chlorpromazine inhibits cysteine oxidation by Fe+++ but not by Cu++ (Jap. J. Pharm.,6, 138 (1957)), since the present experiments elucidate an Fe+++-chlorpromazine interactionbut no reaction with Cu++. See also Yamamoto's later article (Jap. J. Pharm., 10, 38 (1960))reporting chlorpromazine inhibition of enzymes containing Fe+++ but not of enzymes containingCu++ or Fe+"-.

18 The ultraviolet absorption spectra of mepazine and trifluoperazine appear virtually identicalwith that of chlorpromazine (Fig. 4), except that all absorption peaks are shifted -2 m;& towardshorter wavelengths. The ultraviolet absorption spectra of the chromophoric reaction productsfrom both mepazine and trifluoperazine also are essentially congruent with that of the red productfrom chlorpromazine (Fig. 4) except that they are shifted -2 mMA to have their peaks at -272 mM.

19 Michaelis, L., M. P. Schubert, and S. Granick, J. Am. Chem. Soc., 61, 1981 (1939).20Michaelis, L., Ann. N. Y. Acad. Sci., 40, 39 (1940).21 With chromophores generated somewhat differently, in strong H2SO4 in the presence of

Fe+++, Rieder found the major absorption from promazine at 510-512 m.25 This is concordantwith the structure-reactivity correlations drawn from the present data, because promazine isstructurally identical with chlorpromazine except for its lacking chlorination at "R2" (Fig. 1).

22 Nakajima, H., J. Biochem., 46, 559 (1959).23Bernheim, M. L. C., Proc. Soc. Exp. Biol. Med., 102, 660 (1959).24Fels, I. G., and M. Kaufman, Nature, 183, 1392 (1959).25 Rieder, H. P., Medicina Exper., 3, 353 (1960).26 Forrest, F. M., and I. S. Forrest, Am. J. Psych., 113, 931 (1957)."' Forrest, I. S., and F. M. Forrest, Clin. Chem., 6, 11 (1960).28Michaelis, L., M. P. Schubert, and S. Granick, J. Am. Chem. Soc., 62, 204 (1940).29 Granick, S., L. Michaelis, and M. P. Schubert, J. Am. Chem. Soc., 62, 1802 (1940).30 Michaelis, L., S. Granick, and M. P. Schubert, J. Am. Chem. Soc., 63, 351 (1941).31 Granick, S., and L. Michaelis, J. Am. Chem. Soc., 69, 2983 (1947).

INTERACTION OF TRACE METALS WITH PHENOTHIAZINE DRUGDERIVATIVES, H. FORMATION OF FREE RADICALS*

BY DONALD C. BORG AND GEORGE C. COTZIASMEDICAL RESEARCH CENTER, BROOKHAVEN NATIONAL LABORATORY, UPTON, L. I., NEW YORK

Communicated by Donald D. Van Slyke, February 23, 1962

In the preceding paper,' it was reported that several biologically importanttransition-group metals can act on chlorpromazine and its congeners to developcharacteristic chromophores even under mild conditions. The nature of thischemical process and the characterization of the colored reaction products are thesubject of this communication.

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The experiments reported here provide evidence for the identification of thechromophore formed from chlorpromazine (and its congeners) as a semiquinoneradical ion. The redox nature of the reaction and the stability of its reactants andproducts are investigated by means of optical absorption spectrometry, oxidimetrictitrations, and electron spin resonance (ESR) spectrometry.

Materials.-Reagents: Stock solutions were made up with demineralized (Barn-stead cation exchange resin column) distilled water from reagent grade metal salts,sodium hydroxide, and concentrated hydrochloric (sp. gr. 1.188-1.192) and sulfuric(sp. gr. 1.841-1.844) acids. Chlorpromazine hydrochloride (Smith, Kline andFrench Laboratories) and mepazine hydrochloride (Warner-Chilcott Laboratories)were used in the powder form provided by the suppliers, and stock solutions foreach experiment were either freshly made up or stored in the dark for not more thana few days.

Apparatus: The pH meter, recording spectrophotometers, and microburet werelisted previously.1 Data for time curves and dilution curves of the chromophoreswere gathered with a Beckman Model DU spectrophotometer. Potential (emf)measurements were taken with a Beckman Model H-2 meter supplied with a plat-inum electrode and a calomel, saturated-KCl reference electrode. ESR spectrawere recorded at room temperature with a Varian Model V-4500 spectrometer andassociated six-inch magnet, employing both audio frequency (200 cycle) and 100 kcfield modulation. Samples were encapsulated at the ends of sealed soft glasscapillaries of 1.3 mm I.D. and were held by a polyethylene collar at the center ofthe resonant cavity. The effect of ultraviolet light was studied with an essentiallycontinuous ultraviolet illumination spectrum obtained from a high-intensity, water-cooled hydrogen arc lamp that was fitted with a stand to hold sample cuvettes at aconstant distance from its quartz window.

Results.-Oxidizing nature of reactants: In the previous paper, evidence was pre-sented for oxidation of phenothiazine congeners in the formation of a colored prod-uct with metal ions. A requirement for higher valence states of reactive metalcations was shown, whereas some physiologically important metals which do notlend themselves readily to univalent oxido-reduction were inert.' Therefore, inthe present work oxidants not normally present in living material also were tested.

Ceric ion and even the oxidizing anion, persulfate, reacted with chlorpromazineand mepazine to produce chromophores with absorption spectra identical with thoseillustrated in the preceding report.' On the other hand, confirming publishedreports2' I an excess of hydrogen peroxide oxidized both 0.01 M chlorpromazine andmepazine directly to their colorless sulfoxides. H202 produced no color even withexcess thiazine. Ultraviolet absorption spectra of reaction mixtures following addi-tion of H202 revealed a partial conversion of reduced phenothiazine to sulfoxidewith absence of the 274 m, absorption band characteristic of the chromophores.1It should be noted, however, that a red color was observed by Cavanaugh upon thefurther addition of peroxidase or catalase to a mixture of chlorpromazine andH202.4

Identification of absorption spectra: The reported chromogenic reaction betweenmanganese and chlorpromazine produced a slight decrease in the main ultravioletabsorption band of the thiazine concomitantly with the appearance of a new peakat about 274 miu, whereas an inverse change occurred upon bleaching of the color

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with ultraviolet light.' Presumably, the absorption spectrum of chlorpromazinewas transformed into that of the new reaction product, but, with the generating sys-tems used then, the process was incomplete. ' This presumption was confirmed whenpersulfate or ceric ion was used, despite the kinetic peculiarities of these two sub-strates:5 the yield of the colored product was essentially complete when dilutesolutions of either of these oxidants reacted with chlorpromazine. The typicalultraviolet spectrum of the phenothiazine was totally replaced by that of thechromophore. This permitted the quantitative determination of the coloredproduct (Fig. 1). Utilization of this as a standard of reference showed that ferricion converted 10-20 per cent of reduced phenothiazine to the chromophore. Withmanganese, a yield of no more than 10 per cent was observed, and this was usuallyless than 5 per cent when the titration technique was used without oxygenation.'

ABSORPTION SPECTRUM

30,000

Cm 20,000

10,000 -_

0200 400 600 800 1000

WAVELENGTH (my)

FiG. L.-Spectrum of the colored product resulting fromthe action of appropriate oxidants on chlorpromazine (seetext). The curve is a composite of several ultraviolet andvisible spectra recorded at concentrations and optical pathlengths selected to provide measurable optical densities ineach instance. Molar extinction coefficients, em, were cal-culated from reaction with equimolar amounts of Ce4 + indilute solution, wherein the yield of colored product amproached 100% (see text and later figures).

Many years ago, Michaelis and his colleagues proved the existence of semiquinoneradical forms of thiazine dyestuffs in strong acid (5-25 N H2SO4) by means of poten-tiometry.7 8 Subsequently, a red-colored free radical of an N-substituted pheno-thiazine was demonstrated following oxidation by Br2 in 80 per cent acetic acid.9The absorption spectra of all these compounds were complex and relatively inde-

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pendent of the number and nature of side chains. This weak dependence on side-chain substituents was also encountered in the preceding study.' In their details,Michaelis' spectra are similar to those of the chlorpromazine chromophore (Fig. 1)as well as to the spectra of the colored products derived from congeners, as depictedin the preceding report.1 Since these latter spectra were not altered by strongacid1 (although their stability was increased), Figure 1 lends itself to a correlationwith the results of Michaelis et al.

Effect of reducing systems: If the red chromophore were truly a semiquinone,reducing systems should inhibit or reverse chromogenesis. Indeed, excess ofcysteine, reduced glutathione, ascorbic acid, and ferrous ion promptly bleached thered color developed from 0.005 M chlorpromazine by manganese or the other oxi-dants. Furthermore, a reversion of the spectrum of Figure 1 to that of nativechlorpromazinel accompanied the discoloration. Also, these reducing agents, ifpresent initially, suppressed the appearance of color from chlorpromazine alto-gether.

It can be seen from the representative examples of Table 1, (a) and (b), that theabove-mentioned reducing agents reacted stoichiometrically with the chromophorerather than with the metal or the thiazine ions. This supports the hypothesis thatthe chromophore is a free radical.

TABLE 1INHIBITION OF FORMATION OR BLEACHING OF THE COLORED PRODUCT FROM CHLORPROMAZINEIn order to ascertain the stoichiometric relationship between various reductants and the separate components of

the chromogenic reaction, a system providing only fractional yields was selected. For most experiments, themanganese-titration technique' was employed, so as to leave no excess reactant likely to perpetuate the interactionor to react directly with the added reducing agent.

(a) 10 pmoles of chlorpromazine (CPZ) and 5 pmoles of MnCl2 were made up to 3 ml with water and titratedwith NaOH in air to pH 7.0. A few minutes (varying from 2 to 8 in different experiments) after back-titration topH 5 with HCI, the optical density was determined at 523 mp.1 The molar yield of colored product in the controlsolution, C, was calculated by comparing the absorbance measured at 523 my with the molar extinction coefficientof a dilute solution of CPZ completely converted to the chromophoric product by cerium (eM- 7800) (Figs. 1, 7).To other samples, known amounts of ethylenediaminetetraacetic acid (EDTA) or ascorbic acid (AA) were addedprior to the titration step, and hence before generation of color.

MmolesMinimum amount ofreactant requiredexperimentally for

Colored product total inhibition orReactant present in control bleaching of -Molar ratios-tested CPZ Mn solution (C) chromogenesis (M) M/Mn M/CEDTA 10 5 0.26 4.9 0.98 18.85AA 10 5 0.26 0.27 0.05 1.04

(b) Similar to above except that the colored product was first generated by the Mn-CPZ titration reaction, andsubsequently FeSO4 (Fe + +) or AA was added to produce bleaching.

Reactant CPZ Mn C M M/Mn M/CAA 10 5 0.26 0.26 0.05 1.00Fe++ 27 13.5 0.70 0.8 (0.6 < M < 1.0) 0.06 1.1

(c) Fe (NOs)3 was used in place of MnCl2, and the titrations were omitted. EDTA was added subsequent tocolor development to produce bleaching.

Reactant CPZ Fe C M M/Fe M/CEDTA 15 7.5 0.70 7.0 0.95 10.0

Prior sequestration of these metals by appropriate chelating agents preventedcolor formation from chlorpromazine congeners. Equivalent amounts of 1,10-phenanthroline (OP) or 2,2'-dipyrridyl (DP) did not inhibit chromogenesis byferric ion from chlorpromazine and related derivatives and may even have increasedthe yield of the reaction, but equimolar concentrations of ethylenediaminetetra-acetic acid (EDTA) suppressed altogether the appearance of color when either theiron or the manganese-titration systems were used (Table 1, (a) and (c)). Further-

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more, the color from chlorpromazine or trifluoperazine produced by Fe+++ waspromptly quenched by the subsequent addition of EDTA (Table 1 (c)). Similarly,this agent caused a slower bleaching of color after cerium oxidation or manganesetitration. Saturated solutions of 8-hydroxyquinoline (oxine) bleached both theiron and the manganese-induced chromophores. Since EDTA and oxine preferen-tially bind with and stabilize the trivalent forms of iron and manganese," theymight have produced bleaching by shifting far to the left the reaction:'2

(colorless thiazine)n+ + M3+ IM' + (colored semiquinone) + 1)+

In conformity with this concept, EDTA quenched the color of the Fe+++-contain-ing system totally, but only when it was equimolar to the concentration of Fe+++(Table 1 (c)); although OP and DP (which do not preferentially stabilize the highervalence states of iron)'3 failed to bleach the red product.

Potentiometric oxidation-reduction titrations: The semiquinone-radical characterof the forms of thiazine dyes that exist in strong acid was deduced originally byMichaelis et al. on the basis of potentiometric studies.'-'0 More recently, Dusinsk'yand Liskova' reported that stoichiometric titration of chlorpromazine with Ce(S04)2resulted in the formation of a red "semiquinodinic" radical upon univalent oxida-tion, followed by bleaching upon removal of an additional electron.14The tentative identification of the red chromophore from chlorpromazine as a

semiquinone free radical also was supported in this work by titrations of solutions ofthe drug with ceric ion and other oxidizing metal cations. It was desirable thatthe titrations be performed at a constant pH which was sufficiently acid to ensurethe solubility of oxidants such as Ce4+ and Fe3+: namely, pH = 1.2. Since dis-tinct and reproducible curves could not be obtained in the presence of buffer, thesetitrations were carried out in 0.1 N H2S04. This permitted the avoidance of signifi-cant changes in pH and of ionic strength during the procedures. With millimolarchlorpromazine solutions, the greatest concentration of colored product was yieldedby the addition of an equimolar amount of Ce4+ (Fig. 2), as would be expected if asemiquinone free radical were indeed formed. Addition of a second equivalent ofthis electron acceptor quenched all color, and the titration curve reflected the com-pletion of a bivalent oxidation step (Fig. 2). This curve was readily reproducible.The rapid initial change of potential after each addition of oxidant was followed bystabilization with only very slow subsequent change or "drift",'5 indicating that atthis concentration of chlorpromazine the system reflected a reversible redox reac-tion.'6 This was supported by independent evidence: Spectrophotometricanalysis showed that the spectrum obtained by the addition of only one equivalentof Ce4+ to 0.001 M chlorpromazinel7 (Fig. 1) was reverted to that of native chlor-promazine after addition of a reductant (Fe2+ or ascorbic acid). Addition of twoequivalents of Ce4+ yielded a colorless product with the ultraviolet absorptionspectrum of chlorpromazine sulfoxide." 18 Addition of the same reducing agents asused before to this colorless material did not result either in the formation of coloror in any other perceptible spectral change. Subsequent reoxidation with Ce4+produced an abrupt rise in potential without eliciting either the titration curve orthe red color indicated in Figure 2. As will be pointed out in detail, these dataimply that only the colored intermediate and not the sulfoxide can be reversed bythese steps.

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OXIDATIVE TITRATIONS

1100 WITH Ce4

1000 WITH Co3 /

E900 IO*3 M CPZ a/OX IDAT I VE T ITRATIONS 3Omv

- . ~. , 29mv

1100 1/10M Fe WITH Ce4;

700 -6Zjj27mv26 mv

1000 600

0 50 100900 PER CENT OXIDATION

E I03 CPZE 800 0/_M CPZ

I~~~~~~ ~ ~~~~~~~~~~~~~/I_7001,, 4 'O M Fe 7~~~~00;~~~~~ 70

0 4 8 12 16 20 24/ ~~~~~~~~~~~~~~~~EQUIVALENTSOF Fe3'

600

FIG. 3.-Potentiometric titrations~ of chlorpromazine (CPZ) at pH 1.2

500 | - (see Fig. 2 for details), expressed asper cent of a complete oxidation step(upper graph).As with Ce4+ (Fig.

0 2 2), titration with a suspension of Co3 +EQUIVALENTS OF Ce4 (as Na3lCo(C03)3j37 freshly made up

in 0.1 N H2SO4) produced a roseFIG. 2.-Potentiometric titrations with eerie color with its maximum intensity cor-

salts (as (NH4)2 Ce(NOs)6) of 10 ml aliquots in responding to univalent oxidation;0.1 N H2SO4 (pH = 1.20) at 230C. In order to however, six equivalents of Co3+ weremaintain constant pH throughout the titra- required for 100 per cent oxidation.tions, the oxidizing reagent (Ce4+) also was The index potentials at 25 per centmade up to pH / 1.2 with H2SO4. For calcu- and 75 per cent oxidation, as definedlation of emf relative to the standard hydrogen by Michaelis,19 were -29.5 mv forelectrode, the potential of the saturated-KCl CPZ with both Ce4+ and Co3+.calomel electrode was taken as 242mv.16 Dur- Again the titration of Fe++ is givening titration of chlorpromazine (CPZ), the typi- for reference. As anticipated fromcal rose color appeared and gradually darkened the minimal overlap of CPZ and Feuntil one equivalent of oxidant (0.1 ml of 0.1 M oxidation curves (Fig. 2), even a largeCe4+) had been added. Thereafter, color faded, excess of Fe3+ did not convert CPZdisappearing simultaneously with the comple- fully to the univalent intermediatetion of bivalent oxidation to the sulfoxide. The and did not produce bivalent oxida-univalent titration of FeSO4 is depicted for ref- tion to the sulfoxide at all (lowererence. graph).

The relative shape of the titration curve of chlorpromazine at millimolar concen-tration also was typical of an oxidation reaction with a univalent intermediatestep. The curve was essentially symmetrical, and it was steep enough to have anindex potential, E1,19 of about 28.6 mv. This is predicted for the titration curve ofa simple univalent redox system20-22 and approximates the experimental values forthe transfer of a single electron from ferrous iron (Fig. 3). Theoretically the Etfor a direct bivalent oxidation-reduction reaction is 14.3 mv, 5 20-22 whereasMichaelis showed that bivalent oxidations proceeding through the formation of

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detectable semiquinone intermediates will give rise to index potentials of greaterthan 14.3 mv.15, 20-22

Figure 3 also explains why the yield of colored reaction product from chlorpro-mazine was incomplete when Fe"+ was used as the oxidant. Figure 2 shows onlya slight overlap between the curves for chlorpromazine and for the Fe2+-Fe3+ redoxcouple: full oxidation of iron to the trivalent state occurred at an emf below thepoint of 50 per cent oxidation of the thiazine. Since one half of the total oxidationreaction should yield the semiquinone radical, not even a large excess of Fe'+ canbe expected to oxidize the thiazine beyond its intermediate redox form. This isillustrated by Figure 3.When semiquinone formation is the only "complicating factor"15 in a potentio-

metric titration, the curves for different concentrations are congruent when normal-ized to the midpoint of titration, provided that ratios of activity coefficients andother conditions do not change significantly.15' 21, 22 However, Figure 4 shows theshapes of such titration curves to be dependent on the initial concentration of

OXIDATIVE TITRATIONS+200

+150

+1008

+ 50 3 1

EEm.

-150 6 _0 88766 446.5

-200 2* 0 50 100PER CENT OXIDATION WITH Ce4+

FIG. 4.-Potentiometric titrations of differentconcentrations of chlorpromazine (where [CPZ]indicates molarity) at pH 1.2 (experimental con-ditions as in Figure 2 except that Ce4+ was asCe(HSO4)4). Em represents the midpoint potential(50 per cent of total oxidation) relative to thehydrogen electrode.24 At4>7480 per cent oxi-dation, emf's tended to drift slowly to lower valuesafter tie rise following each additional incrementof Ce4+ and the plotted values are the highestrecorded after each addition (Table 2).

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chlorpromazine. This implies the existence of further significant factors in thisreaction. Firstly, the steepness of the initial half of the titration curve increasedsmoothly with dilution (up to a limiting value of the order of 3 X 10-5 M) so that theindex potential at 25 per cent of total oxidation was inconstant (Fig. 4). Secondly,the final portion of the curve became increasingly distorted in shape with greaterdilution, manifesting unstable potentials that drifted toward lower values with time(Fig. 4). The former behavior suggests that the provision of constant activitycoefficients, cited above, does not hold. More specifically, dimerization of the re-duced form of the substrate is denoted, as will be shown in the next section. How-ever, the instability of the titration curves beyond 50 per cent of oxidation mayimplicate an irreversible step following completion of bivalent oxidation, as willalso be discussed further on.

Probable formation of a dimer of chlorpromazine: Because of the inference of di-merization of native chlorpromazine drawn from the concentration dependence ofthe shapes of the oxidimetric titration curves (Fig. 4), other evidence of dimerformation was sought. However, the ultraviolet extinction of chlorpromazine at254 mA (Fig. 9) showed no systematic deviation from Beer's law over a wide rangeof concentrations (10-6 to 2 X 10-2 M), nor was there significant shift in the wave-length of maximum absorbancy over this range. Hence, the putative dimerizationcould not affect appreciably the energy levels underlying the prominent ultravioletabsorption bands of the component thiazine nuclei. Nonetheless, other experi-ments did support the concept of chlorpromazine dimerization.

Following the approach of Clark,'5 Ox represents the oxidant of concentration[Ox] = ox and R the reductant (where [R] = r) of an over-all bivalent oxidation-reduction reaction: i.e., Ox + 2e- = R. Then where S symbolizes total concen-tration of all species of a reactant, S = Sx + Sr. When there is dimerization of thereductant,

Sr = [R] + 2[R2]. (1)The degree of reduction, that is the concentration ratio of reduced forms relative

to all forms of the substrate, is denoted by a = Sr/S. One can also define a dimer-ization constant, Kdr, for the reduced species:

Kdr = [R2. (2)

Clark shows that with formation of a soluble dimer from the reductant, potentio-metric titration curves steepen with decreasing concentration,15 23 especially witha < 0.5. Figure 4 is consistent with this. Clark also derives an expression thatdefines as a function of S and Kdr the departure, AE', of the apparent potential of aredox system with dimerized reductant from the potential of the same oxidation-reduction in the absence of dimer formation, provided that all components remainin solution:"5

AE' = ( ln - In[1+ 1 + 8aSKdr]) (3)

Figure 5 represents the best fit of data from the present experiments to equation3, solved for the midpoint potentials where a = 0.5. In order to avoid possible

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errors due to drift toward lower potentials caused by formation of an irreversiblefinal product, as noted with Figure 4, and to avoid further complication from thedismutation reactions that will be discussed later, data for Figure 5 were not takenfrom actual titration curves. Rather, mid-point potentials were approacheddirectly by admixture of equimolar chlorpromazine and eerie ion (Table 2). Inthis way, the afore-mentioned downward drift of emf took place in one step ratherthan being divided into small portions following each incremental addition of oxi-dant during the titrations. Hence, its presence was accentuated, and it was evenseen clearly at higher concentrations (Table 2). However, at S = 3 X 10-5 M

TABLE 2ELECTRIC POTENTIALS IN 1: 1 SOLUTIONS OF CHLORPROMAZINE AND Ce4+ AS A FUNCTION OF

CONCENTRATION-emf (relative to H2 electrode)-

Maximumvalue

Molarity At 1/2 over 3 min Direction ofof min interval At 3 min drift of emf Ee.50.1.2 AE

reactants (mv) (mv) (mv) with time (mv) (mv)10-2 850 850 832 Down 850 -30.5

3 X 10-3 859 859 841 Down 859 -21.510-3 863 863 845 Down 863 -17.5

3 X 10-4 871 871 851 Down 871 -9.510-4 874 874 860 Slowly down 874 -6.5

3 X 10-5 872 877 at 1 min 872 Changes 877 -3.510-5 852 864 864 Up >864 > (-16.5)

3 X 10-6 829 843 843 UP >843 > (-37.5)10-6 807 819 819 Up >819 > (-61.5)

5 ml dilutions of Ce(H504)4-in-H2S04 at pH = 1.2 denoted by the symbol Ce4 + were added with constant stirringat 220C to 5 ml aliquots of equimolar solutions of chlorpromazine-in-O.1 N H2SO4 at pH = 1.2 (CPZ). Electrodepotentials (emf) were recorded at 30 sec intervals for 3 min. The apparent emf at 50 per cent titration of the CPZby Ce4 + (Figs. 2-4) at pH = 1.2 was taken as the highest value of emf recorded. Referred to the hydrogen stand-ard by taking the emf of the calomel reference electrode as 242 mv,15 these potentials are denoted by Et,,1.2. Byextrapolation to infinite dilution (Figs. 4, 5) the true midpoint potential for the titration of CPZ is calculated as880.5 my = Ema.2. Then AE = Ema.2 - Eh,6o.1.2 (Fig. 5), after the fashion of Clark.'5

there was first a slow rise in potential followed by a drift to lower values; and mix-tures of lower concentrations produced only a steady upward drift (Table 2).Apparently redox equilibrium was established only very slowly at high dilution, sothese data evince behavior sharply divergent from those derived from higher con-centrations (Fig. 5). Nonetheless, Figure 5 shows the fit of the latter data to equa-tion 3 according to Clark's method'5 to be quite satisfactory for an apparent dimer-ization constant of Kdr " 104. This is not entirely surprising, because relatedthiazine dyes have been reported previously to dimerize.'5

Despite the apparent dimerization of the reduced chlorpromazine itself, the redsemiquinone radical was shown to obey Beer's Law over a concentration range ofgreater than an order of magnitude. This was best tested by serial dilutions ofchromophore produced by the manganese-titration system' (Fig. 6). The man-ganese reaction provided sufficiently dilute product, yet any excess or residual metalor substrate remaining after the titration steps would not be reactive subsequently'and hence would not confound the interpretation of the dilution curve (Fig. 6).Since the chromophore's extinction curve followed Beer's Law upon dilution (Fig.6), the yield of semiquinone radical as a function of reactant concentration could befollowed optically in order to confirm the dimerization of chlorpromazine inferredfrom the potentiometric experiments. At higher concentrations of chlorpromazine,the competitive formation of dimers should decrease the apparent activity coef-ficient of the thiazine for the oxidation reaction with Ce4+, resulting in a lower

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Ce4+: CPZ = 1:SKd, FOR Kd, =104 -

-02 l0-, lo, 02 103

310 E f8~~~~~~

-30 AE'=-(t n Z-~-In( I ,+ 8aS Zd, 1<-40 -)

FOR a =0.5 AND T=22°CS= (CPZI

OA(MOLAR)/ [ ITOTAL(

-50

I6 10-5 10-4 io-3 o-2 to-I

.- S-4

FIG. 5.-Relation of AE' to SK& according to theequation of Clark1 for the case wherein the reductantforms a reversible dimer. AE' represents deviationfrom the midpoint potential, Em, for the limiting casein which there is insignificant dimerization. See textfor other symbols. For comparison, the actual datafor 50 per cent oxidation (Table 2) were graphedagainst S, and the limiting potential, Em, wasestimated. The experimental curve (lower scalealong abscissa) was shifted to coincide optimally withthe theoretical curve (upper scale), as suggested byClark;15 and the extent of the shift that was requiredgave the value of Kdr as approximately 104.

DILUTION OF COLORED PRODUCTFROM CHLORPROMAZINE

(.0w

< 0.8

W

:L 0.6

E-JzU- 0.40

0.2

0 I I I0 0.2 0.4 0.6 0.8 1.0

RELATIVE CONCE NTRATION

FIG. 6.-A solution 0.005 M in both Mn++and chlorpromazine was titrated to pH 7.4 with1 N NaOH and was back-titrated with HCl.1Aliquots of the rose-colored stock solution (e623= 1.65) were promptly diluted with water, andtheir absorbance was compared with that ofthe stock before appreciable decay of color (Fig.10) bad occurred.

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yield of the chromophore per mole of chlorpromazine. If the product is measuredby its optical extinction (at 523 mru), the "specific absorption" of the reaction (i.e.,the ratio of specific extinction coefficient of the chromophore to the starting con-centration of chlorpromazine) should fall with increasing concentration of thereactants. The experiments depicted by Figure 7 were in full accord with theseexpectations. Furthermore, if the true molar extinction coefficient of the chromo-phore is taken as that value of specific absorption beyond which there is no increasewith further dilution, then the "break point" in the curve of Figure 7 represents theconcentration at which the apparent activity coefficient became significantly lessthan unity (i.e., the concentration where dimerization becomes important). This"break point" is at a concentration > 5 X 10-5 M but < 10-4M (Fig. 7), which isin good agreement with the dimerization constant (Kdr) of 104 estimated from thepotentiometric analysis (Fig. 5).

Ce4+: CPZ =I:9000 I 11111111 1 11111111 l I

8000

7000

6000-

CONC.5000 - -

4000 -

3000 _

2000 liill11F6 10-4 10 3 2102

CONCENTRATION OF REACTANTS (MOLAR)

FIG. 7.-"Specific absorption" of the chromogenic reaction fromthe admixture of equimolar chlorpromazine (CPZ) and ceric ion as afunction of reactant concentration. For each determination, an ap-propriate dilution of Ce(HS04)4-in-0.1 N H2S04 was added to an ali-quot of equal volume and concentration of CPZ-in-0.1 N H2S04, ab-sorbance at 523 mu was determined over appropriate optical pathlengths at 1 min, and the extinction coefficient was calculated. Toavoid local excesses in Ce4+ concentration which could irreversiblyoxidize some of the CPZ to colorless sulfoxide (see text) and henceproduce suboptimal yields of chromophore, it was necessary to addthe aliquots of Ce4+ to the solutions of CPZ with constant gentlemixing: CPZ added to Ce4+ solutions gave lower and less repro-ducible yields.

The stoichiometry of the preferred reaction complex between chlorpromazine andan appropriate oxidant also should reflect dimer formation. This stoichiometryshould correspond to the ratio of reactant concentrations that produces optimalchromogenesis when the mole fractions of the reactants are varied while the sumof their molarities is kept constant.2 The manganese-titration reaction is useful

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for deriving such a plot (Job's plot2"), because its relatively low yield of chromo-phore should provide measurable optical densities at chlorpromazine concentrationssufficiently high to induce significant dimerization. Figure 8 shows that maximumchromogenicity occurred at a drug: metal ratio of approximately 2:1. This isconsistent with an initial interaction between a metal ion and a soluble dimer ofchlorpromazine.

RATIO Mn: CPZ

3;.1 2:1 1:1 1:2 1:3 1:4 1:62.0I XXi

1.8

Ero)~'1.4-

1.2-

< 1.0

Z 0.8

0-6Q6

[MTI IC +[PZ1 {[M n] +[CPZ] =5.0 m M}

FiG. 8.- Job)'s plot of manganese-chlorpromazine mixturestitrated to pH 7.4 with 1.0 N NaOH and then back-titratedto pH -3 with 1.0 N HC1. The scatter of data points re-flects the strong sensitivity of the reaction to the maximumpH obtained in the alkaline titration step' and the resultantdifficulty in achieving exact uniformity of conditions foreach experimental run. Some rounding of the peak of theJob's plot also may have occurred due to quicker decay ofcolor in more intensely colored samples during the 3 min be-tween reaction and the measurement of optical density.This is to be expected from the second-order reaction kineticsestablished for the spontaneous fading of color (Fig. 10).

Stability of the chromophore: Earlier in this communication and in the previousstudy,' the red product from chlorpromazine was noted to be metastable, bleachingspontaneously over a period of hours and almost instantaneously under ultravioletlight. If the red reaction product is in fact a semiquinone free radical as has beenproposed here, then it is likely that its spontaneous loss of color in the dark repre-sents the classical dismutation reaction of free radicals.202 In dismutation, tworadical molecules-each with an unpaired free electron-interact so as to pair theelectrons on one of the two reactants, converting it to the fully reduced form whileits mate becomes fully oxidized:

2FXr R + Ox. (4)

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For the case at hand, the free radical (Fr) is taken to be the red chromophore,native chlorpromazine is the fully reduced form (R), and chlorpromazine sulfoxideis either the oxidized form (Ox) or is directly (and irreversibly) derived from it.The data of Figure 9 then confirmed that the spontaneous bleaching of the putativesemiquinone resulted from dismutation: As noted earlier, the characteristic ultra-violet spectrum of chlorpromazine (curve 1, Fig. 9) was entirely transformed to

ABSORPTION SPECTRA

40,000 l_

"\2 1 __CPZ

30,000 333 2DAYS1__-(

20,000 ~ ~ /

10,000

0220 240 260 280 300 320 340

WAVELENGTH (mu)

FIG. 9.-Dismutation of the red chromophore from chlorpromazine (CPZ).CPZ was 3.3 X 10- M in 0.1 N H2S04 in each case. The solution corresponding tocurve 2 was rose-colored initially and decayed spontaneously in the dark to a colorlesssolution corresponding to curve 3, recorded 2 days later. Curve 4 was recorded withina few minutes after mixing and is congruent with the spectrum of chlorpromazinesulfoxide (Fig. 4 of reference 1). The points, (o), are calculated and hence representa 1:1 mixture of CPZ and its sulfoxide.

that of the red semiquinone product' (Fig. 1) by 1:1 addition of Ce4+ (curve 2,Fig. 9), provided that reactant concentrations were sufficiently low to avoid inter-fering dimerization (Fig. 7). In contrast to this, reaction of two equivalents ofelectron acceptor with chlorpromazine produced prompt conversion to the sulfoxide(curve 4, Fig. 9), as had been noted during the preceding oxidimetric titrations(Fig. 2). However, if the aliquot of colored radical were allowed to fade in thedark under either nitrogen or air, its ultraviolet absorption corresponded to curve3 of Figure 9. It can be seen that this curve is identical to that which was calculatedfor a 1: 1 mixture of reduced chlorpromazine and chlorpromazine sulfoxide the sumof whose concentrations equaled that of the substrate initially present. This lattermixture is the one theoretically predicted by the dismutation reaction (equation 4).

It is clear from equation 4 that dismutation involves two molecules of free radicaland hence should follow second-order reaction kinetics. If C is the concentrationof a substrate, A, that reacts by 2A -- products, then the decay of C, of initial con-centration Co, is given26 by:

-d = kC2, (5)d

where k is a rate constant. The integrated form is then

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k 1 (Co- C)t (CoXC)(6

Solving equation (6) for t provides the relationship

1 1 1t = - -- = k - constant. (7)

kC kGo, k

Equation (7) is a reciprocal (or hyperbolic) function; so for a second-order reaction,a plot of concentration, C, against time, t, should give a linear regression (straightline) on hyperbolic graph paper.By this criterion, the spontaneous decay of colored chlorpromazine semiquinone

followed second-order kinetics. For the reasons noted above with regard to theexperiments relevant to Figure 6, the manganese-titration technique' was selectedfor the generation of the chromophore. A maximum initial concentration of radicalwas sought that would avoid serious interference from dimerization (Fig. 7), andthe subsequent bleaching was followed optically (Fig. 10). The data conformedwell (Fig. 10) to the reciprocal relationship (equation 7) required by a second-orderdismutation reaction (equation 4).However, under other circumstances the spontaneous bleaching of semiquinone

color followed different kinetics. With Fe+++ oxidation, for example, color per-sisted longer, because the initial conversion of chlorpromazine was incomplete(Fig. 3), and the residual reactants could regenerate the chromophore while it de-cayed by dismutation. On the other hand, while strong acid (> 1 N H2SO4) did notalter the spectra of thiazine radicals produced by univalent oxidants,' it stabilizedthese products7 8 SO that they decayed very slowly or not at all, the latter case re-quiring highly concentrated acids (>6 N H2SO4). The meaning of this stabiliza-tion will be discussed in the next paper.

In addition to the innate instability of the chlorpromazine radical ion in the dark,due to dismutation, photobleaching by ultraviolet light was cited in the precedingarticle.' Experiments with a high-intensity hydrogen arc light source equippedwith interference filters indicated that decay of the red color was most pronouncedat 270 my (Fig. 11), corresponding closely to the major absorption peak of thesemiquinone (Fig. 1). Light with greatest intensity at 254 m/A and 290 m/A pro-duced less bleaching (Fig. 11), although absorption bands of chlorpromazine andits sulfoxide are prominent near these wavelengths (Fig. 9). In a separate experi-ment, strong green light with peak intensity at 523 mg (Fig. 1) failed to acceleratethe decay of color beyond that expected from dismutation alone. The "actionspectrum" of the quenching of color therefore corresponds to a reaction wherein aphoton of ultraviolet light is absorbed directly by the colored chlorpromazine radi-cal, as opposed to its parent or daughter species.

Electron spin resonance (ESR) spectrometry: Direct evidence that the red chro-mophore from chlorpromazine is a free radical should be obtainable by ESR.27From the detailed structure of ESR signals, further conclusions may be drawn re-garding the free electron distribution and the configuration of free radicals in mostcases that can be examined in an uncomplicated chemical environment.28 Hence,in the present studies ESR was used to gain added confirmation of the free radical

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DECAY OF COLORED PRODUCT FROM Mn: CPZTITRATION REACTION

0040

2015

109

7

4 6-j00 5\

4z0

zwU 3-z0

2 I0 100 200 300 400

MINUTESFIG. 10.-With 1.3 ml of 0.010 M chlorpromazine

plus 1.3 ml of 0.010 MnCl2 three drops of 0.1 NNaOH were mixed, followed by three drops of 0.1 NHCl.' The optical density was followed at 523 m1Awith the solution held at 220C in the dark betweenmeasurements. The reference solution was identicalexcept that the HC1 was added prior to the NaOH.1The concentration of chromophore was calculatedby taking the molar extinction coefficient as eM= 7,800 (Figs. 1, 7). The ordinate scale is from re-ciprocal ruling paper, designed to make linear re-gressions for hyperbolic functions.

nature of the chlorpromazine colored reaction product and to seek more detailedknowledge of its structure.

In some experiments, ESR spectra were recorded from chromogenic mixtures ofchlorpromazine titrated with Mn++.I The typical six-line ESR patterns of divalentmanganese aquoions29 30 were then observed, plus a superimposed single resonancepeak with a width of -34 gauss and with a g value near the free electron positionof 2.023 (refs. 31, 32) (Fig. 12a). The intensity (amplitude) of this overlying signalwas found to vary in direct proportion to the absorbance of the red chromophoreand was quenched promptly by ultraviolet light concomitantly with the bleachingof color described previously.' With its g value denoting a free radical, this peakwas therefore attributed to the red reaction product, which was thereby identifiedwith even greater certainty as a semiquinone radical.

In any given experiment, the intensity of the resonance signal corresponding toMn++ itself was the same both before and after formation of the radical by the to-and-fro titration procedure,' nor did it change during gradual decay of color due tothe disputation reaction. This confirmed the previous interpretation that in the

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10080

06 C -290m

40- \254m

001^ \ MAXIMUM0 TRANSPARENCY20 OFI- ~~~~~~~~~~~FILTERzw

100-8

E 6270mls

0; 20 2 4 6 8 10 12 14

MIN OF EXPOSURE TO U.V. (H-ARC)FIG. 11.-Bleaching by ultraviolet light of the

red reaction product from titrated 0.005 MMn-chlorpromazine. Aliquots were irradiated inquartz cuvettes and were shaken after every tenseconds of exposure. Values were comparedwith an unexposed control aliquot, whose opti-cal absorbance dropped from 0.93 to 0.53over the -80 min elapsed (clock) time requiredfor the experiment.

oxidation of phenothiazines to the semiquinone form by the Mn++-titration tech-nique,' the trivalent state was entered only transiently and with a very low steady-state concentration.

Because of possible obfuscation of a more complex pattern by the manganesehyperfine structure (Fig. 12a), the radical signal also was generated by Fe+++,which at room temperature does not manifest an ESR signal with a g value in theregion of free electron resonance.32 Again, a simple and essentially symmetricalresonance peak of -34 gauss width was noted (Fig. 12b). A similar spectrum ofsomewhat narrower width was elicited from the metal-free semiquinone in the solidstate (Fig. 12c). This latterwas produced by mixing chlorpromazine and ammoniumpersulfate in a concentrated slurry followed by quick drying with an air blast. Inthis manner, the mixture was evaporated before all the semiquinone had disap-peared by dismutation or oxidation to the sulfoxide. The residual free radicalcolor remained unchanged for months.

In both manganese and iron experiments, the shape of the radical signal re-mained constant even as its amplitude vanished following spontaneous dismutationor high dilution of the chromophore. No fine structure was observed in the spec-trum when it was scanned at high resolution with a 100 kc modulation frequencyand low modulation amplitude. However, the relatively broad resonance line ob-tained (-30 gauss, Fig. 12) suggests that some secondary fine structure may havebeen missed.6 This could have resulted from exchange coupling28 3 4 with the

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VOL. 48, 1962 BIOCHEAILSTRY: BORG AND COTZIAS 639

DERIVATIVE OF ELECTRON SPIN RESONANCE ABSORPTIONOF CHLORPROMAZINE SEMIQUINONE FREE RADICAL

100 GAUSS

(a)

,/ ~~~~~~~~~Mn+(+

g=2.0--10 GAUSS ,.10 GAUSS

AU Ag=2.0 MAGNETIC FIELD g9=2.0

FIG. 12.-Electron spin resonance spectra. (a) and (b)were recorded from samples in water solution with Mn++titrations1 (denoted by Mn++(+)) and Fe+++ oxidizingsystems, respectively. (c) was taken from a solid samplewith persulfate as the oxidant (see text). Different con-centrations of chlorpromazine alone, or of its sulfoxide,did not produce detectable resonance signals.

high concentrations of paramagnetic ions that existed in the reaction systemsstudied in these ESR experiments27' 5 (Fig. 12a, b).Evidence to support this conclusion was provided by ESR study of the free radical

in the absence of paramagnetic metal ions. If the previous conclusion was correctthat univalent oxidation of chlorpromazine by metal ions produced a red semiqui-none ion, then the same chromophore might be formed at the anode of an electrolysiscell. Platinum electrodes were inserted into a beaker containing 45 ml of a solutionof 1.4 X 10-3 M chlorpromazine in 11.5 N H2S04 (to stabilize the semiquinoneproduct), and the cathode was isolated in a small glass well separated from the bulkof the solution by a sintered glass disk. After 40 minutes of exposure to an appliedpotential of 2.0 volts at a current of -10 milliamperes, the solution was wine-colored. Absorption spectra of aliquots revealed the characteristic spectrum ofthe chlorpromazine semiquinone radical (Fig. 1), and the electron resonance patternof another aliquot is depicted by Figure 13. Figure 13 shows a slightly asymmetri-cal ESR signal that is narrower than those recorded in the presence of 0.005 Mmetal cations (Fig. 12) and which possesses a detailed hyperfine structure.

Effect of ultraviolet light on reduced chlorpromazine: Several years ago Forrest,Forrest, and Berger reported a colorless free radical from chlorpromazine that couldbe prepared by exposure of dilute aqueous solution to a sunlamp for three hours.36No distinct absorption characteristics were found in the visible or ultravioletregions; but in concentrated HCl the product developed a deep-blue color, asopposed to the pale-pink from native chlorpromazine.36 The compound was stablefor months when stored in the dark, in acid solution, and under refrigeration. Itsfree radical character was inferred from ESR spectra taken on solid samples ofdeeply colored nitrite and 2,4-dinitrophenylhydrazine crystalline derivatives:

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640 BIOCHEMISTRY: BORG AND COTZIAS PRoc. N. A. S.

MAGNETIC

FIELD

10 GAUSS

FIG. 13.-Electron spin resonance spectrum of chlorpromazine semiquinone radi-cal formed electrolytically in 11.5 N H2SO4. See text for experimental details.

resonance was observed at the free-electron g value with a line width of 16-23gauss.36The properties of the "free radical" of Forrest et al. are not those of the chromo-

phores discussed here. In order to distinguish further the two products, some ef-fects of ultraviolet light on native (reduced) chlorpromazine were studied. 15-60seconds of exposure to a Hanovia ultraviolet lamp caused no apparent change inthe ultraviolet absorption spectrum, but the irradiated solution reacted differentlywith iron or manganese, even after further storage in the dark for several days.Rather than forming the intensely red product, titrations with Mn++ or additionof Fe+++ to 5 X 10-3 M irradiated chlorpromazine produced a faint blue colorthat faded over several minutes at room temperature while changing its hue gradu-ally from blue to purple, to gray, to dusky yellow. Serial absorption spectra re-vealed a broad band from about 570 mA to about 1,100 mAs with the absorptionmaximum shifting from --.735 m1A to -820 mjA during the decay. Preliminarystudies of the kinetics of the bleaching were complicated by the changing color, butthe rate of color loss appeared generally compatible with a second-order reaction-in the manner of Figure 10.

Longer exposures to ultraviolet light seemed to produce a general degradation ofchlorpromazine. Exposure of four hours gradually turned the colorless solutionsto yellow and finally to ruddy brown. Absorption spectra showed no distinctmaxima or minima from 220 mg to 900 miA, but the characteristic ultraviolet pat-tern of chlorpromazine was slowly transformed into a diffuse nonspecific absorptionlevel. ESR spectra were taken of aliquots irradiated from 15 seconds up to onehour, but no resonance peaks were seen; however, sufficient ultraviolet exposure toturn the chlorpromazine dark brown produced a weak ESR peak of -29 gausswidth and without fine structure.

Clearly the ultraviolet-induced product-be it a long-lived excited state or afree radical form as suggested by Forrest et al.m-is quite distinct from the coloredsemiquinone radicals that are produced by transition metals under mild conditions.'

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Discussion.-In some of the experiments reported in this communication, thereaction conditions employed to characterize and describe the red chromophorefrom chlorpromazine were less physiological than those discussed in the precedingpaper,1 especially when nonphysiological oxidants (e.g., Ce4 , S20) or high acidity(e.g., the pH of 1.2 for potentiometric studies) were involved. Yet the spectralidentity of the products in all instances and the ESR pattern obtained under mildconditions with Mn++ (Fig. 12a) leave little doubt but that the same reactionproduct is involved throughout.

It is also clear from the univalent oxidation step involved in the chromogenic re-action, from the dismutation and lability toward reductants of its products, andfrom the electrochemical and magnetic resonance data that the chromophoricproduct is indeed a free radical, in so far as all compounds (except metals) thatpossess a "free" or unpaired electron are designated free radicals.33 The absorption spectra further identify the radical as belonging to the semiquinone class pre-viously established for phenothiazine dyes.9' 10, 27 36 The observed stabilization ofthe chromophoric radical in strong acid is attributable to resonance involving theheterocyclic nitrogen and sulfur atoms of the thiazine nucleus in the presence of asufficient concentration of hydrogen ions, as put forth some years ago by Michaelisand his co-workers.7-9 This is in accord with the position of the present discussionsas to the cardinal role of the thioether linkage in determining the reaction of pheno-thiazine drug derivatives with oxidizing metal ions.' This interpretation pertainsto the stabilization and not to the generation of free radicals- The latter was shownto be possible under conditions mild enough to merit the adjective "biological."Indeed, correlations of biological findings with the present data, as well as furtherintegration of the latter, are developed in the following paper.6Summary.-Absorption spectrometry, oxidimetric titrations, electrolysis, and

electron spin resonance spectrometry served to identify as a semiquinone free radicalion the chromophore produced from chlorpromazine and its congeners by some metalions and other univalent electron acceptors. Some interactions leading to theformation and disappearance of the radical ions were described. Two concurrentreactions were encountered while defining the complete bivalent oxido-reductionsequence from the native drugs via semiquinone intermediates to the respectivefully oxidized products: a probable dimerization of the reduced chlorpromazineand a tautomerization of the bivalently oxidized quininoid form to the sulfoxide,the latter step being irreversible under the mild reaction conditions of the forwardreactions. A spontaneous dismutation of the free radical also was documentedunder conditions wherein the radical was not stabilized by concentrated acid.

* This work was supported by the U.S. Atomic Energy Commission. Parts of it have beenpresented at the Conference on Biological Aspects of Metal-Binding (University Park, Pa., Sep-tember, 1960) and to the Association for Research in Nervous and Mental Disease (New York,N.Y., December, 1960).

1 Borg, D. C., and G. C. Cotzias, these PROCEEDINGS, 48, 617 (1962).2 Massie, S. P., Chem. Rev., 54, 797 (1954).3 Wechsler, M. B., and I. S. Forrest, J. Neurochen., 4, 366 (1959).4 Cavanaugh, D. J., Science, 125, 1040 (1957).6 In the reaction with potassium or ammonium persulfate there was an initial white clouding of

the solution of mixed colorless reactants, followed within one or two seconds by deepening colora-tion. After about thirty seconds chiomogenesis stopped, and the solution became transparent.

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However, subsequent fading was far more rapid than when similar intensities of color were pro-duced by other oxidizing systems (Fig. 10). The explanation for this may lie in the formation ofperoxy-complexes.

6 Borg, D. C., and G. C. Cotzias, these PROCEEDINGS, 48, 643 (1962).7 Michaelis, L., M. P. Schubert, and S. Granick, J. Am. Chem. Soc., 62, 204 (1940).8 Granick, S., L. Michaelis, and M. P. Schubert, ibid., 62, 1802 (1940).9 Michaelis, L., S. Granick, and M. P. Schubert, ibid., 63, 351 (1941).

10 The term "semiquinone" was applied by Michaelis to radical ions similar to the intermediateformed specifically by semioxidation of a quinone-hydroquinone bivalent redox system. Subse-quently, it has been used to designate any odd electron intermediate in an oxidation-reductionprocess involving transfer of two electrons to or from an aromatic molecule (Bersohn, R., "Electronparamagnetic resonance of organic molecules," in Determination of Organic Structures by Physi-cal Methods, supplementary volume, ed., F. C. Nachod and W. D. Phillips (New York: AcademicPress, in press)).

'1 Williams, R. J. P., The Enzymes, ed., P. D. Boyer, H. Lardy, and K. Myrbick, vol. 1 (NewYork: Academic Press, 1959), p. 391.

12 Williams, R. J. P., Fed. Proc., 20, Suppl. No. 10, 117 (1961).13 Williams, R. J. P., Biol. Revs., 28, 381 (1953).14 Duginsky, G., and 0. Ligkovaf, Chem. Zvesti. 12, 213 (Chem. Abstr., 52, 1268f) (1958).16 Clark, W. M., Oxidation-Reduction Potentials of Organic Systems (Baltimore: Williams and

Wilkins Company, 1960).16 Conant, J. B., Chem. Rev., 3, 1 (1926).17 See also curve 1 of Figure 9 for reference spectrum.18 See curve 4 of Figure 9.19 Et is defined according to the convention introduced by Michaelis (Ann. N.Y. Acad. Sci.,

40, 39 (1940)) as the potential difference between 50 per cent of a complete redox reaction andeither 25 per cent or 75 per cent oxidation.

20 Michaelis, L., J. Biol. Chem., 96, 703 (1932).21 Michaelis, L., Chem. Rev., 16, 243 (1935).22 Michaelis, L., Ann. N.Y. Acad. Sci., 40, 39 (1940).23 Michaelis, L., and E. S. Fetcher, Jr., J. Am. Chem. Soc., 59, 2400 (1937).24 These midpoint potentials may be compared with values previously reported for the somewhat

similar compound, N-methyl phenothiazine, by Michaelis, Granick, and Shubert.9 Oxidimetrictitrations of that substance in 80 per cent or 90 per cent acetic acid gave Em's of 829-882 mv.However, the downward drift of the higher electrode potentials noted here-and even of midpointpotentials upon initial reaction (Table 2)-raises some doubt as to the accuracy of the numericalvalues of Em listed.

25 Job, P., Ann. Chim. (10) 9, 113 (1928).26 Glasstone, S., Elements of Physical Chemistry (New York: Van Nostrand, 1946), p. 592.27 Borg, D. C., Fed. Proc., 20, Suppl. No. 10, 104 (1961).28 Ingram, D. J. E., Free Radicals as Studied by Electron Spin Resonance (London: Butterworths

Scientific Publications, 1958).29 Cohn, M., and J. Townsend, Nature, 173, 1090 (1954).30 van Wieringen, J. S., Farad. Soc. Disc., 19, 118 (1955).31 Pake, G. E., S. I. Weissmann, and J. Townsend, Farad. Soc. Disc., 19, 147 (1955).32 Kozyrev, B. M., Farad. Soc. Disc., 19, 135 (1955).33 Whiffen, D. H., Quart. Rev. (London), 12, 250 (1958).34 Faber, R. J., and M. T. Rogers, J. Am. Chem. Soc., 81, 1849 (1959).35 Cotzias, G. C., and D. C. Borg, Association for Research in Nervous and Mental Disease.

Proceedings, vol. 40 (Baltimore: Williams and Wilkins (in press), 1962).36 Forrest, I. S., F. M. Forrest, and M. Berger, Biochim. Biophys. Acta, 29, 441 (1958).37 Bauer. H. F., and W. C. Drinkard, J. Am. Chem. Soc., 82, 5031 (1960).

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