Analyst, October 1995, Vol. 120 2639

New Tautomerism Suggested by pKa Determinations

Hugues-Olivier Bertranda, Marie-Odile Christenb and Jean-Louis Burgota a UFR des Sciences Pharmaceutiques et Biologiques, Dtpartement d Etudes Physicochimiques et Biocinttiques des Pharmucosyst2mes, Laboratoire de Chimie Analytique, 2 Av. du Pr Leon-Bernard, 35043 Rennes-Cedex, France b SOLVAY-PHARMA (L.T.M.), 42 Rue Rouget de Lisle, 92151 Suresnes-Cedex, France

The UVNIS spectrophotometric PKa determination of (E)- and (2)-5-(1-hydroxyiminomethyl)-4-methyl-1,2- dithiole-3-thione is described; for each isomer, the PKa value was obtained by a non-linear least-squares methodology. This methodology was used to take into account some unusual experimental difficulties (very low solubility in water and E + 2 isomerization). The pKaZ value (5.13) strongly suggests that the resonant basic 2 form is chiefly a thiolate form of a heteropentalene. Spectrophotometric results were confirmed by 13C NMR spectroscopy. A new type of tautomerism is suggested to explain the results. Keywords: 5-(Hydroxyiminoalkyl)-l,2-dithiole-3-thione; pKa measurements; I -oxa-6,6a SN-dithia-2-azapentalene

Introduction 1,2-Dithiole-3-thiones (1) are compounds of scientific interest1 and of growing pharmaceutical interest.2~3 Among them, 5-( 1 -hydroxyiminoalkyl)- 1,2-dithiole-3-thiones(2) have re- cently been patented.4

R2 Jps Rl

OH S-S

I I

Rz Rl

2 1

Oximes (2) exhibit in water an acidic behaviour due to the hydroxyimino group. Since the oxidation of oximes (22) by iodine in alcoholic solution give disulfides, protolytic equilibria must take into account the highly probably occurrence of the tautomeric species 5-mercapto-6,6a S1v-dithia-2-azapentalenes (32), even though these latter forms have not previously been detected by NMR spectroscopy in different solvents.5

32 2z

Heteropentalenes (32) belong to the class of no-bond resonance compounds and they can be written as bicyclic aromatic compounds with a-electron delocalization.6.7 Accord- ing to these results, protolytic equilibria must be written as shown in Scheme 1, where kalZ and kdz are microscopic

ionization constants. Whether the starting acidic form is 2 2 or 32, it is worth noting that the same basic resonant species is obtained by protolysis. Besides, protolytic equilibria in which two tautomeric acid forms give the same mesomeric base have already been the subject of numerous studies.* It is obvious that, if the thiolate forms have a significant contribution to the resonant basic species, (32)- the PKa values of compounds 2 2 must differ significantly from those of 'classical oximes' even if the major component of the tautomeric acidic mixture is 22.

We report the determination of PKa values of oximes 2aE and 2aZ (a: R1 = CH,; R2 = H) in water, by UV/VIS spectrophotometry. The study of the 2aE PKa values was undertaken for the sake of comparison because oximes 2E did not give disulfides in oxidative conditions. As a result, the tautomeric forms 3E of compounds 2E must not be taken into account. However, the basic species (3E)- are also resonant molecules as are (32)- species (see Scheme 2).

This investigation is somewhat complicated by the possible interconversion of E/Z isomers under usual experimental

11

3.2 (=I-

Scheme 1 Ionization scheme of 22 isomers or their tautomeric forms.

s-s

2E s-s

I , ( R2 R1

Scheme 2 Ionization scheme of 2E isomers.

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2640 Analyst, October 1995, Vol. 120

conditions and by the very low solubility of these solutes in water.

Experimental Symbolism

The symbols kalZ, ka2z, and KaE have been introduced above. KaZ is the macroscopic equilibrium constant for the Z form. It is a well known fact that microscopic constants kalz and ka2Z cannot be determined without any supplementary assumptions completing the experimental data,9 and of course, for the Z isomer, only the macroscopic constant KaZ was determined.

Kaz 2aZ + 3aZ (32)- + H+

[(3Z)-l[H+l [2aZ + 3aZ]

with KaZ =

KaZ is related to the microscopic constants by the relation: 1 1 1

Kaz kalz ka2’

--- - +-

For the E isomer, the ionization scheme is:

KaE 2aE (3E)- + H+

Apparatus and Reagents The spectrophotometer used was a Kontron land) Uvikon 930 and the DH-meter was

(Zurich, Switzer- a Solea Tacussel

(Villeurbanne, France) LPH i30 T. All measurements (optical densities and pH values) were performed at 25 “C.

13C NMR spectra were obtained using a Bruker (Billeria, MA, USA) AM 300 WB spectrometer at 75 MHz with sodium 4,4-dimethyl-4-silapentane- 1 -sulfonate (DSS) as external ref- erence.

A mixture of 2aE and 2aZ was prepared as described elsewhere.5 The 2aE and 2aZ isomers were separated by column chromatography on silica gel (0.060-0.200 mm; pore diameter approximately 6 nm; Janssen Supplier Ref. 24 037 78). Both compounds were eluted with heptane-diethyl ether (70 + 30 v/v). The first eluted isomer was 2aE (TLC Kieselgel 60 Kieselguhr F 254 Merck Supplier Art. 6567 with the same mixture Rf = 0.34). Next, 2aZ was eluted (Rf 0.14). However, only 2aE could be isolated in a ‘geometrically’ pure solid state. We were not able to obtain a ‘geometrically’ pure 2aZ because, when the solution was concentrated after the elution, some isomerization into 2aE occurred. The best Z/E ratio that we were able to obtain in the solid state was 84/16 (ratio obtained by 1H NMR in dimethylsulfoxide, DMSO-&). This difficulty did not, however, prevent, the determination of K a Z because, fortunately, as prior UV/VIS spectrophotometry had shown, no appreciable interconversion in water between E and 2 acidic and basic species occurs in dark conditions. Since in solution the (3E) form comes only from the initial solid mixture, it was possible to take its existence into account in our calculations.

The buffers used for spectrophotometry were Britton- Robinson buffers10 and l3C NMR spectra were recorded in molar aqueous Na2C03 (Carlo Erba Code no. 479127; Carlo Erba, Milan, Italy).

Methods The KaZ and KaE values were determined by fitting calculated absorbances (after judicious selection of Ka values) of the

various solutions of 2aE and of 2aZ (containing a small amount of 2aE) with the corresponding experimental densities recorded for different pH values. This was carried out by a non-linear least-squares regression process. For each isomer, solutions with 10 different pH values were made and the experiments were replicated independently seven times. Two different wavelengths were chosen to check the consistency of the results.

For the 2 form, the expression used to calculate the absorbance was:

Ai = E(3E)- [(3E)-li + E(32)- [(3z)-]i EE [Eli EZ [ z ] i (1) where the index i represents the pHi value; [Eli and [Zli are the concentrations of compounds 2aE and 2aZ + 3aZ at this pH; (3E)- and (32)- are the concentrations of the basic forms; EE

ands &WE)- are the molar absorptivity of the acidic and basic E forms; EZ and ~(32)- are the molar absorptivity of the acidic and basic Z forms; and EZ is given by the expression:

where E Z ~ Z and ~ 3 ~ 2 are the unknown molar absorptivities of the 2aZ and 3aZ forms.

For each pHi, the absorbance was calculated according to:

where C o E = [El + [(3E)-] and CoZ = [z] + [(3Z)-]. The first term in the right-hand side of eqn. (2) is the absorbance of the E form present in the mixture. The unknown constants were K a z ,

EZ, E ( ~ z ) - , CoE and COz because of the existence of the E/Z mixture, the ratio of which in water was unknown. This fact precluded the direct use of the Henderson-Hasselbach relation, as a result, a non-linear least-squares methodology was used. The constant K a E was obtained independently by treatment of ionization data of the E pure form (see below). Moreover, on a practical level, the total concentration CoE + C o Z was also considered as an unknown parameter because of some un- certainty in the concentration of the solutes inherent to the very low solubilities of these compounds in water (10-5 moll-l) at room temperature.

According to eqn. (2), only C o E , KaZ and the products CoZtzz, CoZ&(3z)- can be obtained by treatment of ionization data.

For the E isomer, the equation giving the absorbance was the classical one:

where the unknown parameter was K a E . The use of the Henderson-Hasselbach relation was possible but, as before, EE and CoE were treated as supplementary parameters because of the uncertainty of the concentration C o E in aqueous solution. Using this methodology we did not have to use an organic solvent for the dissolution of the acidic forms. Otherwise &BE- was estimated under the experimental conditions.

The solutions studied were prepared by weighing and dissolving amounts of approximately 18 mg of compound 2aE or 2aZ (containing a small amount of 2aE) in 1000 ml of water. The suspension was then stirred and filtered. These procedures were carried out in the dark and Britton-Robinson buffers10 were used. A 50 ml volume of the stock solution of oxime was mixed with 50 ml of each buffer and this mixture was kept in the dark. The pH values were systematically determined before

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Analyst, October 1995, Vol. 120 264 1

absorbance measurements were taken after standardization of the glass electrode with NIST standard reference solutions.

Results and Discussion UV/VIS spectra of 2aE and 2aZ (containing a small amount of 2aE) versus pH are given in Figs. 1 and 2, respectively.

The results of ionization data treatment for E and 2 isomers are summarized in Table 1.

E isomer The values obtained were consistent throughout all of the wavelengths and analytical concentrations (C,) used (Table 1).

0.50

0.40

a c

0.30 8 9

0.20

300 400 500 Wavelengthhm

Fig. 1 UV/VIS spectra of 2aE at different pH values.

This fact can be considered as an indirect proof of the accuracy of our mathematical model and of our experimental data. We considered the mean value of the seven results obtained (Table 1) as the true pK,E, and the lowest and highest values found as the upper and lower limits.

pKaE = 8.28 * 0.07

Treatment of pooled ionization data, a methodology which, according to some authors,'l would have given the best variances and standard deviations, could not be performed

0.40

0.30 0,

C

e 8 0.20 a

0.10

I ( 2 a ~ + 31 (2aZ + 3aZ) + 2aE

I 1

W

300 400 500 Wavelengthhm

Fig. 2 UV/VIS spectra of (2aZ + 3aZ) containing a small amount of 2aE at different pH values.

Table 1 Results of ionization data treatment for the E and Z isomers. (Concentration and absorptivities are given in mol 1-1 and 1 mol-1 cm-1, respectively)

Experi- ment

1

2

5

6

7

Para- meters

pKaE COE

E(3E)-* pKaE COE

E(3E)-* pKaE COE

EE

EE

&E &(3E)-* PKaE COE

E(3E)- pKaE COE

E(3E)-* pKaE COE

EE

EE

EE

&(3E)-* pKaE COE &E

E(3E)-*

pKaE Determination

320 nm 8.26 3.02 x 10-5 9565 2934

8.35 4.46 x 10-5 9417 2934

8.25 3.55 x 10-5 9692 2934

8.21

10031 2934

8.26

3.31 x 10-5

3.10 x 10-5 965 1 2934

8.32 3.08 x 10-5 9644 2934

8.3 1 4.40 x 10-5 9045 2934

* Experimental data

370 nm 8.26 2.78 x 10-5

793 1 1847 8.34 3.99 x 10-5

75 1 1 1847 8.30 3.20 x 10-5

925 1 1847 8.24 3.13 x 10-5

750 11 847 8.25 2.85 x 10-5

73 1 1 1847 8.26 2.82 x 10-5

728 11847 8.26 3.76 x 10-5

957 1 1847

-Experi- ment

1

4

5

6

7

' with

pKaZ Determination

320 nm* 3.38 X 5.09 5.94 x 10-2 0.295 5.78 x 5.15 2.19 X 0.219 5.58 X 5.14 2.28 X 0.214 3.80 X 10-6 5.13 1.87 X 0.152 4.31 x 5.12 2.36 X 10-2 0.192 3.00 X 5.12 4.89 X 10-2 0.245 4.88 x 10-6 5.11 2.53 X 0.202 8.28 9577 2934

430 nm* 6.55 X 10-6 5.07 0.440 0.255 4.55 x 10-6 5.34 0.381 0.222 4.16 X 10-6 5.14 0.374 0.216 2.06 X 10-6 5.11 0.269 0.157 5.67 X 10-6 5.11 0.312 0.173 4.44 x 10-6 5.08 0.383 0.221 4.14 X 10-6 5.13 0.346 0.199 8.28 7015

11 140

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2642 Analyst, October 1995, Vol. 120

because of the differences in the total oxime concentrations among the 'replicates'.

A value of 8.28 value is undoubtedly relatively low for an oxime pKa value.12 For example, E and Z benzaldoximes exhibit values of 10.68 and 11.33 while (E)-4-nitrobenzaldoxime has a value of 10.09 in water at 293 K.13914 It is a well known fact that electron withdrawing groups reduce pKa values. In this context, the pKaE value indicates that the 5-( 1,2-dithiole-3-thione)-yl group is a very strong electron-withdrawing group. This finding is in agreement with our previous electrochemical studies. 15,16 Another explanation for the low pKa value obtained could be the existence of resonant forms of the basic species (3E)-, among which the nitrosothiolate forms could have a predominance (Scheme 2), but this explanation is ruled out by independent 13C NMR spectroscopy in aqueous molar Na2C03 solutions (Fig. 3).

Of great significance is the chemical shift of the carbon of the thiocarbonyl group, the value of which (6 = 215 ppm) is quite normal (Fig. 3.)17 Another indirect argument for the existence of the basic form (3E)- in its oximate structure is the pKaE value calculated with a well grounded relationship12 PKa (oxime)/60H (TMS/DMSO-d6) (TMS = trimethylsilane). Inserting the value of a O H = 12.70 ppm in this relation gives a calculated pKa value of 8.21, which is in good agreement with the experimentally found one. It is worth noting that the 1JC5,-H = 173 Hz coupling constant has the same value in the acidic form 2aE and in the basic form (3E)-. This indicates that the E geometry is preserved in the ionization process.18J9

Z isomer The seven values obtained are consistent (Table 1). The mean value is pKaZ = 5.13 f 0.21. This value was obtained by inserting, into eqn. (2), the values of &E, &(3E)-, and KaE found independently. Interestingly, the same calculations performed with the upper and lower limits of K a E did not change the pKaz values. Only the products C,=E~ and C,ZE~Z)- varied slightly. It can be noted that the given error for the Z isomer is significantly greater than for the E isomer. The reason is that a single pKa value, which seems to be an obvious outlier [Table 1; pKaz determination, experiment 2 (430 nm)].

The value pK,Z = 5.13 is very low. It does not fit at all with the correlation mentioned above." Moreover, 13C NMR spectroscopy (in aqueous molar Na2C03) (see Experimental) does not exhibit the typical chemical shift of the thiocarbonyl group (Fig. 3). A likely explanation for this is that the basic species, (32)-, is represented by a resonant thiolate form.

Also, in agreement with this assertion, is the fact that a PKa value of 5.13 is even lower than that of thiophenol (PKa = 6.52)

k CH3 k CH,

and according to Barlin and Perrin,20 falls in the range of that of trifluorothiophenol. From another standpoint this thiophenol pKa value confirms the n-electron delocalization of hetero- pentalene (32). We note that the 1Jc5,-H coupling constant of 187 Hz is the same for the acidic form (2aZ) and the basic (32)-. This shows that the Zgeometry is preserved (Fig. 3).l*J9 l3C NMR experiments also exhibit the E basic form, the origin of which is the existence of the mixture 2aE-2aZ.

As a result, a new type of tautomerism is suggested by these data.

G. Bouer is thanked for his kind assistance in the preparation of the manuscript and ComitC d'Ille et Vilaine, Nationale contrele Caneir is thanked for its financial support.

References 1 2

3

4 5

6

7 8 9

10 11

12

13 14 15 16

17

18

19

20

Pedersen, C., Th., Adv. Heterocycl. Chem., 1982, 31, 63. Moon, R. C., Rao, K. V. N., Deprisac, C. J., Keloff, J. G., Steele, V. E., and Doody, L. A., Int. J . Oncol., 1994, 4, 651. Egner, P. A., Kensler, T. W., Prestera, T., Talalay, P., Libry, A. H., Joyner, H., and Curphey, T. J., Carcinogenesis (London), 1994, 15, 177. Christen, M. O., and Burgot, J. L., Fr. Pat. Appl., Fr 267057; 1992. Abazid, M., Bertrand, H. O., Christen, M. O., and Burgot, J. L., Phosphorus Sulfur, 1994,88, 195. Dingwall, J. G., Dunn, A. R., Reid, D. H., and Wade, K. O., J. Chem. SOC., Perkin Trans. I , 1972, 1360. Gleiter, R., and Gigax, R., Top. Curr. Chem., 1976, 63,49 Norris, R. K., and Sternhell, S., Tetrahedron Lett., 1967, 2, 97. Edsall, J. T., Martin, R. B., and Hollingworth, B. R., Proc. Natl. Acad. Sci., 1958, 44, 505. Bntton, H. T. S., and Robinson, R. A., J. Chem. SOC., 1931, 458. Draper, N., and Smith, H., Applied Regression Analysis, Wiley, New York, 2nd edn., 1981, p. 33. Peter Kurtz, A., and Themistocles, D. J., J. Pharm. Sci., 1987, 76, 599. Brady, 0. L., and Goldstein, R. F., J . Chem. SOC., 1926, 1923. Meyer, G., Viout, P., Tetrahedron, 1981, 2269. Saidi, M., PhD Thesis, University of Rennes I, France, 1989. Burgot, J. L., Darchen, A., Jehan, P., and Saidi, M., Proceedings of Journte d'Electrochimie, Brest, France, 1991. Plavac, N., Still, I. W. J., Chauchan, M. S., and McKinnon, D. M., Can. J. Chem., 1975, 53, 836. Maciel, G. E., McInver, J. W., Otlund, N. S., andPople, J. A., J. Chem. SOC., 1970, 92, 1. Martin, G. J., and Martin, M. L., Prog. Nucl. Magn. Reson. Spectrosc., 1972,8, 202. Barlin, G. B., and Perrin, D. D., Q. Rev., 1966,20,75.

Fig. 3 (3E)- and (32)- in molar aqueous Na2C03.

13C NMR spectroscopy (6 ppm/DSS; 75 MHz) of basic species Paper 410401 9A

Received June 26,1995 Accepted June 29, I995

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