Polarographic Detection of 5-Pyrimidinyl Disulfides NYDIA G. LUTHY,' Chemistry Department, The University, Manchester, England, and BERTHA LAMB, Chemical laboratory, Tinsfey (Industrial Instruments, lfd.), North Circular Road, london, N. W. IO, England
b Because certain 5-pyrimidinyl disulfides have shown small but significantly inhibitory effects on tumor growth, a method was required for monitoring the syntheses of these compounds in order to ascertain the presence or absence of the disulfide bond. That the reduction wave pattern in alkaline solution i s due to the -4-5- linkage is shown by the disappearance of the waves on complete conversion to the thiol form. Starting materials which were 5-monobromouracil derivatives, intermediate isothioureas, thiols (RSH), and monosulfides (RSR), where R i s a pyrimidinyl group, do not show diffusion-controlled reduction waves under the same conditions. A model compound, di(2,4 dihydroxy 6 methylpyrimidin5-yl) disulfide (6-methyluracil-5-disulfide), was studied quantitatively and the main reduction wave was found to be diffusion-controlled in the region 10-3 to 1 O-4M. Polarograms of other 5-pyrimidinyl disulfides synthesized showed a similar behavior.
-
D
- -
development of preparative methods for certain pyrimidinyl disulfides to be tested as tumor inhibitors] a rapid technique was needed for detecting the presence of the disulfide linkage (1,3). URINQ
OH
electrode] giving polarographic waves that were of a unique pattern and readily identified (Figure 1). A diffusion-controlled reduction wave is produced by the reduction of the -S-Slinkage in simple alkyl disulfides; with aromatic disulfides two waves are produced; and with tri- and tetrasulfides three waves are produced. Karchmer and JJ7alker(8)observed that the most negative of these waves corresponded to the reduction of the -S-Sbond. This latter step was mainly diffusion-controlled, but the remaining two steps behaved in an irregular fashion and were often accompanied by large maxima. No definite conclusion as to the mechanism of the reduction a t the dropping mercury electrode for the polysulfides discussed by them could be drawn. However, of the polarographic waves produced by the pyrimidinyl disulfides, the one with the most negative half-wave potential value was found to be essentially diffusion-controlled and could be used quantitatively for the determination of the -S-Slinkage. It would appear that the similarity in the reduction exhibited by the alkyl and aryl, as well as heterocyclic disulfides examined, was primarily a property of the over-all polarographic behavior of their disulfide moiety.
solution was used. Nitrogen was bubbled through the cells in the absence of mercury to remove dissolved oxygen. A saturated calomel half cell was used as the anode, and the temperature of the system was maintained a t 21.8" =t0.2" c. All the polarograms were taken a t two heights of the mercury reservoir: 36 and 64 cm., and drop times 6.3 and 3.6 seconds, respectively. Both
$Iw 2 a
a
3 V
?H
H
H
I"
Di-(2,4-dihydroxy-6-methy1pyrimidin-5-y1) disulfide ( 6-methyluracil-&disulfide) From the syntheses three products were possible: the thiol (RSH),the disulfide (R-S-S-R), or the monoThe usual colorisulfide (R-S-R). metric tests (11, 1.2) have often failed to distinguish between these products, and ultraviolet and infrared absorption spectroscopy were not specific enough to be of diagnostic value. On the other hand, the polarographic method was well suited for this purpose. Of the expected products, only the uracil derivatives containing the disulfide linkage reduced a t the dropping mercury Present address, 13 University Crescent, Liguanea P. O., Kingston, Jamaica, BWI. 1
1454
ANALYTICAL CHEMISTRY
EXPERIMENTAL
All measurements were made on a Tinsley pen-recording polarograph Mark 15. Fresh stock solutions of 1 X 10-3M di-(2,4-dihydroxy-6-methylpyrimidind-yl) disulfide (6-methyluracil-5-disulfide) were prepared in 0.1M potassium hydroxide (Polaritan grade). From this appropriate dilutions to 5 X and 1 X 10-4M were made for the polarographic measurements. The galvanometer sensitivity was 6 pa. for full scale deflection in all concentrations examined except that of 1 X 10-4M, where the reading of the current was improved by using a sensitivity of 0.6 pa. The work was carried out in the electrolysis stand supplied with the instrument and 10 ml. of each
0-
l
"
2
.
'
3
'
4
5
'
6
~
"
'
8 .9 1.0 APPLIED VOLTS VS. MERCURY POOL ANODE - I
Figure 1. Polarograms obtained from 5-pyrimidinyl disulfides in 0.1 M potassium hydroxide (all concentrations ca.
10-4M ) 1.
2.
3.
4.
Di-(2,4,6-frihydroxypyrimidin-5-yl) disulfide (barbituric acid disulfide) Di-(hexahydro-6-imino-l -methyl-2,4-dioxopyrimidin-5-yl) disulfide (6-amino-1 methyluracil disulfide) Di-(6-amino-2,4-dihydroxypyrimidin 5 yl) disulfide (6-aminouracil disulfide) Di-(2,4-dihydroxy-6-methylpyrimidin 5 yl) disulfide (6-methyluracil disulfide)
-
- - -
damped and undamped waves were obtained; the former were used to measure the diffusion current, while on the latter the half-wave potentials were measured graphically. Derivative waves were also taken to aid in reading the El/, values. DISCUSSION OF RESULTS
As a model compound B-methyluracil&disulfide was selected because it could be synthesized by an independent method (IO),and in alkaline solution it was not converted rapidly to the thiol form, thus making possible a quantitative study of the polarographic waves of the disulfide linkage. Of the three steps shown = -0.1, -0.25, and - 0.58 volt us. S.C.E.) by the compound, the most prominent one ( E l / , = -0.58 volt) was chosen for investigation. That this reduction wave was diffusioncontrolled can be seen from an examination of Table I. Table 1. Polarographic Behavior of Di-(2,4-d ihydroxy-6-methylpyrimidin5-yl) Disulfide (6-Methyluracil-5-disulfide) in 0.1M Potassium Hydroxide
Concn.,
Mole
1 X 10-8
5 X IO-‘ 1 X IO-‘
Diffusion Current, id, ba. At 36 At 64 id36Cm. cm. cm. id64Cm. 2.93 3.91 0.75 1.43 1.87 0.76 0.26 0.34 0.76
The wave height was proportional to the concentration from to lO-4iM, while the El/, value remained constant a t -0.58 volt V.S. S.C.E. Furthermore, a comparison was made with the reduction of cadminum ion whose polarographic wave is known to be diffusion-controlled (91, The wave height of a standard solution of cadmium in 0.1M potassium nitrate was found at two heights of the mercury reservoir, 36 and 64 em. The ratio of these two step heights mas 0.76, which is in fair agreement with the calculated figure of 0.75-i.e., the ratio of the square roots of the two mercury heights chosen. The ratios of the values of the 6-methyluracil-5-disulfide curves were similar to those found for the cadmium ion. Examination of the rest of the polarogram revealed that the smaller reduction wave-EI/z = -0.25 volt us. S.C.E.-was independent of concentration. The first wave, El/, a t approximately -0.1 volt. represents an oxidation and appears to be due to some structural feature of the pyrimidine ring, as it remained even after all the disulfide had been reduced. The same type of anodic step in the region of 0.0 to -0.1 volt has been observed for other non-sulfur-containing uracil derivatives-e.g., 6-aminouracil, 6-amino-
5-bromouracil, and 2-amino-4-hydroxypyrimidine. It was found best to work with freshly made solutions, because in alkali 6-methyluracil-5-disulfide which had stood 24 hours showed a decrease in wave height of approximately 5%. However, the other pyrimidinyl disulfides studied have a much greater tendency to form the thiol after long standing, as shown by the decreasing wave height and increasing positive values for the half-wave potentials. These polarographic results were substantiated by ultraviolet absorption measurements. Of the two absorption maxima exhibited by most of the disulfides synthesized, the one a t the longer wave length (320 to 330 mp) associated with the disulfide gradually disappeared. On the other hand, the maximum between 266 to 268 mp which was common to both disulfide and the thiol, and was undoubtedly the contribution from a simple pyrimidine structure, did not change when these measurements were made in 0.1M alkali solution. As a control measure all starting materials and intermediates were examined polarographically. The following compounds showed no reduction waves in alkaline solution: 6methyluraci1, 6-aminouracil, 6-aminouracil monosulfide [di-(6-amino-2,4-
the pyrimidinyl disulfides whose polarograms are given in Figure 1, uracil disulfide also shows a polarographic behavior similar to that found for the model compound. 6-methyluracild-disulfide. COMPARISON OF DISULFIDES WITH OTHER SULFUR-CONTAINING PYRIMIDINES
I n order t o determine further the specificity of the polarographic waves shown by the 5-pyrimidinyl disulfides, a number of other pyrimidines holding a sulfur atom nere examined. These did not produce the typical disulfide polarogram but gave instead nondiffusion-controlled waves a t much greater negative potentials-just before the decomposition voltage of the solvent, 0.1M sodium hydroxide. The following compounds showed polarographic steps between -1.5 and -1.8 volts (vs. the mercury pool anode) : 5-phenyl-6-thiopyrimidine, 5-chloro-6-thiopyrimidine, 1,3 dimethyl - 6 - thiouracil, 5amino - 6 - mercapto - 2,4 - dihydroxypyrimidine (thiouramil), and 2,CdithioGhydroxypryimidine [prepared by the method of Buttner (4). Analysis: Found, C, 30.45, 30.35: H, 1.35, 2.0; N, 17.85. C4H30NzSz requires 30.2; H, 1.89; h-,17.6%]. In some cases these waves were so much higher than would be expected from the concentration used, that they dihydroxypyrimidin-5-yl)sulfide], 6could be distinguished from the decomamino-1-methyluracil, barbituric acid, position voltage of the supporting cytidylic acid, cytosine, isocytosine, electrolyte only by increasing the 5-bromo-6-aminouraci1, 6-amino-5-broresolution of the instrument in three mo-1-methyluracil, 6mercaptopyrimiways: decreasing the current sensitivity, dine, and 4,6-dimercaptopyrimidine. reducing the rate of applied potential In more acid solution Heath (7’) found from 100 mv. in 20 seconds to 100 mv. no reduction waves for cytidylic acid in 40 seconds, and increasing the and its derivatives, while Cavalieri mercury drop rate. and Lowy ( 5 ) report that isocytosine That the sulfur atom in these comdoes not reduce a t pH less than 7. pounds is held in other than an -SH However 5j5-dibromobarbituric acid linkage is supported also by the ultraand 6-amino-5,5-dibromo-l-methylura- violet absorption data: 1,3-dimethyl-6cil gave non-diffusion-controlled waves thiouracil, 6-thiouraci1, and 6-thiothya t -0.22 and -1.22 volts against the mine have maxima between 320 and mercury pool anode. These waves 360 mp which are scarcely influenced by were presumably due to the easy wide changes in pH (6). This suggests a hydrolysis of one of the bromine I atoms, which may come off as the -C=S (thio) linkage and is in sharp positive bromide ion (Br+). The contrast with “2-thiouracil” derivatives ultraviolet absorption curves of both whose maxima lie between 260 and compounds were also similar, giving 270 mp, and for whose sulfur atom the only end absorption from 220 to 300 mp I thiol (-C-SH) form is assigned ( 5 ) . as distinguished from the monobromo Indeed, the ultraviolet spectra of the derivatives which have typical pyrimilatter compounds are markedly indine absorption around 265 mp. fluenced by pH changes. The reduction waves shown by the Thus the polarographic method can be disulfide linkage were useful not only used not only to ascertain the presence of for following syntheses of 5-pyrimithe disulfide linkage, but also to aid in dinyl disulfides ( 2 ) ,but also for checking the assignment of structure for these the homogeneity of various fractions organic sulfur compounds. after recrystallization. Thus, if a crystallization solvent promoted the ACKNOWLEDGMENT reduction of the disulfide to the thiol, this could be quickly discovered by The authors wish to thank J. F. W. checking the polarogram a t each step of McOmie and his students a t the the purification process. In addition to University of Bristol for the samples of
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c,
VOL. 2 9 , NO. 10, OCTOBER 1957
1455
5-substituted 6-thiopyrimidines. They wish also to thank Juliet Backshall and W, J. Watts for helpful technical assistance in the polarography. Grateful acknowledgment is made to the British Empire Cancer Campaign for financial assistance to N. G. Luthy in this work. LITERATURE CITED
( 1 ) Barker, G. R., Luthy, N. G., Chemistry and Industry 1955,983.
“Polarography,” 2nd ed., Vol. 11, Interscience, Kew York, 1952. (10) Maggiolo, A , Hitchings, G . H.,
(2) Barker, G . R., Luthy, N. G., J. Chem. SOC.1956, 917. (3) Barker, G. R., Luthy, N. G., Dhar, M. M . , Ibid., 1954,4206. ( 4 ) Buttner, Ber. 36, 2234 (1903). (5) Cavalieri, L. F., Lowy, B. A., Arch. Biochem. Biophys. 35, 83 (1952). (6) Elion, G. B., Ide, W. S., Hitchings, G. H., J . Am. Chem. SOC.68, 2137 (1946); 69,2138 (1947). (7) Heath, J. C., Nature 158,23 (1946). (8) Xarchmer, J. H., Walker, M. T., ANAL.CHEM.26, 271 (1954). (9) Kolthoff, I. M., Lingane, J. J.,
J . Am. Chem. SOC.73,4226 (1951). (11) Siggia, S., “Quantitative Organic
Analysis Via Functional Groups,’’ Wiley, Kew York, 1949. (12) Snell, F. D., Snell, C. T., “Colorimetric Methods of Analysis,” p. 257, Van Nostrand, Iiew York, 1937.
RECEIVED for review September 17, 1956. Accepted May 14, 1957.
Anodic Polarographic Estimation of Aliphatic Sulfides in Petroleum HARRY V. DRUSHEL and JAMES F. MILLER Mellon Institute o f lndusfrial Research, Pittsburgh, f a .
b An anodic polarographic procedure is described for the estimation of aliphatic sulfides in petroleum. Stationary platinum wire electrodes are used following a special alternating current treatment prior to polarization. A solvent-electrolyte containing nitrobenzene is used, which readily dissolves petroleum samples and has a relatively high electrical conductivity. A number of pure sulfides were studied by this procedure and the effect of molecular weight upon the apparent rate of diffusion was evaluated. By fractionating the crude oil or petroleum sample into fractions of narrow molecular weight range b y molecular distillation, it was possible to use this information to estimate aliphatic SUIfides. Cyclic and noncyclic alkyl sulfides and alkyl aryl sulfides are determined.
Preliminary separation of petroleum samples by molecular distillation yielded distillates having narrow molecular weight ranges. Information from the anodic polarograms of these fractions and a knowledge of the effect of molecular weight upon the average diffusion coefficients of aliphatic sulfides made possible the estimation of aliphatic sulfide sulfur.
necessary to find a solvent-electrolyte system having a relatively high specific conductivity and good solvent action for crude oil. A 70:30 (by volume) mixture of nitrobenzene and methanol containing hydrochloric acid was found satisfactory (4). The cell resistance was further reduced by using a simplified reference electrode. A silver electrode of special shape coated with silver chloride was introduced directly into the chloride supporting electrolyte near the anode (6, 9). With this arrangement cell resistances between 150 and 350 ohms were obtained.
EXPERIMENTAL
Experimental details have been described (4). The silver-silver chloride reference electrode was formed into a helical coil about 10 mm. in diameter
Residual Current
Curve I Curve 2 Curve 3
I
studies of the polarography of organic sulfur compounds Nicholson (8) solved the fundamental differential equation for cylindrical diffusion and tested the resulting equations for the polarographic current with several aliphatic sulfides. Nicholson ( 7 ) also observed that the -iimax./cvalues varied with concentration and attributed this to a n ohmic potential effect. She found that correction for this effect by the approximate equation of Delahay ( 2 ) or by using iR drop compensators ( 7 ) was adequate. I n order to apply this technique to crude oils, it was necessary to add appropriate solvents such as benzene t o the electrolyte. When this was done, however, the Delahay correction was too large. The polarograph used in the present work was not equipped with a n iR drop compensator; therefore, it was N HER
1456
ANALYTICAL CHEMISTRY
YI
f 0
e
.-
-.-I I
1.00
I
I
I
I
I
E (Volts
I
I
I
0.60
0.80 VS.
I
I
I
C 0
Ag-MCI)
Figure 1. Successive anodic polarograms of thiacyclopentane
0.384 millimole per liter; platinum electrode No. 4
used
