e, 45
O
70
80 ~~
85
Table V. Reproducibility of A, Smallest Detectable Change, Aa, and Experimental Sensitivity, Gexp Minimal signal amplification Maximal signal amplification a = 0” a = lo _______ Sa, Ao VI % Sa, Ao VI % Aa, Gexp Aa, G Z 0.030 58.48 f l .o 0.015 518. 12 f l . O 3.94 0.016 0.021 3.65 *0.7 0.0053 10.75 f0.4 0.013 21.18 0.0063 9.09 0.0092 21.32 0.0026 10.48 *0.2 22.04 0.0038 15.08 f0.5 0.0068 0.0038 10.40 0.0024 5.22 f0.5 0.0031 5.24 k0.4 0.0013 43.74 0.0023 25.13 O
ous rotation and apparent Cotton effects were found, as can be seen from Figure 4. .4lso the troughs for glutamic acid and phenylalanine were no longer detectable when precautions were taken (4). Furthermore, in the higher wavelength region (>300 mp) spurious rotation was not important. The applicability of Equation 14 was tested in the wavelength region from 600 to 220 mp making use of Equation 21. For an angle 8 = 85’ the values of the constant, A , and the standard deviation, sat, as reported in Table IV prove the applicability of Equation 14. An influence of concentration on the constant A was not detectable. The same result applies to other values of 8. The instrument was constructed in such a way that four values of 8 (45’, 70°, 80°, and 85’) could be adjusted reproducibly. This reproducibility should be reflected in the reproducibility of the constant, A . Therefore five independent determinations of A were carried out using minimal signal amplification and three independent measure-
ments using maximal signal amplification. The mean results for both series are shown in Table V. Here v is the mean deviation in ‘4 for each series. It may be concluded that the adjustment of the analyzers shows a reproducibility within 1%. I n Table V an impression of the precision is given by the standard deviation, sa. Making use of the experimental values of A (Table V) the smallest detectable change A a in the rotation can be calculated, a well as the experimental values (GeXp) for the sensitivity as defined in Equation 2. This is shown in Table V for two values of a. As can be seen, these results compare reasonably with the theoretical values of Table 11. ACKNOWLEDGMENT
The authors express their sincere appreciation to Joh. Groot for constructing the instrument. One of us (I.P.D.) thanks Th. J. de Boer for his interest and valuable discussions.
LITERATURE CITED
(1) Carroll, B., Blei, I., Science 142, 200
(1963). (2) De la Provostaye, F., Desains, P., Ann. Chim. Phus. 27, 232 (1849). (3) . D i r k , I. P., Haak, P. J. van der, Sixma, F. L. J., Chem. Weekblad 56, 151 (1960). (4) Dirkx, I. P., Sixma, F. L. J., Rec. Trav. Chim., in press. (5) Djerarlyi, C., “Optical Rotatory Dispersion, McGraw-Hill, New York, 1960. ( 6 ) “Handbook of Chemistry and Physics,” C. D. Hodgman, ed., 40th ed., p. 3017, Chemical Rubber Publishing Co., Cleveland, Ohio, !958. ( 7 ) Leemann, H. G.,, Stich, K., Chimia 17, 184 (1963) (review). (8) Weissberger,,A., ed., “Physical Methods of Organic Chemistry,” 3rd ed., Vol. 1, D . 1514, Interscience, New York, i949. (9) Woldbye, F., Bagger, S., Acta Chem. Scand. 17, 817 (1963). RECEIVEDfor review November 18, 1963. Accepted A ril 2, 1964. Taken in part from the P%D. thesis of I. P. Dirkx, University of Amsterdam, 1962. The authors are grateful to the van’t Hoff Foundation of the Royal Netherlands Academy of Sciences for donating the Glan prism.
Polarography of Some Benzodiazepines B. Z. SENKOWSKI, M. S. LEVIN, J. R. URBIGKIT, and E. G. WOLLISH N. 1.
Analytical Research latioratory, Hoffmann-la Roche Inc., Nutley, The polarography of a number of benzodiazepines has been investigated. Well defined cathodic waves were obtained at the dropping mercury electrode in a mixture of alcoholwater as the solvent. Factors which influence the azomethine reduction, such as the effect of pH or substituents, were studied. The diffusion current was measured and found to b e proportional to concentration, thus permitting a quantitative determination of these compounds.
0
limited data on the polarographic reduction of benzodiazepines have been reported in the literature, primarily concerned with the polarography of cl~lorodiazepoxide (7 - chloro - 2 - methylamino - 5phenyl - 3 H - 1,4 - benzodiazepineNLY
4-0xide)hydrochlorideJ compound XIII, Table I. We have been studying the properties of benzodiazepines for some time, since this class of compounds has assumed great importance in modern drug therapy. The polarography of heterocyclic compounds containing nitrogen has been discussed by Brezina and Zuman (2) and Kolthoff ‘ and Lingane (6). Various investigators have studied the reduction of the aso,methine group, among them Breyer, Buchanan, and Duewell ( I ) , who worked with acridine and substituted acridines. Lingane, Swain, and Fields (6) demonstrated the electrode process for the reduction of the -C=Nmoiety by controlled potential electrolysis of 9-(0-iodophenyl) acridine in basic solution. Two
electrons were consumed in this reaction with the formation of the resultant dihydro compound. Preparative reductions at controlled potential as well as the polarography of a number of compounds containing ‘the asomethine group were investigated by Lund (7‘). A very systematic and thorough study of the reduction of some purines (9), pyrimidines (IO), and related compounds has been reported by Smith’and Elving. These investigators postulated the reaction mechanism for the reduction of these compounds from data obtained by polarography, controlled potential electrolysis, and subsequent chemical testing of the resultant products. Our objective was to investigate the reduction of the azomethine group in the benzodiazepines. VOL. 36, NO. 10, SEPTEMBER 1964
1991
EXPERIMENTAL
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Apparatus. A RIetrohm E-354 rapid drop polarographic assembly and a Polarecord hlodel E-261 recorder (3) were used in conjunction with a silver-silver chloride electrode to obtain the current-voltage curves. A Beckman Model G pH meter was used to measure pH. Cell resistance was determined with a Thomas-Serfass conductance bridge. Chemicals. All solutions were prepared with chemicals of reagent grade. Buffers. Uritton-Robinson buffers were used as the supporting electrolyte and to obtain the desired pH. Sample Stock Solutions. Solutions were accurately prepared in methanol to contain benzodiazepine concentramolar. tions of 2.5 to 7 X PROCEDURE ,\-AQUEOUS-XLCOHOLIC SYSTEM.I 5-ml. aliquot of the stock solution was pipetted into a 25-ml. volumetric flask and diluted to volume with 0.1N hydrochloric acid or buffer of the desired pH. Upon measurement Df the apparent, pH,the resultant solutions. were deaerated with scrubbed purified nitrogen. The drop time was mechanically set a t a constant rat'e of four drops per second and the polarograms were run a t 25.00 -C 0.05" C. PROCEI)URE B. A 5-ml, aliquot of the stock solution and 5 ml. of methanol containing 0.5M ammonium chloride were pipetted into a 50-ml. volunietric flask and diluted to volume with ethanol. The polarography was carried out as described in Procedure A except for pH measurement. The rapid dropping mercury electrode produced constant drop rates and consequently the electrocaIillary curve associated with the usual slow drop rates was not obt,ained. At potentials from 0 to -1.6 volts, measured in 0.4 volt increments, the capillary characteristic did not, vary more than 2'3G and can be ascribed to the mercury flow rate.
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Figure 1. Influence of drop rate on polarographic waves of chlorodiazepoxide hydrochloride in aqueous BrittonRobinson buffer at pH 3.8 Upper wave, 4 seconds per drop Lower wave, 4 drops per second
1992
ANALYTICAL CHEMISTRY
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Figure 2.
ELECTRODE
Illustration of the azomethine reduction
The recent work, carried out by Oelschlager (8) on the polarography of chlorodiazepoxide hydrochloride in aqueous Britton-Robinson buffers, was in accord with data obtained in this laboratory. However. using a conventional drop time of approximately 4.2 seconds per drop, the formation of masima did not occur in our work with this compound when 0 . 1 s hydrochloric acid cont,aining 20c7, ni!ethanol wab the solvent. The maxima observed in aqueous Britton-Robinson buffers at the same drop time were also eliminated by changing the drop characteristic with an induced rapid rate of 4 drops per second (Figure 1). Polarographic data of benzodiazepines containing the >C=Kas
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Figure 3. Solvent effect on the reduction of chlorodiarepoxide hydrochloride (0.53 millimole/liter) B.
RESULTS AND DISCUSSION
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-1.5
-1.2
-0.8
VOLTS
A.
The half-wave potentials were measured againqt a silver-silver chloride electrode in conjunction with a saturated potassium chloride salt bridge containing 2% agar. Conversion of the potentials in Table I to the saturated calomel electrode was made by applying a correction of 0.046 volt. This factor was determined experimentally and represents the difference in the potentials of the standard calomel and silver-silver chlinride electrodes saturated with resuert to riotassium chloride. The apparent p H of the hydrochloric acid-methanol solutions varied from 0.95 to 1.35. Since the change in Eli2 with IIH was linear within this region and in order to have consistency in the comparison of the data, an extrapolation was made to an apparent p H of 1.0.
-0.4
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Compound 111 (dihydro) Compound XI (tetrahydro) Electrolyte (0.1 N hydrochloric acid containing 20% methanol)
C.
D.
0.1 N hydrochloric acid containing 20% methonol, sensitivity 0.05 pa./mm. 0.05M ammonium chloride in ethanol containing 20% methanol, sensitivity 0.05 pa./mm. 0.05M ammonium chloride in dimethylforrnamide containing 20% methanol, sensitivity 0.05 po./mm. 0.05M ammonium chloride in dimethylacetamide containing 20% methanol, sensitivity 0.2 pa./mm.
well as other reducible functional groups are given in Table I (insert). The reduction of the azomethine group in 0.1N hydrochloric acid containing 20% methanol can be seen in the polarograms of Compound 111, 7chloro - 1,3- dihydro - 5 - phenyl - 2H-1,4-benzodiazepin-%one and Csmpound XI, 7 - chloro - 1,3,4,5 - tetrahydro - 5phenyl - 2 H - 1,4 - benzodiazepin - 2one (Figwre 2). Two protons and two electrons may be postulated in this reduction although analysis of the polarographic wave indicated an an, value of 1.4 for an irreversible process. As is evident from Table I, the El for the reduction of the -C=Ngroup is sufficiently separated from the E l l 2 of the N-oxides permitting the differentiation of Y-oxide from non-Noxide derivatives in the hydrochloric acid-methanol system. Since the half-
Table 11.
Compound I I11 VI
VI11 XI1 XI11
wave potentials of the azomethine group in Compounds 111 through VI are essentially the same, the following reaction process may be postulated for the reduction of the N-oxide derivatives.
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