( 5 ) Blodgett, K. B., J . Am. Chem. SOC.57, 1007 (1938). 16) Coster, D., Hof, S., Phusica 7, 655 (1940). ( 7 ) Crisp, R. S., Phil. Mag. 5,1161 (1960). ( 8 ) Crisp, R. S., Tlrilliams, S. E., Ibid., 6, 363 (1961). ( 9 ) Fischer, D. W., Baun, W. L., Spectrochzm. Acta 21, 443 (1965). (10) Ibid., J . Appl. Phys. 36, 534 (1965). 111) Fischer. D. W.. J . Chem. Phus.. 43 ‘ June (1965) (in press). (12) Ibid., J. Appl. Phys., 36 (1965) (in j
,
press). (13) Fisher, F., Crisp, R. S., Williams, S. E., Optica Scta 5, 31 (1958). (14) Furnas, T. C., White, E. W., WADDTR-61-168 (1961). (15) Gwinner, E., Kiessig, H., 2. Physik. 107, 449 (1937). (16) Hautot, A,, Serpe, J., J . Phys. 8, 175 (1937).
(25) Prins, J. A., Takens, A. J., 2. Physik. 77, 795 (1932). (26) Rogers, J. L., Chalklin, F. C., Proc. Roy. SOC.(London) B67, 384 (1954). (27) Schnell, E., Monatsh. Chem. 94, 703
(17) Henke, B. L., “Advances in X-Ray
Analysis,” Vol. 7, Plenum Press, N. Y., 1cl64
(18) Holliday, J. E., J . A p p l . Phys. 33, 3259 f 1962). (19) Ibid., “Handbook of X-Rays,” Chapter 38, McGraw-Hill, New York, 1965. (20) Nicholson, J. B., Wittry, D. B., “Ad-
(196.11.
(28) Siegbahn, K., Contract Report #2,
AF 61-052-795, Institute of Physics, Uppsala University, 1964. (29) Siegbahn, M., Magnusson, T., 2. Physik. 87, 291 (1934). (30) Ibid., 95, 133 (1935). (31) Tomboulian, D. H., Cady, W. M., Phys. Rev. 60, 551 (1941). (32) Tyren, F., Arkiv. Mat. Astron. Fysik. 25A, #32 (1937).
vances in X-Ray Analysis,” Vol. 7, Plenum Press, N. Y., 1964. (21) O’Bryan, H. M., Skinner, H. W. B., Proc. Roy. SOC.(London) A176, 229
(1940). (22) Ong, S. P., 13th Annual Conference
on Applications of X-Ray Analysis, Denver, Colo. August 1964. (23) Pauling, L., “The Nature of the Chemical Bond,” Cornel1 University Press, 3rd Edition, 1960. (24) Piori, E. D., Harvey, G. G., Gyurgy, E. M., Kingston, R. H., Rev. Sci. Instr. 23, 8 (1952).
RECEIVED for review January 27, 1965.
Accepted April 19, 1965. 16th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1, 1965.
Spectrofluorometric Determination and Mechanism of Fluorescence of 5,6-Dihydro-5-(2-imidazolin-2-ylmethyI)-mo r pha nt hridine a nd Related Compounds FREDERICK TISHLER, H. E. HAGMAN, and S. M. BRODY Research Department, ClBA Pharmaceutical Co., Summit, N . J . A spectrofluorometric procedure for
-
-
5,6 - dihydro - 5 (2 - imidazolin - 2 ylmethyl)-morphanthridine and related compounds, based on the fluorogen formed when the compound i s heated with acetic anhydride, i s applicable to blood level studies where as low as 0.2 pg. of the compound per milliliter of blood can be determined. At this level, the recovery from blood ranged from 85 to lO5%, with a mean recovery of 95% and standard deviation of 2 8 % . The product formed in the reaction has been isolated by preparative thin layer chromatography and a mechanism for the acetic anhydride-induced fluorescence i s postulated.
Amino - Bowman spectrophotofluorometer equipped with an oscilloscope. Nuclear magnetic resonance spectra were obtained in CDC13 on a Varian A-60 using tetramethylsilane as a n internal reference standard. A Cary recording spectrophotometer and a
Beckman IR-5 were used to record ultraviolet and infrared spectra, while current-voltage curves were obtained on a Leeds and Northrup recording Chemograph. Reagents. All compounds studied (Table I) were either of reference
H
CO-CH3
T
introduction of therapeutically active imidazolinyl - substituted morphanthridine compounds has made it necessary to develop an analytical procedure capable of detecting microgram quantities of the compound in blood level studies. When the parent compound is heated with acetic anhydride a t 100’ C., a fluorogen is formed, which is the basis of the method described. Isolation of the reaction product by preparative thin layer chromatography and identification by application of infrared, ultraviolet, nuclear magnetic resonance spectra, and current-voltage curves have made it possible to postulate a mechanism for the acetic anhydride-induced fluorescence. HE
EXPERIMENTAL
Excitation and fluorescence spectra were recorded on an X-Y recorder in conjunction with a n Apparatus.
906
ANALYTICAL CHEMISTRY
COCH~
CH I
,A“
[
b Lo I
“
III
t Ht
CHZC-N-CHz-CH2NH
-C’ kOCH3
.Ix
I1
CH3
VI
IV
VI1
/ Figure 1. Postulated mechanism for acetic anhydride-induced fluo-
,I.C-CH2-r;r-CH2CH2-N“3
rescence
VIII
-
LOCH3
standard quality as determined by phase solubility (4) when possible or Table 1. Fluorescence of 5,6-Dihydro-5-(2-imidazolin-2-yl-methyl)-morphanchromatographically pure as determined thridine and Related Compounds by thin layer chromatography. The Structure Fluorescence ( or - ) acetic anhydride was of fluorescent grade quality. Procedure. GEKERAL. All compounds (free bases) were dissolved 1 Excitation 310 in acetic anhydride, and a 15-ml. Fluorescence 410 aliquot (5 pg.) was heated a t 100" C. for 45 minutes. The solutions were cooled to room temperature and diluted to 25 ml. with acetic anhydride. The fluorescence spectra were viewed on 2 the oscilloscope a t various excitation wavelengths. Uncorrected excitation and fluorescence wavelengths of 5,6dihydro - 5 - (2 - irnidazolin - 2 - ylmethyl)-morphanthridine (hereafter referred to as Compound I, Figure !) 3 and related compounds appear in Table I. ISOLATION PROCEDURE. One gram of Compound I was mixed with 25 ml. of acetic anhydride and the mixture 4 was heated a t 100" C. for 1 hour. The acetic anhydride was evaporated in vacuo and the reaction product then isolated by preparative thin layer chromatography on Silica Gel G according t o the procedure described previously 5 (3, 6) using a solvent system composed of benzene-2-propanol-formic acid-water (80: 80: 20 : 10). Each plate, containing about 0.5 gram of material, was developed twice for a better resolution of the mixture. Each developExcitation 310 6 ment took approximately 16 hours. Fluorescence 390 The plate was viewed under ultraviolet light and the major band isolated using a 1 to 1 mixture of chloroform-methanol as the eluent. After evaporation of the solvent, the compound was further 7 lCH3 HC-COOH I purified by chromatographing on a CH2-CHz-CH2-N !I small alumina column. The total reHC-COOH covery from the preparative thin layer 'CH3 procedure was 86%. Using a 10-gram sample, 5.0 grains of the major product and 3.6 grams of the minor products, 8 which consisted in large part of unreacted starting material, were isolated. C-CHJ POLAROGRAPHIC DETERMINATION. The anodic behavior of all compounds was determined according to the procedure of Rehm and Smith ( 5 ) . The N H electrolytic cell consisted of a con9 ventional H-cell with a reference elecCH2-t] I trode of a silver wire, immersed in 0.lM N silver nitrate solution. A 5-mm. length of platinum sealed in a glass tube and rotated a t a constant speed of 500 r.p.m. was used as the anode. Contact 10 with the electrode was made through a column of mercury inside the tube. Oxidation studies were carried out a t a concentration of approximately 10-31W in spectral grade acetonitrile using 10% hydroxide and 20 ml. of ether. The heated in an oven a t 100' C. for 45 sodium perchlorate as the supporting minutes, cooled to room temperature, tubes were shaken for 2 minutes and electrolyte. The current-voltage curves and then diluted to volume with acetic centrifuged, and the ether layer was were then run over the range of 0 to anhydride. The fluorescent intensity withdrawn into a 150-ml. beaker with f1.0 applied volt. was determined a t an excitation of 310 the aid of a IO-ml. hypodermic syringe DETERMINATIOX IN BLOOD.Five mp and a fluorescence of 410 mp. equipped with a 14-gauge, round-nosed, milliliters of distilled water (standard 15-cm. needle. The extraction was reblank), 5.0 ml. of standard solution peated with four 20-ml. portions of (1 pg. per ml. of water), 5.0 ml. of the RESULTS AND DISCUSSION ether. After evaporation to dryness patient's blood prior to medication with the aid of a hair dryer (cold air), The elemental analysis of theyisolated (sample blank), and 5.0 ml. of the the residue was transferred quanticompound indicated that diacetylation sample blood were pipetted into septatively to a 25-ml. volumetric flask of Compound I (Figure 1) had occurred, arate 40-ml. centrifuge tubes. To each with the aid of three 5-ml. portions of followed by the opening of the imidazoacetic anhydride. The flasks were tube were added 2 ml. of 0.2N sodium
+
+
+
Q \:
VOL. 37,
NO. 7, JUNE 1965
0
907
b Figure 3.
Nuclear magnetic resonance spectra A. Compound I 6. Compound Vlll C. Compound 8
2. Ultraviolet absorption spectra of 5,6dihydro-5-(2-imidazolin-2-yl-methyl)-morphanthridine Figure
(0.03mg./ml.)
and related compounds in methanol 1. 2. 3. 4. 5.
Compound I Compound Vlll N-Methylacetanilide Compound 10 Compound 8
line ring, which would ordinarily lead to Compound IX (1). The infrared spectrum, however, showed only a single broad band in the carbonyl region (1630 cm.-I) and not two widely separated bands in the regions of 1790 to 1720 cm.-l and 1710 to 1670 ern.-' which is typical of compounds with an 0 0
11
I
I/
.
imide structure (-C-N-C-) The ultraviolet spectra of Compound I (Figure 1) and related compounds, except Compounds 4 and 8, and N methylacetanilide, showed similar specTable II.
Compound I Compound VI11
tra with maxima between 245 to 255 mp (major) and 295 to 305 mp (minor), which is characteristic of N,N-dialkylsubstituted anilines (2). The spectrum of the isolated compound (Figure 2) did not show the characteristics of an N,N-dialkylarylamine. The polarographic behavior of the compounds in Table I and those with similar structures studied by Rehm and Smith (5) all exhibited well defined oxidation waves (except Compounds 4 and 8), varying from +0.53 to +0.71 volt us. Ag/Ag+ (0.lM) a t the platinum electrode. Compounds 4 and 8 and the
isolated compound exhibited no waves between 0 and $1.0 volt. Since the polarographic behavior of the compounds studied appeared to correlate with the same structural feature as the ultraviolet spectra, the isolated compound probably no longer contained an N,N-dialkyl nitrogen. Further evidence that the structure represented by Compound I X was incorrect was obtained from a study of the nuclear magnetic resonance spectra of the isolated compound and related compounds (Figure 3). In Table I1 are summarized the structural assignments of the isolated compound. From the spectra (APBP pattern), it was evident that the imidazoline ring had opened and that both nitrogens were acetylated. The nuclear magnetic resonance spectra of Compound I and similar compounds clearly showed sharp signals for each of the methylene groups in the morphanthridine nucleus as well as the methylene group which connects the morphanthridine ring to the imidazoline ring. However, the isolated compound, Compound 8, and similar compounds which contained a carbonyl group adjacent to the nitrogen of the morphanthridine nucleus exhibited only one or two sharp or broad signals (depending on the compound studied) and one smeared signal which, on first inspection, might be interpreted as a possible impurity. The integrated area, in each case, indicated the presence of four or six hydrogens, which is consistent for two or three methylene groups. The smeared signal, which is
Structural Assignments from Nuclear Magnetic Resonance Data in Cycles per Second
261 264
241 or 262 200-400
(smear)
241 or 252
272 394
...
215 (ring closed) 207 multiplet 223 multiplet A& pattern (ring open) 325
1li '$nd 118
316 134
I
-N-I Compound 8
908
0
241 (broad)
ANALYTICAL CHEMISTRY
250-360
(smear)
...
...
115
c=o ,*-*\
I
C - ICH2,; -N-
6
I-_.
...
...
m
'I
I
I U
W J U L
. .
_ . . .* . . .
I
?n
. . I . - 0
.
.
. . .
.
I . . I . . .
U
,.._
I -
.
U
. . . , I
. . . .
.
I
.
..
. . . . . . I . .
u
..
I #-..I
W
I
.
.
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.
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,A. U
VOL. 37, NO. 7,JUNE 1965
e
909
Table 111. Recovery of 1 pg. of 5,6Dihydro 5 (2 imidazolin 2 ylmethyl)-morphanthridine from 5 MI. of Blood
- - -
Sample“
- -
Found, p g . 0.87
Difference, p g . -0.13
1.05 1.00
+0.05 0.00 -0.07
2-B
0.93 1.04
4-B 5-E3
0.85
-0.07 +0.04 -0.08 -0.15 -0.05
1-A 2-A 3-A 4-A
5-A
I-B 3-B
0.93 0.97 -0.03 Mean 0.96 Std. dev. 0.08 0.92
0.95 Mean 0.94 Std. dev. 0.07 Samples 1-A to 5-A were determined using blood from Patient A; samples 1-B to 5-B represent blood from Patient B. 5
attributed to the ring methylene group
\
/ \
connected to the nitrogen (CH2-N),
showed up as a sharp signal when spectra were obtained a t higher temperatures. (A more detailed discussion will be presented in the future describing this effect together with an explanation of the various shifts of the methylene groups.) Final proof that the carbonyl group was adjacent t o the nitrogen of the seven-membered ring was obtained from the spectra of the hydrolyzed product of Compound I--namely, Compound 10. This compound exhibited normal ultraviolet (Figure 3) and nuclear magnetic resonance spectra as well as a well-defined oxidation wave.
Based on all of the above data, it can now be inferred that the isolated compound has the structure assigned to Compound VI11 in Figure 1. Compound VI11 is probably formed in a manner as represented by scheme 1 in Figure 1. A species such as that given by Compound IV would probably be responsible for the fluorescent characteristic of Compound I. A similar mechanism can be applied to Compound 6 (Table I), thereby explaining its fluorescent properties. Since this mechanism can also be applied to Compounds 2 and 9, it can be seen that a second prerequisite is needednamely, a dihydromorphanthridie nucleus. A plot of fluorescent intensity us. time indicated that optimum fluorescence was obtained when the compound was heated with acetic anhydride a t 100” C. for 45 minutes. At temperatures lower than 100” C., the reaction proceeded very slowly. A plot of fluorescent intensity us. concentration of an extracted standard was found to be linear up to a t least 5 pg. per 25 ml. of final solution. Since this would encounter the expected concentration in blood, higher concentrations were not studied. Standards, blood samples, and blood blanks were always run in duplicate when possible, and duplicate intensity readings never differed by more than 5%. At all levels studied the “noise” of the instrument was of no significance. Recoveries of 85 to 105% were obtained from blood a t the level of 0.2 pg. per ml. Table I11 summarizes the recovery of Compound I a t this level.
A blood blank, although not necessary most of the time, was run in the same manner as the sampb, since it was found that the blood of several patients and animals contained compounds which tended to give higher results. The interference could clearly be seen by comparing the fluorescent spectra of the standard and sample on the oscilloscope. The highest blood blank observed would be equivalent to 0.2 pg. of drug per ml. of blood. However, since the fluorescent intensity reading is sufficiently high for acceptable precision and accuracy at this low level, it is felt that such a high blank is no drawback in this procedure. ACKNOWLEDGMENT
The authors thank Keville Finch,
L. H. Werner, and W. E. Rosen for their advice and interest in this work and Louis Dorfman and his group for their assistance in interpreting the nuclear magnetic resonance spectra. LITERATURE CITED
(1) Hofman, K., “Chemistry of Hetero-
cyclic Compounds, Imidazole and Its Derivatives,” Part I, p. 221, Interscience, New York, 1953. (2) Kamlet, M. ,!., “Organic Electronic Spectral Data, Vol. I, p. 216, Interscience, New York, 1960. (3) Korzun, B. P., Dorfman, L,, Brody, S. M., ANAL.CHEM.35, 950 (1963). (4) Mader, W. J., “Organic Analysis,” Vol. 2, p. 253, Interscience, New York, 1954. (5) Rehm, C. R., Smith, J. B., CIBA Pharmaceutical Co., Summit, N. J., unpublished work, July 1963. (6) Tishler, F., Brody, S. M., J . Pharm. Sci. 53, 161 (1964). RECEIVED for review November 3, 1964. Accepted April 12, 1965.
A Study of the Effect of Tetracyanoethylene, TrinitrofIuorenone, and Other Pi Acceptors on the Fluorescence of Aromatic Carbons GEORGE H. SCHENK and NORMAN RADKE Department of Chemistry, Wayne State University, Detroit,
b Tetracyanoethylene (TCNE), 2,4,7trinitrofluorenone (TNF), 7,7,8,8-tetracyanoquinodimethane (TCNQ), and other 7r acids or acceptors were studied to determine their relative quenching efficiency and their effect on the fluorescence excitation and emission maxima of aromatic hydrocarbons such as anthracene and pyrene. TCNE does not eliminate the fluorescence of anthracene via the Diels-Alder reaction since fluorometric excitation wavelengths at 365 mp cause photodecomposition of the TCNE-anthracene adduct. Both TCNQ and TNF are more effective 910
ANALYTICAL CHEMISTRY
Mich. 48202
quenchers of pyrene than TCNE; this correlates with the magnitude of their ground state formation constants. TCNQ, TCNE, and TNF appear to exert a small effect on the fluorescence excitation and emission maxima of the aromatic hydrocarbons themselves.
P
ACCEPTORS or acids such as tetracyanoethylene (TCNE) (16) and 2,4,7-trinitrofluorenone(TNF) (16) have been studied for their potential use in spectrophotometric methods for n donors or bases. Another potential I
analytical use for these compounds is that of quenching or modifying the fluorescence of aromatic hydrocarbons. It is our object to summarize the pertinent literature and present analytical studies. A distinction must be made between the action of the A acceptor in quenching the fluorescence emission of the n donor itself, and that of the 7r acceptor in forming a n complex which also emits fluorescent light, but a t longer wavelengths. If the R acceptor is strong enough, the fluorescence emission of the P complex w ill occur far enough away
