Studies on the Structure of “Boun” Dmorphine*

Studies on the Structure of “Bound” Morphine* By JAMES M. FUJIMOTOt and E. LEONG WAY Crystalline “bound” morphine isolated from the urine of addicts was subjected to various physical and chemical tests. On the basis of X-ray diffraction, infrared, ultraviolet, and electrometric data, the compound was identified to be 3-morphineglucuronide i n the zwitterion form.

N A PREVIOUS COMMUNICATION

the isolation and

I c rystallization of conjugated morphine from the urine of human addicts was reported (1). It was suggested that this morphine metabolite is the principal if not t h e only form of “bound” morphine excreted i n the urine. Indirect evidence for the existence of such a metabolite of morphine was presented earlier (2, 3). Subsequently, Woods (4) isolated a morphine conjugate from the urine a n d bile of dogs a n d reported it t o be morphine-monoglucuronide dihydrate. The present communication describes experiments leading t o the establishment of the identity of the “bound” morphine form the urine of human addicts. EXPERIMENTAL Two samples of conjugated morphine from different sources, as well as a n authentic sample of morphine, were subjected to various physical and chemical tests as described below. One sample of conjugated morphine was obtained from the urine of human addicts’ in crystalline form a s previously described (1) and the other conjugated morphine obtained from dog urine.’ The morphine content was measured b y the method of Fujimoto, Way, and Hine (5) after effecting the release of free morphine from the conjugated form by acid-pressure hydrolysis. The method, which is highly specific for morphine, depends upon separation of morphine from other phenolic substances by its selective extraction from a strongly alkaline solution, followed by estimating i t with a phenolic reagent. The glucuronide content of “bound” morphine was measured by the glucuronic acid method (6) with naphthoresorcinol or that of Dische (7) using carbazole. Either menthyl glucuronide or glucuronolactone were used as the standards. Powder X-ray diffraction patterns (chromium Ka) were obtained on the dinitrophenol ether derivatives of the morphine liberated from each sample

*

Received July 13, 1957 from Department of Pharmacology, Schools of Pharmacy and Medicine, University of California, San Francisco. Supported by a research grant (USPHS RG-1839) by the National Institutes of Health. f Present address: Department of Pharmacology, School of Medicine, Tulane University, New Orleanr, La. 1 The urine from addicts was obtained through the courtesy of Drs. Harris Isbell, H. Frazer, and Ann Eisenman, Addiction Research Center, Lexington, Ky. * Kindly furnished to us by Dr. L. A. Woods of the University of Michigan.

by acid hydrolysis.8 Preparation and purification of the dinitrophenol ether derivatives were carried out according to the procedure used by Adler and Shaw (8). Ultraviolet absorption studies on the samples were carried out in acid and in alkali, using a spectrophotometer (Beckman, model DU). The spectra were first obtained on 3 ml. aliquots of each sample dissolved in 0.5 N HC1 to make a concentration of 154 mcg./ml., following which 0.5 ml. of 50% potassium hydroxide was a.dded and the spectra again determined. Infrared spectra were obtained on samples either in a n oil mull or as evaporated residues with a double-beam spectrophotometer (Perkin-Elmer, model 21). The titration curves of the samples were C3btained with 14.5 mg. or 2.9 X 10-3 milliequivalents (using 499 as the molecular weight of morphine monoglucuronide dihydrate) of the metabolite dissolved in 5 ml. of distilled water. Higher concentrations of the material were not used because of the low solubility of the compounds. The course of the titration was followed with a pH meter (Beclman, model H-2), adding standard acid and base by means of a microburette. The initial pH was 5.8. RESUL’TS

Table I shows that the morphine content of the crystalline bound morphine isolated from urine of human addicts is the same as that TABLE I.-MORPHINE

AND GLUCURONIC ACIDCONTENT OF THE METABOLITESISOLATED FROM HUMAN URINEAN:D FROM DOGURINE

MORPHINE

Test

Metabolite from Human Urine

Metabolite from Dog Urine

Morphine 5 6 . 8 f 3 . 0 56.8zk2.2 Content (% of total weight) Glucuronic 42.8 =!= 1.0 41.8 =k 0 . 4 Acid Content (% of total weight)

Tbeoretical Values”

57.2

38.9

From Woods (4) for morphine monoglucuronide dihydrate. We are indebted to Dr. D. H . Templeton of the D e p a t ment of Chemistry, University ,of California. Berkeley, for the powder X-ray diffraction Fmattern determinations and interpretations.

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for the conjugated morphine obtained from dog urine. The values obtained are consistent with calculated values for the morphine monoglucuronide dihydrate postulated by Woods (4). The presence of morphine as an essential component of conjugated morphine from urine of human addicts was further established by the powder X-ray diffraction studies made on the dinitrophenol derivative of the hydrolyzed morphine conjugate. The pattern (Fig. 1) obtained showed lines corresponding in position and intensity with those obtained from authentic morphine dinitrophenyl ether. The pattern of the latter agrees with those of the ether derivative of authentic and biosynthetic morphine (8). The pattern of the dinitrophenol ether of the hydro-

Fig. 1.-X-ray diffraction pattern for dinitrophenyl ether derivation of morphine from crystalline bound morphine.

lyzed morphine conjugate derived from dog urine was also the same as the other patterns. This procedure is highly specific for morphine and is capable of distinguishing slight differences between ethers of morphine and dihydromorphine where the usual colorimetric methods fail. The presence of the glucuronic acid moiety in the bound morphine rests on two types of evidence. In the infrared curves shown in Fig. 2 the

I'd. XLVII, No. 4

very strong band in the 3b region is in agreement with the OH stretch frequency broadened by association to be expected from a polyhydroxy glucuronide compound. The strong absorption in the 6 fi-7 f i region is in agreement with the presence of a carboxylate ion. Also, the compound gives the typical color reaction for glucuronic acid by both the naphthoresorcinol and carbazole methods for the estimation of glucuronic acid type compounds. It should be noted, however, that the glucuronide values given in Table I are somewhat higher than theoretical. This is probably attributable to a higher color index for the morphine monoglucuronide as compared with the standards. This is not surprising since higher values for other glucuronic acid conjugates as compared to glucuronolactone are known to exist. I n all probability this morphine conjugate is a P-Dglucuronide but this point was not established. The molecular site of conjugation of bound morphine with glucuronic acid appears to be at the 3-phenolic position since the phenol reagent used in the morphine determination procedure did not give a color with the conjugated material until after acid pressure hydrolysis. Moreover, the ultraviolet absorption characteristics of the compound in acid and base give no evidence as to the presence of a free phenol (Fig. 3). Morphine and its congeners which have a free phenol group a t the 3 position (such as nalorphine, normorphine

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Fig. 2.--Infrared absorption characteristics of bound morphine from human addict urine (upper curve) and from-dog bile (lower curve).

Fig. 3.-Ultraviolet absorption curves for bound morphine in acid and base. Morphine monoglucuronide, 154 microgm./ml. distilled water.

April 1958

SCIENTIFIC EDITION

dilaudid, and metopon) show a bathochromic shift with an increase in pH as is generally known for phenols ( 5 ) . Since the curve for the morphine conjugate is very similar to that obtained for codeine in which the phenol group is masked, the evidence is consistent with the postulate that the conjugation of morphine with glucuronic acid occurs at the 3- position of morphine. Further evidence to support the conclusion that morphine conjugate from addict urine is a glucuronide was obtained by establishing its identity with morphine monoglucuronide dihydrate obtained by Woods (4) from dog urine and bile. Proof was based on elemental analyses and assays for morphine and glucuronide content. As previously described, our assays of morphine and glucuronide content are compatible with such a structure and, furthermore, the infrared absorption spectra indicate clearly that the two bound morphines from different sources are identical (Fig. 2). The slight difference in the shape of the curves in the region of 3 I.( can be explained as due either to the presence of differing amounts of associated water of crystallization or to a minor difference in crystal structure. The infrared spectra4 of bound morphine also yield information that indicates the compound exists as a zwitterion. The infrared spectra show a maximum at 6.2 I.( with no band between 5.6 and 6.2 p . This can be interpreted to mean that the carboxyl group of the glucuronic acid moiety is present in an ionized form. Such an interpretation would in turn necessitate the presence of a positive charge on the piperdine nitrogen. Thus the crystal would have to be an internal salt or zwitterion. The possibility that bound morphine might exist as an ampholyte was mentioned by Oberst (9). The titration curve, Fig. 4, gives additional evidence for the zwitterion formulation as well as an estimate of the pK values for the two groups which make up this zwitterion. Note that there are two pK values, one on each side of the initial starting point of the titration. If these pK values are estimated as occurring at the halfway point of the titration, that is at 1.45 X milliequivalents of acid and base, a pK1 of 3.2 and pK2of 8.1 are obtained. The corresponding dissociation constants would then be 6.3 X and 0.8 X lo", respectively. Since the latter value is the constant for the conjugate acid, its basic dissociation constant would be 1 X This value is in very good agreement with the 4 We are grateful t o Dr. L. A. Strait and Mr. Michael Hrenoff Spectrographic Laboratory, School of Pharmacy, University of California Medical Center, San Francisco, for the infrared determinations and their comments.

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Fig. 4.-Titration

curve for morphine monoglucuronide and approximate pK values. Formula for zwitterion.

value theoretically predicted by Kumler (10) for the basic dissociation constant of this compound. The value of 6.3 X for Ki is also in excellent agreement with the predicted value of lo-' for the Kd value for this compound (10). K 1 is the dissociation constant for the structure with a positive charge on the nitrogen, while Kd is for the form without a positive charge. According to Kumler ( l l ) , K 1 will be somewhat larger than K d because of the positive charge on the nitrogen. While the positive charge on the nitrogen is acid-strengthening, it could not be responsible for the observed increase of acidity of 0.8 of a pKa unit by its inductive effect through nine intervening atoms. However, molecular models of the compound show that the positively charge nitrogen and the carboxyl group can approach within four Angstroms of one another and the resulting field effect directly through space could account for an increase of acidity corresponding to a few tenths of a pKa unit. Based on Kumler's calculations, a t pH 5.8, 99.99% of morphine monoglucuronide dihydrate is in the zwitterion form (10) with the structure as shown in Fig. 4. REFERENCES (1) Fujimoto, J. M.. and Way, E. L., J . Pharmacol. Ezpll. Therap. 121 340(1957). (2 Gross, E. G.', and Thompson, V., ibid., 68,413(1940).

31 Oberst F. W. rbid 69 240(1940). 14) W?odi L. A.;ibid.y lli, 158(1954) (5) FUJlmOtO, J. M., w a y , E. L., and' Hine, C. H.. J . Lab. Clan. Mcd., 44 627(1054). ( 6 ) Maughan, d.B., Eve1 K. A,, and Browne, 1. S. L.. J . B i d Chem 126 567(1038qP' (7) 'Dische: 2..:bid., 167, 189(1047). (8) Adler, T.K., and Shaw, F. H., J . Pharmacol. Exp?:. Therap 104 l(1952). (Q 'bbedt F. W. ibid 73 401(1941). (101 Kumlcr, W. 6..J.'brg: Chcm., 20,700(1955). (11) Kumler, W. D., personal communication.