Journal of Medicinal Chemistry, 1978, Vol. 21, No. 7 649
N - Hydroxyacetaminophen (a) Address correspondence to this author a t SISA Inc., Cambridge, Mass. 02138; (b) deceased. For a recent review, see E. F. Elslager, Prog. Drug Res., 18, 99 (1974); for a detailed discussion, see E. A. Steck, “The Chemotherapy of Protozoan Diseases”, Walter Reed Army Institute of Research, 1971. T. S. Osdene, P. B. Russel, and L. Rane, J. Med. Chem., 10, 431 (1967). The compounds were tested under the auspices of the Walter Reed Army Institute of Research at the Dr. Leo Rane Laboratory, University of Miami, Miami, Fla. E. A. Nodiff, A. J. Saggiomo, M. Shinbo, E. H. Chen, H. Otomasu, Y. Kondo, T. Kikuchi, B. L. Verma, S. M. Matsuura, K. Tanabe, M. P. Tyagi, and S. Morosawa, J. Med. Chem., 15, 775 (1972). German Patent 954599; Chem. Abstr., 53, P6257i (1959). Second International Symposium on Pharmaceutical Chemistry, Munster, Germany, July 22-26, 1968.
(8) F. Bossert, Justus Liebigs Ann. Chem., 680, 40 (1964). (9) W. Baker, G. G. Clarke, and J. B. Harborne, J. Chem. SOC., 998 (1954), used a similar procedure for the conversion of flavones to thionoflavones. (10) A. Schonberg and K. Junghaus, Chem. Ber., 99,1015 (1966). (11) Beilstein, 2nd ed, 17, 395 (1952). (12) Where analyses are indicated only by symbols of the elements, analytical results obtained for those elements were within *0.4% of the theoretical values. (13) L. Thorp and E. R. Brunskill, J . Am. Chem. SOC., 37,1258 (1915). (14) J. M. Strayley and A. C. Adams, “Organic Syntheses”, Collect. Vol. IV, Wiley, New York, N.Y., 1963, p 415. (15) T. Y. Shen, E. F. Rogers, and L. H. Sarett, U.S. Patent 3089876; Chem. Abstr., 59, 12822 (1963). (16) L. B. Clapp, J. Am. Chem. SOC.,70, 184 (1948). Jpn., 24, (17) M. Eto and Y. Oshima, Bull. Agric. Chem. SOC. 473 (1960); Chem. Abstr., 55, 25598 (1961).
Synthesis of N-Hydroxyacetaminophen, a Postulated Toxic Metabolite of Acetaminophen, and Its Phenolic Sulfate Conjugate M a r k W. Gemborys, Gordon W. Gribble, Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755
and Gilbert H. Mudge* Departments of Pharmacology and Toxicology and of Medicine, Dartmouth Medical School, Hanover, New Hampshire 03755. Received November 17. 1977 The synthesis of N-hydroxyacetaminophen (N-acetyl-N-hydroxy-p-aminophenol, 4), a postulated toxic metabolite of acetaminophen (N-acetyl-p-aminophenol, 3), and its phenolic sulfate conjugate (potassium N-acetyl-Nhydroxy-p-aminophenyl sulfate) (13) is described. Potassium p-nitrophenyl sulfate was reduced to the hydroxylamine, acetylated, and treated with sulfatase to yield N-hydroxyacetaminophen. The structures assigned are supported by the spectral data (IR, UV, MS, lH NMR, and 13CNMR). N-Hydroxyacetaminophen was found to be moderately unstable a t physiological pH and temperature, whereas its phenolic sulfate conjugate was stable. Acetaminophen (N-acetyl-p-aminophenol, 3) is a widely used mild analgesic. It is largely metabolized to glucuronide and sulfate conjugates which are readily excreted by the kidney and are considered nontoxic. The possibility that a small fraction of the administered dose of acetaminophen might also be metabolized to a toxic metabolite arose f r o m t h e high incidence of severe hepatotoxicity resulting from suicidal overdosage in man.l The probable mechanism of toxicity has emerged from studies in animals i n which i t has b e e n shown that acetaminophen is converted to a metabolite which depletes hepatic glutathione and then covalently b i n d s t o tissue macromolecules.2 Depletion of h e p a t i c glutathione and the s u b s e q u e n t covalent binding of radio-labeled acetaminophen to hepatic protein are enhanced b y pretreatment with agents known t o stimulate the Cyt P-450 mixed function oxidase system and decreased b y inhibitors of d r u g metabolism.2 The p a t h w a y that has been postulated to account for the covalent binding is shown i n S c h e m e I and involves the formation of N - h y d r o x y a c e t a m i n o p h e n (N-acetyl-Nhydroxy-p-aminophenol, 4) or its dehydration product, N-acetyl-p-benzoquinone imine (6), which then reacts with the sulfhydryl g r o u p of glutathione (7) or o t h e r cellular nucleophiles. More recently i t has been proposed that the s a m e biochemical mechanism m a y underly acetaminophen-induced a c u t e renal necrosis as well as t h e nep h r o p a t h y of chronic analgesic a b u ~ e . ~Although ,~ Nhydroxyacetaminophen is a key compound in t h e proposed
Scheme I
mechanism of toxicity, i t has not been synthesized. M a n y questions involving either hepatic or renal toxicity require the availability of N-hydroxyacetaminophen for toxico-
0022-2623/78/l821-0649$01.00/0 0 1978 American Chemical Society
650 Journal of Medicinal Chemzstrq, 1978, Vol. 21, No. 7
5-6-0 -
Table I. Decomposition of 4 and 13 at 37 " C as a Function of pH
Scheme I1 1.
so,
2.KOH
OH
IO
Zn
AcCl
Compd ( 2 mM) pH"
\
0 S03K
0S0,K
I/ *
-
Gemborys, Gribble, Mudge
4
12
7.0 7.2
0
3
A
8.0
,OH
OSOSK
13
1.5 5.0 6.6
Aryl -Sulfotore
-L
t~! 2 , b Compd
min
(2 mM) pHa
1100
150 66
1.5 5.0 7.0 8.5
21
12.7
C
13
tl!?b
min
910 c C
13000d C
9.8
12.7 C a Buffer is 100 mM (Na,HPO, for pH 6.6, 7.2, 8.0, and and 8.5; AcONa for pH 5.0; HC1 for pH 1 . 5 ; and NaOH for pH 12.7). The slope was obtained from a linear regression analysis of the experimental points obtained in the first 3 half-lives or in the course of the experiment (6 h). The slope was not significantly different from zero at p < 0.05. The decomposition was followed for 24 h.
logical studies, e.g., the detailed examination of the reactions between 4 and cellular nucleophiles or the renal mechanisms by which 4 might become concentrated within relatively weak molecular ions and ions at M - 16, M -- 59, the tubular fluid and parenchyma of the kidney to produce and M - 88; however, a satisfactory mass spectrum of 13 papillary lesions. was not obtained using electron impact or field desorption. In this paper, we are reporting the first synthesis5 of 4, The stability of 4 and 13 in the range of physiological a sulfate conjugate of 2 (potassium N-acetyl-N-hypH was determined by following their decomposition with droxy-p-aminophenyl sulfate, 131,and an analogue of 2 a ferric chloride assay.16 Aliquots of a solution of 4 or 13 (N-acetyl-p-methoxyphenylhydroxylamine, 16). We are in buffer were periodically withdrawn and assayed for the also reporting the stability of 4 and 13 in the range of amount of N-hydroxy compound present. Two factors physiological pH and temperature. interfered with the assays and were appropriately conSyntheses. Potassium N-acetyl-N-hydroxy-p-amino- trolled. First, the decomposition of 4 and 13 led to phenyl sulfate was synthesized since it is a likely phenolic products which increased the absorbance at the wavelength sulfate conjugate of 4. As illustrated in Scheme 11, POmonitored. Second, if 4 was present in the aliquot assay, tassium p-nitrophenyl sulfate (11) was prepared from the complex formed with ferric chloride was unstable, in p-nitrophenol(10) by the method of Burkhardt and Wood6 contrast to 13 and 16,and the absorbance decreased with and then reduced to the hydroxylamine 12 by the method time. of Kamm,7 which was then acetylated with acetyl chloride The rate of decomposition of 4 and 13 (Table I) was by the method of Smissman and Corbett8 to 13. The pseudo-first-order. In an acidic solution, 4 and 13 slowly product (13)was purified by column chromatography using decomposed. In the range of physiological pH, 13 was Sephadex G10 as an absorbent and as a molecular sieve. stable but the rate of decomposition of 4 increased as the The yield of 13 from 11 varied from 25 to 45%. pH increased. Both 4 and 13 were stable in alkaline soThe synthetic route to N-hydroxyacetaminophen (4)is lutions. also outlined in Scheme 11. An aqueous solution of 13 was Discussion added to a buffered solution of aryl sulfatase. When back At low pH, N-hydroxyacetaminophen and N-acetyltitration with dilute base indicated that all of 13 had been N-hydroxy-p-aminophenyl sulfate probably decompose by converted to 4,4was extracted from the aqueous solution the same mechanism proposed for other N-acetyl-Nwith ether. Initially, the sulfatase was Sigma Type Vi9 of aryl hydroxylamine^,^^,^^ Le., decomposition to a nitrenium which one unit hydrolyzes 1kmol of p-nitrophenyl sulfate ion (top of Scheme I). Unlike other N-acetyl-N-arylper minute at pH 7.1 and 37 "C. However, very low yields hydroxylamines, N-hydroxyacetaminophen is unstable in of 4 (
