ill)
o-Methoxy Derivatives of the Carcinogen, N-2-Fluorenylacetariiide. Potential Latent Biological Arylating Agents'
Receiced August 10, 1965 Revzsed Jfanuscript Received A p r i l 29, 1966
The two o-methoxy derivatives of the carcinogen, N-2-fluorenylacetamide, aiz., N-( 1-methoxy-2-flrioreny1)acetamide ( 1b ) and N-(3-methoxy-2-fluorenyl)acetamide(2b), as well as t,he corresponding amines, l-methoxy2-fluorenamine (IC) and 3-methoxy-2-flriorenamine (2c), were synthesized starting from indene and indan-1-one, mid tested for carcinogenic activity. The synthetic path leading to the 3-methoxy derivative is amenable to incorporation of a I4C label in the fluorene ring, heretofore not possible by ot'her routes. Compormds l b and IC hydrochloride were moderately carcinogenic for the rat on feeding, but 2b and 2c hydrochloride were inactive.
A large body of experimental data supports the vienthat binding of chemical agents, or metabolites thereof, to proteins may play an etiological role in chemical carcinogenesk2 >lode1 studies on the binding of the carcinogen, K-2-fluorenylacetamideJ to proteins suggested a mechanism involving the arylation of cellular proteins by the o-fluorenoquinone imines, 2-imino-l,2fluorenoquinone and 2-iniino-2,3-fluorenoquinone. These highly reactive o-quinone imines result from the deacetylation of the ortho-hydroxylated metabolites of N-2-fluorenylacetamide, viz., N- (1-hydroxy-2-fluoreny1)acetamide and N-(3-hydroxy-2-fluorenyl)acetamide (2f), t o the corresponding o-aniinofluorenols which are then oxidized e n ~ y m a t i c a l l y . ~However, the relevance of these reactions for carcinogenesis by N-2-fluorenylacetaniide has remained in doubt, mainly because N-( 1-hydroxy-2-fluoreny1)acetamideand the isomeric 3-hydroxy derivative (2f) 1% ere either weakly or not a t all carcinogenic when administered orally, intraperitoneally or by bladder implantation. While these o-amidofluorenols lacked carcinogenic activity, it is known that the carcinogenicities of many aromatic hydroxylated compounds, which include 2-aniino-lnaphthol, 3-hydroxy-4-dimethylaminoazobenzene, benzo [a]pyren-3-01, and N-(i-hydroxy-2-fluorenyl)acetaniide, are greatly enhanced by methylating their phenolic hydroxyl gr0ups.j The increase in biological activity may be attributable to a greater lipid solubility of the methylated derivatives which would facilitate their entry into the Moreover, the conjugation (1) Supported in part b y U. 9.Public Health Service Grant CA 02571. ( 2 ) (a) E . C. nlillerand J. A. Miller, J . "iatl. CancerInst., 15, 1571 (1955); (b) C. Heidelberger in Ciba Foundation Symposium on Carcinogenesis: hlechanisrns of Action, G. E. \V. JTolstenholme and .\I. O'Connor, Ed., Little, Brown and Co., Boston, Mass., 1958, p 1T9; (e) D. B. Clayson, "Chemical Carcinogenesis," Little Hroivn and Co., Boston, hlass., 1962, p p 372-395; (d) H. C. Pitot and C. Heidelberger, Cancer Res,, 23, 1C94 (1963). (3) (a) H. T. Sagasawa and H. R. Gutmann, J . B i d Chem., 234, 1593 (1959); (b) H . R. Gutmann aud H. T . Kagasawa, zbid., 236, 3466 (1960); ( c ) H. R. Gutmann, U. S.Seal, and C. C. I r i i n g , Cancer Res., 20, 10i2 (1960); (d) C. h l . King, H. R. Gutmann, and S. F. Chang, J . Bioi. Chem., 238, 2199 i1963j; (e) H. T. h-agasawa and A . J. Osteraas, Bzochem. Pharmacol., 13, 713 (1964). (4) (a) H. P. Morris, C . A Valet, B. P. Wagner, >I. Dahlgard. and F. E . R a y , J . Natl. Cancer Inst., 24, 149 (1960); (b) E. C . hliller, J. A. Miller. and H . A . Hartrnann, Cancer Res., 21, 815 (1961); (c) ref 2c. p 222; (d) C. C . Irving, H. R. Gutmann, and L). 31. Laruon. Cancer Res., 23, 1782 (1968). ( 5 ) (a) I). 13. ('layson. J. \V. ,Jull, and G. hl. I%onser,B r i t . J . Cancer, 12, 222 (1958); (b) J. A. Miller, E. C. Miller, and G. C. Finger, Cancer Res., 17, 387 (1957); ( c j .J. A . Miller and E. C. Miller, ibid.. 21, 1068 (1961); (d) J. \V. Cook a n d R. Schoental, B r i t . J . Cancer, 6 , 400 (1952); (e) G. 11. Bonser. L. I3radsliam, D. U. Clayson, and J. \V. Jull, ref Pb, p 215.
to glucosiduronic acids or sulfates and the subsequent excretion of the conjugates by the kidney would be delayed until the methyl ether groups had been cleaved metabolically or until the compound had been further hydroxylated. On the basis of these considerations, the o-methoxy derivatives of S-2-fluorenylacetamide and of 2-fluorenamine were desired for carcinogenicity tests (lb, IC, 2b, and 2c, Table I). These derivatives would not only be expected to be lipophilic, but enzymatic 0-demethylation (and S-deacetylation when applicable) would release o-aininofluorenols capable of arylating cellular constituents oxidatively via the o-fluorenoquinon imines, i e . , these compounds could act as latent, enzyme-activated arylating agents. The preparation of S-(l-niethoxy-2-fluorenyl)acetamide (1 c) for carcinogenicity tests required large quantities of fluoren-1-01 (If) as starting material. Previously, If had been available froin fluoranthene in six steps7 or from indene in seven step^.^^^ The convenient synthesis shown in Scheme I afforded the fluorenol If in three steps. Indenylsodiuiii was condensed with 4-bromobutyronitrile to 4-(3-indenyl)butyronitrile (3). This compound, descaribed previously by Howell and Taylor,*a had not been conipletely characterized. The attachment of the butyronitrile side chain of 3 to the 3 position of indene was confirmed by its nmr spectrum which exhibited a multiplet centered a t 6 = 6.17 ppm due to a single (6) B. I3. nrudie in "Absorption and Distribution of Drugs," T . I%. Ijinns, E d . , E. and S . Liringstone Ltd., London, 1964, pp 16-48. ( 7 ) E . K. Keisburger and J . H . Keisburger, J . Org. Chem., 18, 861 ( 1 9 5 3 ) . (8) (a) F. H . Hoxell and D. A . H. Taylor, J . Chem. Soc., 3011 (1957): (b) h l . A. Morgan a n d H. R. Gutmann, J . OrQ. Chem., 24, 1163 (1959); (e) C. C. Irving and R. F. T\-illard, ibid.,27, 2260 (1962). (9) Traces of residual acid during the isolation of 8-(3-indenyl)pro1)anol ( i ) , a n intermediate in the seven-step syntliesis, catalyzes the cyclization u f
i 11
...
111
tile alcohol to spiro [tetrahydrofuran-2,l'-indan] (ii), a heterocyclic s y s t e m not previously described. The nmr spectrum of this product is consistent x i t h this assignment and rules o u t the isomeric indenopyran structure iii.
720
li I
Scr1carc I
f 0
Br(CH,),CN
3
4
vitiyl protoii.l(' The iiitrile 3 \v:ih tlieii c*yelizetl i i i polyphoxphoric :wid to 3,1-clihydrofluoreii-1(2I-I)-onc (4). The over-all yield of the lietoiie 4 from irideiiylsodiuin wab 67yc. Catalytic clehydrogenation of 4 with I'd in refluxing fluoreiic~E')or catalytic* tr:iii4cr dehydrogenatioiiil gave fluoreii-1-01 (If). C'oinpouiicl If was then nitrated t o 2-nitrofluoren-1-01 (lg)' and 5 a , R = H . X Cl the latter \vas methylated to 1-rnethoxy-2-nitrofluob, R=CH,,X I rene (la), 1-;\lethoxyfluorenr n licii iiitrated under the cbonditions employed for the nitration of fluorc>iimcooc2H5 1-01 (If) was recovered uric8h:inged. Rcduct ivc :icc.tylatioii of l a afforded S-(l-iiietIios.-2-flilol.cllyl):lc.c't OH amide (lb) while catalytic, 1iydrogcn:itioIi gnvc. 1 6a methor;y-2-fluoreriai~iii~e (IC). The isomeric N-(X-lne t hoxy-2-fluorenyl) :mt:iiiiidt~ (2b) aiid 3-methoxy-2-fluoreriai~ii1ic( 2 ~were ) prep:~retl in a similar mariner hy rrductive acrtglatioii w i i d catalytic hydrogenation of ~~-inetho~y-2-1iitrofluore1i~~ (2a).I2 The latter (2a) IT prcyared hy nitration of 7 ;3-i~irthoxyfluoreiieand its structure was (,onfirmed by denicthylation t o tlie I m n w 2-nitrofluoren-3-01.'" The preparation of t h e 3-niethoxyfluorcne derivatives ( I O ) Tlie at taclirnent of the butyronitrile side chain t u the :3-positiun of indene is expected from the studies of .\,-N. ]Yeidler, Acta Chem. Scand.. 17, 2724 (1963), \rho showed t h a t in tlie alkylation of indenylsodiiim, a 1alkylindene is first formed which ralidly rearranges t o t i i n tliermodynami
