Chem. Res. Toxicol. 1992,5, 797-801
797
Mutagenic Decomposition Products of Nitrosated 4- Chloroindoles Nigel K. Brown,tJ Thanh T. Nguyen,t98 Koli Taghizadeh,? John S. Wishnok,? and Steven R. Tannenbaum'9tIll Division of Toxicology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received December 13, 1991
4-Chloro-6-methoxyindole,a constituent of fava beans, forms a potent direct-acting mutagen, 4-chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one oxime,when nitrosated. In order to better understand the properties of this mutagen, we have studied a readily-available analog, 4-ChlOrO2-hydroxy-1-nitrosoindolin-3-one oxime, prepared by nitrosation of 4-chloroindole. This analog is also mutagenic, and both mutagens decompose rapidly a t neutral or higher pH to yield in each case a new, less potent mutagen which then reacts further to form a nonmutagenic final oxime, on product. The two products arising from 4-chloro-2-hydroxy-l-nitrosoindolin-3-one the basis of comparison of spectroscopic and chromatographic evidence with that from authentic standards, are 4-chloro-N-nitrosodioxindoleand 4-chloroisatin; those arising from 4-chloro-6methoxy-2-hydroxy-l-nitrosoindolin-3-one oxime appear to be the corresponding 6-methoxy analogs. The interplay of these pathways with respect to net biological activity, especially under gastric conditions, remains to be described. Introduction Studies presented in the 1970s (1) highlighted the existence of a population in Colombia found to be at especially high risk for developing stomach cancer with an estimated, age-adjusted incidence rate of 150 per 100 000population. Nitrite ions from bacterial reduction of dietary nitrate were hypothesized as one causative factor (2)yielding activated N-nitroso compounds from suitable nitrogenous precursors under gastric conditions (3-5).This laboratory demonstrated that fava beans, a prominent component of the Colombian diet, yielded a fraction mutagenic to Salmonella typhimurium TM677 when treated with nitrite under simulated gastric conditions (6). The promutagen was later identified as 4-chloro-6methoxyindole (CMI;I Figure 1,I) (7). Nitrosation of this compound yielded 4-chloro-6-methoxy-2-hydroxy-l-nitrosoindolin-3-one oxime (NCMI; Figure 1, 11),a directacting exceedingly potent mutagen of even greater activity (MNNG), a than N-methyl-N-nitroso-N'-nitroguanidine known gastric carcinogen in the rat (8, 9). NCMI can induce sister chromatid exchange and forward mutations in the HGPRT locus in V79 Chinese hamster cells and can inhibit gap-juctional intercellular communication,which is an index of potential tumor-promoting ability (IO). 4-Chloroindole (4CI; Figure 1,111)can be nitrosated in an * Address correspondence to this author.
Division of Toxicology. Present address: Upjohn, Ltd., Fleming Way, Crawley, West Sussex RHlO 2NJ. Eneland. 8 Present ad8ress: Universal Foods Corp., 6143N 60th St., Milwaukee, WI 53218. 11 Department of Chemistry. Abbreviations: MNNG, N-methyl-N-nitro-N'-nitrosoguanidine; HGPRT, hypoxanthine guanine phosphoribosyltransferase;EI, electron ionization; PCI, positive chemical ionization; FTIR, Fourier-transform infrared spectrometry; 4NQO,4-nitroquinolineN-oxide; DMSO, dimethyl sulfoxide; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; CMI,4-chlorc-6-methoxyindole; 4CI,4-chloroindole;NCMI, 4-chlorc6-methoxy-2-hydroxy-l-nitrosoindolin-3-one oxime; NICI, 4-chloro-2hydroxy-1-nitrosoindolin-3-one oxime; CNDI, 4-chloro-N-nitrosodioxindole; CDI, 4-chlorodioxindole; CIS, 4-chloroisatin; CMIs, 4-chloro-6methoxyisatin; CMNDI, 4-chloro-6-methoxy-N-nitrosodioxindole. t t
C' R
c'
02 H
NOH
R HO@ NO
I: R = O M e 111: R = H
11: R = O M e IV:R=H
R'
R'
V: R = R' = H VII: R H R' = NO VIII: R = OMe; R' = N O
VI: R = R ' = H IX: R = OMe; R' = H
Figure 1. Structures. equivalent fashion (11) to yield a highly mutagenic a-hydroxy-N-nitrosamine, 4-chloro-2-hydroxy-l-nitrosoindolin-&one oxime (N4CI; Figure 1,IV), which has been useful as a model for the methoxy analog due the ready commercial availability of the precursor. Although these a-hydroxy-N-nitrosaminesare relatively stable at low pH, we now present evidence demonstrating that they decompose rapidly in buffer solutions at neutral or higher pH, or in nonbuffered alkaline solution, to yield further mutagenic and nonmutagenic products. These products are identified,and the implications of this process with respect to possible mutagenic events in vivo are discussed.
Experimental Procedures Caution: 4-Chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3one oxime, 4-chloro-2-hydroxy-l-nitrosoindolin-3-one oxime, 4-chlorodioxindole, and 4-chloro-6-methoxydioxinindoleare mutagenic and potentially carcinogenic and should be handled carefully. Protectiue clothingshould be wornand manipulations should be carried out in hoods and with specifically designated 0 1992 American Chemical Society
Brown et al.
798 Chem. Res. Toxicol., Vol. 5, No. 6, 1992 equipment (e.g., balances). They can be decomposed in aqueous media by the careful addition of base. Materials. Sodium nitrite and all HPLC solvents were purchased from Mallinckrodt (Paris, KY). '80-Labeled water, 15N-labeled sodium nitrite, and all NMR solvents were from Cambridge Isotope Laboratories (Woburn, MA). 4-Chloro-6methoxyindole was synthesized by Toronto Chemical Research Inc. (Ontario, Canada). All other reagents were purchased from the Aldrich Chemical Co. (Milwaukee, WI). Synthesis of 4-Chloro-2-hydroxy-l-nitrosoindolin-3-one Oxime and 4-Chloro-6-methoxy-2-hydroxyl-nitrosoindolin%one Oxime and Their 1 8 0 - and 'EN-Labeled Analogs. The a-hydroxy-N-nitrosoindoleswere prepared according to the method of Biichi et al. ( 1 1 ) with modifications as follow. In a typical synthesis, 201 mg of 4CI was dissolved in 0.5 mL of ethanol and then mixed with 265 mL of aqueous NaN02 (0.05 M) which had been brought to pH 2.5 with HC1. This solution was stirred in the dark at room temperature for 1 h and the product then extracted into ethyl acetate. The solvent was removed under vacuum and the sample reconstituted in methanol. Purification was done by HPLC using a pBondapak C18 column (Waters, MA) and eluting with a mixture of methanol in water (10-50% over 20 min, then 50-100% over 10 min) at a flow rate of 3 mL/ min. The stable isotopomers were prepared by substitution of H2180 for HzO, and Na16N02 for NaN02. Decomposition of Nitrosated Chloroindoles. Purified NCMI or N4CI was dissolved in methanol to a concentration of 10 pg/pL. Ten-microliter aliquots of these solutions were then mixed with 390 pL ofwater, or buffer solution, and 20-pL aliquots were analyzed by reverse-phase HPLC (as described above) after various time periods. Quantitation was based on peak areas for the chromatograms generated by absorption at 262 nm (Amm for NICI, log e = 4.2; I I ) , which gave good sensitivity for both starting materials and decomposition products. For analytical preparation of the decomposition products, the a-hydroxy-N-nitrosoindoles were treated with 50 mM NH4OH solution. Mutation Assay. Forward mutation assays were carried out with 5'. typhimurium strain TM677 (uvrB, rfa, pKM101, gal-, bio-, his+)in which resistance to the purine analog 8-azaguanine is used as a genetic marker (12). The bacteria were treated in triplicate with doses ranging from 0.1 to 1 pmol/plate test compound without microsomal activation. Survivors/plate and mutants/plate were measured separately, and mutagenicity was expressed as a ratio of mutants to survivors, i.e., mutant fraction (12). DMSO was the negative control and 4NQ0 the positive contro1. Synthesis of 4-Chloroisatin. 4-Chloroisatin was prepared according to the method of Marvel and Heirs (13) modified to employ 3-chloroaniline as a starting material. Chloral hydrate (9 g) and water (120 mL) were placed in a 500-mL round-bottom flask followed in order by crystalline NazS04 (130g), a solution of 3-chloroaniline (6.5 g) in 30 mL of aqueous HC1, and finally, a solution of hydroxylamine hydrochloride (11g) in water (50 mL). This mixture was slowly (over a period of about 45 min) heated to boiling, kept boiling for 1-2 min, and then cooled under running water to precipitate a mixture of Na~S04and the intermediate isonitrosochloroacetanilide. This intermediate was cyclized by treatment with concentrated sulfuric acid as follows: 33 mL of concentrated HzSO4 were warmed to 50 "C in a roundbottom flask with rapid magnetic stirring, and 18.5 g of the crude precipitate of NazS04 and isonitrosochloroacetanilide was added at such a rate as to keep the temperature at 60-70 "C. After the addition was complete, the solution was heated to 80 "C, kept at that temperature for 10 min, and then allowed to cool to room temperature. The cooled solution was poured over 400 mL of cracked ice and allowed to stand for 30 min during which a mixture (approximately 1:l)of 4-chloroisatin and 6-chloroisatin precipitated. The isatins were collected by filtration and resolved by fractional precipitation from 2 M NaOH by addition of glacial acetic acid according to the method of Sadler (14), with the 4-chloroisatin crystallizing first. The overall yield, based on 3-chloroaniline, was 87 %
.
Synthesis of 4-Chlorodioxindole. This was effected by reduction of CISas described in ref 15 except that dithionite was the reducing agent, typically on 100-mg lots of CIS. Nitrosation of Morpholine. 4-Chloroindole (200 mg) was dissolved in 270 p L of 0.05 M NaNOz at pH 2.5, stirred in the dark for 1hat room temperature, and then extracted with EtOAc. The solvent was removed under vacuum and the product taken up in methanol for purification by HPLC as described below. The peak corresponding to A1 was collected and added directly to a solution of morpholine (approximately 1% ) in methanol. This solution was allowed to stand for 1h at room temperature, and an aliquot was then injected directly into the GC-MS. NMR Spectra. 'H-NMR spectra in DMSO or D20 were recorded on a Varian VXR-500 (500-MHz) spectrometer. Residual solvent peaks, e.g., the methyl resonance of DMSO, were used as references. Mass Spectra. Low-resolution electron ionization (EI) and positive chemical ionization (PCI) spectra were obtained on a Hewlett Packard Model 5971A mass-selective detector or a Hewlett Packard Model 5989A in series with 5890 gas chromatographs fitted with 15- or 30-m capillary DB-17 columns. The PCI reagent gas was methane. The mass spectrometer was typically operated with autotune values at a scan range of 50400 D. The GC was typically operated in the splitless mode, with an injection time of 0.5 min at 60 "C followed by a temperature ramp of 20 OC/min to 250 "C. Helium was the carrier gas at a flow of about 2 mL/min. Ultraviolet Spectra. UV spectra were recorded in water or phosphate buffer on a Hewlett Packard Model 8450A UV/vis spectrophotometer or acquired as diode-array spectra during HPLC analysis on a Hewlett Packard Model 1090 liquid chromatograph. GC-FTIR. Spectra were recorded on a Hewlett Packard Model 5965A infrared detector in series with a Hewlett Packard Model 5890 gas chromatograph fitted with a 15-m capillary DB-17 column. Preparative HPLC. 4-Chloro-2-hydroxy-l-nitrosoindolin%one oxime (7.5mg/mL) was incubated in 0.5 mM NaOH for 3.5 h. Aliquots of the resulting solution were then chromatographed on a semipreparative pBondapak C-18column (30 cm X 0.78 cm; Waters) with an elution gradient of 10-100% (water/methanol) over 20 min with a flow rate of 2 mL/min. Fractions containing compound A1 (based on the diode-array UV spectra) were collected once and then reinjected and collected again for additional purification. Removal of the solvent under vacuum left a yellow solid that was then stored in a sealed vial at -80 "C until further analysis. Attempts to isolate pure compound B1 were unsuccessful due to contamination from degradation products.
Results As judged by HPLC, both N4CI and NCMI decomposed in water to give initially one major compound each, A1 and A2, respectively. UV spectra obtained on the HPLC diode-array detector demonstrated that A1 and A2 possessed the same chromophore, with essentially identical spectra including an absorption maximum at 386 nm, consistent with an N-nitrosoindole, Figure 2 shows the UV spectrum of A l . The decompositions in water are summarized in Table I; Figure 3 shows the relative stabilities in 20 m M HEPES buffer (pH = 7.2). I n both water and HEPES buffer, NCMI was more stable than N4C1, although t h e rates of decomposition were far greater in the buffer for both compounds. For decomposition in water, the pH decreased from 6.5immediately after mixing t o about 4.5 after 40 min and remained at 4.5-4.4 for longer incubation times. T h e decomposition is also rapid in 20 m M sodium succinate (pH = 6.5), 100 mM phosphate buffered saline (pH = 7.4), a n d 100mM sodium carbonate
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 799
Decomposition of Nitrosated 4-Chloroindoles
Table 11. PMR Data for N4C1, CDI, Al, and B1* NH
C(2)- C(2)- C(3)- C(3)- C(3)- C(5)- C(6)- C(7)H OH H OH NOH H H H
N4CI 20-
J (Hz) with Dz0 A1
6.52 5.19
9.78
(d) (d)
(8)
5.2
5.2
(s)
exch.
300 Uavel.ng%h
(d)
(d) 9.3
8.0
8.0
8.0
with DzO
(s)
exch
(dl
(t)
(d)
10.4
4.84 6.20
Table I. Decomposition of NCI and NCMI in Water. time time (min) N4CI A1 NCMI A2 (min) N4CI A1 NCMI 0 100 25 100 24 240 17 89 64 77 50 300 nm nm 39 20 72 35 80 43 45 nm nm 360 nm nm 16 120 35 55 73 62 420 nm nm 12 160 20 65 nm nm
6.71 7.18 6.93
(d)
(d)
(d)
(t)
(d)
J (Hz)
8.5
8.8
8.0
8.0
8.0
withDzO exch B1 n.0.
(8)
exch
(d)
(t)
(dl
6.82
7.51 7.02
(8)
initial and final decomposition products, respectively, of 4-chloro2-hydroxy-1-nitrosoindolin-3-one oxime.
(d)
J (Hz) with DzO A2 96 82 65 47
0 Time course for the decomposition of N4CI and NCMI with formation of 4-chloro-N-nitrosodioxindole(Al) and of 4-chloro-6methoxy-N-nitrosodioxindole(A2), respectively, in water. Initial concentrations of N4CI or NCMI were 1pM. Values are expressed as % of initial readings for these compounds. nm = not measured.
(d) 8.3
9.3
(nm)
Figure 2. Ultraviolet spectra of compounds A1 and B1, the
(d) 8.3
J (Hz) CDI
400
(d) 8.3
exch (d) (t) (d) 7.05 7.60 7.24 (d) (t) (d)
5.35 7.02
10-
250
7.40 7.51 7.87
7.5
(t)
7.5
(d) 7.5
(d) (t) (d) a n.0. = not observed; exch = signal lost, exchangable proton; (s) = singlet; (d) = doublet; (t) = triplet. Table 111. E1 Mass Spectral Data for NICI, NDI, Al, and B1* compound M+ base ion other ions (intensity, %) N4CI 227 (0.5) 180 197 (9), 163 (15), 149 (18) A1
d
NDI
183 (74) 181(59)
B1 a
137 (181,125 (9), 111(10) 183 (74), 155 (26), 90 (16) 155 (26),90 (16) 126 (60), 90 (15)
127 127 153
Only 36Cl isotopes are listed. d = denitrosates in injector.
Scheme I. Formation and Decomposition of 4-Chloro-2-hydroxy-l-nitrosoindolin-3-one Oxime and 4-Chloro-6-methoxy-2-hydroxy-1-nitrosoindolin-3-one Oxime
1
Physiological pH
I
0 4 0
IS
30
60
Time (Minutes)
Figure 3. Relative stabilities of 4-chloro-2-hydroxy-l-nitrosoindoh-Sone oxime and 4-chlor0-6-methoxy-2-hydroxy-l-nitrosoindolind-one oxime in 20 mM HEPES buffer at pH 7.2. (pH = 8.3; data not shown). Complete decomposition of N4CI to A1 could be achieved within 1 min on treatment with 50 mM ammonium hydroxide solution. Compounds A1 and A2 were shown to decomposefurther to give compounds B1 and B2, respectively, which also had similar UV spectra; that for B1 is shown in Figure 2. Compounds A1 and B1 were isolated by HPLC as described above, and their mutagenic activity was measured. While B1 showed no activity, A1 yielded a mutant fraction of -1 X 10YpM with S. typhimurium TM677 (cf. -1.3 X 10-2/pM for NCMI). A base-treated sample of NCMI had similar activity. Proton NMR analysis of A1 and B1 is summarized in Table 11. The spectrum of B1 contained three aromatic resonances, while that of A1 had an additional two doublets at 7.02 ppm (exchangeable in D2O) and 5.35 ppm (collapsing to a singlet in D2O) consistent with a >CH(OH) function. GC-FTIR analysis of these compounds showed distinct bands at 1783 cm-l for A1 and 1755 and 1790 cm-l
("'1
I
No
for B1, highly characteristic of one and two ketone functions, respectively, within a five-membered ring system, along with prominent bands at 1612, 1458, and 1155 cm-l for A1 and 1622,1474,1321, and 1174 cm-1 for B1. Mass spectral analysis (see Table 111)showed strong ions at mlz = 183 and m/z= 181 in the E1 spectra of A1 and B1, respectively. The corresponding ions in their PCI spectra were at mlz = 184 and mlz = 182. These data were consistent with the identity of A1 as a derivative of 4-chlorodioxindole (CDI; Figure 1, V) and B1 as 4-chloroisatin (CIS;Figure 1, VI). Synthetic samples of CDI and CIS were prepared, and B1 was confirmed to be CIS on the basis of identical NMR, GC-FTIR, W, mutagenic, mass spectral,and chromatographicproperties. However, although the GC-FTIR and mass spectral characteristics of A1 were identical to those of CDI, the
800 Chem. Res. Toxicol., Vol. 5,No.6,1992
Brown et al.
Scheme 11. Reaction of 4-Chloro-2-hydroxy-l-nitrosoindolin-3-one Oxime with DNA
c*
NOH
H
DNA
NMR, UV, and mutagenic properties were at variance. Since the UV data suggested a nitrosated structure, we postulated that A1 was 4-chloro-N-nitrosodioxindole (CNDI; Figure 1,VII) which denitrosated in the injector port of the gas chromatograph. Although not conclusive, the presence of a labile N-nitroso group in this compound is supported by the formationof N-nitrosomorpholinefrom reaction of A1 with morpholine, presumably by transnitrosation (16). These observations are summarized in Scheme I. 1SN-labeled A1 and B1 (prepared from 15N-labeled nitrite) showed no change in their mass spectra,consistent with 4-chloro-N-nitrosodioxindole(after denitrosation in the injector port) and 4-chloroisatin. Synthesis of l80labeled A1 and B1 (achieved by decomposition in 180labeled water) resulted in incorporation of an additional two mass unite for both compounds. These data are consistentwith the proposed structures and suggest attack of water at C-3 of N4CI with retention of oxygen at C-2. Although unequivocal identification of A2 and B2 was not possible because of the limited quantity of CMI and the unavailability of synthetic precursors (e.g., 3-chloro5-methoxyaniline),we tentatively identify A2 and B2 as 4-chloro-6-methoxy-N-nitrosodioxindole (Figure 1,VIII) and 4-chloro-6-methoxyisatin(Figure 1,IX),respectively, on the basis of the similarity of UV spectra and HPLC behavior with A1 and B1, in addition to the chemical similarities that have already been reported (II,l7).
Discussion After human consumption of fava beans, it has been postulated that 4-chloro-6-methoxyindoleis released into the stomach and may undergo nitrosation to form highly mutagenic NCMI. This, in turn, may react with the gastric mucosa where it can initiate mutagenic events, which may eventuallylead to gastric cancer. It now appears, however, that NCMI may decompose rapidly at the intracellular pH experienced at the gastricmuma, yielding the putative 4-chloro-6-methoxy-N-nitrosodioxindole. Decomposition rates in model buffer systems suggest that decomposition
of NCMI would be essentially complete within 1 h. Although the mutagenicity of N4CI is approximately 100 in the S. times greater than 4-chloro-N-nitrosodioxindole typhimurium TM677 assay,this may bear no relationship to their relative carcinogenicpotential, which has yet to be determined. Furthermore, this bioassay is conducted at physiological pH, making it difficult to apportion the overall result to particular components of this dynamic system. The ultimate decomposition product, CIS,has no mutagenic activity. There were no signals in the NMR spectrum of partially decomposed NCMI that would indicate whether the mechanism of conversion to CISwas concerted or may have followed two sequential steps of denitrosation and oxidation (in either order). From the standpoint of carcinogenic potential, the inimechanism by which 4-chloro-N-nitrosodioxindole tiates mutagenic events may be important. There is evidencethat the mutagenicity of N4CI can be attributed to formation of a benzenediazonium ion which reacts with nucleophilic sites on cellular macromolecules (18). With adenine, we have demonstrated the formation of 6-hydrazinopurine after reaction with N4CI and subsequent reduction with sodium borohydride and hydrolysis (19, 20; see Scheme 11). CNDI, however, does not possess the m-hydroxy function necessary for this reaction. Further studies to elucidate this mechanism are consequently underway. Acknowledgment. This work was supported by NIH Grants CA26731(NCI) andES02109 (NIEHS). We thank Woody Bishop for conducting the mutagenicity assays, Billy Day for running the NMR spectra, and Peter DesChamps and Arthur LaFleur for use of the GC-FTIR instrument (purchased under NIEHS Grants EHS-5P30ES02109-10 and EHS-5P01-ES01640-11). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.
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Chem. Res. Toxicol., Vol. 5, No. 6, 1992 801 pound in fava beans (vicia faba). 11th International Meeting on N-nitroso compounds, Kailua-Kona, HI, November 1-2, 1991, International Agency for Research on Cancer, Lyon. (11) BUchi, G., Lee, G. C. M., Yang, D., and Tannenbaum, S. R. (1986) Direct acting, highly mutagenic, a-hydroxy-N-nitroinea from 4-chloroindoles. J. Am. Chem. SOC. 108, 4115-4119. (12) Skopek,T. R.,Liber,H. L., Kroweleki,J. J.,andThilly, W. G. (1978) Quantitative foward mutation assay in Salmonella typhimurium using &azaguanineresistanceas a geneticmarker. h o c . Natl.Acad. Sci. U.S.A. 75, 410-414. (13) Marvel, C. S., and Hiere, G. S. (1941) Isatin. Organic Syntheses (Blatt, A. H., Ed.) Collect. Vol. 1,2nd ed., pp 327-328, Wdey and Sons,New York. (14) Sadler, P. W. (1956) Absorption Spectra of Indigo Dyes. J. Org. Chem. 21, 316-318. (15) Marschalk, C. (1912) Uberfilhrung des Oxindola in Ieocumarinon. Ber. 45,582-585. (16) Singer, S. S.,Lijinaky, W., and Singer, G .M. (1977)Transnitroeation by aliphatic nitrosamines. Tetrahedron Lett. 19,1613-1616. (17) Brown, N. K., Nguyen, T. T., Taghizadeh, K., Sahali, Y.,Tannenbaum, S. R., and Wishnok, J. S. (1991)Decomposition of nitroeated 4-chloroindoles. Proceedings of the 39th ASMS Conferenceon Mass Spectrometry, and Allied Topics, Nashville, TN, May 19-24,1991, American Society for Mass Spectrometry, Santa Fe. (18) Chin, A., Hung, M. H., and Stock, L. M. (1981) Reaction of benzenediazonium ions with adenine and its derivatives. J. Org. Chem. 46, 2203-2207. (19) Kroeger-Koeppky, M. B., Smith, R. H., Jr., Gocdnow, E. A., Brasheara,J., Kratz, L.,Andrews,A. W.,Alvard, W. G.,andMichejdn, C. J. (1991) 1,3-Dialkyltriazenes: reactive intermediates and DNA alkylation. Chem. Res. Toxicol. 4, 334-340. (20) Nguyen, T. T., Wishnok, J. S., and Tannenbaum, S. R. (1990) Reactions of Nitrosated 4-substituted Indoles with DNA. h o c . Am. Assoc. Cancer Res. 31,91.
Registry No. 11,99684-91-0;111,25235-85-2;IV, 93490-32-5; VI, 6344-05-4; VII, 143618-15-9; VIII, 143631-75-8; IX,14361% 17-1; 3-chloroaniline, 108-42-9.
