Chem. Res. Toxicol. 1993,6, 480-485
480
Synthesis of N-(Purin-8-y1)arylamines David H. Swenson,*p+Karam El-Bayoumy,* Gong-Huey Shuie,* Stephen S. Hecht,* Emerich Fiala,* Fred F. Kadlubar,s and J. Pat Freemans Karkinos Biochem, Inc., 3245 North Washington Street, Chandler, Arizona 85225,School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803,American Health Foundation, One Dana Road, Valhalla, New York 10595,and National Center for Toxicological Research, Jefferson,Arkansas 72079 Received February 22,1993
Methods for direct synthesis of N-(purin-8-y1)arylamineswere investigated. N-(Purin-& y1)arylamines are adducts from reaction of electrophilic metabolites of arylamines with DNA and have not been readily available by direct synthesis. Ability to generate significant quantities of this class of DNA adduct by synthetic means would facilitate basic research in molecular toxicology. Two routes with common thiourea intermediates were developed for this purpose. In the first route, 6-hydroxy-2,4,5-triaminopyrimidine was caused to react in aqueous medium with dithiocarbamate derivatives of 0-,m-,or p- toluidine to form corresponding 2,4-diamino5-(tolylthioureido)aminopyrimidin-6-ones in 60-70 % yields. The thiourea intermediates were converted to carbodiimides and isolated in 20-25% yields after treatment with HgO in dimethylformamide. The carbodiimides were cyclized at 125 "C in dimethylformamide under Nz for 45 h to yield N-(guanin-8-yl)(o-, m-,or p-)toluidines in 65-75% yields. In the second route, direct reaction of p-tolyl isothiocyanate with 6-hydroxy-2,4,5-triaminopyrimidine or with 4,5,64riaminopyrimidine in dimethylformamide and triethylamine led to the formation of 2,4diamino-b(p- tolylthioureido)aminopyrimidin-6-oneand 4,6-diamino-5-(tolylthioureido)aminopyrimidine, respectively, in 77-79 % yields. Methylation of the two thiourea derivatives by methyl iodide in dimethylformamide gave the corresponding methylisothiuronium derivatives, which were isolated in 71% and 84% yields (as HI salts), respectively. Conversion of the methylisothiuronium derivatives to N-(guanin-8-yl)-p- toluidine or N-(adenin-&yl)-p- toluidine was accomplished by heating in dimethylformamide for 5-7 h at 80-90 "C, in 70% and 62% yields, respectively. The most efficient synthesis of the thioureas would appear to proceed by coupling the triaminopyrimidine precursor with the easily prepared dithiocarbamate derivatives of the arylamine followed by conversion to a methylisothiuronium and thermal cyclization of the methylated intermediate. These routes should be directly applicable to polynuclear arylamines and suitably blocked diaminoarenes.
Introduction The carcinogenicity of arylamines and their derivatives has been well established in laboratory animal models and in humans (1-3). In general, mononuclear arylamines produce fewer DNA adducts and are less potent as carcinogens than are those with two ring systems (3). Consequently, the single-ring compounds have received less research emphasis than have the more complex arylamines. However, more recent studies of occupational carcinogenesis in the rubber industry have suggested the importance of single-ring arylamines as potential human carcinogens (4). A body of evidence has accumulated showing that arylamine carcinogens are not active per se but are metabolized in one or more steps to electrophilic,ultimate carcinogenicspecies (5). These electrophilicspecies appear to exert their carcinogenic effects by covalently binding to nucleophilic sites in DNA (5), particularly at the C-8, possibly from initial reactions at N-7 (61, and at the exocyclic amino positions of purines (7-11). The resulting
* To whom correspondence and reprint requests should be addressed.
t Karkinos Biochem, Inc.; present address: School of Veterinary Medicine, Louisiana State University. t American Health Foundation. $National Center for Toxicological Research.
0893-228x/93/2706-0480$04.00/0
DNA adducts, especially those not removed by cellular repair processes, may result in point or deletion mutations in the DNA. Mutations in suppressor genes or in oncogenes may result in altered activity of the gene products, leading to neoplasia (11,12). The structures of many binuclear arylamines have been elucidated. C-8-Deoxyguanosineadducts are generallythe predominant products, and N2-deoxyguanosinesare minor products, while deoxyadenosine adducts have generally been reported as minor products both in vivo and in vitro (7,11,13).The lack of information on structures of DNA adducts derived from mononuclear arylamines, and the limited number of synthetic methods to generate significant quantities of such adducts, prompted this study on methods for generation of (purin-8-y1)arylamineadducts. With the availability of high specific activity tracer methods (111,with the 32P-postlabelingtechnique (141,or with antibody-based assays (15),it is possible to quantitatively characterize the formation of specific DNA adducts that exist at very low levels, but unambiguous identification of the materials detected has been hampered by a paucity of reference standards. Once purinearylamine base adducts have been prepared, they can be converted to deoxynucleosidesand deoxynucleotideswith appropriate blocking/deblocking strategies and existing chemical glycosylationand phosphorylation schemes (160 1993 American Chemical Society
Chem. Res. Toxicol., Vol. 6,No.4,1993 481
Synthesis of N-(Purin-8-yl)arylamines
Table I. NMR Data for Products in Scheme I* NMR resonances for compd no. ~~
4
proton Ar-CHs H-Nl H2-N2 H2-N4 H-N6 H-N-tolyl H-N9 aromatic 0
a
b
10.2b 5.8 6.2 8.lb 8.9b
2.3 10.0b 5.8 6.2 8.lb 9.lb
2.2
7.0-7.3b,s
6.8-7.4m
2.3 10.1b 5.8 6.2
2.3 9.2 6.0 6.5
b 2.3 10.1 5.9 6.5
2.2
10.1 5.9 6.5
8.0
9.lb 7.0d (2H)b 6.9-7.313 (3H) 7.4d (2H)b 7.8-8.0b,d (1H)
a
C
7.0-7.7m
2.3 10.6b 6.1
b 2.3 10.4b 6.0
C
2.2
10.4b 6.0
8.8b 8.0b 8.9b 11.2b 11.2b 10.9b 7.0d (2H)b 6.7-7.3m (3H) 6.6-6.8d (1H) 7.0d (2H)b 7.4d (2H)b 7.9-8.lb,d (1H) 7.0-7.5m (3H) 7.5d (2H)b
-
Values are ppm from tetramethylsilane internal standard; resonances are singlets except as noted; b = broad, d = doublet, m = multiplet, 8 Hz.
s = singlet. b J
18);enzymatic methods at this time appear to be ineffective for C-8-substituted purines (19). Current methods for generation of such adducts from arylamines include direct reaction of electrophilicsynthons with nucleic acids, nucleosides, or nucleotides followed by isolation and characterization of the individual adducts (6-8,ll).An alternative procedure for generation of (guan8-y1)arylamine adducts is based on the reaction of esters of guanine 3-N-oxide with arylamines (101,but this procedure is not applicable to generation of adenine adducts. A preliminary report from our laboratory suggested that thiourea intermediates prepared from coupling of triaminopyrimidine precursors with p-tolyl isothiocyanate could be thermally cyclized to (purin-8-y1)tolylamines (20). More recent studies from our laboratory have indicated that this procedure is not sufficiently reproducible to be useful as a general method for generation of this type of adduct.' We describe in this report two improved routes, starting from thiourea intermediates, for synthesis of C-8 purine adducts derived from toluidines. The toluidines represent potential human carcinogens ( 4 ) and serve as models for other arylamines in these synthetic schemes.
Experimental Procedures Hazardous Materials: Arylamines should be treated as potential carcinogens and handled accordingly. These and other reagents or solvents should be handled and disposed according to instructions in their respective "Material SafetyData Sheets". 0-,m-, and p-toluidines (la-c, respectively), p-tolyl isothiocyanate (S),4,5,6- triaminopyrimidine sulfate (lo),and 6-hydroxy2,4,5-triaminopyrimidine sulfate (3) were from Aldrich Chemical Co. (Milwaukee, WI). Other chemicals were reagent grade in purity. Mass spectra were obtained on a Finnigan 4023, upgraded to a 4500, using a Finnigan direct-exposure probe and electron ionization or chemical ionization with methane or ammonia as the reagent gas. Some molecular ions were confirmed by fastatom-bombardment mass spectrometry on a Kratos MS 50 using a thioglycerol matrix. TLC was carried out on 250-pm layers of silica gel in one of three solvents: A, n-BuOH/HzO/acetic acid (4/2/1); B, H2O-saturated n-BuOH; C, iPrOH/H20/NHdOH (7/ 2/11. Products and intermediates were judged to be >95% pure by TLC, except for the dithiocarbamates, which were not analyzed by TLC and were used immediately upon isolation. NMR were obtained in dimethyl-de sulfoxide with a Bruker AM400 or a Jeol Model FX9OQ NMR spectrometer. NMR data have been consolidated in tabular form (Tables I and 11) to facilitate comparisons. FT-IR were obtained with a Nicolet 2OSXC FTIR system, at 2-cm-1 resolution. Elemental analysis was carried 1
6
5
a
C
D. H. Swenson, unpublished results.
Table 11. NMR Data for Products in Scheme 11. compd no. proton 9 11 12 13 Ar-CH3 2.32,2.356 2.3 2.3b 2.3 H-N' 10.6b H-C2 7.9 8.2b 8.3 H2-N2 6.5b H2-N4 6.6b 6.7bC -d H-N6 9.5b, 9.4bb 8.4b 4 H2-Ne 6.7bC -d 7.7b -d 10.lb H-N-tolyl 10.3b, 10.4bb 9.8b S-CH3 2.65, 2.60b 2.6b H-Ne 11.3 aromatic (7.ld, 7.27d)e 7.ld (2H)g 7.0-7.3m,b 7.2dg (7.32d, 7.4d)f 7.5s,b (2H) 7.5dg 0 Values are ppm from tetramethylsilane internal standard, and resonances are singlets except as noted; b = broad, d = doublet, m = multiplet, s = singlet. b In the spectrum of compound 9, the S-CHS, the N6-H, the tolyl-N-H, and the aromatic tolyl protons suggest the presence of two tautomers for the methylisothiuronium structure, with the predominant structure accounting for about 80% of the total, the first resonance listed for these protons is the 80 % tautomer, the second is the 20% tautomer. H2-N' and Hz-NB are identical in compound 11and integrate for 4 H; in compound 13Hz-Neintegrates for 2 H. d Not observed between 0 and 10 ppm; presumed rapid exchange with HI. e J 8 Hz, 80% of aromatics. f J 8 Hz, 20% of aromatics. J 8 Hz. ~
~
- -
~
-
out by MHW Labs, Phoenix, AZ. Melting points (uncorrected) were obtained with a Fisher-Johns melting point apparatus. Two related synthetic schemes, which had common intermediates, were used to generate (purin-8-y1)toluidine derivatives (Schemes I and 11). Scheme I: Dithiocarbamate/Carbodiimide Route. (A) Ammonium Tolyldithiocarbamates 2a-c. The tolyldithiocarbamates were synthesized by standard literature procedures (21). Thus, into a stirring mixture of CS2 (1.67 g, 22 mmol) and NHdOH (30%, 3 mL) was added 2.74 g (20 mmol) of 0 - , m-,or p-toluidine, la-c, respectively, in a dropwise fashion. After complete addition, the reaction was stirred for an additional 30 min, during which a precipitate was gradually formed. After standing at room temperature for 30 min, the precipitate was filtered to yield the ammonium tolyldithiocarbamate as a white powder in 60-70% yields. The material was used in subsequent steps without further purification. Mass spectra (mlz, relative abundance, interpretation): 2a, 183,8%,M+;168,5%,M+- CHa; 149,34%,M-H&117,12%; 107,64%,M-CS2;106,79%,M+ - CS2H; 91, 13%, tropylium; 76, loo%, CS2+. A good mass spectrum of 2b was not obtained due to decomposition on storage or handling, apparently to the bis-tolylthiourea (mlz = 256, M+ and corresponding fragments from loss of H2S and N-C bond cleavage). 2c, 183,1%,M+; 149,8%,M - H2S; 107,61%, M+CS2;106,84%,M+ - CS2H; 91,6%,tropylium; 76, loo%,CS2+. Chemical ionization (CH3 mass spectrometry results confirmed molecular ions for 2a and 2c. FT-IR (cm-l): 2a, 3416,3378,3125, 2906, 2697, 1488, 1400, 1284, 988, 840,750, 712, 634, 597; 2b,
482
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Swenson et al.
3406,3281,3144,3062,2969,2922,2944,2797,1622,1609,1503, 1360,1341,1218,1203,1131,962,850,766,744,700,697,575,562, 453,403;4b, 3483,3391,3350,3200, 3344,3312,3250-3156,1634, NH, H N A s - A, 1594,1562,1516,1494,1465,1384,1353,1281,1203,1156,1006, 891,769,668,528,422;4c, 3465,3350,3142,3072,2973,2903, &R1 , CS2,NH40H 2847,2802,2734,1632,1556,1511,1463,1384,1370,1342,1311, I 0-5'C * 1216,1013,853,771,667,404. Melting points: 4a, decomposition T R 2 I beginning at 265 "C; 4b, decomposition beginning at 249 "C; 4c, R3 R3 decomposition beginning at 246 "C; none melted
