Syntheses of two potential food mutagens

Article abstract of DOI:10.1002/jhet.5570400403

The syntheses of the potential heterocyclic amine food mutagens 1,4,6-trimethyl-2-aminoimidazo[4,5-c]pyridine and 1,5,7-trimethyl-2 -aminoimidazo [4,5-b]pyridine are described.

Full text of DOI:10.1002/jhet.5570400403

Jul-Aug 2003
Syntheses of Two Potential Food Mutagens
569
M. J. Tanga,* W. W. Bradford, J. E. Bupp, and J. A. Kozocas
SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025
Received January 23, 2003
The syntheses of the potential heterocyclic amine food mutagens 1,4,6-trimethyl-2-aminoimidazo[4,5-c]-
pyridine and 1,5,7-trimethyl-2-aminoimidazo[4,5-b]pyridine are described.
J. Heterocyclic Chem., 40, 569(2003).
Our diet is a complex mixture that provides nutritional
[13]. These data are consistent with increased risk of
cancer from ingestion of heterocyclic amines [1,4,11-13].
To understand the risk that consuming heterocyclic
amines formed during cooking meat and fish poses to
humans, it is essential to isolate, identify, and synthesize
these compounds. Although some of these compounds
have been identified and synthesized, one that contributes
10-15% of the total mutagenic activity of a fried meat sam-
ple [14-17] has only been identified by mass spectra to have
a molecular weight of 176 [14,16,17]. From the available
preliminary data, the mutagenic compound has been deter-
mined to be one of the twelve isomers of 2-amino-
trimethylimidazopyridine (TMIP) [14,17]. To investigate
the biological risk associated with ingesting this unidenti-
fied compound, we have previously synthesized six of the
possible isomers: 1,5,6-trimethyl-2-aminoimidazo[4,5-b]-
pyridine; 3,5,6-trimethyl-2-aminoimidazo[4,5-b]pyridine
[11]; 3,5,7-trimethyl-2-aminoimidazo[4,5-b]pyridine;
1,4,7-trimethyl-2-aminoimidazo[4,5-c]pyridine; 1,6,7-
trimethyl-2-aminoimidazo[4,5-c]pyridine; and 3,4,6-
trimethyl-2-aminoimidazo[4,5-c]pyridine [18,19]. We now
report the syntheses of two other possible isomers-1,4,6-
trimethyl-2-aminoimidazo[4,5-c]pyridine (5) and 1,5,7-
trimethyl-2-aminoimidazo[4,5-b]pyridine (14)-so that they
can be compared and tested against the unknown mutagen
[20]. The syntheses of the two compounds have not previ-
ously been reported in the literature.
sustenance but may also be important in the causation,
modulation, and prevention of human disease. Cooking,
heat processing, and pyrolysis of protein-rich foods
induce formation of mutagenic and carcinogenic hetero-
cyclic amines through chemical reactions involving
amino acids, creatine, and proteins [1,2]. The formation
of these compounds requires high temperatures, such as
are found in radiative or conductive cooking (e.g., grilling
or frying) but usually not in indirect convective cooking
(e.g., roasting) or boiling methods (e.g., poaching or
steaming) [3,4]. The dietary concentration of heterocyclic
amines can be reduced by making changes to cooking
methods, for example marinating meat before grilling [5],
and eating foods containing catechins, flavonoids, and
caffeic acid [6,7].
Heterocyclic amines are potent mutagens in the
Ames/Salmonella assay and are also carcinogens in labo-
ratory animals [8,9] and suspected carcinogens in humans
[10]. Recent epidemiological studies have shown that
these substances are ingested by humans in cooked meats
and fish products [1,4,11]. A positive association has been
reported between the preference for well-done red meat
and the risk of postmenopausal breast cancer [11,12]. A
recent study demonstrated that heterocyclic amines are
bioavailable to the human colon following defined dietary-
relevant doses, and DNA and protein adducts are formed

570
M. J. Tanga, W. W. Bradford, J. E. Bupp, and J. A. Kozocas
Vol. 40
The successful synthesis of 1,4,6-trimethyl-2-aminoimi-
aminoimidazo[4,5-c]pyridine (5), was accomplished in
good yield (75%) by using anhydrous hydrofluoric acid
with heat. We explored alternative methods for the cleav-
age of the sulfonamide 4, such as using potassium carbon-
ate in aqueous methanol with heat, concentrated sodium
hydroxide in aqueous methanol with heat, concentrated
sulfuric acid with heat, concentrated sulfuric acid in aque-
ous methanol with heat, and finally hydrofluoric acid in
pyridine with heat. None of these methods gave the
desired product 5, and usually unreacted starting material
4 was recovered.
The synthesis of the potential food mutagen 1,5,7-trimethyl-
2-imidazo[4,5b-]pyridine (14) is shown in Scheme II. The
product 14 has been prepared by the cyanogen bromide
method as well as by the sulfonamide method. Bromination of
commercially available 2-amino-4,6-dimethylpyridine (6) in
glacial acetic acid gave the desired 2-amino-5-bromo-4,6-
dimethylpyridine (7) plus a small amount of 2-amino-3,5-
dibromo-4,6-dimethylpyridine. Trituration in hexane sepa-
rated the two compounds, which were formed in a 12:5 ratio.
dazo[4,5-c]pyridine (5) is outlined in Scheme I. Previous
attempts to synthesize 5 resulted in the synthesis of 1,4,6-
trimethyl-7-aminoimidazo[4,5c-]pyridine or in the inability
to ring close 3-amino-2,6-dimethyl-4-(methylamino)-
pyridine (3) by methods such as cyanogen bromide [19].
The success of this synthesis is based on the use of the
reagent N-dichloromethylene-p-toluenesulfonamide (2) [21]
to accomplish the ring closure and introduce the desired
amino function at the 2-position. It is a facile way to form
the desired heterocycle 5, and purification is simplified.
The reagent N-dichloromethylene-p-toluenesulfon-
amide (2) was formed in good yield (83%) from commer-
cially available N-[bis(methylthio)methylene]-p-toluene-
sulfonamide (1) by treatment with excess chlorine gas in
glacial acetic acid [21]. One equivalent each of the
diamine 3 and the reagent 2 in dichloromethane forms a
solution at 40°, and after 5 minutes the product 4 begins to
precipitate as the hydrochloride salt. Cleavage of the sul-
fonamide 4 to give the desired product, 1,4,6-trimethyl-2-

Jul-Aug 2003
Syntheses of Two Potential Food Mutagens
571
N-Dichloromethylene-p-toluenesulfonamide (2) [21].
The amine 6 was brominated to direct the nitration to the 3-
position. Nitration of the amine 7 with potassium nitrate in
sulfuric acid gave the nitro compound 8 in 64% yield.
Reduction and dehydrohalogenation of 8 in one step, using
hydrogen with 10% palladium on carbon as the catalyst in
ammonium hydroxide and methanol at 50°, yielded 2,3-
diamino-4,6-dimethylpyridine (9) in 98% yield. Treatment of
A suspension of 25.0 g (90.8 mmoles) of N-[bis(methylthio)-
methylene]-p-toluenesulfonamide (1) in 170 mL of glacial acetic
acid was cooled to 15°. A stream of chlorine gas was slowly
introduced over 1 hour, keeping the reaction temperature at 15°.
The resulting solution was stirred at 10° for 1 hour. The sample
was evaporated under vacuum and the solid residue sublimed at
70-75° (5 mm of mercury) to give 19.1 g (83%) of pure product
(2), mp 80-83°; 1H nmr (deuteriochloroform): d 2.46 (s, 3H),
7.38 (d, 2H, J = 8.45 Hz).
3
the diamine 9 with benzyl chloroformate (10) yielded N -ben-
zyloxycarbonyl-2,3-diamino-4,6-dimethylpyridine (11) in
49% yield with a small amount of the bis-benzylated material
(9%), which was separated by column chromatography.
Reduction of 11 with lithium aluminum hydride gave 2-
amino-4,6-dimethyl-3-(methylamino)pyridine (12), which
was immediately used in the ring cyclization reaction with
cyanogen bromide in ethanol in a bomb at 120° to give the
desired final product 14 in 10% yield. In an effort to synthe-
size greater quantities of product 14, which could be more
readily purified, the sulfonamide 13 was prepared from the
diamine 12 and N-dichloromethylenep--toluenesulfonamide
(2) in 51% yield. The sulfonamide 13 was cleaved using
anhydrous hydrofluoric acid to give the product 14 in 40%
yield, which was identical to 14 synthesized using the
cyanogen bromide method. The final products of these syn-
theses are undergoing biological testing [17].
In conclusion, two potential food mutagens were synthe-
sized for biological evaluation using an approach that is
applicable to the preparation of similar heterocyclic com-
pounds. In all, ten of the twelve isomers of 2-
aminotrimethylimidazopyridine (TMIP) are presently
available as reference materials from the National Cancer
Institute [20].
2-Amino-N-p-toluenesulfonamide-1,4,6-trimethylimidazo[4,5-
c]pyridine (4).
A sample (392 mg, 2.59 mmoles) of 3-amino-2,6-dimethyl-4-
(methylamino)pyridine (3) in 10 mL of dichloromethane was
treated with 653 mg (2.59 mmoles) of N-dichloromethylene-p-
toluenesulfonamide (2). The reaction solution was heated to 40°
for 18 hours, then treated with 5 g of anhydrous sodium sulfate
and stirred overnight to form the free base. The sample was fil-
tered through a MilliporeÒ filter and evaporated to give 1.15 g of
crude product (4). The filtrate was filtered through 5 g of silica
gel eluting with 125 mL of chloroform/methanol/triethylamine
(94.5/5/0.5). Evaporation of the eluent gave 653 mg (76%) of
1
desired product (4); mp 239-241°; H nmr (deuteriochloroform):
d 2.40 (s, 3H), 2.58 (s, 3H), 2.60 (s, 3H), 3.45 (s, 3H), 6.80 (s,
1H), 7.27 (d, 2H, J = 8.28 Hz), 7.86 (d, 2H, J = 8.28 Hz); 13C nmr
(deuteriochloroform): d 12.70, 14.46, 17.61, 21.57, 94.36,
115.22, 119.04, 122.34, 130.04, 132.50, 133.18, 135.64, 143.17,
145.49; ir (potassium bromide) 3258, 2920, 2365, 1578, 1542,
1522, 1458, 1402, 1375, 1331, 1277, 1200, 1132, 1088, 1040,
986, 918, 831, 806, 788, 696, 666, 570, 540 cm-1; uv (methanol)
l
max 311 nm (e 2,347), 286 (7,410), 242 (27,072), 220 (19,809).
1,4,6-Trimethyl-2-aminoimidazo[4,5-c]pyridine (5).
In a TeflonÒ lined bomb, 327 mg (0.990 mmole) of 2-amino-
N-p-toluenesulfonamide-1,4,6-trimethylimidazo[4,5-c]pyridine
(4) in 10 mL of anhydrous hydrofluoric acid was heated to 100°
for 1 hour. After cooling to 0°, the bomb was carefully opened
and the hydrofluoric acid evaporated with a gentle stream of
argon. The solid residue was rinsed into a continuous extraction
apparatus with 1 mL of 4 N sodium hydroxide solution and 100
mL of water, extracted with chloroform overnight, and then dried
(sodium sulfate), filtered, and evaporated to give 280 mg of crude
product (5). This material (5) was triturated with 3 x 5 mL of
warm hexanes and then with 3 x 2 mL of ethyl ether to give 130
EXPERIMENTAL
Melting points (uncorrected) were obtained using a Thomas-
Hoover melting point apparatus. The ir spectra were recorded on
a Perkin Elmer 1600 FTIR spectrophotometer, the uv spectra on a
Varian DMS-90 spectrometer, and the nmr spectra on a Varian
Mercury VX-300 MHz spectrometer. All chemical shifts are
reported in parts per million (d) downfield from tetramethylsi-
lane. The nmr multiplicities are reported using the following
abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, mul-
tiplet; and b, broad. Column chromatography was carried out
using E. Merck silica gel 40 (70-230 mesh, ASTM). All solvents
were dried over 3 Å molecular sieves, except tetrahydrofuran,
which was dried by refluxing over sodium with benzophenone
ketyl as an indicator. Microanalyses were performed by Atlantic
Microlab, Inc., Norcross, GA.
After extensive effort we were unable to obtain reasonable ele-
mental analysis for compounds 5 and 14. It is our experience that
these compounds do not give reasonable elemental analysis.
Analysis by HPLC shows the compounds to be greater than 95%
pure.
Since 1,4,6-trimethyl-2-aminoimidazo[4,5-c]pyridine (5) and
1,5,7-trimethyl-2-aminoimidazo[4,5-b]pyridine (14) are poten -
tial carcinogens and mutagens, direct contact should be avoided.
1
mg (75%) of pure product (5); mp 244-248°; H nmr (deuteri-
omethanol): d 2.41 (s, 3H), 2.50 (s, 3H), 3.46 (s, 3H), 6.55 (br s,
2H), 6.89 (s, 1H); 13C nmr (deuteriomethanol): d 16.86, 17.36,
34.18, 128.97, 132.07, 132.89, 139.84, 140.09, 148.24; ir (potas-
sium bromide) 3342, 3246, 3068, 1665, 1615, 1582, 1454, 1413,
1259, 1125 cm-1; uv (95% ethanol) l max 281 nm (e 4,745), 253
(5,468), 215 (30,725); ms (DCI-NH3) m/z (relative intensity) 177
(100, molecular ion + H), 123 (10), 78 (10); hrms Calcd. for
C9H12N4: 176.1062. Found: 176.1050.
2-Amino-5-bromo-4,6-dimethylpyridine (7).
A solution of 25.6 g (0.21 mole) of 2-amino-4,6-dimethylpyri-
dine (6) in 50 mL of glacial acetic acid under argon was treated
with a solution of 33.5 g (0.21 mole) of bromine in 25 mL of
glacial acetic acid over 20 minutes with water bath cooling

572
M. J. Tanga, W. W. Bradford, J. E. Bupp, and J. A. Kozocas
Vol. 40
keeping the temperature below 20°. The solution became a solid
mass and was allowed to stand at room temperature for 1 hour.
After cooling in an ice bath, the material was treated with 500 mL
of cold 20% sodium hydroxide solution. The mixture was
extracted with 3 x 250 mL of dichloromethane, dried (sodium
sulfate), filtered, and evaporated to provide 44.3 g of crude prod-
uct (7). Trituration of the material (7) with 3 x 250 mL of hot
form to give 100 mg (9%) of biscarbamate and 370 mg (49%) of
the desired product (11); mp 155-157°; 1H nmr (deuteriochloro-
form): d 2.12 (br s, 3H), 2.31 (s, 3H), 4.62 (br s, 2H), 5.18 (s,
2H), 6.11 (br s, 1H), 6.42 (s, 1H), 7.30-7.45 (m, 5H); 13C nmr
(deuteriochloroform): d 18.5, 24.7, 68.5, 114.6, 117.1, 129.1,
129.2, 129.4, 129.5, 129.6, 137.1, 156.2, 156.4; ir (potassium
bromide) 3411, 3276, 3156, 2964, 2371, 1686, 1638, 1605, 1567,
1523, 1474, 1458, 1371, 1254, 1235, 1061, 848, 812, 774, 747,
693, 576, 483 cm-1; ms (70 eV, electron impact) m/z (relative
intensity) 271 (25, molecular ion), 136 (51), 91 (100).
1
hexane gave 24.7 g (59%) of pure product (7); mp 134-137°; H
nmr (deuteriochloroform): d 2.27 (s, 3H), 2.50 (s, 3H), 4.40 (br s,
2H), 6.24 (s, 1H); 13C nmr (deuteriochloroform): d 24.3, 26.2,
109.0, 113.4, 149.6, 156.5, 157.4; ir (potassium bromide) 3431,
3310, 3174, 2367, 2343, 1637, 1592, 1543, 1506, 1458, 1419,
1260, 1210, 1020, 960, 942, 740, 671, 621, 578, 525 cm-1.
Anal. Calcd. for C7H9N2Br: C, 41.82; H, 4.51; N, 13.93.
Found: C, 42.10; H, 4.51; N, 13.90.
2-Amino-4,6-dimethyl-3-(methylamino)pyridine (12).
A solution of 349 mg (1.29 mmoles) of N3-benzyloxycar-
bonyl-2,3-diamino-4,5-dimethylpyridine (11) in 10 mL of dry
tetrahydrofuran under argon was treated with 340 mg (8.96
mmoles) of lithium aluminum hydride. The reaction mixture was
refluxed for 1 hour. The mixture was cooled to 0° and a solution
of 5 mL of 50% tetrahydrofuran/50% water was very slowly
added, followed by the addition of 1 mL of 15% sodium hydrox-
ide solution. The resulting mixture was filtered and the residue
washed with water and tetrahydrofuran. The tetrahydrofuran was
removed under vacuum and the aqueous suspension continuously
extracted with chloroform overnight. The extract was dried
(sodium sulfate), filtered, and evaporated to give 255 mg of crude
material, which was triturated into hot hexane (3 x 200 mL) and
evaporated to give 140 mg (99%) of crude product (12); 1H nmr
(deuteriochloroform): d 2.19 (s, 3H), 2.31 (s, 3H), 2.67 (s, 3H),
6.35 (s, 1H); ir (potassium bromide) 3362, 3028, 2922, 2858,
2360, 1513, 1476, 1452, 1410, 1381, 1238, 1206, 1136, 1022,
2-Amino-5-bromo-4,6-dimethyl-3-nitropyridine (8).
A sample of 7.04 g (69.66 mmoles) of potassium nitrate was
carefully added over 30 minutes to a stirring solution of 10.00 g
(49.76 mmoles) of 2-amino-5-bromo-4,6-dimethylpyridine (7) in
30 mL of concentrated sulfuric acid. The mixture was stirred for
1 hour at room temperature and then poured onto 200 g of ice.
To the solution was added 300 mL of chloroform, and the mix-
ture was neutralized with sodium carbonate, filtered through
Celite, extracted into chloroform (2 x 300 mL), dried (sodium
sulfate), filtered, and concentrated to afford 7.89 g (64%) of pure
1
product (8); mp 148-151°; H nmr (deuteriochloroform): d 2.56
(s, 3H), 2.57 (s, 3H), 4.51 (br s, 2H); 13C nmr (deuteriochloro-
form): d 21.26, 26.55, 112.72, 118.25, 144.37, 150.45, 161.22; ir
(potassium bromide) 3446, 3296, 3171, 1635, 1573, 1543, 1496,
1434, 1378, 1324, 1243, 1122, 1022, 1002, 949, 849, 777, 672,
540, 496 cm-1.
735, 698 cm-1; uv (methanol) l
299 nm (e 2,604), 230
max
(2,453), 203 (10,697).
1,5,7-Trimethyl-2-aminoimidazo[4,5-b]pyridine (14).
2,3-Diamino-4,5-dimethylpyridine (9).
To 650 mg (4.30 mmoles) of 2-amino-4,6-dimethyl-3-(methyl-
amino)pyridine (12) was added 1.37 g (12.91 mmoles) of
cyanogen bromide in 6 mL of methanol. The mixture was sealed
in a TeflonÒ lined bomb under argon and heated at 120° for 6
hours with stirring. The mixture was cooled, evaporated to dry-
ness, redissolved in 2 mL of methanol, and purified by prepara-
tive reverse phase C 18 hplc column eluting with 25%
methanol/75% water with 0.5% triethylamine. From the chro-
matography was obtained 75 mg (10%) of a white solid product
To 3.00 g (12.20 mmoles) of 2-amino-5-bromo-4,5-dimethyl-
3-nitropyridine (8) in 200 mL of methanol with 6 mL of ammo-
nium hydroxide was added 300 mg of 10% palladium on carbon.
The reaction mixture was hydrogenated for 18 hours at 50 psi of
hydrogen and 50°. The mixture was filtered through Celite, con-
centrated, suspended in 30 mL of water, and treated with sodium
carbonate until basic. The material was extracted into chloro-
form (3 x 200 mL), dried (sodium carbonate), filtered, and evap-
orated to afford 1.64 g (98%) of a yellow solid (9); mp 157-160°;
1H nmr (deuteriochloroform): d 2.14 (s, 3H), 2.30 (s, 3H), 3.11
(br s, 2H), 4.12 (br s, 2H), 6.40 (s, 1H); 13C nmr (deuteriochloro-
form): d 16.69, 23.05, 117.78, 124.56, 131.69, 146.20, 148.15; ir
(potassium bromide) 3319, 3162, 2914, 2374, 1652, 1636, 1559,
1476, 1244, 1152, 1116, 1030, 932, 830, 756, 613, 528 cm-1; ms
(electrospray ionization) m/z (relative intensity) 138 (100, mole-
cular ion + H), 121 (26), 105 (16).
1
(14); mp 237°; H nmr (deuteriodimethylsulfoxide): d 2.33 (s,
3H), 2.49 (s, 3H), 3.65 (s, 3H), 6.44 (s, 1H), 6.62 (br s, 2H); 13
C
nmr (deuteriodimethylsulfoxide): d 16.7, 23.6, 30.7, 115.9,
123.6, 126.0, 148.6, 155.8, 157.1; ir (potassium bromide) 3323,
3171, 1647, 1620, 1544, 1473, 1419, 1392, 1121 cm-1; uv (95%
ethanol) l max 299 nm (e 12,178), 209 (28,307); ms (70 eV, elec-
tron impact) m/z (relative intensity) 176 (53, molecular ion), 161
(14), 148 (7), 133 (9), 106 (7), 92 (8), 77 (18), 65 (60), 51 (43),
42 (100); hmrs Calcd. for C9H12N4: 176.1062. Found:
176.1062.
N3-Benzyloxycarbonyl-2,3-diamino-4,6-dimethylpyridine (11).
A suspension of 381 mg (2.77 mmoles) of 2,3-diamino-4,6-
dimethylpyridine (9) in 10 mL of acetone at 0° under argon was
treated with 1.53 g (11.1 mmoles) of potassium carbonate, and
473 mg (2.77 mmoles) of benzyl chloroformate (10) was added
dropwise. The reaction mixture was stirred at room temperature
for 18 hours. The solvent was removed under vacuum and the
residue extracted with 3 x 200 mL of chloroform. Evaporation
left approximately 1 g of crude material, which was chro-
matographed on silica gel eluting with 5% methanol/95% chloro-
2-Amino-N-p-toluenesulfonamide-1,5,7-trimethylimidazo-
[4,5-b]pyridine (13).
A solution of 430 mg (2.85 mmoles) of 2-amino-4,6-dimethyl-3-
(methylamino)pyridine (12) in 8 mL of dichloromethane was
treated with 720 mg (2.85 mmoles) of N-dichloromethylene-p-
toluenesulfonamide (2). The reaction solution was heated at 40° for
1 hour under argon. After the mixture was cooled to 0°, 0.57 mL
(5.7 mmoles) of 10.0 M sodium hydroxide solution was added. The

Jul-Aug 2003
Syntheses of Two Potential Food Mutagens
573
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mixture was evaporated to dryness to give a brown gum, which was
triturated with 20 mL of dichloromethane, placed on a 2 cm x 30 cm
silica gel column, and eluted with 97% dichloromethane/3%
methanol and a trace of triethylamine. The product fractions were
combined and evaporated to yield 477 mg (51%) of desired product
1
(13); H nmr (deuteriochloroform): d 2.39 (s, 3H), 2.50 (s, 3H),
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2.56 (s, 3H), 3.69 (s, 3H), 6.74 (s, 1H), 7.23-7.33 (m, 2H), 7.83 (m,
2H); ir (potassium bromide) 2922, 2847, 2360, 2339, 1587, 1450,
1263, 1141, 1085, 840, 812 cm-1; uv (methanol) l max 338 nm (e ;
ms (atmospheric pressure chemical ionization) m/z (relative inten-
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[4,5-b]pyridine (14).
In a TeflonÒ lined bomb, a solution of 477 mg (1.44 mmoles)
of 2-amino-N-p-toluenesulfonamide-1,5,7-trimethylimidazo[4,5-
b]pyridine (13) in 10 mL of anhydrous hydrofluoric acid under
argon was heated at 100° for 1 hour. After cooling to 0°, the
bomb was carefully opened and the hydrofluoric acid evaporated
under a gentle stream of argon. The solid residue was treated
with 4 mL of 15% sodium hydroxide solution. The mixture was
evaporated to dryness and then refluxed in 400 mL of chloroform
overnight to dissolve the product away from any salts. The
organic extract was dried (sodium sulfate), filtered, and evapo-
rated to give 230 mg of material. This residue was triturated with
3 x 5 mL of ethyl ether and the insoluble residue chro-
matographed on a 1 cm x 27 cm silica gel column eluting with
80% chloroform/19% methanol/1% triethylamine. The product
fractions were combined and evaporated to give 102 mg (40%) of
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Land, and K. W. Turteltaub, Cancer Epidemiol. Biomarkers
Prevention 8, 507 (1999).
[14] J. S. Felton, M. G. Knize, N. H. Shen, B. D. Andersen, L.
F. Bjeldanes, and F. T. Hatch, Environ. Health Perspect., 67, 17
(1986).
[15] J. S. Felton, M. G. Knize, M. Roper, E. Fultz, N. H. Shen,
and K. W. Turteltaub, Cancer Res., 52 (Suppl.), 2103 (1992).
[16] G. Becher, M. G. Knize, I. F. Nes, and J. S. Felton,
Carcinogenesis, 9, 247 (1988).
[17] J. S. Felton, M. G. Knize, F. T. Hatch, M. J. Tanga, and M.
E. Colvin, Cancer Lett., 143, 127 (1999).
1
desired product (14); mp 237°; H nmr (deuteriochloroform): d
2.33 (s, 3H), 2.49 (s, 3H), 3.65 (s, 3H), 6.46 (s, 1H), 6.65 (br s,
2H); 13C nmr (deuteriochloroform): d 16.6, 23.4, 30.7, 115.9,
123.7, 126.3, 148.4, 155.6, 157.0; ir (potassium bromide) 3342,
3167, 1653, 1623, 1547, 1481, 1426, 1397, 1310, 1282, 1225,
1121, 1028 cm-1; uv (methanol) l max 299 nm (e12,968), 208
(30,182); ms (direct chemical ionization-ammonia) m/z (relative
intensity) 177 (100, molecular ion); hrms Calcd. for C9H12N4:
176.1062. Found: 176.1062.
[18] M. J. Tanga, J. E. Bupp, and T. K. Tochimoto, J.
Heterocyclic Chem., 31, 1641 (1994).
[19] M. J. Tanga, J. E. Bupp, and T. K. Tochimoto, J.
Heterocyclic Chem., 34, 717 (1997).
Acknowledgments.
This work was supported by the National Cancer Institute
under Contract No. N01-CB-67257 and Grant No. PO1-
CA55861. We would also like to thank Dr. M. G. Knize,
Lawrence Livermore National Laboratory, Biomedical Sciences
Division, University of California, for his assistance.
[20] The ten isomers 1,5,6-trimethyl-2-aminoimidazo[4,5-b]-
pyridine; 3,5,6-trimethyl-2-aminoimidazo[4,5-b]pyridine; 3,5,7-
trimethyl-2-aminoimidazo[4,5-b]pyridine; 1,4,7-trimethyl-2-amino-
imidazo[4,5-c]pyridine; 1,6,7-trimethyl-2-aminoimidazo[4,5-c]pyri-
dine; 3,4,6-trimethyl-2-aminoimidazo[4,5-c]pyridine; 1,4,6-
trimethyl-2-aminoimidazo[4,5-c]pyridine; 1,5,7-trimethyl-2-amino-
imidazo[4,5-b]pyridine; 3,6,7-trimethyl-2-aminoimidazo[4,5-b]pyri-
dine; and 3,6,7-trimethyl-2-aminoimidazo[4,5-c]pyridine are
presently available as reference materials from Dr. Harold Seifried,
National Cancer Institute, 6130 Executive Boulevard, EPN 3158,
Bethesda, MD 20852.
REFERENCES AND NOTES
[1] D. W. Layton, K. T. Bogen, M. G. Knize, F. T. Hatch, V.
M. Johnson, and J. S. Felton, Carcinogenesis, 16, 39 (1995).
[2] F. T. Hatch, M. G. Knize , S. K. Healy, T. Slezak, and J. S.
Felton, Environ. Mol. Mutagen., 12, 1 (1988).
[21] R. Gompper and R. Kunz, Chem, Ber., 99, 2900 (1966).