Synthesis and mutagenic potency of structural isomers of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Article abstract of DOI:10.1002/jhet.5570450614

(Chemical Equation Presented) Synthesis of 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP), three structural isomers, and two desphenyl PhIP congeners has been carried out. Mutagenic potency was evaluated using S. typhimurium strain TA98 in the Ames test. Mutagenic potency increased in relation to structural features in these heterocyclic amines that allow extended resonance between the phenyl and imidazo[4,5-b]pyridine N²-amino substituents. By contrast, PhIP isomers, whose substitution disallows involvement of the phenyl group in their ammoimidazo resonance hybrids, and desphenyl congeners were from 86- to 234-fold less mutagenic than PhIP.

Full text of DOI:10.1002/jhet.5570450614

Nov-Dec 2008
1641
Synthesis and Mutagenic Potency of Structural Isomers of
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
W. Chrisman, M. G. Knize§, and M. J. Tanga*
Bioscience Division, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025
§Chemistry, Materials and Life Sciences Directorate, Lawrence Livermore National Laboratory,
7000 East Avenue, L-452, Livermore, CA 94550
E-mail: mary.tanga@sri.com
Received March 28, 2008
N
N
N
N
3-Me-5-Ph-IP
NH2
NH2
N
N
2500 ± 250
TA98 revertant
counts/μg
CH3
CH3
CH3
CH3
1-Me-5-Ph-IP
N
N
NH2
NH2
N
N
N
N
97 ± 7.30
TA 98 revertant
counts/μg
Synthesis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), three structural isomers, and
two desphenyl PhIP congeners has been carried out. Mutagenic potency was evaluated using S.
typhimurium strain TA98 in the Ames test. Mutagenic potency increased in relation to structural features in
these heterocyclic amines that allow extended resonance between the phenyl and imidazo[4,5-b]pyridine
N2-amino substituents. By contrast, PhIP isomers, whose substitution disallows involvement of the phenyl
group in their aminoimidazo resonance hybrids, and desphenyl congeners were from 86- to 234-fold less
mutagenic than PhIP.
J. Heterocyclic Chem., 45, 1641 (2008).
CH3
INTRODUCTION
R
N
N
R
N
NH2
NH2
N
Amino acid pyrolysates, including heterocyclic amines
(HAs), are found in fried meat and fish at levels of ng/g;
in particular, the imidazopyridine PhIP (1) commonly is
the most abundant HA in meat and fish [1]. PhIP is a
suspected pro-carcinogen [2] that undergoes metabolism
to proximate mutagens including predominantly N-
hydroxy-PhIP [3]. It is well established that PhIP is a
substrate for xenobiotic oxidation by cytochrome P450
enzymes en route to either N-glucuronide conjugates or
heterolytic N-O bond cleavage furnishing the PhIP-
nitrenium ion [4]. The impact on human health from PhIP
and other HAs [5] and the quantification of HAs obtained
from dietary sources [6] are topics of research interest.
Previously we compared PhIP and 3-Me-6-Ph-IP (3)
following activation by Aroclor induced rat liver, and
found that PhIP was 86-fold more mutagenic against S.
typhimurium TA98 in the Ames test [7]. The difference in
potency between these two HAs raised structure-activity
questions. Consequently, we have prepared a set of
isomeric HAs (Figure 1): PhIP (1); PhIP isomers 3-Me-6-
Ph-IP (3), 3-Me-5-Ph-IP (5), and 1-Me-5-Ph-IP (6); and
the desphenyl PhIP congeners 1-Me-IP (2) and 3-Me-IP
(4). Our studies have resulted in efficient, not previously
reported syntheses of PhIP isomers 5 and 6.
N
N
CH3
PhIP (1): R = Ph
3-Me-6-Ph-IP (3): R = Ph
3-Me-IP (4): R = H
1-Me-IP (2): R = H
CH3
N
N
NH2
NH2
N
N
Ph
N
Ph
N
CH3
3-Me-5-Ph-IP (5)
1-Me-5-Ph-IP (6)
PhIP (1) and isomers 3-Me-6-Ph-IP (3), 3-Me-5-Ph-IP (5), 1-Me-5-Ph-
IP (6), and desphenyl PhIP congeners 1-Me-IP (2), and 3-Me-IP (4)
Figure 1
Tests with these six HAs have assessed their relative
potency against S. typhimurium TA98 and elucidated the
possible structure activity relationship (SAR) between the
four PhIP isomers and mutagenicity.
RESULTS AND DISCUSSION
I.
Synthesis. Synthesis of PhIP (1) (Scheme 1)
began with 3-phenylpyridine 7 that was aminated in a
Chichibabin reaction furnishing 2-aminopyridine 8 in

1642
W. Chrisman, M. G. Knize, and M. J. Tanga
Vol 45
52% yield. Bromination in acetic acid at 0 °C gave a 72%
yield of 3-bromopyridine 9, which underwent desamino-
bromination at 200 °C by the action of aqueous
methylamine in the presence of cupric sulfate furnishing
unpurified diaminopyridine 10. Condensing crude
diaminopyridine 10 with cyanogens bromide under a
pyridine 13 [10], which was reduced using lithium
aluminum hydride in tetrahydrofuran at reflux to provide
N3-methylaminopyridine 14 in 22% yield over two steps.
Cyclization with aqueous cyanogen bromide at 40 °C,
gave the corresponding imidazo derivative 14 in 43%
yield of 1-Me-IP (2) after C-18 flash chromatography.
The PhIP isomer 3-Me-6-Ph-IP (3) was prepared from
3-phenylpyridine via amination with sodium amide
(Scheme 3), giving a 52% yield of 2-aminopyridine 8.
Conversion to the corresponding 2-bromopyridine 15
under Sandmeyer reaction conditions gave a 95% yield; it
was followed by copper mediated desaminobromination
of 15 using aqueous methylamine to furnish a mixture of
2-methylaminopyridine 16 in 63% yield, along with a
dehydrobromination by-product. Bromination in acetic
acid led to a 97% yield of 3-bromopyridine 17, which was
aminated by the copper-mediated action of aqueous
ammonia at 200 °C to give diaminopyridine 18 in 82%
yield plus a dehydrobromination by-product. Unpurified
diamine 18 and dichloro-N-tosylmethanimine (11) were
condensed in dioxane at 85 °C to give a 96% yield of
sulfonamidoimidazo[4,5-b]pyridine 19. Unmasking the
amine moiety with anhydrous hydrogen fluoride at 100 °C
gave 3-Me-6-Ph-IP in a 43% yield.
Scheme 1
Ph
iii
R
Ph
iv
ii
72%
92%
N
NH2
N
R
9: R = Br
7: R = H
i
10: R = NHCH3
8: R = NH2
CH3
CH3
Ph
N
N
N
v
NHTs
NH2
38%
N
N
N
12
PhIP (1)
Reagents: (i) NaNH2, toluene, reflux, 52%; (ii) Br2, AcOH, 0-20 oC;
(iii) CH3NH2(aq), CuSO4, 200 oC, 79%; (iv) Cl2C=NTs (11) , K2CO3,
dioxane, 85 oC; (v) HF, 100 o
C
variety of conditions led to low yields of PhIP, plus by-
products and recovered starting material. By contrast,
when impure 10 and dichloro-N-tosylmethanimine (11)
[8] were condensed in dioxane at 85 °C, the sulfonamido-
imidazo[4,5-b]pyridine 12 was obtained in 73% yield
over two steps from 9. Attempted detosylation of 12 by
standard methods was unsuccessful; e.g., hydrogen
bromide in acetic acid resulted in recovered starting
material, and hydrolysis in concentrated sulfuric acid at
100 °C or treatment with excess butyl lithium in
tetrahydrofuran from –78 °C to 20 °C gave tars [9]. N-
Toluenesulfonyl removal with anhydrous hydrogen
fluoride at 100 °C led to PhIP in 38% yield, although C-
18 flash chromatography followed by trituration from
ethanol was needed for separation of by-products.
Scheme 3
Ph
R
Ph
Ph
N
N
vi
iv
NHR
96%
97%
N
NHCH3
N
N
R
CH3
R = H
i
ii
17: R = Br
18: R = NH2
19: R = Ts
3: R = H
v
vii
8: R = NH2
15: R = Br
iii
16: R = NHCH3
Reagents: (i) NaNH2, toluene, reflux, 52%; (ii) HBr, HCl, Br2, NaNO2, 0 oC,
95%; (iii) CH3NH2(aq), CuSO4, 200 oC, 63%; (iv) Br2, AcOH, 0-20 oC,
82%; (v) NH4OH(aq), CuSO4, 200 oC, 82%; (vi) Cl2C=NTs (11), K2CO3,
dioxane, 85 oC; (vii) HF, 100 oC, 43%
Synthesis of 3-Me-IP (4) and 3-Me-5-Ph-IP (5) began
with 3-amino-2-chloropyridine (20). Copper-mediated
deaminochlorination with aqueous methylamine at 200 °C
led to a mixture of diaminopyridine 21 (Scheme 4) in
Scheme 2
CH3
53% yield and
a
dehydrochlorination by-product.
R
NH2
NH2
NHR
NH2
N
i
iii
Condensation of 21 and dichloro-N-tosylmethanimine
(11) under the standard conditions furnished a 45% yield
of sulfonamidoimidazo[4,5-b]pyridine 22. Deprotection
using anhydrous hydrogen fluoride at 100 °C left the free
amine 3-Me-IP in 68% yield after flash chromatography.
Bromination of 1-Me-IP (2) in acetic acid at 100 °C
regiospecifically led to 6-bromopyridine 23 (Scheme 2) in
15% yield, without substitution at the pyridine C-5 or C-7
positions; this result is attributable to participation of ꢀ-
complex resonance stabilization from the N2-amino group.
By contrast, an imidazomethylamine moiety positioned at
NH2
25%
43%
N
N
N
N
13: R = Cbz
14: R = CH3
1-Me-IP (2); R = H
23: R = Br
ii
iv
Reagents: (i) PhCH2OC(O)Cl, THF-pyridine, 0-20 oC; (ii) LiAlH4, THF,
reflux, 87%; (iii) BrCN, H2O, 40 oC; (iv) Br, AcOH, 100 oC, 15%
Synthesis of desphenyl PhIP congener 1-Me-IP (2)
from 2,3-diaminopyridine (Scheme 2) began with a
moderately regioselective conversion to N3-benzylamino-

Nov-Dec 2008
Synthesis and Mutagenic Potency of Structural Isomers
1643
N3 in 3-Me-IP directs bromination toward C-5. It follows
that our synthesis of PhIP isomer 5 involved using a
Suzuki cross-coupling (Scheme 4) of 5-bromo-3-Me-IP
(24) with phenylboronic acid.
Conditions employed for bromination of 1-Me-IP when
applied to 3-Me-IP produced exclusively 5-bromopyridine
24. Under Pd(0)-mediated cross-coupling conditions, 24
was allowed to react with either phenyl- or perdeutero-
phenylboronic acid, resulting in 5 and 25 in yields of 59%
and 71%, respectively.
3,5-dibromo-6-phenylpyridine, and recovered starting
material in nearly equal parts.
Directed lithium-halogen exchange was utilized to
bypass this issue in the bromination of aminopyridine 27.
Thus, the amine was converted to pivalamide 28 that is
sparingly soluble in diethyl ether, tetrahydrofuran, or 1,2-
dimethoxyethane. Initially, ortho-directed lithiation of 2-
pivaloylaminopyridine 28 using butyllithium, sec-BuLi,
or tert-BuLi in tetrahydrofuran-hexane or lithium
diisopropylamide in tetrahydrofuran failed under standard
methods [12]. However, the action of butyllithium
complexed with tetramethylethylenediamine (TMEDA) in
diethyl ether allowed regiospecific metalation of 28.
Dilithiated species 29 as a heterogeneous paste in diethyl
ether was treated with tetrabromomethane to give a 70%
yield of 2-pivaloylamido-3-bromopyridine 30. Hydrolysis
of the pivaloylamide moiety furnished 3-bromopyridine
31 in 38% yield over three steps from 27.
Deaminobromination with aqueous methylamine under
the usual conditions led to 2,3,6-trisubstituted 32 in about
80% yield, along with a dehydrobromination by-product.
Unpurified diaminopyridine 32 was allowed to react with
cyanogen bromide in ethanol at 34 °C for 20 hours, giving
1-Me-5-Ph-IP (6) in 19% yield after flash
chromatography and trituration with ethanol.
Scheme 4
N
N
NH2
R
N
N
ii
iv
NH2
NHR
45%
77%
R
N
N
N
CH3
CH3
24: R = Br
5: R = Ph
20: R = Cl
22: R = Ts
3-Me-IP (4): R = H
v
i
iii
21: R = NHCH3
vi
25: R = D5-Ph
Reagents: (i) CH3NH2(aq), CuSO4, 200 oC, 53%; (ii) Cl2C=NTs (11), K2CO3,
dioxane, 85 oC; (iii) HF, 100 oC, 68%; (iv) Br2, AcOH, 100 oC; (v) [Pd].
ethanol-dioxane, 85 oC, PhB(OH)2 59% for 5, or D5-PhB(OH)2 71% for 25
Synthesis of PhIP isomer 6 began with amination of 2-
II. NMR Spectra. 1H and 13C nmr spectra obtained for
all synthetic compounds were in accord with their
structural assignments. The positions of phenyl and
imidazo N-Me substituents for each PhIP isomer were
phenylpyridine (26) employing the Chichibabin reaction
conditions used for preparing 2-aminopyridine 8.
Refluxing 2-phenylpyridine, toluene, and sodium amide
in xylenes (50 wt% leads unexpectedly to secondary
amine bis(6-phenylpyridin-2-yl)amine [11]. By contrast,
substituting N,N-dimethylaniline for toluene and using dry
sodium amide (Scheme 5), the amination led to 2-
1
confirmed by H PFGSE nOe experiments recorded at
ambient temperature for dilute samples in degassed
dimethyl sulfoxide-d6 (freeze-pump-thaw). NOe mixing
times were on the order of T1 (longitudinal relaxation) for
pyridine H-7 that was in the range of 1.5-2.6 seconds.
The 1H nmr spectrum of 3-Me-5-Ph-IP was
insufficiently resolved between 7.38 and 7.49 ppm to
confirm the phenyl position at pyridine C-5. By contrast,
the spectra of 2H5-phenyl congener 25 displayed doublets,
J = 8.1 Hz, for vicinal protons H-6 and H-7 in accord with
ortho-substitution. NOe spectra of 5 showed strong
enhancement of the N2-amino, ꢀ 6.84 (broad singlet), and
medium enhancement of the phenyl H-2ꢁ and H-6ꢁ
resonances, ꢀ 8.05 (app. d, J = 7.9 Hz), upon irradiation of
the N3-Me, ꢀ 3.60 (singlet). 1H nmr spectra of 1-Me-5-Ph-
IP did not clarify the position of the phenyl substituent
because H-6, H-7, and phenyl resonances were
Scheme 5
Li
N
R
OLi
t-Bu
iii
70%
Ph
N
Ph
N
NHR'
Ph
i
N
R
26: R = H
30: R = Br, R' = Piv
31: R = Br, R' = H
29
iv
27: R = NH2
28: R = NHPiv
ii
CH3
NHCH3
NH2
N
v
vi
19%
NH2
80%
Ph
N
N
N
Ph
32
1-Me-5-Ph-IP (6)
coincident. The nOe spectra of
6 showed strong
Reagents: (i) NaNH2, N,N-dimethylaniline, 170 oC, 81%; (ii) PivCl, NEt3, THF,
0-20 oC, 56%, (iii) BuLi-TMEDA, Et2O, -70 oC, then CBr4; (iv) HCl(aq), 70 oC,
enhancement of both the N2-amino, ꢀ 6.89 (broad singlet),
and the doublet arising from H-7, ꢀ 7.43 (app. d, J = 8.0
Hz), when the N1-Me, ꢀ 3.52 (singlet), was irradiated.
96%; (v) CH3NH2(aq), CuSO4, 200 oC, 80%; (vi) BrCN, ethanol, 34 o
C
1
In H nmr spectra of 2-amino-5-bromoimidazo[4,5-b]-
pyridine (24), obtained via bromination of 3-Me-IP,
doublets occurred at ꢀ 7.33 (d, J = 7.9 Hz, H-6) and ꢀ
7.10 (d, J = 8.1 Hz, H-7). Coupling of 8.0 Hz between
H-6 and H-7 correlates with vicinal protons in 24,
aminopyridine 27 in good yield. When treated with 1.1
equivalent of bromine in acetic acid at 0 °C, 2-
aminopyridine 27 was unfortunately converted to a
mixture of 2-amino-5-bromo-6-phenylpyridine, 2-amino-

1644
W. Chrisman, M. G. Knize, and M. J. Tanga
Vol 45
whereas the spectrum of 2-amino-1-methyl-6-bromo-
imidazo[4,5-b]pyridine, showed doublets at ꢀ 7.89 (d, J =
1.9 Hz, H-5) and ꢀ 7.56 (d, J = 1.9 Hz, H-7) [13].
Table 1
TA98 Revertants/μg [a]
Compound
III. Mutagenic Activity. PhIP and structural isomer 3,
which we have previously compared [7], were
synthesized and re-evaluated in this study of the four HA
isomers, to ensure that data were self-consistent. While
the four isomers 1, 3, 5, and 6 are a complete set for PhIP,
two remaining isomers 1-Me-7-Ph-IP and 3-Me-7-Ph-IP
have not been reported. PhIP isomers 1, 3, 5, and 6 are
linear structures versus tricyclic angular HAs exemplified
by 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) [4b],
which would be structurally more closely related to either
of the 7-Ph-PhIP isomers.
Mutagenic activity was determined using the standard
plate incorporation assay described by Ames et al. [14],
with S. typhimurium strain TA98 (a gift from Professor
Bruce Ames, University of California, Berkeley). Two
milligrams of Aroclor-induced rat liver S9 protein were
added per plate for metabolic activation, and HAs were
plated in serial doses to give a linear response covering a
range from 0.1 to 100 μg/plate. An HA positive control
3-Me-5-Ph-IP (5)
PhIP (1)
1-Me-5-Ph-IP (6)
3-Me-6-PH-IP (3)
3-Me-IP (4)
2500 ± 252
1800 ± 138
97 ± 7.30
20 ± 0.78
8.2 ± 0.22
7.9 ± 0.39
1-Me-IP (1)
[a] Five sequential concentrations were used; data points
were measured in duplicate; r = 0.93-0.99.
Hydroxylamine metabolites (proximate mutagens) of
PhIP give rise to nitrenium ions (primary mutagens)
forming covalent bonds with DNA bases or undergoing
metabolic decomposition [15]. During these steps an
increase in mutagenicity and carcinogenicity frequently
accompanies HAs that stabilize positive character on the
aminoimidazo nitrogen associated with formation of an
HA nitrenium ion, e.g., one of the electrophilic
metabolites of PhIP [1b,e].
IQ gave 1200-1500 revertants per
5
ng dose.
Dimethylsulfoxide (DMSO) was included in the negative
controls (spontaneous revertant counts) and led to 25-60
revertant colonies per plate.
CH3
CH3
N
N
NH2
NH2
N
N
N
N
N
A minimum of four dose points from duplicate platings
was used, and from the slope of the linear portion of the
curve, the number of revertant colonies per microgram
HA was calculated taking the standard error of the linear
fit as delta. The Ames test results comparing 1, 3, 5, and
6, plus desphenyl PhIP congeners 2 and 4, ranged from
2500 ± 252 to 7.9 ± 0.39 revertant counts per microgram
of HA (Table 1).
PhIP (1)
33
N
N
N
N
NH2
NH2
N
CH3
CH3
3-Me-6-Ph-IP (3)
34
35
N
N
N
That the position of the imidazo[4,5-b]pyridine N-
methyl substituent strongly influences the pyridine ring by
increasing electron density at C-6 and C-5 of 1-Me-IP (2)
and 3-Me-IP (4), respectively, was demonstrated by the
outcome of bromination of 2 and 4. The electronic effect
is preserved in PhIP and 3-Me-5-Ph-IP, extending via
resonance involvement of the phenyl substituent, and we
propose that this effect is implicated in their mutagenicity.
Isomer 5 was two-fold more mutagenic than PhIP, and
both HAs favor valence-bond delocalization between the
phenyl and 2-aminoimidazo[4,5-b]pyridine amine
substituents illustrated in resonance hybrids 33 and 35
(Figure 2). By contrast, isomers 3 and 6, whose
substitution pattern disallows involvement of the phenyl
group in resonance with the amine substituent, shown in
resonance forms 34 and 36, respectively, were 86-fold
and 200-fold less mutagenic than PhIP. Desphenyl N-Me-
IP congeners 2 and 4, lacking phenyl contribution to
resonance stabilization, were approximately 234-fold less
mutagenic than PhIP.
NH2
NH2
N
N
N
CH3
CH3
3-Me-5-Ph-IP (5)
CH3
N
CH3
N
NH2
NH2
N
N
N
N
36
1-Me-5-Ph-IP (6)
Figure 2. Delocalization between the 2-aminoimidazo[4,5-b] amine
and phenyl substituents is facilitated by the structure of PhIP and
3-Me-5-Ph-IP.
Despite shortcomings in drawing a SAR using the
nonmetabolically activated parent HAs, our data revealed
that relative mutagenic activities for these six HAs agree
well with an extended resonance effect. Structural isomers
that facilitate charge delocalization between the phenyl
and 2-aminoimidazo[4,5-b]pyridine amine substituents

Nov-Dec 2008
Synthesis and Mutagenic Potency of Structural Isomers
1645
1
°C; H nmr (dimethyl sulfoxide-d6) ꢀ 8.23 (d, J = 2.0 Hz, 1H),
7.68 (dd, J = 2.6, 8.5 Hz, 1H), 7.54 (m, 2H), 7.38 (app. t, J = 7.7
Hz, 2H), 7.25 (app. tt, J = 1.2, 7.3 Hz, 1H), 6.52 (dd, J = 0.6, 8.5
Hz, 1H), 6.02 (br. d, J = 4.1 Hz, NH2); 13C nmr (chloroform-d) ꢀ
157.9, 146.4, 138.4, 136.6, 129.0, 127.3, 127.0, 126.4, 108.6;
ms: m/z 172(14), 171(100, M+ +H), 154(11), 145(3).
led to increased mutagenicity. Although the higher
mutagenicity for PhIP and in particular 3-Me-5-Ph-IP
toward TA98 correlates well with this structural model,
the details of the mechanism involved are an open
question. It is not clear whether the higher mutagenicity
of PhIP and in particular 3-Me-5-Ph-IP results from: (i)
facilitated metabolism to HA proximate and ultimate
mutagens, or (ii) higher conversion efficiencies of
resonance stabilized HA metabolites into DNA-
conjugates [16], or (iii) possibly the functional effects of
the modified DNA unrelated to electronics of the parent
HA [17].
2-Amino-3-bromo-5-phenylpyridine (9). Aminopyridine 8
(3.2 g, 18.8 mmol) was combined with acetic acid (30 mL),
cooled to 0 °C, stirred, and treated with bromine (1.1 mL) in
acetic acid (10 mL) over 5 minutes, then warmed to 20 °C. The
slurry was stirred for an additional 30 minutes, concentrated to a
solid, and dissolved in water. Sodium hydroxide was added, and
the solution was extracted with chloroform. The extract was
dried and filtered, and then concentrated to a tan solid, 3.44 g
(73%), tlc Rf = 0.34, methanol:chloroform (1:99); 1H nmr
(dimethyl sulfoxide-d6) ꢀ 8.28 (d, J = 2.1 Hz), 8.03 (d, J = 2.1
Hz), 7.58 (m), 7.40 (app. t, J = 8.2 Hz, H-3', H-5'), 7.29 (app. tt,
J = 1.8, 7.3 Hz, H-4'), 6.31 (br. s, J = NH2); 13C nmr (dimethyl
sulfoxide-d6) ꢀ 155.6, 144.9, 137.9, 137.8, 136.4, 135.7, 128.9,
127.7, 126.9, 126.0, 125.7, 124.7, 120.1, 103.4; ms: m/z =
252(10), 251(100, M+ +H, 81Br), 250(11), 249(98, M+ +H, 79Br).
2-Amino-3-methylamino-5-phenylpyridine (10). Bromo-
pyridine 9 (3.89 g, 15.6 mmol) was combined with cupric sulfate
(0.91 g, 3.64 mmol) plus aqueous methylamine (30 mL, 40
wt%) in a Parr vessel and kept at 200 °C for 48 hours, then
cooled, poured into water, and extracted with chloroform. The
extract was dried and filtered, and then concentrated to an
impure blue-green residue that was carried on without
purification, 2.89 g (ca. 79%), tlc Rf = 0.34, methanol:
chloroform (1:20); ms: m/z 201(12), 200(100, M+ +H).
Acknowledgement. The authors thank Rebekah Wu for
performing the Ames/Salmonella tests; this work was
performed under the auspices of the U.S. Department of
Energy by the University of California, Lawrence
Livermore National Laboratory, under Contract No. W-
7405-Eng-48 and was supported by NCI grant CA55861.
The authors thank Dr. Pavlovich, UCSB Department of
Chemistry Mass Spectrometry Facility, University of
California, Santa Barbara, for high resolution mass
spectra. Financial support from NCI under Contract No.
NO2-CB-37126 is acknowledged with thanks. The
synthesized HAs (Table 1) are available within the NCI
Chemical Carcinogen Reference Standards Repository
operated under contract No. N02-CB-07008 by Midwest
Research Institute, 425 Volker Blvd., Kansas City, MO.
1-Methyl-6-phenyl-2-N-(p-toluenesulfonyl)aminoimidazo-
[4,5-b]pyridine (12). Impure diaminopyridine 10 (2.0 g, ca. 8
mmol) was combined with dichloro-N-tosylmethanimine (11)
(2.42 g, 9.60 mmol), potassium carbonate (2.80 g, 20.3 mmol),
and dioxane (80 mL). The stirred mixture was heated to 85 °C
for 20 hours, cooled, diluted with water, and then extracted with
chloroform. The extract was dried and filtered, and then
concentrated. Flash chromatograpy on silica gel eluted by
methanol:chloroform (1:99) gave a tan solid, 2.75 g (92%), tlc
Rf = 0.70, methanol:chloroform (1:20), mp = 315-319 °C (dec);
1H nmr (dimethyl sulfoxide-d6) ꢀ 12.34 (br. s, NH), 8.46 (d, J =
1.8 Hz), 8.08 (d, J = 1.8 Hz), 7.86 (d, J = 8.1 Hz, 2H), 7.75 (app.
d, J = 7.9 Hz, 2H), 7.49 (app. t, J = 7.5 Hz, 1H), 7.39 (m), 7.32
(d, J = 7.8 Hz), 3.49 (s, N-CH3), 2.34 (s, CH3); 13C nmr
(dimethyl sulfoxide-d6) ꢀ 149.2, 143.3, 142.2, 141.9, 140.6,
129.3, 125.7, 122.8, 118.5, 118.5, 26.8, 20.8; ir: 3448, 3068,
1591, 1461, 1376, 1272, 1378, 1272, 1138, 1086, 924, 837 cm-1.
HRMS: C20H19N4O2S (M+ +H) requires m/z 379.1229; found,
379.1237.
EXPERIMENTAL
Glassware was dried at 120 °C for 12 hours before use.
Procedures employed Schlenck techniques under a nitrogen
atmosphere. Solvents were distilled from calcium hydride or
sodium/benzophenone, and organic extracts were dried over
anhydrous sodium sulfate. All tlc was performed using
ANALTECH No. 02521 silica gel uniplates, with tlc and flash
chromatography eluents given as
a volume ratio. Flash
chromatography utilized EM Merck 220–400 mesh, 32–63 μm
60 Å silica gel, or JT Baker octadecyl C-18, 40 μm prep-LC
silica gel. Commercially available starting materials were used
without further purification. Melting points (uncorrected) were
determined on a Mel-Temp II. Nmr spectra were recorded on a
1
Varian MercuryPlus 300VX (300 MHz for H and 75 MHz for
13C). Low resolution mass spectra were recorded on a Finnigan
LCQ-DUO. All uv spectra were recorded on a Varian Cary-50,
and ir spectra (potassium bromide pellets) were obtained on a
Perkin-Elmer 1600.
1-Methyl-6-phenyl-2-aminoimidazo[4,5-b]pyridine; PhIP
(1). Sulfonamide 12 (0.43 g, 1.14 mmol) was combined with
hydrogen fluoride (8 mL) in a Teflon-lined Parr vessel, heated to
100 °C, and held at that temperature for 2.5 hours. The vessel
was cooled and opened, and then concentrated under nitrogen.
The residue was washed with benzene, taken up in aqueous
sodium hydroxide, and flash chromatographed on C18 silica gel
eluted by a methanol:water gradient (1:50-1:5) to yield an
impure tan solid. Crystallization from ethanol gave an off a
white solid, 96 mg (38%), tlc Rf = 0.26, methanol:chloroform
2-Amino-5-phenylpyridine (8). Sodium amide in xylenes
(32.3 g, 50 wt%) was combined with toluene (300 mL) and 3-
phenylpyridine (15.26 g, 97.1 mmol) was added. The stirred
mixture was refluxed for 36 hours, cooled, treated with aqueous
potassium hydroxide, and then extracted with ethyl acetate. The
extract was dried and filtered, and then concentrated to a dark
oil. Flash chromatography on silica gel eluted with
1
methanol:chloroform (1:80-1:20) yielded
a
tan solid.
(1:20), mp = 315-317 °C (dec); H nmr (methanol-d4) ꢀ 8.21 (d,
Crystallization from ethyl acetate provided tan flakes, 8.66 g
(52%), tlc Rf = 0.28, methanol:chloroform (1:50), mp = 135-136
J = 1.8 Hz, 1H), 7.70 (d, J = 1.8 Hz, 1H), 7.59 (app. d, J = 8.0
Hz, 2H), 7.40 (app. t, J = 7.6 Hz, 2H), 7.29 (app. tt, J = 1.2, 7.2

1646
W. Chrisman, M. G. Knize, and M. J. Tanga
Vol 45
Hz, 1H), 3.56 (s, N-CH3); 13C nmr (dimethyl sulfoxide-d6) ꢀ
158.0, 156.3, 139.3, 139.1, 128.9, 127.9, 126.6, 126.4, 126.2,
111.7, 28.4; ir: 3294, 3087, 1668, 1593, 1549, 1470, 1440, 1270,
1102, 914, 881, 759 cm-1. HRMS: C13H13N4 (M+ +H) requires
m/z 225.1135; found 225.1136.
(1:9) yielded a white solid, 0.51 g (15%), tlc Rf = 0.22,
methanol:chloroform (1:9), mp = 178-179 °C; 1H nmr (dimethyl
sulfoxide-d6) ꢀ 7.98 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 2.2 Hz,
1H), 7.04 (br s, NH2), 3.49 (s, N-CH3); 13C nmr (methanol-d4)
ꢀ159.9, 155.7, 142.3, 130.5, 118.4, 110.6, 29.0; ir: 3299, 3134,
1669, 1621, 1588, 1544, 1461, 1428, 1296, 1263, 1103, 938,
861, 773 cm-1. HRMS: C7H7BrN4 (M+ +H, 79Br) requires m/z
226.9934; found 225.9926.
2-Bromo-5-phenylpyridine (15). Aminopyridine 8 (3.1 g,
18.21 mmol) was combined with aqueous hydrogen bromide
(10.75 mL, 48 wt%) at –5 °C. The stirred solution was treated
with bromine (1.0 mL, 19.5 mmol) and aqueous hydrogen
chloride (4.5 mL), followed by dropwise addition of sodium
nitrite (4.8 g, 69.6 mmol) in water (20 mL) over 15 minutes.
After stirring at –5 °C for 1 hour, aqueous sodium hydroxide
(11.0 mL, 40 wt%) was added, followed by extraction with
chloroform. The extract was dried and filtered, and then
concentrated to an impure brown solid, 4.10 g (95%), tlc Rf =
0.65, chloroform; 1H nmr (dimethyl sulfoxide-d6) ꢀ 8.70 (dd, J =
0.7, 2.6 Hz, 1H), 8.03 (dd, J = 2.6, 8.2 Hz, 1H), 7.71 (m, 2H),
7.53-7.40 (m, 4H); 13C nmr (dimethyl sulfoxide-d6) ꢀ 148.3,
140.3, 137.5, 135.7, 135.3, 129.2, 128.6, 128.1, 126.9; ms: m/z
237(9), 236(96, M+ +H, 81Br), 235(10), 234(100, M+ +H, 79Br),
172(10), 156(12).
N2-Amino-N3-benzyloxyamido-pyridine (13). 2,3-Diamino-
pyridine (8.73 g, 79.8 mmol) was combined with pyridine (20
mL) in tetrahydrofuran (200 mL), cooled to 0 °C, and stirred.
Benzyl chloroformate (20 mL, 130.0 mmol) in tetrahydrofuran
(20 mL) was added over 30 minutes, and the mixture was
warmed to 20 °C and then stirred overnight. Water was added,
followed by extraction with ethyl acetate. The extract was dried
and filtered, then concentrated. Flash chromatography on silica
gel eluted by methanol:chloroform (1:20-1:10) yielded an
impure tan solid. Trituration from chloroform left a white solid,
4.82 g (25%), tlc Rf = 0.35, methanol:chloroform (1:20), mp =
1
178-179 °C; H nmr (dimethyl sulfoxide-d6) ꢀ 8.78 (br. s, 1H),
7.73 (dd, J = 1.5, 4.8 Hz, 1H), 7.33 (br. d, J = 7.6 Hz, 1H), 7.40-
7.32 (m, 5H), 6.55 (dd, J = 4.8, 7.7 Hz, 1H), 5.76 (br. s, NH2),
5.13 (s, CH2-O); 13C nmr (dimethyl sulfoxide-d6) ꢀ 154.2, 152.7
(br. s), 143.4, 136.6, 130.1 (br. s), 128.4, 128.0, 118.5, 112.3,
65.9; ms: m/z 245(15), 244(100, M+ +H), 228(2), 226(9), 200(2).
2-Amino-3-methylaminopyridine (14). Benzyloxyamido-
pyridine 13 (0.98 g, 4.03 mmol) in tetrahydrofuran (85 mL) was
cooled to 0 °C, combined with lithium aluminum hydride (20
mL, 1.0 M tetrahydrofuran), and stirred at 0 °C for 20 minutes.
The mixture was refluxed for 3 hours, then cooled and treated
with ethyl acetate. Water was added and the solution was
acidified with aqueous hydrogen chloride, then washed with
diethyl ether. The aqueous phase was cooled and treated with
solid potassium hydroxide, and then extracted with diethyl ether.
The extract was dried and filtered, then concentrated to yield a
tan solid, 0.43 g (87%), tlc Rf = 0.25, methanol:chloroform
(1:30), mp = 178-179 °C; 1H nmr (dimethyl sulfoxide-d6) ꢀ 7.27
(dd, J = 4.9, 5.0 Hz, 1H), 6.53 (dd, J = 1.8, 7.6 Hz, 1H), 6.47
(dd, J = 4.7, 7.6 Hz, 1H), 5.31 (br. s, NH2), 4.83 (br. app. q, J =
4.5 Hz, NH), 2.67 (s, N-CH3); 13C nmr (chloroform-d) ꢀ 148.9,
136.4, 133.5, 116.4, 116.2, 30.7; ms: m/z 125(6), 124(100, M+
+H), 117(5), 109(2), 97(4), 85(4), 65(1).
1-Methyl-2-aminoimidazo[4,5-b]pyridine; 1-Me-IP (2). Di-
aminopyridine 14 (0.80 g, 6.49 mmol) was combined with
cyanogen bromide (0.83 g, 7.83 mmol) in water (20 mL) and
stirred at 40 °C for 5 hours before addition of a second quantity
of cyanogen bromide (0.25 g, 2.36 mmol). The solution was
stirred at 40 °C overnight, then concentrated. Flash
chromatography on C18 silica gel eluted by methanol:water
(1:3) yielded an off-white solid, 0.41 g (43%), mp = 284-286 °C
after crystallization from methanol; 1H nmr (methanol-d4) ꢀ 7.94
(dd, J = 1.4, 5.2 Hz, 1H), 7.43 (dd, J = 1.3, 7.7 Hz, 1H), 6.92
(dd, J = 5.3, 7.7 Hz, 1H), 3.52 (s, h/2 = 1.1 Hz, N-CH3); 13C nmr
(methanol-d4) ꢀ 159.2, 156.6, 141.8, 129.5, 115.8, 115.8, 28.8;
ir: 3259, 3079, 1680, 1630, 1595, 1546, 1472, 1432, 1267, 1246,
1126, 927, 910, 771 cm-1. HRMS: C7H9N4 (M+ +H) requires m/z
149.0821; found 149.0821.
2-Methylamino-5-phenylpyridine (16). Bromopyridine 15
(3.83 g, ca. 16 mmol) was combined with cupric sulfate (0.82 g,
3.28 mmol) plus aqueous methylamine (23 mL, 40 wt%) in a
Parr vessel and kept at 200 °C for 48 hours, then cooled and
poured into water, and then extracted with chloroform. The
extract was dried and filtered, and then concentrated. Flash
chromatography on silica gel eluted by methanol:chloroform
(1:50) yielded a tan solid, 1.85 g (63%), tlc Rf = 0.31,
methanol:chloroform (1:90), mp
=
120-122 °C; 1H nmr
(dimethyl sulfoxide-d6) ꢀ 8.31 (dd, J = 0.6, 2.6 Hz, 1H), 7.69
(dd, J = 2.6, 8.7 Hz, 1H), 7.55 (m, 2H), 7.39 (app. t, J = 8.1 Hz,
2H), 7.25 (app. tt, J = 1.8, 7.3 Hz, 1H), 6.58 (br. q, J = 4.3 Hz,
NH), 6.52 (dd, J = 0.7, 8.7 Hz, 1H), 2.79 (br. s, N-CH3); 13C nmr
(dimethyl sulfoxide-d6) ꢀ 158.9, 158.8, 145.6, 138.2, 134.9,
128.9, 126.2, 125.3, 123.4, 107.7, 28.0, 27.9; ms: m/z 186(13),
185(100, M+ +H), 178(2), 160(6).
2-Methylamino-3-bromo-5-phenylpyridine (17). Methyl-
aminopyridine 16 (1.05 g, 5.69 mmol) in acetic acid (25 mL)
was cooled to 0 °C and treated with bromine (0.35 mL, 6.83
mmol) in acetic acid (10 mL), then warmed from 0 °C to 20 °C
over 30 minutes, and then concentrated. Cold aqueous sodium
hydroxide was added, and the solution was extracted with
chloroform. The extract was dried and filtered, then
concentrated. Flash chromatography on silica gel eluted with
chloroform yielded a tan solid, 1.46 g (97%), tlc Rf = 0.56,
1
chloroform, mp = 68-69 °C; H nmr (dimethyl sulfoxide-d6) ꢀ
8.38 (d, J = 2.4 Hz, 1H), 8.03 (d, J = 2.1 Hz, 1H), 7.58 (m, 2H),
7.40 (app. t, J = 8.2 Hz, 2H), 7.28 (app. tt, J = 1.8, 7.3 Hz, 1H),
6.46 (br. q, J = 4.6 Hz, NH), 2.88 (d, J = 4.7 Hz, N-CH3); 13C
nmr (dimethyl sulfoxide-d6) ꢀ 154.4, 144.5, 137.4, 136.5, 129.0,
126.8, 125.7, 125.1, 104.9, 28.7. HRMS: C12H12N2 (M+ +H, 79Br)
requires m/z 263.0184; found 263.0192.
2-Methylamino-3-amino-5-phenylpyridine (18). Bromo-
pyridine 17 (1.50 g, 5.69 mmol) was combined with cupric
sulfate (0.14 g, 0.56 mmol) plus aqueous ammonia (35 mL, 28
wt%) in a stainless steel Parr vessel. The mixture was heated at
200 °C for 48 hours, then cooled and poured into water, and then
extracted with chloroform. The extract was dried and filtered,
6-Bromo-2-aminoimidazo[4,5-b]pyridine (23). 1-Me-IP
(2.22 g, 14.98 mmol) in acetic acid (120 mL) at 100 °C was
treated with bromine (5.58 g, 34.9 mmol) in acetic acid (10 mL)
and the mixture was stirred for 2 hours, then cooled to 50 °C and
concentrated. Saturated aqueous potassium carbonate was
added, and the solution was extracted with ethyl acetate. The
extract was dried and filtered, then concentrated. Flash
chromatography on silica gel eluted with methanol:chloroform

Nov-Dec 2008
Synthesis and Mutagenic Potency of Structural Isomers
1647
and then concentrated to yield an impure blue-green solid that
was carried on without purification, 1.08 g (ca. 82%), tlc Rf =
0.40, methanol:chloroform (1:20); H nmr (dimethyl sulfoxide-
d6) ꢀ 7.72 (d, J = 1.8 Hz, 1H), 7.48 (app. d, J = 7.2 Hz, 2H), 7.37
(app. t, J = 7.2, 2H), 7.23 (app. t, J = 7.3 Hz, 1H), 6.98 (d, J =
1.8 Hz, 1H), 5.76 (br. d, J = 4.8 Hz, NH), 4.73 (app. br. d, J =
7.4 Hz, NH2), 2.88 (d, J = 4.7 Hz, N-CH3); ms: m/z 201(12),
200(100, M+ +H).
combined with dichloro-N-tosylmethanimine (11) (8.41 g, 33.4
mmol) and potassium carbonate (10.92 g, 79.6 mmol) in dioxane
(120 mL) and stirred at 85 °C for 20 hours. The mixture was
cooled and diluted with water, and then extracted with
chloroform. The extract was dried and filtered, and then
concentrated. Flash chromatography on silica gel eluted by
methanol:chloroform (1:99) provided a tan solid, 4.16 g (45%),
tlc Rf = 0.27, methanol:chloroform (1:99), mp = 208-209 °C; 1H
nmr (dimethyl sulfoxide-d6) ꢀ 11.91 (br. s, NH), 8.14 (dd, J =
5.1, 1.3 Hz, 1H), 7.81 (app. d, J = 6.8 Hz, 2H), 7.66 (dd, J = 7.9,
1.5 Hz, 1H), 7.32 (app. d, J = 7.9 Hz, 2H), 7.19 (dd, J = 7.8, 5.1
Hz, 1H), 3.41 (s, N-CH3), 2.33 (s, CH3); 13C nmr (dimethyl
sulfoxide-d6) ꢀ 149.2, 143.3, 142.2, 141.9, 140.6, 129.3, 125.7,
122.8, 118.5, 118.5, 26.8, 20.8; ir: 3294, 1628, 1594, 1492,
1416, 1300, 1282, 1138, 1087, 1003, 897, 833, 786 cm-1; ms:
m/z = 325.10 (M+ +Na).
1
3-Methyl-6-phenyl-2-N-(p-toluenesulfonyl)-aminoimidazo-
[4,5-b]pyridine (19). Impure diaminopyridine 18 (1.08 g) was
combined with dichloro-N-tosylmethanimine (11) (1.76, 6.98
mmol) and potassium carbonate (1.31 g, 13.8 mmol) in dioxane
(80 mL), and the solution was stirred at 85 °C for 14 hours. The
cooled solution was poured into water and extracted with
chloroform. The extract was dried and filtered, and then
concentrated. Flash chromatography on silica gel eluted by
methanol:chloroform (1:99) yielded a white solid, 1.57 g (ca.
96%), tlc Rf = 0.33, methanol:chloroform (1:99), mp = 228-230
°C (dec); 1H nmr (chloroform-d) ꢀ 10.52 (br. s, NH), 8.40 (d, J =
1.8 Hz, 1H), 7.89 (app. d, J = 8.4 Hz, 2H), 7.66 (d, J = 1.8 Hz,
1H), 7.55-7.41 (app. m), 7.29 (app. d, J = 8.1 Hz, 2H), 3.60 (s,
N-CH3), 2.40 (s, CH3); 13C nmr (chloroform-d) ꢀ 150.5, 143.8,
143.3, 143.0, 141.9, 140.4, 139.4, 137.9, 133.1, 129.9, 129.7,
129.4, 128.2, 127.5, 126.7, 126.3, 122.8, 116.7, 27.4, 21.7; ir:
3357, 3261, 2924, 1598, 1489, 1340, 1304, 1254, 1159, 1133,
1086, 1004, 837, 814 cm-1. HRMS: C20H19N4O2S (M+ +H)
requires m/z 379.1229; found 379.1237.
3-Methyl-6-phenyl-2-aminoimidazo[4,5-b]pyridine; 3-Me-
6-Ph-IP (3). Sulfonamide 19 (1.34 g, 3.54 mmol) was combined
with hydrogen fluoride (10 mL) in a Teflon-lined Parr vessel
and held at 100 °C for 2.5 hours. The flask was cooled and
opened, and then evaporated under nitrogen. The residue was
washed with benzene, taken up in aqueous sodium hydroxide,
then flash chromatographed on C18 silica gel eluted by
methanol:water (1:50-1:5) to yield a tan solid, 0.34 g (43%), mp
= 222-223 °C after trituration from methanol; 1H nmr (dimethyl
sulfoxide-d6) ꢀ 8.14 (d, J = 1.8 Hz, 1H), 7.66 (app. d, J = 1.3 Hz,
1H), 7.63 (app. d, J = 7.8 Hz, 2H), 7.44 (app. t, J = 7.9 Hz, 2H),
7.33 (app. tt, J = 2.4, 7.5 Hz, 1H), 6.83 (br. s, NH2), 3.53 (s, N-
CH3); 13C nmr (dimethyl sulfoxide-d6) ꢀ 156.7, 147.8, 139.1,
136.1, 136.0, 129.7, 128.9, 126.8, 118.3, 27.0; ir: 3445, 3029,
1652, 1547, 1494, 1464, 1410, 1361, 760 cm-1; ms: 226(15),
225(100, M+ +H). HRMS: C13H13N4 (M+ +H) requires m/z
225.1134; found 225.1131.
3-Methyl-2-aminoimidazo[4,5-b]pyridine;
3-Me-IP
(4).
Sulfonamide 22 (0.95 g, 3.14 mmol) was combined with
hydrogen fluoride (8 mL) in a Teflon-lined Parr vessel and held
at 100 °C for 2.5 hours. The vessel was cooled and opened, and
then concentrated under nitrogen. The residue was washed with
benzene, taken up in aqueous sodium hydroxide, then flash
chromatographed on C18 silica gel eluted by methanol:water
(1:50-1:5) to yield a tan solid, 0.32 g (68%), mp = 220-221 °C
1
after trituration with ethanol; H nmr (dimethyl sulfoxide-d6) ꢀ
7.83 (dd, J = 1.4, 5.1 Hz, 1H), 7.37 (dd, J = 1.2, 7.6 Hz, 1H),
6.93 (dd, J = 5.1, 7.7 Hz, 1H), 6.73 (br. s, NH2), 3.49 (s, N-CH3);
13C nmr (dimethyl sulfoxide-d6) ꢀ 155.9, 148.0, 137.4, 135.6,
120.0, 116.6, 26.9; ir: 3317, 3135, 1655, 1609, 1552, 1493,
1407, 1290, 1217, 1112, 930, 766 cm-1; ms: m/z 149(100),
135(1), 134(5), 132(2). HRMS: C7H9N4 (M+ +H) requires m/z
149.0821; found 149.0816.
5-Bromo-3-methyl-2-aminoimidazo[4,5-b]pyridine (24). 3-
Me-IP (0.32 g, 2.16 mmol) was combined with acetic acid (35
mL), and the stirred solution was heated at 100 °C. Bromine
(0.30 mL, 5.8 mmol) in acetic acid (10 mL) was added. The
mixture was stirred for 1 hour, concentrated, taken up in
aqueous potassium carbonate, and then extracted with ethyl
acetate. The extract was dried, filtered, and then concentrated.
Flash chromatography on silica gel eluted by methanol:
chloroform (1:20) yielded a tan solid, 0.38 g (77%), tlc Rf =
1
0.25, methanol:chloroform (1:20), mp = 198-201 °C (dec); H
nmr (dimethyl sulfoxide-d6) ꢀ 7.33 (d, J = 7.9 Hz, 1H), 7.10 (d, J
= 8.1 Hz, 1H), 6.91 (br. s, NH2), 3.47 (s, N-CH3); 13C nmr
(methanol-d4) ꢀ157.9, 148.9, 135.5, 130.4, 124.2, 121.6, 27.6; ir:
3450, 3308, 3084, 2282, 1659, 1626, 1548, 1496, 1396, 1257,
1115, 1090, 939, 831, 806, 763 cm-1. HRMS: C7H8N4Br (M+ +
H, 79Br) requires m/z 226.9932; found 226.9929.
3-Methyl-5-phenyl-2-aminoimidazo[4,5-b]pyridine; 3-Me-
5-Ph-IP (5). Bromoimidazopyridine 24 (1.11g, 4.89 mmol) was
combined with phenylboronic acid (1.37 g, 11.21 mmol),
tetrakis(triphenylphosphine)palladium (0.60 g, 5.19 x 10-4 mol)
and dioxane (70 mL). Potassium carbonate (2.81 g, 20.3 mmol)
in water (15 mL) and ethanol (18 mL) was added, and the
mixture was heated overnight at 85 °C. The mixture was cooled
and diluted with brine, then extracted with ethyl acetate. The
extract was dried and filtered, then concentrated. Flash
chromatography on silica gel eluted by methanol:chloroform
(1:20) yielded a tan solid, 0.65 g (59%), tlc Rf = 0.26,
2-Methylamino-3-aminopyridine (21). Chloropyridine 20
(7.72 g, 60.0 mmol) was combined with cupric sulfate (1.49 g,
6.0 mmol) and aqueous methylamine (45 mL, 40 wt%) in a
stainless steel Parr vessel, and the mixture was held at 170 °C
for 24 hours. The cooled mixture was treated with aqueous
potassium carbonate and continuously extracted for 24 hours
using diethyl ether. The extract was dried and filtered, and then
concentrated. Flash chromatography on silica gel eluted by ethyl
acetate:hexane (2:3) yielded a tan solid, 3.91 g (53%), tlc Rf =
1
0.34, methanol:chloroform (1:20); H nmr (dimethyl sulfoxide-
d6) ꢀ 7.38 (dd, J = 5.1, 1.6 Hz, 1H), 6.65 (dd, J = 7.26, 1.6 Hz,
1H), 6.32 (dd, J = 7.5, 4.8 Hz, 1H), 5.54 (br. app. d, J = 4.2 Hz,
NH), 4.58 (br. s, NH2), 2.81 (d, J = 4.8 Hz, N-CH3); 13C nmr
(dimethyl sulfoxide-d6) ꢀ 148.6, 135.0, 130.2, 117.1, 112.0,
28.1; ir: 3318, 3091, 1657, 1606, 1520, 1475, 1442, 1412, 1371,
1302, 1259, 1156, 1126, 1085, 786 cm-1; ms: m/z 124 (M+ +H).
3-Methyl-2-N-(p-toluenesulfonyl)-aminoimidazo[4,5-b]py-
ridine (22). Diaminopyridine 21 (3.76 g, 30.5 mmol) was
methanol:chloroform (1:20), mp
1
=
209-210 °C after
crystallization from methanol; H nmr (dimethyl sulfoxide-d6) ꢀ
8.05 (app. d, J = 7.9 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.44

1648
W. Chrisman, M. G. Knize, and M. J. Tanga
Vol 45
(app. d, J = 8.2 Hz, 1H), 7.45-7.39 (m, 2H), 7.30 (app. tt, J =
0.9, 7.3 Hz, 1H), 6.84 (br. s, NH2) 3.60 (s, N-CH3); 13C nmr
(dimethyl sulfoxide-d6) ꢀ 156.6, 148.1, 144.7, 139.7, 135.4,
128.5, 127.3, 125.7, 120.6, 113.5, 26.9; ir: 3442, 3312, 3056,
1654, 1542, 1495, 1409, 1300, 1247, 1126, 828, 758 cm-1.
HRMS: C13H13N4 (M+ +H) requires m/z 225.1140; found
225.1143.
ethylenediamine (0.21 mL) and diethyl ether (5.0 mL), and the
stirred solution was cooled to –70 °C, then treated with butyl
lithium (0.81 mL, 1.6 M in hexanes). The resulting paste was
warmed to 20 °C over 1.5 hours, recooled to –70 °C, then
treated with tetrabromomethane (0.42 g, 1.25 mmol) in diethyl
ether (4.0 mL). After 15 minutes at –70 °C, the mixture was
slowly warmed to 20 °C, poured into water, diluted with
aqueous sodium hydroxide, and then extracted with ethyl
acetate. The extract was dried and filtered, and then
concentrated. Flash chromatography on silica gel eluted by ethyl
acetate:hexane (1:10) yielded white flakes, 0.11 g (70%), tlc Rf
3-Methyl-5-(phenyl-2H5)-2-aminoimidazo[4,5-b]pyridine
(25). The procedure for 5 was employed with bromopyridine 24
(0.33 g, 1.45 mmol), phenyl-(2H5)-boronic acid (0.37 g, 2.95
mmol), tetrakis(triphenylphosphine)palladium (0.14 g, 1.19 x
10-4 mol), and dioxane (35 mL). Flash chromatography on silica
gel eluted by methanol:chloroform (1:20) yielded a tan solid,
0.23 g (71%), tlc Rf = 0.24-0.29, methanol:chloroform (1:20),
1
= 0.23, ethyl acetate:hexane (1:10); mp = 110-111 °C; H nmr
(dimethyl sulfoxide-d6) ꢀ 9.78 (br. s, NH), 8.18 (d, J = 8.2 Hz,
1H), 8.03 (m, 2H), 7.81 (d, 8.4 Hz, 1H), 7.42-7.53 (app. m, 3H),
1.26 (s, tert-butyl); 13C nmr (dimethyl sulfoxide-d6) ꢀ 176.5,
154.4, 149.6, 142.7, 137.0, 129.5, 128.8, 126.5, 119.8, 117.4,
38.7, 27.1; ir: 3312, 2966, 2871, 1666, 1573, 1506, 1437, 1174,
1026, 934, 830, 772 cm-1. HRMS: C16H18N2OBr (M+ +H, 79Br)
requires m/z 333.0597; found 333.0587.
1
mp = 209-210 °C after crystallization from methanol; H nmr
(dimethyl sulfoxide-d6) ꢀ 7.55 (app. d, J = 8.1 Hz, 1H), 7.44
(app. d, J = 8.0 Hz, 1H), 6.84 (br. s, NH2) 3.60 (s, N-CH3); 13C
nmr (dimethyl sulfoxide-d6) ꢀ 156.6, 148.1, 144.6, 139.6, 135.4,
129.0 (t, JCD = 23.2 Hz), 126.8 (t, JCD = 22.3 Hz), 125.3 (t, JCD
=
22.9 Hz), 120.6, 113.5, 26.9; ir: 3442, 3048, 1653, 1539, 1495,
1494, 1414, 1382, 1316, 1286, 1227, 1125, 819, 762 cm-1.
2-Amino-3-bromo-6-phenylpyridine (31). Pivalylamide 30
(0.21 g, 0.63 mmol) in aqueous hydrochloric acid (15 mL, 3.0
M) was stirred at 75 °C for 12 hours, then cooled and poured
over ice. Water was added, and the solution was treated with
aqueous ammonium hydroxide then extracted with chloroform.
The extract was dried and filtered, and then concentrated. Flash
chromatography on silica gel eluted by ethyl acetate:hexane
(1:10) yielded a white solid, 0.15 g (96%), tlc Rf = 0.46, ethyl
HRMS: C13H8 H5N4 (M+ +H) requires m/z 230.1454; found
2
230.1446.
2-Amino-6-phenylpyridine (27). The solvent was decanted
off sodium amide in xylenes, leaving a semidry solid (11.7 g,
ca. 0.30 mol), which was combined with phenylpyridine 26
(9.71 g, 62.5 mmol) in N,N-dimethylaniline (30 mL) and
stirred at 170 °C for 4 hours under a slow flow of nitrogen.
The cooled mixture was treated with ice followed by aqueous
potassium hydroxide, and extracted with diethyl ether. The
extract was dried and filtered, then concentrated. Flash
1
acetate:hexane (1:10); mp = 97-98 °C; H nmr (chloroform-d) ꢀ
7.91 (dd, J = 1.8, 8.4 Hz, 2H), 7.70 (d, J = 7.9 Hz, 1H), 7.28-
7.47 (app. m, 3H), 6.99 (d, J = 7.9 Hz, 1H), 4.97 (br. s, NH2);
13C nmr (chloroform-d) ꢀ 155.5, 155.3, 141.1, 129.2, 128.9,
126.9, 112.3, 103.2; ir: 3482, 3316, 3176, 3056, 1963, 1778,
1628, 1565, 1457, 1322, 1276, 1151, 1053, 1024, 995, 804, 765
cm-1. HRMS: C11H10N2Br (M+ +H, 79Br) requires m/z 249.0020;
found 249.0020.
chromatography on silica gel eluted by
a
ethyl
acetate:hexane:methanol (1:5:0.15) yielded a tan solid, 8.62 g
(81%), tlc Rf = 0.33, ethyl acetate:hexane (1:5), mp = 64-65
1
°C; H nmr (dimethyl sulfoxide-d6) ꢀ 7.95 (m, J = 1.4, 8.0 Hz,
2H), 7.31-7.47 (m, 4H), 7.03 (dd, J = 0.8, 7.4 Hz, 1H), 6.43
(dd, J = 0.7, 8.0 Hz, 1H), 5.94 (br. s, NH2); 13C nmr (dimethyl
sulfoxide-d6) ꢀ 159.6, 154.4, 139.4, 138.0, 128.5, 128.4, 126.3,
108.4, 107.1; ir: 3469, 3383, 3330, 3188, 3055, 1955, 1613,
1572, 1466, 1449, 1344, 1274, 984, 805, 762 cm-1; ms: m/z
172(12), 171(100, M+ +H).
2-Amino-3-methylamino-6-phenylpyridine (32). Bromo-
pyridine 31 (0.78 g, 3.13 mmol) was combined with cupric
sulfate (0.31 g, 1.24 mmol) plus aqueous methylamine (35
mL, 40 wt%) in a stainless steel Parr vessel. The mixture
was held at 200 °C for 60 hours, cooled, poured into water,
then extracted with chloroform. The extract was dried and
filtered, then concentrated to yield an impure blue-green
solid that was carried on without purification, 0.74 g (ca.
80%), tlc Rf = 0.52, methanol:chloroform (1:20); ms: m/z
201(12), 200(100, M+ +H).
2-Pivalylamido-6-phenylpyridine (28). Aminopyridine 27
(0.78 g, 4.58 mmol) was combined with dry tetrahydrofuran (20
mL) and triethylamine (6.5 mL) and cooled to 0 °C. The solution
was treated with pivaloyl chloride (2.14 g, 17.7 mmol) at 0 °C,
then stirred at 20 °C for 5 hours. Methylene chloride was added,
and the solution was diluted with aqueous sodium hydroxide
then extracted with chloroform. The extract was dried and
filtered, and then concentrated. Flash chromatography on silica
gel eluted by ethyl acetate:hexane (1:5) provided a white solid,
0.65 g (56%), tlc Rf = 0.65, ethyl acetate:hexane (1:5); mp = 93-
1-Methyl-5-phenyl-2-aminoimidazo[4,5-b]pyridine; 1-Me-
5-Ph-IP (6). Impure diaminopyridine 32 (0.81 g, ca. 3.4 mmol)
was combined with cyanogen bromide( 0.34 g3.2 mmol) in
ethanol (40 mL) and stirred at 32 °C for 8 hours. Additional
cyanogen bromide (0.46 g, 4.3 mmol) was added, and the
mixture was stirred for 12 hours then concentrated. Flash
chromatography on silica gel eluted by methanol:chloroform
(1:10) yielded a brown solid, 0.15 g (19%), tlc Rf = 0.32,
methanol:chloroform (1:10); mp = 272-273 °C (dec) after
1
94 °C; H nmr (dimethyl sulfoxide-d6) ꢀ 9.58 (br. s, NH), 8.11
(app. dd, J = 1.6, 8.2 Hz, 2H), 7.99 (dd, J = 0.6, 8.2 Hz, 1H),
7.84 (app. t, J = 7.9 Hz, 1H), 7.65 (dd, J = 0.9, 7.6 Hz, 1H),
7.39-7.55 (br. m, 3H), 1.27 (s, tert-butyl); 13C nmr (dimethyl
sulfoxide-d6) ꢀ 177.1, 154.5, 151.9, 138.9, 138.1, 129.0, 128.5,
126.6, 115.5, 112.9, 39.3, 26.9; ir: 3510, 3285, 2967, 2874,
1952, 1763, 1664, 1573, 1569, 1441, 1382, 1291, 1158, 933, 759
cm-1. HRMS: C16H19N2O2 (M+ +H) requires m/z 255.1482; found
225.1482.
1
trituration from ethanol; H nmr (dimethyl sulfoxide-d6) ꢀ 8.01
(d, J = 8.4 Hz, 2H), 7.39-7.50 (app. m, 4H), 7.30 (app. tt, J =
0.6, 1.8 Hz, 1H), 6.92 (br. s, NH2), 3.52 (s, N-CH3); 13C nmr
(dimethyl sulfoxide-d6) ꢀ 157.9, 156.6, 148.0, 140.4, 128.4,
127.3, 127.1, 126.0, 114.0, 110.4, 28.4; ir: 3443, 3314, 3125,
1664, 1545, 1477, 1422, 1274, 1238, 1147, 1101, 936, 818, 764
cm-1. HRMS: C13H13N4 (M+ +H) requires m/z 225.1140; found
225.1135.
2-Pivalylamido-3-bromo-6-phenylpyridine (30).Pivaloyl-
amide 28 (0.12 g, 0.47 mmol) was combined with tetramethyl

Nov-Dec 2008
Synthesis and Mutagenic Potency of Structural Isomers
1649
[10] (a) Zecchini, G.P.; Torrini, I.; Paradisi, M.P. J. Heterocyclic
Chem. 1985, 22, 313; (b) Zecchini, G.P.; I. Torrini, I.; Paradisi, M.P. J.
Heterocyclic Chem. 1985, 22, 1061.
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[11] bis(6-Phenylpyridin-2-yl)amine (C22H17N3); tlc Rf = 0.31,
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sulfoxide-d6) ꢀ 9.77 (br. s, h/2 = 8.2 Hz, NH), 8.07-8.10 (m, 4H), 7.76-
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