Synthesis and potent cytotoxicity of some novel imidazopyridine derivatives against MCF-7 human breast adenocarcinoma cell line

Article abstract of DOI:10.1007/s10593-015-1765-7

[Figure not available: see fulltext.] A series of novel 2-phenyl-3H-imidazo[4,5-b]pyridines and 2-phenyl-3H-imidazo[4,5-c]pyridines and their precursors were synthesized. Their in vitro cytotoxicity against MCF-7 human breast adenocarcinoma cell line has been investigated, and some of the tested compounds have shown high cytotoxic activity against MCF-7 cells. N-Hydroxy-4-(3H-imidazo[4,5-b]pyridin-2-yl)benzenecarboximidamide was the most active compound with IC₅₀ equal to 0.082 μM, which is an activity almost as high as that of a commonly used anticancer drugs docetaxel and imatinib mesylate.

Full text of DOI:10.1007/s10593-015-1765-7

DOI 10.1007/s10593-015-1765-7
Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
Synthesis and potent cytotoxicity of some novel imidazopyridine
derivatives against MCF-7 human breast adenocarcinoma cell line
Mustafa Orhan Püsküllü1, Cigdem Karaaslan2, Filiz Bakar3, Hakan Göker2*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Erciyes University,
38039 Kayseri, Turkey; e-mail: puskullu@erciyes.edu.tr
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University,
06100 Tandogan, Ankara, Turkey; e-mail: goker@ankara.edu.tr
Department of Biochemistry, Faculty of Pharmacy, Ankara University,
06100 Tandogan, Ankara, Turkey; e-mail: flzbakar@yahoo.com
3
Published in Khimiya Geterotsiklicheskikh Soedinenii,
2015, 51(8), 723–733
Submitted July 30, 2015
Accepted August 4, 2015
R1
R3
R1
R3
N
NH2
HO
+
Y
Y
N
NHR2
X
NaO3S
X
R2
X = CH, Y = N; X = N, Y = CH
A series of novel 2-phenyl-3H-imidazo[4,5-b]pyridines and 2-phenyl-3H-imidazo[4,5-c]pyridines and their precursors were synthesized.
Their in vitro cytotoxicity against MCF-7 human breast adenocarcinoma cell line has been investigated, and some of the tested com-
pounds have shown high cytotoxic activity against MCF-7 cells. N-Hydroxy-4-(3H-imidazo[4,5-b]pyridin-2-yl)benzenecarboximidamide
was the most active compound with IC50 equal to 0.082 μM, which is an activity almost as high as that of a commonly used anticancer
drugs docetaxel and imatinib mesylate.
Keywords: carboxamidine, 3H-imidazo[4,5-b]pyridine , 3H-imidazo[4,5-c]pyridine, cytotoxicity, MCF-7 cell line, steric hindrance.
Cancer is the biggest health issue in the world. Breast
carcinoma is the second most common cancer in women
and remains as the major cause of malignancy-associated
deaths in humans. In the last 60 years, the development of
novel chemotherapeutic agents has significantly prog-
ressed, however, the treatment of most types of solid
tumors (e.g., breast and ovarian) is still a problem.1 In
addition, resistance against many existing anticancer drugs
is developing, therefore, development of new potent, safe,
and selective antitumor agents is strongly needed.
The imidazopyridine nucleus is considered as a bio-
isostere to the purine nucleus and easily interacts with large
molecules such as DNA, RNA, or some proteins.2 Hence
imidazopyridines exhibit promising cytotoxic activity by
inhibiting some kinase enzymes. As it is well known,
kinases are involved in cellular signaling pathways that
control various cellular functions, including cell division,
growth, metabolism, differentiation, and survival, through
reversible phosphorylation of the hydroxyl groups of
tyrosine residues in proteins.3 Cyclin-dependent kinases
(CDKs) are the major regulators of the cell cycle and trans-
cription. Inhibition of this enzyme by a series of imidazo-
[4,5-b]pyridine and 4-heteroarylpyrimidine derivatives as
anticancer agents has been reported.4 Bavetsias et al.5–8
reported the identification of the novel imidazo[4,5-b]-
pyridine derivatives as potent inhibitors of some Aurora
kinases. Aurora proteins A, B, and C, a small family of
serine/threonine kinases, play distinct role in the regulation
of mitosis. In recent years, these proteins have been
actively pursued as targets for the discovery of new
anticancer chemotherapeutics. As a result, several simple
inhibitors of Aurora kinases identified and some of them
have reached clinical evaluation.5–8 Furthermore, very
recently some imidazopyridine derivatives were published
as selective potent inhibitors of TANK-binding kinase
inhibitors (TBK1).9,10 In another study, the discovery of
new imidazo[4,5-b]pyridine derivatives, potent, highly
selective inhibitors of CDK4/6 kinases, was reported,11 in
addition potent c-Met kinase inhibitory activity was also
reported for new analog of 3H-imidazo[4,5-b]pyridine12
(Fig. 1).
Prompted by the findings from the studies mentioned
above, we set out to develop and synthesize novel imid-
azo[4,5-b]- or imidazo[4,5-c]pyridine derivatives and
investigate their inhibitory activity against the MCF-7 cell
line. It is well known that carboxamides, carboxamidines,
and combinations of both are present in the structure of a
variety of antitumor agents, such as DNA minor groove-
0009-3122/15/5108-0723©2015 Springer Science+Business Media New York
723

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
Me
Cl
O
N
O
N
S
N
H
N
N
N
N
N
Me
N
Cl
Me
Cl
N
N
Br
N
N
N
N
N
NH
N
H
Me
N
H
N
N
N
H
Me
N
Aurora kinase inhibitor5
Aurora kinase inhibitor6
FLT3 kinase and Aurora kinase inhibitor7
H
N
Me
O
Me
HN
N
Me
Br
N
S
O
N
N
N
H
O
Me
N
H
N
TBK1/IKK kinase inhibitor9
TBK1/IKK kinase inhibitor10
Figure 1. Previously reported imidazo[4,5-b]pyridines possessing anticancer activity.
binding agents netropsin, DAPI, distamycin.13–15 In conti-
nuation16–18 of our efforts in search of potent molecules
exhibiting antimicrobial and anticancer activities, we have
synthesized carboxamidine and carboxamidoxime deriva-
tives of imidazopyridine and tested their anticancer
activity, which we wish report in this paper.
By converting chlorine atom in 2-chloro-3-nitropyridine
(1) and 5-bromo-2-chloro-3-nitropyridine (2) to alkylamino
group in an aromatic nucleophilic substitution reaction, and
the subsequent reduction of the nitro group in compounds 3
and 5, pyridinediamines 4 and 6 were prepared (Scheme 1)
which, like the commercially available compounds 7–10,
served as starting compounds for the synthesis of imidazo-
pyridines 11–26. The latter were obtained by the
condensation of the respective substituted pyridinediamines
4,6–10 with the corresponding Na2S2O5-adducts of various
arylaldehydes in DMF (Scheme 2).19
The attemt to apply the general method to the reaction of
diamine 4 with sodium bisulfite adduct of 2-formyl-
benzenesulfonic acid failed to produce the expected imidazo-
pyridine derivative. Using harsher conditions (130°C, 7 h)
resulted in 2,3-dihydro intermediate 27 and the expected
aromatic product 28 as a mixture having similar peak areas
in HPLC-MS chromatogram, but neither of the two com-
pounds could be isolated (Scheme 3). Probably, because of
the strong steric congestion between SO3H and benzyl
groups, the conversion of intermediate 27 has not been
complete. Hence, in order to obtain only compound 27, the
same reaction was carried out under milder conditions (in
EtOH, at 75°C). This time, the mixture of three compounds
Scheme 1
R1
R1
R1
R1
R1
NO2
Cl
NH2
NO2
NO2
NH2
BnNH2
DMF
H2, Pd/C
EtOH
n-BuNH2
Zn, HCl
H2O, EtOH
97°C, 1 h
DMF
N
N
N
1, 2
N
N
NHBn
NHBn
NHBu-n
NHBu
-n
110°C, 5 h
100°C, 3 h
4
3
5
6
1, 3, 4 R1 = H; 2, 5, 6 R1 = Br
Scheme 2
7
R1
1
R3
R1
R3
6
N
NH2
NHR2
HO
2
3
+
Y
Y
N
5
DMF, 110°C, 4 h
X
NaO3S
X
R2
4
11–26
4, 6–10
X
N
Y
R1 R2 R3
X
N
Y
R1 R2
R3
X
Y
R1 R2
R3
X
Y
R1 R2
23
CH
N
CH
CH
H
H
H
H
H
H
H
4-CN
4-CN
3-CN
11
CH
N
CH
N
CH
CH Cl
H
H
H
H
H
H
H
H
H
H
H
2-COOH
2-COOH
3-COOH
3-COOH
4-COOH
4-COOH
17
18
19
20
21
22
N
N
N
N
N
N
CH
CH
CH
H
H
H
Bn 3-COOH
Bn 2-COOH
24 CH
12 CH
7
N
CH
N
CH Cl
CH Br
H
H
H
H
H
H
25
26
N
N
H
3-SO3H
2-SO3H
8
13
N
CH
N
Bn 4-CN
CH Br
CH
CH Br n-Bu 2-SO3H
H
9
14 CH
N
10
H
Bn 3-SO3H
15
16
N
N
724

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
Scheme 3
NaO3S
HO
NaO3S
N
SO3Na
NaO3S
N
4'
H
N
6
3'
2'
5'
6'
5
NaO3S
2
NH
N
N
+
4
+
4
N
3
N
N
EtOH, 75°C, 30 h
NaO3S
OHC
27 (Na salt) (42%)*
28 (Na salt) (19%)*
29 (13%)*
HCl, EtOH, H2O
EtOH
50°C, 24 h
O
SO3H
HO3S
29
7
H
N
3
N
6'
N
6
2
5'
O
5
N
N
3'
N
4'
EtOH, 75°C, 6 h
* LC-MS data.
27
28
27 and 28 (both in form of sodium salt), and an open-chain
Schiff base 29 has been identified chromatographically.
Since compound 27 was the major product, it has been
isolated by crystallization from the reaction medium; the
other minor products could not be isolated. Therefore,
compound 29 was prepared separately by the reaction of
compound 4 and sodium 2-formylbenzenesulfonate only
for the purpose of identification. Compound 27 was con-
verted to compound 28 by using 1,4-benzoquinone as the
aromatizing agent.
When we have attempted to carry out the reaction
between compound 3020 and sodium 2-formylbenzene-
sulfonate or its bisulfite adduct (Scheme 4), only com-
pound 31 and a trace amount of compound 32 have been
observed, but not the corresponding Schiff base. As an
mechanistic alternative to the involvement of Schiff base in
the metabisulfite-mediated reaction, Ridley et al.19 sugges-
ted that it proceeds by way of formation of an α-amino-
sulfonic acid derivative I (Scheme 5), the product normally
formed in the condensation of amines and aldehyde
Scheme 4
NaO3S
SO3Na
SO3Na
N
H
N
NH2
NH
N
N
N
N
X
N
+
EtOH, 70°C, 30 h
30
31 (Na salt)
HCl, H2O, EtOH
SO3H
32 (Na salt)
(traces)
SO3H
H
N
N
N
N
N
PhNO2
X = CHO, CH(OH)SO3Na
N
200°C, 4 h
31
32
Scheme 5
HO
H
N
R'
H
N
NH2
SO3Na
R'
N
H
NaO3S
R'
N
R
R'
NH
R
N
R
NH
R
I
II
725

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
bisulfite adducts, followed by the intramolecular nucleo-
philic displacement of sodium sulfite with the closure of
imidazole cycle giving benzimidazoline II. Compound 31
was isolated in good yield, the corrresponding monoimine
(Schiff base) has not detected, and a trace amount of
pyridoimidazole derivative 32 (as sodium salt) has been
identified in LC-MS chromatogram. In order to obtain
compound 32 preparatively, nitrobenzene has been used as
the aromatizing agent instead of 1,4-benzoquinone, since
some unexpected impurities have been occured using
1,4-benzoquinone. Our results suggest that both
mechanisms described above could be possible depending
on the properties of the starting pyridinediamines.
In compounds 18, 22, 28, and 32, the steric hindrance
between the benzylic methylene group and the COOH and
SO3H groups of the o-substituted 2-phenyl moiety was
1
confirmed by their H NMR spectra. In the spectrum of
compound 18 (in CD3OD at room temperature), benzylic
protons are observed as two overlapping singlets in contrast
to expected as one singlet (Fig. 2a). However, when the
temperature was raised to 42°C the two signals coalesced
into one (Fig. 2b). In the same solvent, benzylic protons of
compound 28 have been seen separately as broad singlets
at 5.21 and 5.77 ppm (Fig. 3), and these peaks did not
merge even at 40°C. Finally, in the spectrum of 2,3-di-
hydro derivatives 27 and 31 these protons were observed as
two sharp doublets (Fig. 4). (This shows us that SO3H
group causes greater steric hindrance than COOH group.)
The analogous reaction of o-phenylenediamine with
aldehydes, yielding benzimidazoles, has been investigated
using NMR spectroscopy.21 It has been established that the
reaction begins with the formation of monoimines (isolated
in condensations with some aromatic aldehydes) which are
cyclized to the corresponding benzimidazolines, and these
compounds are converted into 2-substituted benzimidazo-
les. The structures of monoimines and benzimidazolines
were proved unequivocally by the presence of a signal for
the resonance of the azomethine and the 2-CH proton of
appropriate intensity at 8.0–8.5 and 5.0–6.0 ppm, respec-
tively.21 The corresponding proton signals in the isomeric
structures 29 and 27 have been observed at 9.60 and 7.69 ppm,
respectively. However, the chemical shift value of the
2-CH in benzimidazolines is highly dependent on the NMR
solvent used, e.g., the 2-CH signal of 2-phenylbenzim-
1
Figure 2. A fragment of the H NMR spectrum of compound 18
at (a) rt and (b) 42°C.
Figure 3. A fragment of the 1H NMR spectrum of compound 28.
21
idazoline has been observed at 5.6 ppm in DMSO-d6 and
at 8.4 ppm in CCl4.22 In another study, monoimines and
benzimidazolines have been shown exitisting in a tauto-
meric equilibrium23 or they have been postulated as
sequential intermediates in the synthesis of benzimidazoles
from hydrazones.24
No added oxidizer was needed to obtain compounds
without steric congestion (11–26), since the aromatic
cyclization (rapid dehydrogenation) is taking place
spontaneously (the oxidizer probably being O2 from air or
bisulfite anion19) with sodium metabisulfite adducts of
aldehydes. However, under same conditions it was not
possible to obtain compounds 28 and 32. This means that,
the more bulky SO3H group, but not carboxyl group (in
compound 18), inhibits the last step of the reacion because
Figure 4. A fragment of the 1H NMR spectrum of compound 27.
726

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
Figure 5. The aliphatic part of the 1H NMR spectrum of compound 22 in DMSO-d6.
of its steric effect. However, a similar problem has not been
observed in the synthesis of compound 22, since there is
less steric congestion between SO3H and butyl groups than
using hydroxylamine hydrochloride producing compounds
33–35 (Scheme 6). The cyano group of compounds 23–26
was also converted, using the Pinner method,25 to the
imidate esters which were not purified and characterized
due to their instability, but were instead reacted with
ammonia or isopropylamine to yield the corresponding
carboximidamides 36–39.
1
between SO3H and benzyl groups. Nevertheless, in the H
NMR spectrum of compound 22 (in DMSO-d6), N-methylene
protons of the n-butyl substituent are observed as a multiplet
instead of the expected triplet (Fig. 5).
As a further step of functionalization, the cyano group of
compounds 23–25 was converted to amidoxime group
The antiproliferative activity of compounds 11–22, 28,
33–39 was assessed against MCF-7 human breast cancer
Scheme 6
NH2OHHCl
NEt(i-Pr)2
N
N
NH
NH
1) HCl, EtOH, 6 days
23
NH
2 2HCl
N OH
H
2) NH3, EtOH, 60°C
3) HCl, EtOH
N
H
EtOH, , 6 h
N
N
N
33
34
36
NH2OHHCl
NEt(i-Pr)2
N
N
NH
NH
1) HCl, EtOH, 6 days
Me
Me
24
25
N
N
N OH
H
2HCl
N
2) N(i-Pr)2, EtOH, 60°C
EtOH, , 6 h
N
H
N
H
H
37
6'
5'
N
NH2OHHCl
NEt(i-Pr)2
N
N
4'
1) HCl, EtOH, 6 days
N
H
N
N
2'
NH2HCl
2) NH3, EtOH, 60°C
3) HCl, EtOH
EtOH, , 6 h
NH
2 2HCl
HO N
H
35
HN
38
N
N
NH
CN
Me
Me
N
H
1) HCl, EtOH, 6 days
N
N
N
N
2HCl
2) N(i-Pr)2, EtOH, 60°C
3) HCl, EtOH
26
39
727

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
cells using the so-called 48h-MTT cytotoxicity assay,26,27
Compounds containing N-hydroxycarboximidamide group
at the phenyl ring showed the highest in vitro cytotoxic
activity against human breast adenocarcinoma MCF-7 cell
line. This result makes these compounds worthy of a
further investigation.
and the results are summarized in Table 1. Among the first
series of compounds 11–22, 28, only compound 28 showed
a limited activity with the IC50 value of 0.907 µM towards
the MCF-7 cell line. To investigate the effect of carbox-
imidamide and N-hydroxycarboximidamide groups, we
tested a series of imidazopyridines 33–39. It is noteworthy
that almost all of them exhibited potent cytotoxic activity
against the MCF-7 cell line. Among them, compound 33
showed the highest activity with the IC50 value of 0.082 µM
(Table 1). This means that N-hydroxycarboximidamide
group presents the best inhibitory profile. Similarly,
compounds 34 (IC50 0.097 µM) and 35 (IC50 0.100 µM)
containing N-hydroxycarboximidamide group displayed
potent cytotoxic activity. N-Alkyl substitution of carbox-
amidine group and imidazole moiety, caused a dramatic
reduction of the cytotoxic effect (compounds 37 and 39). In
the light of the observed promising antiproliferative
activity, the mechanism of action of compounds 33–35 will
be evaluated later with some biochemical assays.
Experimental
1H and 13C NMR spectra (400 and 100 MHz, respecti-
vely) were recorded on a Varian Mercury 400 FT spectro-
meter, internal standard – TMS. The NMR signals were
assigned with COSY (compound 32), HSQC (compounds
17, 22, 26, 28, 32–36, 38–39) and HMBC (compounds 34,
36) methods. To eliminate the tautomeric effect of imid-
1
azole ring in the H NMR spectra, some of the compounds
were dissolved in DMSO-d6, a tiny amount of dry NaH and
2–3 drops of D2O were added into the NMR tube, stirred
well, and observed visually for a while. If there was any
turbidity remaining, the solution was centrifuged. Mass
spectra were recorded on a Waters Micromass ZQ
spectrometer connected with a Waters Alliance HPLC
instrument, using a C-18 column and a mixture MeCN–
MeOH–0.1 % HCOOH in MeCN–H2O, 65:10:10:15, as
eluent; ionization method – ESI(+) or ESI(–). The
calculated element contents were adjusted for the solvents
In summary, a new series of 2-phenylimidazopyridines
have been successfully synthesized starting from 2,3- or
3,4-pyridinediamines, and sodium bisulphite and aromatic
aldehydes. The structure of all new compounds has been
1
1
confirmed by H and 13C NMR, mass spectroscopy, and
visible in H NMR and water, as the compounds were
elemental analysis. The steric interaction between the o-sulfo
or o-carboxyl group with N-alkyl groups was discussed.
hygroscopic. Elemental analysis was performed on a Leco
CHNS-932 instrument. Melting points were measured on
an Büchi B-540 capillary melting point apparatus and were
not corrected. Commercially available starting materials,
reagents, and dry solvents were purchased from Aldrich-
Sigma and Merck. Compound 30 was prepared according
to a published procedure.20
Table 1. Cytotoxicity expressed as IC50 of compounds 11–22,
28, 33–39 in the 48h-MTT assay
Com-
pound
X
Y
R1
R2
R3
IC50,* μM
N-Benzyl-3-nitropyridin-2-amine (3). A mixture of com-
pound 1 (1.58 g, 10 mmol) and benzylamine (1.07 g, 10 mmol)
in DMF (1 ml) was heated for 5 h at 110°C. The mixture
was allowed to cool, and water was added. The resultant
yellow precipitate was filtered off, washed with water, and
crystallized from ethanol. Yield 1.35 g (59 %). Mp 80–81°C
(mp 78°C (EtOH)28).
N
CH
N
CH
N
H
H
H
H
H
Cl
H
H
H
Br
H
Br
H
H
H
H
H
H
H
H
H
H
2-COOH
2-COOH
3.395
3.492
2.998
0.212
3.011
1.416
3.996
2.604
0.277
0.401
6.221
3.498
0.907
0.082
0.097
0.100
1.103
11
12
CH
N
H
3-COOH
13
CH
N
H
3-COOH
14
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
H
4-COOH
15
N
H
4-COOH
16
N2-Benzyl-2,3-pyridinediamine (4). Compound 3 (0.65 g,
3.26 mmol) in ethanol (30 ml) was subjected to hydro-
genation using 40 psi of H2 and 10 % Pd/C until the uptake
of H2 had ceased. The catalyst was filtered off on a bed of
Celite and washed with ethanol. The filtrate was
concentrated in vacuum. The powder-like residue was used
for the subsequent steps without crystallization. Yield 0.58 g
(90 %). Mp 86–88°C (mp 88°C28).
N
Bn
Bn
H
3-COOH
17
N
2-COOH
18
N
3-SO3H
19
N
H
2-SO3H
20
N
Bn
n-Bu
Bn
H
3-SO3H
21
N
2-SO3H
22
N
2-SO3H
28
5-Bromo-N-butyl-3-nitropyridin-2-amine (5). A mixture
of compound 2 (1.19 g, 5.02 mmol) and n-butylamine
(0.74 g, 10 mmol) in DMF (1 ml) was heated for 3 h at 100°C.
The mixture was allowed to cool, and water was added.
The resultant yellow precipitate was filtered off, washed
with water, and crystallized from ethanol. Yield 0.76 g (55 %).
N
4-C(=NH)NHOH
4-C(=NH)NHOH
3-C(=NH)NHOH
4-C(=NH)NH2
33
CH
N
H
34
CH
CH
N
H
35
N
H
36
CH
N
H
4-C(=NH)NHCH(CH3)2 2.611
3-C(=NH)NH2 0.119
37
CH
CH
H
1
38
Mp 63–64°C. H NMR spectrum (CDCl3), δ, ppm (J, Hz):
N
Bn 4-C(=NH)NHCH(CH3)2 0.430
39
0.96 (3H, t, J = 7.2, CH3); 1.40–1.46 (2H, m, CH2CH3);
1.62–1.67 (2H, m, CH2CH2CH3); 3.57–3.62 (2H, m,
NHCH2); 8.20 (1H, br. s, NH); 8.41 (1H, d, J = 2.4, H-6);
8.51 (1H, d, J = 2.4, H-4). 13C NMR spectrum (CDCl3),
δ, ppm: 156.7; 151.5; 136.9; 128.1; 104.3; 41.4; 31.6; 20.4;
Docetaxel
0.013
0.049
Imatinib mesylate
* IC50 values were calculated from the cell growth inhibition percentages
obtained with ten different concentrations.
728

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
14.0. Mass spectrum, m/z (Irel, %): 276 [M(81Br)+H]+ (98), 276
J = 4.8, H-7); 8.26 (1H, d, J = 7.6, H-4'); 8.77 (1H, s, H-2').
13C NMR spectrum (DMSO-d6 + NaH + D2O), δ, ppm: 171.1;
159.5; 157.8; 141.6; 140.5; 136.5; 133.5; 130.4; 128.4; 128.3;
128.2; 123.2; 116.1. Mass spectrum, m/z (Irel, %): 240 [M+H]+
(100). Found, %: C 64.40; H 3.78; N 17.28. C13H9N3O2.
Calculated (with 1.9% H2O), %: C 64.06; H 3.92; N 17.24.
3-(3H-Imidazo[4,5-c]pyridin-2-yl)benzoic acid (14).
Yield 0.172 g (72%). Mp >370°C (EtOH). 1H NMR
spectrum (DMSO-d6), δ, ppm (J, Hz): 7.60 (1H, br. d,
J = 4.0, H-7); 7.71 (1H, t, J = 7.2, H-5'); 8.09 (1H, d, J = 7.6,
H-6'); 8.31 (1H, d, J = 6.0, H-6); 8.46 (1H, d, J = 8.0, H-4');
8.82 (1H, s, H-2'); 8.96 (1H, s, H-4). Mass spectrum, m/z
(Irel, %): 240 [M+H]+ (100). Found, %: C 61.65; H 4.14;
N 16.58. C13H9N3O2. Calculated (with 5.3% H2O), %: C 61.77;
H 4.19; N 16.63.
[M(79Br)+H]+ (100). Found, %: C 39.17; H 4.37; N 15.32.
C9H12BrN3O2.Calculated, %: C 39.43; H 4.41; N 15.33.
5-Bromo-N2-(n-butyl)pyridine-2,3-diamine (6). The mixture
of compound 5 (0.6 g, 2.19 mmol) in 25% HCl (15 ml) and
EtOH (10 ml) was stirred vigorously. Zinc dust (1.5 g,
23 mmol) was added to this mixture in several portions at
room temperature. After the addition of zinc dust was
completed, the mixture was heated on a water bath at 97°C
for 1 h. Reaction mixture was cooled and made alkaline
with 10% NaOH solution, then extracted with EtOAc. The
extract was washed with water, dried over anhydrous
Na2SO4, and evaporated. Residue was purified by column
chromatography (eluent n-hexane–AcOEt, 1:3). Yield 0.26 g
1
(49%). Dark-purple powder. H NMR spectrum (CDCl3),
δ, ppm (J, Hz): 0.87 (3H, t, J = 7.2, CH3); 1.31–1.39 (2H,
m, CH2CH3); 1.50–1.58 (2H, m, CH2CH2CH3); 3.20 (2H,
br. s, NH2); 3.30 (2H, t, J = 7.6, NHCH2); 4.04 (1H, br. s,
NH); 6.86 (1H, d, J = 2.4, H-6); 7.68 (1H, d, J = 2.4, H-4).
13C NMR spectrum (CDCl3), δ, ppm: 149.4; 139.2; 129.7;
124.0; 107.4; 41.9; 32.0; 20.5; 14.2. Mass spectrum, m/z
(Irel, %): 246 [M(81Br)+H]+ (98), 244 [M(79Br)+H]+ (100).
Synthesis of compounds 11–26 (General method). An
aromatic aldehyde (6.0 mmol) was dissolved in EtOH (20 ml),
and sodium metabisulfite (0.64 g, 3.37 mmol) in H2O
(3 ml) was added in portions. The reaction mixture was
stirred vigorously and more EtOH was added. The mixture
was kept in a refrigerator for several hours. The precipitate
was filtered and dried. The mixture of the obtained sodium
aryl(hydroxy)methanesulfonate (1.0 mmol) and pyridine-
diamine 4, 6–10 (1.0 mmol) in DMF (1–2 ml) was heated
at 110°C for 4 h. The reaction mixture was cooled, poured
into water, and the crude product was filtered off if it was
solid, or, alternatively, it was extracted with chloroform, dried
over anhydrous Na2SO4, evaporated, then crystallized from
appropriate solvent, unless stated otherwise.
4-(3H-Imidazo[4,5-b]pyridin-2-yl)benzoic acid (15).
Yield 0.158 g (66%). Mp >370°C (EtOH). 1H NMR
spectrum (DMSO-d6 + D2O), δ, ppm (J, Hz): 7.28 (1H, dd,
J = 8.4, J = 4.8, H-6); 8.02 (1H, br. d, J = 7.6, H-7); 8.08
(2H, dd, J = 6.8, J = 1.6, H-2',6'); 8.26 (2H, dd, J = 6.8,
J = 1.6, H-3',5'); 8.34 (1H, br. dd, J = 4.4, J = 0.8, H-5).
Mass spectrum, m/z (Irel, %): 240 [M+H]+ (100). Found, %:
C 60.14; H 4.48; N 16.01. C13H9N3O2. Calculated (with
7.7% H2O), %: C 60.27; H 4.36; N 16.22.
4-(6-Chloro-3H-imidazo[4,5-b]pyridin-2-yl)benzoic acid
(16). Yield 0.126 g (46%). Mp >370°C (DMF–EtOH,
5:95). 1H NMR spectrum (DMSO-d6 + NaH + D2O),
δ, ppm (J, Hz): 7.68 (1H, d, J = 2.8, H-7); 7.87 (2H, d, J = 8.0,
H-2',6'); 7.90 (1H, d, J = 2.8, H-5); 8.16 (2H, d, J = 8.4,
H-3',5'). 13C NMR spectrum (DMSO-d6 + NaH + D2O),
δ, ppm: 171.2; 164.5; 159.7; 140.1; 139.7; 137.7; 137.3;
129.6; 126.4; 121.6; 120.7. Mass spectrum, m/z (Irel, %):
272 [M–H]+ (100), 274 [M–H+2]+ (33). Found, %: C 56.63;
H 2.82; N 15.44. C13H8ClN3O2. Calculated (with 0.7%
H2O), %: C 56.67; H 3.00; N 15.25.
3-(3-Benzyl-3H-imidazo[4,5-b]pyridin-2-yl)benzoic acid
(17). Yield 0.151 g (46%). Mp 218–220°C (2-PrOH).
1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 5.62 (2H,
s, PhCH2); 6.97–6.99 (2H, m, H-3,5 Ph); 7.18–7.25 (3H, m,
H-2,4,6 Ph); 7.36 (1H, dd, J = 8.4, J = 5.2, H-6); 7.63 (1H,
t, J = 8.0, H-5'); 7.98 (1H, d, J = 8.0, H-6'); 8.07 (1H, d, J = 8.0,
H-4'); 8.17 (1H, dd, J = 8.0, J = 1.6, H-7); 8.30 (1H, s,
H-2'); 8.39 (1H, dd, J = 5.2, J = 1.2, H-5). 13C NMR
spectrum (DMSO-d6), δ, ppm: 167.3 (C=O); 153.7; 149.3;
144.9 (C-5); 137.5; 135.2; 133.5 (C-6'); 132.1; 131.5
(C-4'); 130.8; 130.4 (C-2'); 130.0 (C-5'); 129.4 (C-2,6 Ph);
128.1 (C-4 Ph); 127.9 (C-7); 126.9 (C-3,5 Ph); 119.7 (C-6);
46.7(CH2). Mass spectrum, m/z (Irel, %): 330 [M+H]+
(100). Found, %: C 70.30; H 4.92; N 11.90. C20H15N3O2.
Calculated (with 2.7% H2O and 4.4% 2-PrOH), %:
C 70.52; H 5.13; N 11.89.
2-(3H-Imidazo[4,5-b]pyridin-2-yl)benzoic acid (11).
Yield 0.065 g (27%). Mp 269–270°C (DMF–EtOH, 5:95).
1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 7.23 (1H,
dd, J = 8.0, J = 4.8, H-6); 7.62 (1H, td, J = 7.6, J = 1.6,
H-5'); 7.68 (1H, td, J = 7.6, J = 1.2, H-4'); 7.77 (1H, dd,
J = 7.6, J = 1.2, H-6'); 7.83 (1H, dd, J = 7.6, J = 1.2, H-3');
7.97 (1H, dd, J = 8.0, J = 1.6, H-7); 8.32 (1H, dd, J = 4.8,
J = 1.6, H-5). Mass spectrum, m/z (Irel, %): 240 [M+H]+
(100). Found, %: C 63.34; H 4.22; N 16.56. C13H9N3O2.
Calculated (with 3.6% H2O), %: C 62.89; H 4.06; N 16.92.
2-(3H-Imidazo[4,5-c]pyridin-2-yl)benzoic acid (12).
Yield 0.057 g (24%). Mp 271–272°C (DMF–EtOH, 5:95).
1H NMR spectrum (CDCl3), δ, ppm (J, Hz): 7.67–7.69 (2H,
m, H-4',5'); 7.80 (1H, d, J = 5.6, H-7); 7.88–7.91 (1H, m,
H-6'); 8.04–8.06 (1H, m, H-3'); 8.38 (1H, d, J = 6.0, H-6);
9.01 (1H, s, H-4). Mass spectrum, m/z (Irel, %): 240 [M+H]+
(100). Found, %: C 59.87; H 4.70; N 16.44. C13H9N3O2.
Calculated (with 7.7% H2O), %: C 60.27; H 4.36; N 16.22.
3-(3H-Imidazo[4,5-b]pyridin-2-yl)benzoic acid (13).
2-(3-Benzyl-1H-imidazo[4,5-b]pyridin-2-yl)benzoic acid
(18). Purified by column chromatography, eluent CH2Cl2–
MeOH–AcOH, 50:10:0.2. Yield 0.072 g (22%). Mp 226–
1
228°C. H NMR spectrum (CD3OD), δ, ppm (J, Hz): 4.92
1
Yield 0.187 g (78%). Mp >370°C (EtOH). H NMR spect-
and 4.93 (2H total, 2s, PhCH2); 6.89–6.91 (2H, m, H-3,5
Ph); 7.11–7.16 (3H, m, H-2,4,6 Ph); 7.22 (1H, d, J = 7.2,
H-6'); 7.37 (1H, dd, J = 8.0, J = 4.8, H-6); 7.56 (1H, td,
J = 7.2, J = 0.8, H-5'); 7.66 (1H, td, J = 7.6, J = 0.8, H-4');
rum (DMSO-d6 + NaH + D2O), δ, ppm (J, Hz): 7.01–7.04
(1H, m, H-6); 7.42 (1H, t, J = 7.6, H-5'); 7.87 (1H, d,
J = 8.0, H-5); 7.94 (1H, d, J = 7.6, H-6'); 8.14 (1H, d,
729

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
8.07 (1H, dd, J = 8.0, J = 1.6, H-7); 8.18 (1H, d, J = 8.0,
112.9; 43.3 (NCH2); 31.2 (CH2CH2CH3); 19.9 (CH2CH3);
13.9 (CH3). Mass spectrum, m/z (Irel, %): 410 [M(81Br)–H]+
(99), 408 [M(79Br)–H]+ (100). Found, %: C 42.55;
H 3.99; N 9.50; S 6.76. C16H16BrN3O3S. Calculated (with
7.8% H2O and 3.2% AcOH), %: C 42.9; H 4.58; N 9.11;
S 6.95.
H-3'); 8.41 (1H, dd, J = 4.8, J = 1.2, H-5). 13C NMR
spectrum (CD3OD), δ, ppm: 167.3; 115.6; 147.6; 144.0;
136.3; 134.3; 132.1; 131.5 (2C); 130.8 (2C); 130.6; 128.3;
127.6; 127.3; 126.8; 118.8; 46.3. Mass spectrum, m/z (Irel, %):
330 [M+H]+ (100). Found, %: C 71.47; H 4.44; N 12.09.
C20H15N3O2. Calculated (with 2.1% H2O), %: C 71.37;
H 4.73; N 12.48.
4-(3H-Imidazo[4,5-b]pyridin-2-yl)benzonitrile (23).
Yield 0.105 g (48%). Mp >370°C. (MeOH). 1H NMR
spectrum (DMSO-d6 + NaH + D2O), δ, ppm (J, Hz): 6.90
(1H, dd, J = 7.6, J = 4.8, H-6); 7.76 (1H, dd, J = 8.0, J = 0.8,
H-7); 7.80 (2H, d, J = 8.0, H-2',6'); 8.05 (1H, d, J = 4.8,
H-5); 8.39 (2H, d, J = 8.0, H-3',5'). Mass spectrum, m/z
(Irel, %): 221 [M+H]+ (100). Found, %: C 69.93; H 3.72;
N 25.24. C13H8N4. Calculated (with 1.6% H2O), %: C 69.75;
H 3.70; N 25.03.
3-(3H-Imidazo[4,5-b]pyridin-2-yl)benzenesulfonic acid
1
(19). Yield 0.58 g (56%). Mp >340°C (2-PrOH). H NMR
spectrum (CD3OD + NaH + D2O), δ, ppm (J, Hz): 6.98
(1H, dd, J = 7.6, J = 5.2, H-6); 7.50 (1H, t, J = 8, H-5'); 7.83
(1H, dt, J = 8, J = 1.6, H-6'); 7.86 (1H, dd, J = 8.0, J = 1.6,
H-7); 8.07 (1H, dd, J = 4.8, J = 1.6, H-5); 8.35 (1H, dt,
J = 8.4, J = 1.2, H-4'); 8.77 (1H, t, J = 1.6, H-2'). 13C NMR
spectrum (CD3OD + NaH + D2O), δ, ppm: 162.7; 159.5;
145.1; 140.0; 139.1; 136.2; 129.0; 128.2; 125.6; 124.7;
123.5; 114.8. Mass spectrum, m/z (Irel, %): 274 [M–H]+
(100). Found, %: C 42.06; H 4.15; N 12.53; S 9.29.
C12H9N3O3S. Calculated (with 19.1% H2O), %: C 42.37;
H 4.80; N 12.36; S 9.42.
4-(3H-Imidazo[4,5-c]pyridin-2-yl)benzonitrile (24). Yield
1
0.095 g (43%). Mp >370°C. (MeOH). H NMR spectrum
(DMSO-d6), δ, ppm (J, Hz): 7.69 (1H, d, J = 5.2, H-7);
8.07 (2H, d, J = 8.0, H-2',6'); 8.35 (1H, d, J = 5.6, H-5);
8.40 (2H, d, J = 8.0, H-3',5'); 9.02 (1H, s, H-4). Mass
spectrum, m/z (Irel, %): 221 [M+H]+ (100). Found, %:
C 66.37; H 3.80; N 23.31. C13H8N4. Calculated (with 6.9%
H2O), %: C 66.04; H 4.18; N 23.69.
2-(6-Bromo-3H-imidazo[4,5-b]pyridin-2-yl)benzene-
sulfonic acid (20). Yield 0.127 g (36%). Mp 257–261°C
1
(2-PrOH). H NMR spectrum (DMSO-d6), δ, ppm (J, Hz):
3-(3H-Imidazo[4,5-b]pyridin-2-yl)benzonitrile (25).
1
7.31–7.39 (2H, m, H-4',5'); 7.61 (1H, dd, J = 7.6, J = 1.6,
H-6'); 7.85 (1H, d, J = 2.4, H-7); 7.93–7.96 (2H, m,
H-3',5). 13C NMR spectrum (DMSO-d6), δ, ppm: 166.7;
159.6; 145.2; 140.6; 138.6; 136.2; 133.0; 129.3; 128.6;
127.5; 124.8; 108.6. Mass spectrum, m/z (Irel, %): 352 [M–H]+
(98), 354 [M–H+2]+ (100). Found, %: C 36.01; H 3.17;
N 10.67; S 8.12. C12H8BrN3O3S. Calculated (with 11.3%
H2O), %: C 36.10; H 3.28; N 10.52; S 8.03.
Yield 0.113 g (51%). Mp 342–344°C. (MeOH). H NMR
spectrum (DMSO-d6), δ, ppm (J, Hz): 7.29 (1H, dd, J = 8.4,
J =4.8, H-6); 7.81 (1H, t, J = 8.0, H-5'); 8.01 (1H, dt,
J = 6.4, J = 1.2, H-6'); 8.08 (1H, br. d, J = 7.6, H-7); 8.4
(1H, br. d, J = 4.0, H-5); 8.54 (1H, dt, J = 6.4, J = 1.2,
H-4'); 8.61 (1H, t, J = 1.2, H-2'); 13.5 (1H, br. s, NH).
Mass spectrum, m/z (Irel, %): 221 [M+H]+ (100). Found, %:
C 70.60; H 3.65; N 25.25. C13H8N4. Calculated, %: C 70.90;
H 3.66; N 25.44.
3-(3-Benzyl-3H-imidazo[4,5-b]pyridin-2-yl)benzene-
sulfonic acid (21). Yield 0.120 g (33%). Mp 304–306°C
4-(3-Benzyl-3H-imidazo[4,5-b]pyridin-2-yl)benzonitrile
(26). Yield 0.197 g (64%). Mp 182–183°C. (MeOH).
1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 5.72 (2H,
s, PhCH2); 6.85–7.05 (2H, m, H-3,5 Ph); 7.20–7.28 (3H, m,
H-2,4,6 Ph); 7.41 (1H, dd, J = 8.4, J = 4.8, H-6); 7.96 (2H,
d, J = 8.4, H-2',6'); 7.99 (2H, d, J = 8.4, H-3',5'); 8.23 (1H,
dd, J = 8.0, J = 1.2, H-7); 8.45 (1H, dd, J = 4.8, J = 1.6,
H-5). 13C NMR spectrum (DMSO-d6), δ, ppm: 152.8;
149.2; 145.4 (C-5); 137.4; 135.2; 134.8; 133.4 (C-3',5');
130.3 (C-2',6'); 129.4 (C-2,6 Ph); 128.3 (C-7); 128.2
(C-4 Ph); 126.9 (C-3,5 Ph); 119.9 (C-6); 118.9; 113.4; 46.8
(CH2). Mass spectrum, m/z (Irel, %): 311 [M+H]+ (100).
Found, %: C 75.33; H 4.30; N 17.44. C20H14N4. Calculated
(with 2.8% H2O), %: C 75.22; H 4.73; N 17.54.
1
(MeOH). H NMR spectrum (CD3OD), δ, ppm (J, Hz):
5.69 (2H, s, PhCH2); 6.99–7.02 (2H, m, H-3,5 Ph); 7.19–
7.22 (3H, m, H-2,4,6 Ph); 7.41 (1H, dd, J = 8.0, J = 4.8,
H-6); 7.56 (1H, t, J = 8.0, H-5'); 7.68 (1H, d, J = 8.0, H-6');
8.02 (1H, dt, J = 8.0, J = 1.2, H-4'); 8.13 (1H, dd, J = 7.6,
J = 1.2, H-7); 8.24–8.25 (1H, m, H-2'); 8.44 (1H, dd,
J = 4.8, J = 1.6, H-5). 13C NMR spectrum (CD3OD),
δ, ppm: 154.5; 148.4; 146.3; 144.5; 136.6; 134.6; 130.8; 129.8;
128.9; 128.6; 127.9; 127.6; 127.2; 126.7 (2C); 119.3; 46.5.
Mass spectrum, m/z (Irel, %): 364 [M–H]+ (98). Found, %:
C 54.27; H 4.87; N 10.38; S 7.72. C19H15N3O3S. Calculated
(with 12.9 % H2O), % : C 54.40; H 5.04 ; N 10.02 ; S 7.64.
2-(6-Bromo-3-butyl-3H-imidazo[4,5-b]pyridin-2-yl)-
benzenesulfonic acid (22). Purified by column chromato-
graphy, eluent CH2Cl2–MeOH–AcOH, 50:10:0.2, and after-
wards recrystallized from MeOH. Yield 0.121 g (30%).
Mp 260–270°C (decomp.). 1H NMR spectrum (DMSO-d6),
δ, ppm (J, Hz): 0.66 (3H, t, J = 7.6, CH3); 1.03–1.06 (2H,
m, CH2CH3) ; 1.48–1.52 (2H m, CH2CH2CH3); 4.07–4.12
(2H, m, NCH2); 7.35 (1H, d, J = 7.2, H-6'); 7.51 (1H, t,
J = 7.2, H-5'); 7.60 (1H, t, J = 7.6, H-4'); 7.92 (1H, d,
J = 8.0, H-3'); 8.29 (1H, d, J = 2.0, H-7); 8.43 (1H, d,
J = 2.0, H-5). 13C NMR spectrum (DMSO-d6), δ, ppm:
157.5; 148.1; 147.3; 143.4 (C-5); 136.5; 131.6 (C-6'); 130.6
(C-4'); 129.2 (C-7); 129.1 (C-5'); 128.5 (C-3'); 127.4;
2-(3-Benzyl-2,3-dihydro-1H-imidazo[4,5-b]pyridin-2-yl)-
benzenesulfonic acid (27). A mixture of compound 4
(0.20 g, 1.0 mmol) and sodium metabisulfite adduct of
sodium 2-formylbenzenesulfonate (0.312 g, 1.00 mmol),
prepared as in the general method above, in EtOH (10 ml)
was stirred at 75°C for 30 h. The reaction mixture was then
cooled to room temperature. The solid was filtered off,
suspended in MeOH–H2O, a few drops of 1 N HCl was
added, and precipitate was filtered off and recrystallized
1
from MeOH. Yield 0.120 g (33%). Mp 269–270°C. H
NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 4.37 (1H, d,
J =16.4) and 4.87 (1H, d, J = 16.4, PhCH2); 6.43 (1H, d,
730

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
J = 7.2, H-5); 6.58 (1H, t, J = 7.6, H-6); 7.02 (1H, d, J = 6.4,
recrystallized from MeOH. Yield 0.265 g (72%). Mp 285–
1
H-7); 7.20–7.33 (7H, m, H Ar); 7.47–7.49 (1H, m, H Ar);
7.69 (1H, s, 2-CH); 7.74–7.76 (1H, m, H-3'); 7.8 (1H, br. s,
NH, D2O-exchangeable). 13C NMR spectrum (DMSO-d6),
δ, ppm: 149.11; 146.8; 138.8; 135.4; 135.3; 130.3; 129.3;
129.2; 128.3 (2C); 128.2; 127.1; 120.1; 114.9; 107.4;
78.03; 46.8. Mass spectrum, m/z (Irel, %): 366 [M–H]+
(100). Found, %: C 61.07; H 4.88; N 11.25; S 8.44.
C19H17N3O3S. Calculated (with 1.2% H2O), %: C 61.36;
H 4.74; N 11.29; S 8.62.
290°C. H NMR spectrum (DMSO-d6), δ, ppm (J, Hz):
4.20 (1H, d, J = 16.4) and 4.61 (1H, d, J = 16.4, PhCH2);
6.39 (1H, d, J = 7.2, H-5); 6.51 (1H, t, J = 6.8, H-6); 6.90
(1H, d, J = 6.8, H-7); 7.20–7.30 (5H, m, H Ar); 7.37–7.47
(2H, m, H Ar); 7.55 (1H, dd, J = 7.6, J = 1.6, H-6'); 7.69
(1H, s, 2-CH); 7.82 (1H, dd, J = 7.6, J = 1.6, H-3'); 9.76
(1H, br. s, NH, D2O-exchangeable); 12.8 (1H, br. s). 13C
NMR spectrum (DMSO-d6), δ, ppm: 149.1; 146.8; 138.8;
135.4; 135.3; 130.3; 129.3; 129.2; 128.3 (2C); 128.2;
127.1; 120.1; 114.9; 107.4; 78.03; 46.8. Mass spectrum, m/z
(Irel, %): 366 [M–H]+ (100). Found, %: C 61.07; H 4.88;
N 11.25; S 8.44. C19H17N3O3S. Calculated (with 1.2%
H2O), %: C 61.35; H 4.74; N 11.29; S 8.62.
2-(1-Benzyl-1H-imidazo[4,5-b]pyridin-2-yl)benzene-
sulfonic acid (32). A solution of compound 31 (0.092 g,
0.25 mmol) in nitrobenzene (2 ml) was heated at 200°C for
4 h. The precipitate was filtered off and recrystallized from
n-hexane–2-PrOH, 1:1, twice. Yield 0.019 g (21%). Mp
>300°C. 1H NMR spectrum (CD3OD), δ, ppm (J, Hz): 5.36
(2H, br. s, PhCH2); 7.01–7.04 (2H, m, H-3,5 Ph); 7.22–7.24
(3H, m, H-2,4,6 Ph); 7.29 (1H, dd, J = 8.4, J = 4.8, H-6);
7.33 (1H, dd, J = 7.2, J = 0.8, H-3'); 7.49 (1H, td, J = 7.2,
J = 0.8, H-4'); 7.65 (1H, td, J = 7.2, J = 0.8, H-5'); 7.82
(1H, dd, J = 8.4, J = 1.6, H-7); 8.13 (1H, dd, J = 7.6,
J = 1.2, H-6'); 8.41 (1H, dd, J = 4.8, J = 1.6, H-5).
13C NMR spectrum (CD3OD), δ, ppm: 157.6; 155.6; 146.2;
145.1 (C-5); 137.3; 132.8; 131.9 (C-5'); 131.2 (C-4'); 129.8
(C-2,6 Ph); 129.2 (C-7,6'); 129.0 (C-4 Ph); 128.3 (C-3,5
Ph); 127.8 (C-3'); 121.6 (C-7); 119.6 (C-6); 50.1 (CH2).
Mass spectrum, m/z (Irel, %): 366 [M+H]+ (100). Found, %:
C 60.33; H 4.96; N 11.40; S 8.41. C19H15N3O3S. Calculated
(with 3.4% H2O and 1.6% 2-PrOH), %: C 60.36; H 4.51;
N 10.94; S 8.35.
2-(3-Benzyl-3H-imidazo[4,5-b]pyridin-2-yl)benzene-
sulfonic acid (28). A mixture of compound 27 (0.184 g,
0.50 mmol) and 1,4-benzoquinone (0.054 g, 0.50 mmol) in
EtOH (20 ml) was heated at 75°C for 6 h. The reaction
mixture was cooled to room temperature, concentrated,
acidified with dilute acetic acid (5%), and diluted with
some water. The solid was filtered off and recrystallized
from n-hexane–2-PrOH, 1:1. Yield 0.148 g (81%). Mp 317–
1
318°C. H NMR spectrum (CD3OD), δ, ppm (J, Hz): 5.21
(1H, br. s) and 5.76 (1H, br. s, PhCH2); 6.89–6.91 (2H, m,
H-3,5 Ph); 7.015 (1H, d, J = 7.2, H-6'); 7.13–7.18 (3H, m,
H-2,4,6 Ph); 7.35 (1H, td, J = 7.2, J = 0.8, H-5'); 7.39 (1H, dd,
J = 8.4, J = 5.2, H-6); 7.63 (1H, td, J = 7.6, J = 0.8, H-4'); 8.09–
8.14 (2H, m, H-7,3'); 8.41 (1H, dd, J = 4.8, J = 1.2, H-5). 13C
NMR spectrum (CD3OD), δ, ppm: 155.9; 148.7; 145.9; 145.5
(H-5); 137.9; 134.9; 132.9 (H-6'); 131.9 (H-4'); 131.1 (C-6);
129.6 (C-2,6 Ph); 129.1 (C-7); 128.7 (C-4 Ph); 128.6 (C-3,5
Ph); 127.9 (C-3'); 127.7; 120.2 (C-5'); 47.8 (CH2). Mass
spectrum, m/z (Irel, %): 364 [M–H]+ (100). Found, %: C 62.55;
H 4.11; N 11.6; S 8.66. C19H15N3O3S. Calculated, %: C 62.5;
H 4.14; N 11.5; S 8.77.
Sodium 2-({[2-(benzylamino)pyridin-3-yl]imino}methyl)-
benzenesulfonate (29). A mixture of compound 4 (0.20 g,
1.0 mmol) and sodium 2-formylbenzenesulfonate (0.208 g,
1.00 mmol) in EtOH (10 ml) was stirred at 50°C for 24 h.
The reaction mixture was cooled to room temperature,
EtOH was evaporated, and the solid was recrystallized
from MeOH–EtOAc, 1:1. Yield 0.31 g (84 %). Yellow
solid. Mp 280–285°C. 1H NMR spectrum (DMSO-d6),
δ, ppm (J, Hz): 4.64 (2H, d, J = 6.4, PhCH2); 6.56 (1H, dd,
J = 7.2, J = 5.2, H-5'); 6.77 (1H, t, J = 6.4, CH2NH,
D2O-exchangeable); 7.14 (1H, dd, J = 7.2, J = 1.6, H-4');
7.19–7.83 (8H, m, H Ar); 7.87 (1H, dd, J = 5.2, J = 1.6,
H-6'); 8.34 (1H, m, H-6); 9.60 (1H, s, CH=N). 13C NMR
spectrum (DMSO-d6), δ, ppm: 160.1; 153.6; 148.3; 145.5;
141.2; 132.4; 132.2; 130.1; 128.7; 127.9; 127.2; 126.9;
126.6; 126.2; 122.6; 111.8; 43.6. Mass spectrum, m/z (Irel, %):
366 [M–H]+ (100). Found, %: C 49.15; H 5.03; N 9.05;
S 6.48. C19H16N3O3SNa. Calculated (with 15.6% H2O), %:
C 49.34; H 5.42; N 9.09; S 6.93.
2-(1-Benzyl-2,3-dihydro-1H-imidazo[4,5-b]pyridin-2-yl)-
benzenesulfonic acid (31). A mixture of compound 3020
(0.20 g, 1.0 mmol) and sodium metabisulfite adduct of
sodium 2-formylbenzenesulfonate (0.312 g, 1.00 mmol) (or
sodium 2-formylbenzenesulfonate (0.208 g, 1.00 mmol)) in
EtOH (10 ml) were stirred at 70°C for 30 h. The reaction
mixture was cooled to room temperature. The solid was
filtered off, suspended in MeOH–H2O, 1:1, and a few drops
of 1 N HCl was added, and precipitate was filtered and
Synthesis of compounds 33–35 (General method).
Hydroxylamine hydrochloride (0.10 g, 1.43 mmol) followed
by N,N-diisopropylethylamine (0.184 g, 1.43 mmol) was
added to a stirred solution of compound 23–25 (1.0 mmol)
in ethanol (35 ml). The solution was heated to reflux for
6 h and then concentrated. Water was added, and the
precipitate was washed with water and crystallized from
ethanol. In order to obtain the HCl salts of compounds 34
and 35, the free bases were dissolved in ethanol, and dry
HCl gas was passed through the solution, and the precipi-
tate was filtered off and dried in vacuum.
N-Hydroxy-4-(3H-imidazo[4,5-b]pyridin-2-yl)benzene-
carboximidamide (33). This compound was obtained in
the base form. Yield 0.142 g (56%). Mp 293–294°C
1
(EtOH). H NMR spectrum (DMSO-d6 + NaH + D2O),
δ, ppm (J, Hz): 6.87 (1H, dd, J = 7.6, J = 4.8, H-6); 7.68
(2H, d, J = 8.0, H-2',6'); 7.75 (1H, dd, J = 8.0, J = 0.8,
H-7); 7.98 (1H, dd, J = 4.8, J = 1.6, H-5); 8.23 (2H, d,
J = 8.0, H-3',5'). 13C NMR spectrum (DMSO-d6), δ, ppm:
162.6; 160.5; 151.2; 139.8 (C-5); 139.6; 136.1; 134.1;
127.1 (C-3',5'); 124.8 (C-2',6'); 123.1 (C-7); 114.5 (H-6).
Mass spectrum, m/z (Irel, %): 254 [M+H]+ (100). Found, %:
C 61.11; H 4.29; N 27.29. C13H11N5O. Calculated (with
0.7% H2O), %: C 61.21; H 4.42; N 27.45.
N-Hydroxy-4-(3H-imidazo[4,5-c]pyridin-2-yl)benzene-
731

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
carboximidamide dihydrochloride (34). Yield 0.121 g
J = 8.0, H-2',6'); 7.97 (1H, d, J = 5.8, H-6); 8.30 (2H, d,
J = 8.0, H-3',5'); 8.73 (1H, s, H-4). 13C NMR spectrum
(CD3OD + NaH + D2O), δ, ppm: 163.6; 163.4; 151.5; 144.4;
137.7; 137.4; 137.2; 137.1; 127.4; 126.7; 111.5; 43.5; 21.6.
Mass spectrum, m/z (Irel, %): 280 [M+H]+ (100). Found, %:
C 55.69; H 6.97; N 19.21. C16H17N5. Calculated (with 16.2%
H2O and 9.6% MeOH), %: C 55.87; H 7.45; N 19.16.
(37%). Mp 325–330°C (decomp.). 1H NMR spectrum
(D2O), δ, ppm (J, Hz): 7.85 (2H, d, J = 8.0, H-2',6'); 8.07
(1H, d, J = 6.8, H-7); 8.21 (2H, d, J = 8.0, H-3',5'); 8.45
(1H, d, J = 6.8, H-6); 9.14 (1H, s, H-4). 13C NMR spectrum
(D2O), δ, ppm: 160.2 (C-2); 158.4 (C-1'); 146.6 (C-7a);
138.3 (C-3a); 133.6 (C-6); 132.6 (C-4); 131.5 (C-4'); 128.7
(C-2',6'); 128.6 (C-3',5'); 128.5 (CNO); 111.3 (C-7). Mass
spectrum, m/z (Irel, %): 254 [M+H]+ (100). Found, %:
C 47.17; H 3.88; N 21.32. C13H11N5O·2HCl. Calculated
(with 1.4% H2O), %: C 47.21; H 4.11; N 21.18.
3-(3H-Imidazo[4,5-b]pyridin-2-yl)benzenecarboximid-
amide dihydrochloride (38). Yield 0.142 g (45 %). Mp
327–330°C. 1H NMR spectrum (D2O), δ, ppm (J, Hz): 7.68
(1H, dd, J = 8.0, J = 6.0, H-6); 7.79 (1H, t, J = 7.6, H-5');
7.97–7.99 (1H, m, H-6'); 8.35–8.38 (1H, m, H-4'); 8.47
(1H, t, J = 2.0, H-2'); 8.48 (1H, dd, J = 6.0, J = 1.2, H-7);
8.53 (1H, dd, J = 8.0, J = 1.2, H-5). 13C NMR spectrum
(D2O), δ, ppm: 169.1; 160.0; 150.8; 139.7 (C-7); 135.9 (C-4');
134.7 (C-6'); 134.6; 133.7 (C-5'); 132.5; 132.0 (C-5); 130.9;
130.3 (C-2'); 122.4 (C-6). Mass spectrum, m/z (Irel, %): 238
[M+H]+ (100). Found, %: C 50.06; H 4.29; N 22.20.
C13H11N5·2HCl. Calculated, %: C 50.34; H 4.23; N 22.58.
N-Isopropyl-4-(3-benzyl-3H-imidazo[4,5-b]pyridin-
2-yl)benzenecarboximidamide dihydrochloride (39).
Yield 0.126 g (28 %). Mp 95–100°C (bubbling); 290–295°C.
1H NMR spectrum (CD3OD), δ, ppm (J, Hz): 1.39 (6H, d,
J = 6.8, CH(CH3)2); 4.03–4.06 (1H, m CH(CH3)2); 5.73
(2H, s, PhCH2); 6.99 (2H, d, J = 8.0, H-3,5 Ph); 7.20–7.25
(3H, m, H-2,6,4 Ph); 7.45 (1H, dd, J = 8.4, J = 4.8, H-6);
7.86 (2H, d, J = 8.4, H-2',6'); 7.93 (2H, d, J = 8.4, H-3',5');
8.18 (1H, dd, J = 8.0, J = 1.2, H-7); 8.46 (1H, dd, J = 4.8,
J = 1.2, H-5). 13C NMR spectrum (CD3OD), δ, ppm: 162.9;
153.6; 148.5; 145.1 (C-5); 136.7; 134.7; 134.3; 131.6;
129.8 (C-3',5'); 128.7 (C-2,6 Ph or C-4 Ph); 128.5 (C-2',6');
127.7 (C-2,6 Ph or C-4 Ph); 127.5 (H-7); 126.3 (H-3'',5'');
119.6 (H-6); 46.5 (CH2); 46.0 (C(CH3)2); 20.3 ((CH(CH3)2).
Mass spectrum, m/z (Irel, %): 370 [M+H]+ (100). Found, %:
C 61.25; H 6.04; N 15.87. C23H23N5·2HCl. Calculated
(with 1.6% H2O), %: C 61.20; H 5.81; N 15.51.
Cytotoxicity Assay. The synthesized compounds were
dissolved in ethanol (70%) to provide 1×10–2 mol/l stock
solutions, these solutions were diluted within the range be-
tween 1×10–3 to 1×10–10 mol/l. The human breast cancer
cell line MCF-7 (HTB 22) was purchased from the Ame-
rican Type Culture Collection (LGC Promochem). The
cells were cultured in Dulbecco's modified Eagle's medium
(DMEM, PAA Laboratories GmbH) with 10% fetal bovine
serum (FBS, Lonza), 1% antibiotics (peniciline and strepto-
mycin, PAA) and 1% L-glutamine (PAA) at humidified
atmosphere (37°C and 5% CO2).
The effects of test samples on cell viability were
determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay by the method of
Mossman26 in modification by Kuzma et al.27 A 180 μl
volume of the cell suspension containing 5×104 cells was
seeded to each well of a 96-well microtiter plate, and the
cells were grown in a humidified atmosphere of 5% CO2 in
air at 37°C. The test samples were added to a final concen-
tration ranged between 100 μM to 10 nM, and the plates
were incubated for 48 h. Control experiments were per-
formed under the same conditions with addition of solvent
(70% EtOH). Following incubation, the culture medium
N-Hydroxy-3-(3H-imidazo[4,5-b]pyridin-2-yl)benzene-
carboximidamide dihydrochloride (35). Yield 0.120 g
(37%). Mp 282–285°C (decomp.). 1H NMR spectrum
(D2O), δ, ppm (J, Hz):7.72 (1H, dd, J = 8.0, J = 5.6, H-6);
7.83 (1H, t, J = 7.6, H-5'); 7.96 (1H, d, J = 7.6, H-6'); 8.34
(1H, d, J = 7.6, H-4'); 8.41 (1H, br. s, H-2'); 8.52–8.55 (2H,
m, H-5,7). 13C NMR spectrum (D2O), δ, ppm: 163.4;
159.5; 150.7; 139.9 (C-7); 135.4 (C-4'); 134.4 (C-6');
134.3; 133.7 (C-5'); 131.8 (C-5); 130.9; 129.9 (C-2');
129.4; 122.4 (C-6). Mass spectrum, m/z (Irel, %): 254 [M+H]+
(100). Found, %: C 47.90; H 3.99; N 21.50. C13H11N5O·2HCl.
Calculated, %: C 47.86; H 4.01; N 21.47.
Synthesis of compounds 36–39 (General method). Com-
pound 23–26 (1.0 mmol) was suspended in absolute EtOH,
cooled in an ice–salt bath, and dry HCl gas was passed
through the solution for 40 min. The solution was stirred in
a stoppered flask at room temperature for 6 days and then
diluted with dry ether. The precipitated imidate ester
hydrochloride was washed with ether, dried under vacuum
at room temperature, and used immediately without
characterization. A suspension of imidate ester hydro-
chloride in absolute EtOH (10 ml) was stirred with NH3
(NH3 gas let into the reaction mixture) or isopropylamine
(1.5–2-fold excess) overnight at 60°C. The reaction
mixture was evaporated and treated with dilute Na2CO3
solution. In order to obtain the HCl salts of compounds 38
and 39, the free bases were dissolved in ethanol and dry
HCl gas was passed through the solution, and the
precipitate was filtered off and dried in vacuum.
4-(3H-Imidazo[4,5-b]pyridin-2-yl)benzenecarboximid-
amide dihydrochloride (36). Yield 0.128 g (41%). Mp
337–339°C. 1H NMR spectrum (D2O), δ, ppm (J, Hz):7.59
(1H, dd, J = 8.4, J = 5.6, H-6); 7.86 (2H, d, J = 8.0,
H-2',6'); 8.12 (2H, d, J = 8.0, H-3',5'); 8.35 (1H, dd, J = 8.0,
J = 0.8, H-7); 8.44 (1H, dd, J = 5.6, J = 1.2, H-5).
13C NMR spectrum (D2O), δ, ppm: 168.4 (C-2); 158.1
(C-1'); 151.4 (C-3a); 141.9 (C-5); 134.7 (C-4'); 134.0
(C-7a); 133.5 (CNO-carboxamidine); 131.5 (CH-2',6');
131.1 (CH-3',5'); 130.7 (CH-7); 122.5 (CH-6). Mass
spectrum, m/z (Irel, %): 238 [M+H]+ (100). Found, %:
C 46.89; H 4.38; N 20.94. C13H11N5·2HCl. Calculated
(with 7.5% H2O), %: C 46.55; H 4.74; N 20.88.
N-Isopropyl-4-(3H-imidazo[4,5-c]pyridin-2-yl)benzene-
carboximidamide (37). It was crystallized from EtOAc–
MeOH, 3:1. Yield 0.177 g (53.5%). Mp 265–270°C
1
(bubbling). H NMR spectrum (CD3OD + NaH + D2O),
δ, ppm (J, Hz): 1.26 (6H, d, J = 6, CH(CH3)2 ); 3.92–3.95
(1H, m, CH(CH3)2); 7.51 (1H, d, J = 5.4, H-7); 7.67 (2H, d,
732

Chemistry of Heterocyclic Compounds 2015, 51(8), 723–733
Kosmopoulou, M.; Joshi, A.; Atrash, B.; Pérez-Fuertes, Y.;
was removed and exchanged for a fresh one. MTT solution
(20 μl, 5 mg/ml in phosphate buffer solution, Sigma) was
added per well and incubated at 37°C for 2 h. The
metabolically active cells reduced MTT dye to formazan
crystals. The medium was then removed, and the blue
MTT-formazan was dissolved in DMSO (Merck). The
extent of the reduction of MTT within the cells was quanti-
fied by measuring the absorbance at 540 nm on a Thermo
MultiSkan GO microplate reader and compared with con-
trolled, untreated cells. The experiments were performed
within triplicates, and the concentration of test compounds
required to reduce survival of cells by 50% (IC50) was
determined from the graph of the amount of visible cells
plotted against the test compound concentration.
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1
Supporting material to this article containing H NMR
spectra of compounds 5, 6, 11–29, 31–39 and 13C NMR
spectra of compounds 5, 6, 13, 16–22, 25–29, 31–39 is
available for the authorized users.
The Central Laboratory of Ankara University, Faculty
of Pharmacy, provided support for the acquisition of the
NMR and mass spectra and elemental analysis.
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