Regioselective N–alkylation of 2– (3,4–dimethoxyphenyl)imidazo[4,5–b] and [4,5–c]pyridine oxide derivatives: Synthesis and structure elucidation by NMR

Article abstract of DOI:10.1016/j.molstruc.2019.07.058

Imidazopyridines can exist in several tautomeric forms such as benzimidazole or purine condensed systems. Regioselectivities were determined for N-alkylations of 2-(3,4-dimethoxyphenyl)- imidazopyridines and their 4 and 5-oxides (2–4, 13, 14) with n-butyl and 4-fluorobenzyl bromides under basic conditions (K₂CO₃ in DMF). It was observed that N-4 (5–8) and N-5 (15–17) regioisomers were mainly formed. Compounds 7 (N⁴) and 7a (N¹) were separated from the mixtures of regioisomers in a 50: 1 ratio. Their structural assignments were made with the use of two-dimensional ¹H-¹H NOE (nuclear overhauser effect spectroscopy [NOESY]) enhancements between the N-CH₂ and protons on the C-4, 5, 6, and 7 positions of the pyridine moiety. To verify the NOESY data, synthesis of compounds 7a and 7b was achieved by the selective method. Complementary structural information was provided by 2D-HMBC spectra of the compounds.

Full text of DOI:10.1016/j.molstruc.2019.07.058

Accepted Manuscript
Regioselective N–alkylation of 2– (3,4–dimethoxyphenyl)imidazo[4,5–b] and [4,5–
c]pyridine oxide derivatives : Synthesis and structure elucidation by NMR
Hakan Göker, Seçkin Özden
PII:
S0022-2860(19)30902-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.07.058
MOLSTR 26811
Reference:
To appear in: Journal of Molecular Structure
Received Date: 6 March 2019
Revised Date: 8 July 2019
Accepted Date: 12 July 2019
Please cite this article as: H. Göker, Seç. Özden, Regioselective N–alkylation of 2– (3,4–
dimethoxyphenyl)imidazo[4,5–b] and [4,5–c]pyridine oxide derivatives : Synthesis and structure
elucidation by NMR, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.07.058.
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ACCEPTED MANUSCRIPT
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OMe
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N
N
N
N
N
N
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OMe
N
N
H
F
2
F
6a (2 %)
6 (88 %)
6b (9.5 %)

ACCEPTED MANUSCRIPT
Regioselective N-alkylation of 2-(3,4-dimethoxyphenyl)imidazo[4,5-b] and
[4,5-c]pyridine oxide derivatives: Synthesis and structure elucidation by NMR
Hakan Gökera*, Seçkin Özdenb
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University,
06100 Tandogan, Ankara-Turkey
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Lokman Hekim University,
Söğütözü, Ankara-Turkey
ABSTRACT
Imidazopyridines can exist in several tautomeric forms such as benzimidazole or purine condensed
systems. Regioselectivities were determined for N-alkylations of 2-(3,4-dimethoxyphenyl)-
imidazopyridines and their 4 and 5-oxides (2–4, 13, 14) with n-butyl and 4-fluorobenzyl bromides
under basic conditions (K2CO3 in DMF). It was observed that N-4 (5–8) and N-5 (15–17)
regioisomers were mainly formed. Compounds 7 (N4) and 7a (N1) were separated from the mixtures
of regioisomers in a 50 : 1 ratio. Their structural assignments were made with the use of two-
1
dimensional H-1H NOE (nuclear overhauser effect spectroscopy [NOESY]) enhancements between
the N-CH2 and protons on the C-4, 5, 6, and 7 positions of the pyridine moiety. To verify the
NOESY data, synthesis of compounds 7a and 7b was achieved by the selective method.
Complementary structural information was provided by 2D-HMBC spectra of the compounds.
Keywords:
Imidazo[4,5-b]pyridine-4-oxide
Imidazo[4,5-c]pyridine-5-oxide
Tautomerism
NOESY (nuclear overhauser effect spectroscopy)
HMBC (heteronuclear multiple bond correlation)
_____________________
*Corresponding author
E-mail address: goker@ankara.edu.tr
1

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Introduction
Imidazopyridines are attractive hetereocycles for medicinal chemists as their derivatives have been
shown to exhibit several biological activities. In 2017, a review reported the pharmacological profile
of imidazopyridines with their anti-tumoral, anti-microbial, anti-inflammatory, anti-diabetic, anti-
hypertensive, and other pharmacological properties [1]. In addition, the use of imidazopyridines in
some neurodegenerative disorders [2], as well as their anti-lipidemic [3] and antifungal [4,5]
activities, has recently been reported.
Imidazopyridines are comprised of an imidazole ring fused with a pyridine moiety and have
various isomeric forms such as imidazo[4,5-b]pyridines, imidazo[4,5-c]pyridines, imidazo[1,5-a]
pyridines, and imidazo[1,2-a]pyridines. The NH group present in imidazopyridines is strongly acidic
and weakly basic. This group exhibits fast prototrophic tautomerism, which leads to equilibrium
mixtures. All three possible structures and numbering of imidazopyridines are depicted in Schemes 1
and 4 for compounds 2–4, 13, and 14. It was reported that [6], in the gas phase, 3H forms are more
stable than other forms. Existence of this tautomerism in imidazo[4,5-b]pyridines [7,8], imidazo
[4,5-c]pyridine [9], and imidazo[4,5-b]pyridine-4-oxide [10,11] has been shown with spectral data,
mainly nuclear magnetic resonance (NMR) spectroscopy. This migration is not observed when the
imidazole hydrogen is replaced by other substituents such as an alkyl group or under special
circumstances in which hydrogen migration is affected by inter- and/or intramolecular hydrogen
bonding [7,9,12].
In our previous studies [13,14], we reported the synthesis of some regioisomers of
benzimidazoles and their structural elucidation was achieved by selective synthesis and/or 2D-NMR
data. As a further contribution to this field, here we report the synthesis of a series of 2-(3,4-
dimethoxyphenyl)imidazopyridines and their oxides with n-butyl and 4-fluorobenzyl groups on the
N
4,5,3,1-positions and their tautomeric behaviours. Following isolation of the pure regiosomers, 1H-1H
NOE, nuclear overhauser effect spectroscopy (NOESY), and heteronuclear multiple bond correlation
(HMBC) experiments were used for structural elucidations.
Experimental
Uncorrected melting points were measured using a Büchi B-540 capillary melting point apparatus.
All NMR experiments were carried out using VARIAN (Agilent) MERCURY 400MHz (Varian,
Palo Alto, CA, USA) at a proton resonance frequency of 400 and 100 MHz for carbon. The NMR
spectrum optimisation was conducted using Agilent VnmrJ version 3.2 revision A software and all
parameters were set in it. The samples (5–15 mg) were prepared in 0.7 ml of DMSO-d6, CDCl3, and
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CD3OD. TMS was used as an internal standart. Because of the tautomeric effect of the imidazole
ring, NMR spectra of compounds 4 and 14 did not give satisfactory results. To eliminate the
tautomeric effect, the related imidazopyridines were dissolved in DMSO-d6, followed by the addition
of a tiny amount of dry NaH and 2–3 drops of D2O to the NMR tube. This effectively eliminated the
tautomeric effect and produced satisfactory results. The liquid chromatography mass spectrometry
(LC-MS) spectra were taken on a Waters Micromass ZQ connected with Waters Alliance HPLC
(Waters Corporation, Milford, MA, USA), using the ESI(+) method with a C-18 column (XTerra®,
4.6 × 250 mm, 5 ꢀm). Analytical conditions of mass spectrometry were as follows: capillary voltage,
3.11 kV; cone voltage, 29V; source temperature, 100°C; and desolvation temperature, 300°C.
Compounds 1–3, 13 [15] and 11-12 [16], were prepared according to the literature methods. The
HCl salt of compounds 5 and 15 were prepared using methanolic HCl.
2-(3,4-Dimethoxyphenyl)-1,(3),(4)H-imidazo[4,5-b]pyridine 4-oxide (4)
To a stirred solution of 2 (2 mmol, 0.510 g) in acetic acid (2.5 mL), hydrogen peroxide (35%, 1 mL)
was slowly added. The solution was warmed to 70°C for 2 h and allowed to stir at room temperature
(RT) overnight. The reaction mixture was diluted with water and the resulting precipitate was
collected by filtration, washed with water, and dried. Crystallisation from MeOH yielded 4, a white
coloured powder, with melting point (mp) > 295°C and yield 0.325 g (60%). 1H-NMR (DMSO-d6) δ
ppm: 3.85 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 7.15–7.19 (m, 2H), 7.53 (br.s, 1H), 7.79 (br.s, 2H),
13
8.14 (d, 1H,
J = 6Hz); C-NMR (DMSO-d6): 152.5, 151.2, 148.9, 123.6, 121.1, 120.2, 118.5,
111.9, 110.1, 55.6, and 55.5. 1H-NMR (DMSO-d6 + NaH + D2O) δ ppm: 3.77 (s, 3H, 4’-OCH3), 3.84
(s, 3H, 3’-OCH3), 6.83 (dd, 1H, J = 7.2 and 6 Hz, H-6), 6.94 (d, 1H, J = 8.4 Hz, H-5’) 7.49 (d, 1H, J
= 7.6 and 1.2 Hz, H-7), 7.81 (dd, 1H, J = 6.4 and 0.8 Hz, H-5), 7.86 (d, 1H, J = 2Hz, H-2’), 7.88 (dd,
1H, J = 5.6 and 1.6 Hz, H-5); COSY: [H-5': H-6'], [H-6: H-7], [H-6: H-5]; NOESY: [H-5’: 4’-
13
OCH3], [H-2’: 3’-OCH3]; C-NMR, HSQC, and HMBC (DMSO-d6 + NaH + D2O) δ ppm: 162.9
(C-2), 150.7 (C-3a), 149.6 (C-4’), 148.75 (C-3’), 143.6 (C-7a), 128.9 (C-1’), 128.75 (C-5H), 120.4
(C-2'H), 116.8 (C-7H), 113.6 (C-6H), 111.8 (C-5’H), 111.1 (C-6’H), 55.92, and 55.9(OCH3) (See
Supp. Info. pg 1-3) ; MS (ESI+) m/z: 272 (M + H, %100), C14H13N3O3.
4-Butyl-2-(3,4-dimethoxyphenyl)-4H-imidazo[4,5-b]pyridine HCl (5)
Only the N-4 regioisomer was obtained. K2CO3 (0.1105 g, 0.8 mmol) was added to a suspension of 2
(0.127 g, 0.5 mmol) in DMF (1 mL) and stirred. After 1 h, butyl bromide (0.082 g, 0.6 mmol) was
added. After overnight stirring at 35°C, water was added, CH2Cl2 extraction was performed, and
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solvent was evaporated. The residue was subjected to silica gel column chromatography and eluted
with CH2Cl2: MeOH (97: 3–5) gradient, oily pure 5 was obtained, which was converted to HCl salt,
mp 130–138°C (bubbling), hygroscopic, yield 0.12 g (41%). 1H-NMR (CD3OD) δ ppm: 1.04 (t, 3H),
1.47 (m, 2H), 2.11 (m, 2H), 3.94 (s, 3H, 4’-OCH3), 3.97 (s, 3H, 3’-OCH3), 4.91 (t, 2H), 7.17 (d, 1H,
J = 8.4 Hz, H-5’), 7.68 (dd, 1H, J = 8 and 6.4 Hz, H-6), 7.86 (d, 1H, J = 1.6 Hz, H-2’), 7.95 (dd, 1H,
J = 8.4 and 2 Hz, H-6’), 8.5 (dd, 1H, J = 8 and 0.8 Hz, H-7), 8.63 (dd, 1H, J = 6 and 0.8 Hz, H-5);
COSY: [H-5': H-6'], [H-6: H-7], [H-6: H-5]; [N-3 butyl group as expected]; NOESY: [H-5’: 4’-
13
OCH3], [H-2’: 3’-OCH3]; [N-CH2-: H-5]; C-NMR, HSQC, and HMBC (CD3OD) δ ppm: 161.14
(C-2), 155.2
(C-4’), 151.2 (C-3’), 151.14 (C-3a), 138 (C-5H), 134.1 (C-7a), 127.7 (C-7H), 123.9
(C-6'H), 120.8 (C-1’), 119.6 (C-6H), 113.1 (C-5’H), 112.3 (C-2’H), 56.8 and 56.7 (OCH3), 55.6 (N-
CH2), 33
(N-CH2CH2), 20.7 (N-CH2CH2CH2), 13.8 (N…-CH3) (See Supp. Info. pg 4-9); MS
.
(ESI+) m/z: 312 (M+H, %100), C18H21N3O2 HCl.
General synthesis of compounds 6 – 8
K2CO3 (0.138 g, 1 mmol) was added to a suspension of 2 – 4 (0.8 mmol) in DMF (1 mL) and stirred.
After 1 h, 4-fluorobenzyl bromide (0.15 g, 0.8 mmol) was added. After overnight stirring at 35°C,
water was added and the precipitate was filtered ; if precipitation did not occur, it was extracted with
CH2Cl2.
2-(3,4-Dimethoxyphenyl)-3-(4-fluorobenzyl)-3H-imidazo[4,5-b]pyridine (6b) and
2-(3,4-Dimethoxyphenyl)-4-(4-fluorobenzyl)-4H-imidazo[4,5-b]pyridine (6) from 2.
N-3 and N-4 regioisomers were obtained. The crude precipitated powder mixture was purified by
column chromatography using CH2Cl2: MeOH (95:5) as eluant to give the following products in the
following order of elution.
Compound 6b (first eluting fraction): mp, 153–155°C, yield 0.010 g (2.7%). Selective synthesis
method for 6b: The mixture of 12 (0.434 g, 2 mmol) and Na2S2O5 adduct of 3,4-dimethoxybenz-
aldehyde (0.542 g, 2 mmol) in DMF (1.5 mL) was heated at 120°C for 3 h. The reaction mixture was
cooled, water was added, and the resulting precipitate was collected by filtration and crystallised
from EtOH: water, mp 154–156°C, yield 0.47 g (64.5%). 1H-NMR (CDCl3) δ ppm: 3.76 (s, 3H, 3’-
OCH3), 3.93 (s, 3H, 4’-OCH3), 5.58 (s, 2H, N-CH2), 6.92 (d, 1H, J = 8 Hz, H-5’), 6.99 (t, 2H, J =
8.8 Hz,
H-,3’’,5’’), 7.1–7.14 (m, 2H, H-2’’,6’’), 7.2 (d, 1H, J = 1.6 Hz, H-2’), 7.23 (dd, 1H, J =
7.6 and 2 Hz, H-6’), 7.27 (dd, 1H, J = 8.4 and 4.8 Hz, H-6), 8.1 (dd, 1H, J = 8.4 and 1.6 Hz, H-7),
8.54 (dd, 1H, J = 4.8 and 1.2 Hz, H-5); COSY: [H-5': H-6'], [H-6: H-7], [H-6: H-5], [H-2’’6’’: H-
13
3’’,5’’]; NOESY: [H-5’: 4’-OCH3], [H-2’: 3’-OCH3]; [N-CH2-: H-2’], [N-CH2-: H-2’’,6’’]; C-
4

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NMR, HSQC and HMBC (CDCl3) δ ppm: 162.2 (d, J = 245 Hz, C-4’’), 154.8 (C-2), 150.9 (C-4’),
149.2 (C-3’), 148.9 (C-3a), 144.1 (C-5H), 135.0 (C-7a), 132.7 (d, J = 3.2 Hz, C-1’’), 128.2 (d, J = 7.7
Hz, C-2’’,6’’H), 127.1 (C-7H), 122.1 (C-1’), 122.0 (C-6’H), 118.95 (C-6H), 115.8 (d, J = 21 Hz, C-
3’’,5’’H), 112.1 (C-2’H), 111 (C-5’H), 55.98 (4’-OCH3), 55.8 (3’-OCH3), 46.3 (N-CH2) (See Supp.
Info. pg 24-30); MS (ESI+) m/z: 364 (M+H, %100), C21H18FN3O2. 6b prepared by the selective
methods was found to be identical in analytical and spectral properties to the product (6b) obtained
during the synthesis reaction of 6.
Compound 6 (second eluting fraction): Hygroscopic, yellow coloured powder, mp 80–83°C, yield
0.15 g (51.7%); 1H-NMR (DMSO-d6) δ ppm: 3.84 (s, 3H, 4’-OCH3), 3.88 (s, 3H, 3’-OCH3), 5.87 (s,
2H, N-CH2), 7.08 (d, 1H, J = 8.4 Hz, H-5’), 7.15–7.22 (m, 3H, H-3’’, 5’’, 6 overlapped), 7.64 (m,
2H, H-2’’, 6’’), 7.94 (d, 1H, J = 1.6 Hz, H-2’), 7.98 (dd, 1H, J = 8.4 and 2 Hz, H-6’), 8.12 (d, 1H, J =
7.6 Hz, H-7), 8.23 (d, 1H, J = 6.4 Hz, H-5); COSY: [H-5': H-6'], [H-6: H-7], [H-6: H-5]; NOESY:
13
[H-5’: 4’-OCH3], [H-2’: 3’-OCH3]; [N-CH2-: H-5], [N-CH2-: H-2’’, 6’’]; C-NMR, HSQC, and
HMBC (DMSO-d6) δ ppm: 168 (C-2), 162 (d, J = 243 Hz, C-4’’), 153.8 (C-3a), 150.4 (C-4’), 148.6
(C-3’), 145.1 (C-7a), 132.1 (d, J = 3.2 Hz, C-1’’), 130.7 (d, J = 9 Hz, C-2’’, 6’’H), 130.4 (C-5H),
127.3
(C-1’), 126.6 (C-7H), 120.8 (C-6'H), 115.5 (d, J = 21 Hz, C-3’’, 5’’H), 112.8 (C-6H),
111.6 (C-5’H), 110.7 (C-2’H), 55.5 (4’-OCH3), 55.4 (3’-OCH3), 54.9 (N-CH2) (See Supp. Info. pg
10-15) ; MS (ESI+) m/z: 364 (M+H, %100), C21H18FN3O2.
2-(3,4-Dimethoxyphenyl)-4-(4-fluorobenzyl)-4H-imidazo[4,5-b]pyridine 4-oxide (7) and
2-(3,4-Dimethoxyphenyl)-1-(4-fluorobenzyl)-1H-imidazo[4,5-b]pyridine 4-oxide (7a) from 4.
N-4 and N-1 regioisomers were obtained. Crystallisation of the resulting precipitate from EtOH: H2O
(50%) gave pure compound 7, mp 80–84°C, yield 0.14 g (73%). 1H-NMR (DMSO-d6) δ ppm: 3.85
(s, 3H, 4’-OCH3), 3.89 (s, 3H, 3’-OCH3), 5.73 (s, 2H, N-CH2), 7.08 (t, 1H, J = 6.8 Hz, H-6), 7.11 (d,
1H, J = 8.4 Hz, H-5’), 7.23–7.28 (m, 2H, H-3’’, 5’’), 7.59–7.62 (m, 2H, H-2’’, 6’’), 7.96 (d, 1H, J =
1.6 Hz, H-2’), 8.01 (dd, 1H, J = 8.4 and 1.6 Hz, H-6’), 8.35 (d, 1H, J = 8Hz, H-7), 8.85 (d, 1H, J =
6.8 Hz, H-5); COSY: [H-5': H-6'], [H-3’’, 5’’: H-2’’, 6’’], [H-6: H-7], [H-6: H-5]; NOESY: [H-5’:
13
4’-OCH3], [H-2’: 3’-OCH3]; [N-CH2-: H-5 (weak)], [N-CH2-: H-2’’, 6’’]; C-NMR, HSQC, and
HMBC (DMSO-d6) δ ppm : 168.4 (C-2), 162.7 (d, J = 245 Hz, C-4’’), 150.6 (C-4’), 149.4 (C-3a),
148.6 (C-3’), 148.0 (C-7a), 132.4 (d, J = 9 Hz, C-2’’, 6’’-H), 129.6 (d, J = 2.6 Hz, C-1’’), 128.2
(C-5H), 126.9 (C-1’), 126.2 (C-7H), 121.1 (C-6'H), 115.5 (d, J = 22 Hz, C-3’’, 5’’H), 112 (C-6H),
5

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111.6 (C-5’H), 110.7 (C-2’H), 79.1 (N-CH2), 55.5 (4’-OCH3), 55.4 (3’-OCH3) (See Supp. Info.
pg 31-37) ; MS (ESI+) m/z: 380 (M+H, %100), C21H18FN3O3.
After precipitation of 7, the supernatant liquid was evaporated and the residue was purified by
column chromatography (CH2Cl2 : MeOH, 93:7) gave the pure compound 7a (0.006 g). Selective
synthesis method for 7a: To a stirred solution of 6a (0.19 g, 0.5 mmol) in acetic acid (1 mL),
hydrogen peroxide (35 %, 0.1 mL) was slowly added. The solution was warmed to 40°C by stirring
for 36 h. The reaction mixture was diluted with water, partly neutralised with dilute K2CO3 solution,
extracted with CH2Cl2, and evaporated. The residue was subjected to silica gel column
chromatography. First, unreacted starting material was removed by eluting with EtOAc: MeOH
(100:2), then desired compound 7a was obtained by the mixture of dichloromethane: MeOH (93:7)
1
as eluant, mp 169–172°C, yield 0.095 g (47.3%). H-NMR (DMSO-d6) δ ppm: 3.71 (s, 3H, 3’-
OCH3), 3.83 (s, 3H, 4’-OCH3), 5.65 (s, 2H,
N-CH2), 7.09–7.18 (m, 5H, H-5’,3’’,5’’,2’’,6’’,
overlapped), 7.2 (dd, 1H, J = 6.4 and 8 Hz, H-6), 7.28 (d, 1H, J = 2 Hz, H-2’), 8.01 (dd, 1H, J = 8.4
and 2 Hz, H-6’), 7.55 (d, 1H, J = 8 Hz, H-7), 8.2 (d, 1H, J = 6.4 Hz, H-5); COSY: [H-5': H-6'], [H-6:
H-7], [H-6: H-5
]
; NOESY: [H-5’: 4’-OCH3], [H-2’:
3’-OCH3]; [N-CH2-: H-7], [N-CH2-: H-2’],
13
[N-CH2-: H-2’’,6’’]; C-NMR, HSQC, and HMBC (DMSO-d6) δ ppm: 161.4 (d, J = 242 Hz, C-
4’’), 153.9 (C-2), 150.8 (C-4’), 148.7 (C-3’), 146.0
(d, J = 3.2 Hz, C-1’’), 128.2 (d, J = 8.3 Hz, C-2’’,6’’H), 122.1 (C-6'H), 120.6 (C-1’), 118.9 (C-6H),
115.7 (d, J = 21 Hz, C-3’’,5’’H), 112.3 (C-2’H), 111.8 (C-5’H), 109.3 (C-7H), 55.6 (4’-OCH3),
(C-3a), 133.4 (C-5H), 132.7 (C-7a), 132.3
55.4 (3’-OCH3), 47.7 (N-CH2) (See Supp. Info. pg 38-44); MS (ESI+) m/z: 380 (M+H, %100),
C21H18FN3O3. 7a prepared by the selective method was found to be identical in analytical and
spectral properties to the product (7a) obtained during the synthesis reaction of 7.
3-(4-Fluorobenzylidene)amino]pyridin-2-amine (9)
To a suspension of 2,3-diaminopyridine (0.36 g, 3.3 mmol) in THF (33 mL), dried molecular sieves
(2 g, 4A°) and 4-fluorobenzaldehyde (0.5 g, 4 mmol) were added. After refluxing for 4 h, the
mixture was stirred overnight. The reaction mixture was filtered and concentrated, the residue was
purified by silica gel column chromatography, and eluted with n-hexane: EtOAc (60 : 40–100)
1
gradient, mp
128-132°C, yield 0.30 g (50 %). H-NMR (CDCl3) δ ppm: 5.23 (br s, 2H, NH2),
6.66 (dd, 1H, J = 5.6 and 5.2 Hz, H-5), 7.18 (t, 2H, J = 8 Hz, H-3’,5’), 7.23 (d, 1H, J = 8 Hz, H-4),
7.88–7.94 (m, 3H,
H-2’,6’,6), 8.48 (s, 1H, N = CH); NOESY: [N = CH:H-4] [N = CH:H-
13
2’,6’]; C-NMR (CDCl3) δ ppm: 164.9 (d, J = 251 Hz), 157.9, 154.7, 145.7, 132.3 (d, J = 3.1 Hz),
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132.1, 130.7 (d, J = 9 Hz), 123.5, 116.0 (d, J = 22 Hz), 113.9 (See Supp. Info. pg 64-68); MS (ESI+)
m/z: 216 (M+H, %100), C12H10FN3.
N3-(4-Fluorobenzyl)pyridine-2,3-diamine (10)
Compound 9 (0.27 g, 1.26 mmol) was dissolved in ethanol (15 mL) and treated with sodium
borohydride (0.3 g, 7.93 mmol). After refluxing for 8 h, water was added, and a light yellow
coloured powder was filtered, dried and used for the next step without crystallisation ; yield 0.25 g
(91.6%).
1H-NMR (CDCl3) δ ppm: 3.58 (br.t, 1H, NH-CH2), 4.24–4.26 (m, 4H, NH-CH2 and 2-
NH2), 6.66 (dd, 1H, J = 7.6 and 5.2 Hz, H-5), 6.78 (dd, 1H, J = 7.6 and 1.2 Hz, H-4), 7.04 (t, 2H, J =
8.4 Hz, H-3’,5’), 7.26–7.35 (m, 2H, J = 8.4 and 5.6 Hz, H-2’,6’), 7.61 (dd, 1H, J = 5.2 and 1.6 Hz, H-
13
6), The values are consistent with literature [17] ; NOESY: [NH-CH2:H-4] [NH-CH2:H-2’,6’]; C-
NMR (CDCl3) δ ppm: 162.17 (d, J = 244 Hz), 148.8, 137.12, 134.1 (d, J = 2.6 Hz), 131.7, 129.2 (d,
J = 8.4 Hz), 117.6, 115.9, 115.5 (d, J = 21 Hz), 47.5 (See Supp. Info. pg 69-74); MS (ESI+) m/z: 218
(M+H, %100), C12H12FN3.
2-(3,4-Dimethoxyphenyl)-1-(4-fluorobenzyl)-1H-imidazo[4,5-b]pyridine (6a)
A mixture of 10 (0.218 g, 1 mmol) and the Na2S2O5 adduct of 3,4-dimethoxybenzaldehyde (0.27 g, 1
mmol) in DMF (1 mL) was heated at 120°C for 2.5 h. The solvent was evaporated and the oily
residue was purified with column chromatography using EtOAc-MeOH (100:1.5) as eluant, mp 151–
1
153°C, yield 0.21 g (57.8%). H-NMR (benzene-d6, 15°C) δ ppm: 3.24 (s, 3H, 3’-OCH3), 3.28 (s,
3H,
4’-OCH3), 4.7 (s, 2H, N-CH2), 6.34 (d, 1H, J = 8 Hz, H-5’), 6.43–6.47 (m, 2H, H-
2’’,6’’), 6.65 (t, 2H, H-3’’,5’’), 6.68 (dd, 1H, J = 4.8 and 8 Hz, H-6), 6.76 (dd, 1H, J = 1.6 and 8 Hz,
H-7), 7.17 (dd, 1H, J = 8.4 and 1.6 Hz, H-6’), 7.34 (d, 1H, J = 1.6 Hz, H-2’), 8.64 (dd, 1H, J = 4.8
and 1.6 Hz, H-5); COSY: [H-5':H-6'], [H-6:H-7], [H-6:H-5]; NOESY: [H-5’:4’-OCH3], [H-2’:3’-
13
OCH3]; [N-CH2-:H-7],
[N-CH2-:H-6’], [N-CH2-:H-2’’,6’’]; C-NMR, HSQC, and HMBC
(benzene-d6, 15°C) δ ppm: 162.3 (d, J = 244 Hz, C-4’’), 157.6 (C-3a), 156.6 (C-2), 152.1 (C-4’),
150.5 (C-3’), 145.9 (C-5H), 132.8 (d, J = 3.2 Hz, C-1’’), 129.35 (C-7a), 127.9 (d, J = 8.3 Hz, C-
2’’,6’’H), 122.8 (C-1’), 122.2 (C-6'H), 118.1 (C-6H), 117.8 (C-7H), 116.5 (d, J = 21.8 Hz, C-
3’’,5’’H), 113.5 (C-2’H), 111.8 (C-5’H), 55.77
(4’-OCH3), 55.56 (3’-OCH3), 48.0(N-CH2)
(See Supp. Info. pg 16-23); MS (ESI+) m/z: 364 (M+H, %100), C21H18FN3O2.
2-(3,4-Dimethoxyphenyl)-3-(4-fluorobenzyl)-3H-imidazo[4,5-b]pyridine 4-oxide (7b)
7

ACCEPTED MANUSCRIPT
To a stirred solution of 6b (0.19 g, 0.52 mmol) in acetic acid (1 mL), hydrogen peroxide (35%, 0.3
mL) was slowly added. The solution was warmed to 40°C by stirring overnight. The reaction mixture
was diluted with water, extracted with CH2Cl2, and concentrated. The residue was purified by
column chromatography with CH2Cl2: MeOH (100:2–5 gradient), mp 105-110°C, yield 0.08 g (40
%).
1H-NMR (DMSO-d6) δ ppm: 3.68 (s, 3H, 3’-OCH3), 3.83 (s, 3H, 4’-OCH3), 6.1 (s, 2H,
N-CH2), 6.98–7.02 (m, 2H, H-2’’,6’’), 7.045–7.18 (m, 3H, H-5’,3’’,5’’), 7.15 (d, 1H, J = 1.6 Hz,
H-2’), 7.25 (dd, 1H, J = 8.4 and 2 Hz, H-6’), 7.4 (dd, 1H, J = 6.4 and 6.8 Hz, H-6), 7.76 (d, 1H, J =
7.2 Hz, H-7), 8.21 (d, 1H, J = 6.4 Hz, H-5); COSY: [H-5':H-6'], [H-6:H-7], [H-6:H-5]; NOESY:
[H-5’:4’-OCH3], [H-2’:3’-OCH3], [N-CH2-:H-2’], [N-CH2-:H-2’’,6’’]; 13C-NMR, HSQC, and
HMBC (DMSO-d6) δ ppm: 161.2 (d, J = 243 Hz, C-4’’), 155.01 (C-2), 150.7 (C-4’), 148.6 (C-3’),
139.3 (C-7a), 137.6
2’’,6’’H), 122.4 (C-6'H), 120.8 (C-1’), 119.4 (C-6H), 118-3 (C-7H), 115.3 (d, J = 23 Hz, C-
3’’,5’’H), 112.5 (C-2’H), 111.7 (C-5’H), 55.6 (4’-OCH3), 55.4 (3’-OCH3), 48.4 (N-CH2) (See
(C-3a), 134.6 (C-5H), 134.19 (d, J = 3.2 Hz,C-1’’), 128.4 (d, J = 8.3 Hz, C-
Supp. Info. pg 45-51) ; MS (ESI+) m/z: 380 (M+H, %100), C21H18FN3O3.
6-Bromo-2-(3,4-dimethoxyphenyl)-3-(4-fluorobenzyl)-3H-imidazo[4,5-b]pyridine (8a) and
6-bromo-2-(3,4-dimethoxyphenyl)-4-(4-fluorobenzyl)-4H-imidazo[4,5-b]pyridine (8) from 3.
N3 and N4 regioisomers were obtained. Following the addition of water to the reaction medium, the
aqueous solution was extracted with CH2Cl2 and concentrated. The crude mixture was subjected to
chromatography (CH2Cl2: MeOH, 96:4) to give the following products in the following order of
elution.
1
Compound 8a (first eluting fraction) : mp 192–194°C, yield 0.022 g (6.2%). H-NMR (CDCl3) δ
ppm: 3.77 (s, 3H, 3’-OCH3), 3.94 (s, 3H, 4’-OCH3), 5.54 (s, 2H, N-CH2), 6.93 (d, 1H, J = 8 Hz, H-
5’), 6.99 (t, 2H, H-3’’,5’’), 7.08–7.12 (m, 2H, H-2’’,6’’), 7.19 (d, 1H, H-2’), 7.21 (dd, 1H, J = 7.2
and 1.6 Hz, H-6’), 8.2 (d, 1H, J = 2 Hz, H-7), 8.42 (d, 1H, J = 2 Hz, H-5); COSY: [H-5':H-6'], [H-
3’’,5’’:
H-2’’,6’’], [H-5:H-7 (secondary)]; NOESY : [H-5’:4’-OCH3], [H-2’:3’-OCH3]; [N-
13
CH2-: H-2’], [N-CH2-:H-2’’,6’’]; C-NMR, HSQC, and HMBC (CDCl3) δ ppm: 162.3 (d, J =
245 Hz, C-4’’), 156 (C-2), 151.2 (C-4’), 149.3 (C-3’), 147.6 (C-3a), 144.7 (C-5H), 136.2 (C-7a),
132.3 (d, J = 3.2 Hz,
121.7 (C-1’), 115.9 (d, J = 21 Hz, C-3’’,5’’H), 114.4 (C-6), 112 (C-2’H), 111 (C-5’H), 56 (4’-
OCH3), 55.8 (3’-OCH3), 46.5 (N-CH2) (See Supp. Info. pg 58-63) ; MS (ESI+) m/z: 442
(M+H, %100), 444 (M + H + 2, %100) C21H17BrFN3O2.
C-1’’), 129.4 (C-7H), 128.1 (d, J = 7.7 Hz, C-2’’,6’’H), 122.1 (C-6'H),
8

ACCEPTED MANUSCRIPT
Compound 8 (second eluting fraction): mp 172–174°C, yield 0.092 g (26%), light yellow coloured
1
powder. H-NMR (DMSO-d6) δ ppm: 3.84 (s, 3H, 4’-OCH3), 3.87 (s, 3H, 3’-OCH3), 5.83 (s, 2H,
N-CH2), 7.0 (d, 1H, J = 8 Hz, H-5’), 7.2 (t, 2H, H-3’’,5’’), 7.67–7.71 (m, 2H, H-2’’,6’’), 7.92 (d, 1H,
H-2’), 7.99 (dd, 1H, J = 8 and 1.6 Hz, H-6’), 8.3 (d, 1H, J = 1.6 Hz, H-7), 8.42 (d, 1H, J = 1.6 Hz,
H-5); COSY: [H-5':H-6'], [H-3’’,5’’:H-2’’,6’’], [H-5:H-7 (secondary)]; NOESY: [H-5’:4’-OCH3],
13
[H-2’:3’-OCH3]; [N-CH2-:H-5], [N-CH2-:H-2’’,6’’]; C-NMR, HSQC, and HMBC (DMSO-d6) δ
ppm: 169.8 (C-2), 162.1 (d, J = 243 Hz, C-4’’), 153.3 (C-3a), 150.8 (C-4’), 148.7 (C-3’), 146.1
(C-7a), 131.7 (d, J = 3 Hz, C-1’’), 131 (d, J = 9.2 Hz, C-2’’,6’’H), 130.1 (C-5H), 128.6 (C-7H),
126.7 (C-1'H), 121.3 (C-6’H), 115.5 (d, J = 21 Hz, C-3’’,5’’H), 111.6 (C-5’H), 110.8 (C-2’H), 105.1
(C-6), 55.6 (4’-OCH3), 55.5 (3’-OCH3), 55.3 (N-CH2) (See Supp. Info. pg 52-57) ; MS (ESI+) m/z :
442 (M + H, %100), 444 (M + H + 2, %100) C21H17BrFN3O2.
2-(3,4-Dimethoxyphenyl)-1,(3),(5)H-imidazo[4,5-c]pyridine 5-oxide (14)
This compound was prepared as described in 4, starting from 13 (2 mmol, 0.510 g). The powder
precipitate was purified by silica gel column chromatography. First, unreacted 13 was collected by a
mixture of CH2Cl2: EtOH: NH3 (100:20:0.5), and pure compound 14 was obtained by a mixture of
CH2Cl2: EtOH: NH3 (100:20:1) as eluant, mp > 295°C, yield 0.17 g (31%). 1H-NMR (DMSO-d6) δ
ppm: 3.83 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 7.15 (d, 1H, J = 8.4 Hz, H-5’), 7.54 (br.d, 1H), 7.7–
13
7.75 (m, 2H), 7.99 (dd, 1H, J = 6.8 and 1.2 Hz), 8.6 (br.s, 1H); C-NMR (DMSO-d6): 151.11,
1
148.9, 133.9, 121.2, 120.0, 111.9, 110.0, 55.6, and 55.59. H-NMR (DMSO-d6 +NaH+D2O) δ ppm:
3.81 (s, 3H,
4’-OCH3), 3.86 (s, 3H, 3’-OCH3), 7.0 (d, 1H, J = 8.4 Hz, H-5’), 7.36 (d, 1H, J = 8.4
Hz, H-7), 7.71 (dd, 1H, J = 6.4 and 1.6 Hz, 1H,H-6), 7.82 (dd, 1H, J = 8.4 and 2 Hz, 1H, H-6’), 7.89
(d, 1H, J = 2 Hz, H-2’), 8.36 (d, 1H, J = 2 Hz, H-4); COSY: [H-5':H-6'], [H-6:H-7], [H-6:H-4
(secondary)]; NOESY : [H-5’:4’-OCH3],
[H-2’:3’-OCH3]; 13C-NMR, HSQC, and HMBC
(DMSO-d6 + NaH + D2O) δ ppm: 166.6 (C-2), 149.5 (C-4’), 148.8 (C-3’), 147.4 (C-7a), 144.9 (C-
3a), 129.2 (C-1’), 128.8 (C-6H), 126.9 (C-4H), 120.2 (C-6'H), 111.9 (C-5’H), 111.5 (C-7H), 111.1
(C-2’H), 55.92 (4’-OCH3), 55.8
272 (M + H, %100), C14H13N3O3.
(3’-OCH3) (See Supp. Info. pg 75-77) ; MS (ESI+) m/z:
5-Butyl-2-(3,4-dimethoxyphenyl)-5H-imidazo[4,5-c]pyridine HCl (15)
Only the N-5 regioisomer was obtained. K2CO3 (0.1105 g, 0.8 mmol) was added to a suspension of
13 (0.127 g, 0.5 mmol) in DMF (0.5 mL) and stirred. After 1 h, butyl bromide (0.082 g, 0.6 mmol)
9

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was added. After overnight stirring at 35°C, water was added, crystallisation of the precipitate from
MeOH: H2O gave pure 15, mp 95–97°C, yield 0.1 g (64.5%), mp of HCl salt, 200–205°C (bubbling):
1H-NMR (CD3OD) δ ppm: 1.03 (t, 3H), 1.46 (m, 2H), 2.06 (m, 2H), 3.94 (s, 3H, 4’-OCH3), 3.97 (s,
3H,
3’-OCH3), 4.69 (t, 2H), 7.18 (d, 1H, J = 8.4 Hz, H-5’), 7.84 (d, 1H, J = 1.6 Hz, H-2’),
7.87 (dd, 1H, J = 8.4 and 2 Hz, H-6’), 8.08 (d, 1H, J = 6.8 Hz, H-7), 8.66 (d, 1H, J = 6.8 Hz, H-6),
9.34 (s, 1H, H-4) ; COSY: [H-5':H-6'], [H-6:H-7], [H-6:H-4 (secondary)]; [N-5 butyl group as
expected]; NOESY:
[H-5’:4’-OCH3], [H-2’:3’-OCH3]; [N-CH2-:H-4 and 6]; 13C-NMR, HSQC,
and HMBC (CD3OD) δ ppm: 161.9 (C-2), 155 (C-4’), 151.2 (C-3’), 147.6 (C-7a), 140.1 (C-3a),
138.6 (C-6), 135.3 (C-4H), 123.5 (C-6'H), 120.2 (C-1’), 113.1 (C-5’H), 112.5 (C-7H), 112.2 (C-
2’H), 62.4 (N-CH2), 56.9 and 56.8 (OCH3), 34.9 (N-CH2CH2), 20.6 (N-CH2CH2CH2), 13.95 (N…-
.
CH3) (See Supp. Info. pg 78-83) ; MS (ESI+) m/z: 312 (M + H, %100), C18H21N3O2 HCl.
2-(3,4-Dimethoxyphenyl)-5-(4-fluorobenzyl)-5H-imidazo[4,5-c]pyridine (16)
Only the N5 regioisomer was obtained. K2CO3 (0.1105 g, 0.8 mmol) was added to a suspension of 13
(0.127 g, 0.5 mmol) in DMF (0.5 mL) and stirred. After 1 h, p-fluorobenzyl bromide (0.6 mmol) was
added. After overnight stirring at 35°C, water was added, and the crude powder was purified by
crystallisation with EtOH: H2O (50%) to give 16, mp 77–80°C (bubbling), hygroscopic, yield 0.14 g
1
(77.7%). H-NMR (DMSO-d6) δ ppm: 3.82 (s, 3H, 4’-OCH3), 3.86 (s, 3H, 3’-OCH3), 5.63 (s, 2H,
N-CH2), 7.05 (d, 1H, J = 9.2 Hz, H-5’), 7.25 (t, 2H, J = 8.8 Hz, H-3’’,5’’), 7.535–7.57 (m, 2H,
H-2’’,6’’), 7.68 (d, 1H, J = 6.8 Hz, H-7), 7.94–7.96 (m, 2H, H-2’,6’, overlapped), 8.16 (dd, 1H, J =
7.2 and 1.6 Hz, H-6), 9.02 (d, 1H, J = 1.6 Hz, H-4); COSY: [H-5':H-6'], [H-3’’,5’’:H-2’’,6’’], [H-
6:H-7], [H-6: H-4 (secondary)]; NOESY: [H-5’:4’-OCH3], [H-2’:3’-OCH3]; [N-CH2-:H-4], [N-CH2-
13
:H-6], [N-CH2-:H-2’’,6’’]; C-NMR, HSQC, and HMBC (DMSO-d6) δ ppm: 171.44 (C-2), 162.3
(d, J = 243 Hz, C-4’’), 155.9 (C-7a), 150.0 (C-4’), 148.5 (C-3’), 145.6 (C-3a), 132.8 (d, J = 2.6 Hz,
C-1’’), 130.7 (C-6H), 130.45 (C-4H), 130.4 (d, J = 8.4 Hz, C-2’’,6’’H), 127.8 (C-1’), 120.6 (C-6'H),
115.8 (d, J = 21 Hz, C-3’’,5’’H), 111.8 (C-7H), 111.5 (C-5’H), 110.7 (C-2’H), 60.2 (N-CH2), 55.5
(4’-OCH3), 55.3 (3’-OCH3) (See Supp. Info. pg 84-89); MS (ESI+) m/z: 364 (M + H, %100),
C21H18FN3O2.
2-(3,4-Dimethoxyphenyl)-5-(4-fluorobenzyl)-5H-imidazo[4,5-c]pyridine 5-oxide (17)
This compound was prepared as described in 16, starting from 14 (0.135 g, 0.5 mmol), mp 165-
1
167oC, yield 0.155g, 81 %. H-NMR (DMSO-d6) δ ppm : 3.82 (s,3H, 4’-OCH3), 3.86 (s,3H,3’-
OCH3), 5.54 (s, 2H, N-CH2), 7.05(d, 1H ,J = 8.4Hz ,H-5’), 7.26(t, 2H, J= 8.8 Hz, H-3’’,5’’), 7.56-
10

ACCEPTED MANUSCRIPT
7.6 (m, 2H,
H-2’’,6’’), 7.65(d, 1H, J = 6.8 Hz, H-7), 7.93-7.96 (m ,2H, H-2’,6’, overlapped),
8.3(dd, 1H, J = 7.2 and 2.4 Hz, H-6), 9.16(d,1H,J = 2 Hz, H-4) ; COSY : [H-5' : H-6'], [H-3’’,5’’ :
H-2’’,6’’], [H-6 : H-7], [H-6 : H-4(secondary) ; NOESY : [H-5’ : 4’-OCH3], [H-2’ : 3’-
OCH3] ; [N-CH2- : H-4],
[N-CH2- : H-6], [N-CH2- : H-2’’,6’’] ; 13C-NMR, HSQC
]
and HMBC (DMSO-d6) δ ppm : 172.3 (C-2), 165.4 (d, J = 244Hz, C-4’’), 155.45 (C-7a), 150.2
(C-4’), 148.5 (C-3’), 144.6 (C-3a), 132.4 (d, J = 9 Hz, C-2’’,6’’H), 129.1 (d, J = 2.6Hz, C-1’’),
127.8 (C-1’), 127.6 (C-6H), 127.3 (C-4H), 120.6 (C-6'H), 115.6 (d, J = 21 Hz, C-3’’,5’’H), 111.5(C-
5’H), 110.7(C-2’H), 110.5(C-7H), 81.4(N-CH2), 55.5 (4’-OCH3), 55.3(3’-OCH3) (See Supp. Info.
pg 90-95) ; MS (ESI+) m/z : 380 (M+H, %100), C21H18FN3O3.
2-(3,4-Dimethoxyphenyl)-5-(4-fluorobenzyl)(4-2H)-5H-imidazo[4,5-c]pyridine 5-oxide (17a)
0.015g of 17 was kept in room temperature in 0.8 ml of CD3OD for 24 h and solvent evaporated.
1H-NMR (CD3OD) δ ppm : 3.89 (s, 3H, 4’-OCH3), 3.94 (s, 3H, 3’-OCH3), 5.5 2(s, 2H, N-CH2),
7.06 (d,1H,J = 8.8 Hz, H-5’), 7.13(t,2H,J=8.8Hz,H-3’’,5’’), 7.45-7.49(m,2H,H-2’’,6’’), 7.64(d,
1H, J = 7.2 Hz, H-7), 7.89-7.93 (m, 2H, H-2’,6’, overlapped), 8.3 (d, 1H, J = 7.2 Hz, H-6) ; COSY
: [H-5' : H-6'],
OCH3] ;
[H-3’’,5’’ : H-2’’,6’’], [H-6 : H-7] ; NOESY : [H-5’ : 4’-OCH3], [H-2’ : 3’-
[N-CH2- : H-6], [N-CH2- : H-2’’,6’’] ; 13C-NMR, HSQC and HMBC (CD3OD) δ
ppm : 173.8
(C-2), 165.4 (d, J = 246 Hz, C-4’’), 156.4 (C-7a), 152.7 (C-4’), 150.6 (C-3’), 144.95
(C-3a), 133.7(d, J = 9 Hz, C-2’’,6’’H), 130.2 (C-6H), 130.1 (d, J = 3.2Hz, C-1’’), 128.6 (t, J = 29
Hz,C-4D), 127.6 (C-1'H), 122.9 (C-6’H), 116.9 (d, J= 22Hz, C-3’’,5’’H), 112.8 (C-2’H), 112.1
(C-5’H), 83.7 (C-7H), 83.9 (N-CH2), 56.55 (4’-OCH3), 56.53 (3’-OCH3) (See Supp. Info. pg 96-
100) ; MS (ESI+) m/z : 381(M+H, %100), C21H17DFN3O3.
Results and Discussion
Compounds 1–17 were prepared using the methods outlined in Schemes 1 and 4. Cyclisation of
2,3 or 3,4-diaminopyridines with the sodyum metabisulfite adduct of 3,4-dimethoxybenzaldehyde (1)
gave the corresponding imidazopyridines 2, 3, and 13. In this condensed system, the nitrogen bears a
hydrogen atom (N1,3) that behaves as a pyrrole-like N-atom; the other (N4,5) resembles a pyridine-like
N-atom. The hydrogen atom attached to the nitrogen in the 1-position readily tautomerise as 1H, 3H,
4H, and 5H as in compounds 2, 3, and 13 in Schemes 1 and 4. Because of these tautomeric forms,
11

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1
both H and 13C-NMR spectra of unsubstituted imidazopyridines cannot be clear unless
hexamethylphosphoramide-d18 (HMPA-d18) [18] is used as the deuterated solvent. The N-4 and N-5
oxide derivatives (4 and 14) were prepared from 2 and 13 using H2O2 [10,11].
In this study, the 1H and 13C NMR signals of 4 and 14 were assigned by inspection on the basis
of chemical shifts and coupling constants with and without tautomerism. Initially, their NMR spectra
were run under normal conditions in DMSO-d6, followed by the elimination of tautomerism (by
adding a tiny amount of dry NaH and 2–3 drops of D2O). In tautomeric forms, some proton signals
1
appeared as broad peaks in their H-NMR spectra and some carbon peaks disappeared in their 13C-
NMR spectra, whereas in non-tautomeric forms, all of the protons signals with expected splitting
patterns and carbon atoms including C3a/C7a, C4/C7, and C5/C6 were observed with sharp peaks.
Hence, very fine NMR assignments were determined using both 1D and 2D NMR techniques.
Elimination of the NH proton followed by substitution of this nitrogen atom would prevent
rapid tautomerism and lead to a separable mixture of 1H, 3H, 4H, and 5H substituted regioisomers
[7–9,11]. Alkylations of imidazopyridines frequently give different yields of both regioisomers.
Khanna et al. [7] reported that benzylation of imidazo[4,5-b]pyridine with benzylbromide in the
presence of NaH afforded all three possible N-alkylated products, in a ratio of N-1 (1), N-3 (3.6) and
N-4 (1.6) with a 72% yield. Because of the difficulties of this method; e.g., tedious chromatographic
separation procedure and lack of large scale preparation, regioselective methods have been suggested
for both regioisomers.
Macdonald et al. [19] reported that protection of the parent imidazo[4,5-b] pyridine with
2-methoxyethoxymethyl chloride (MEM-chloride) gave a significant amount (55–69%) of the N-3
regioisomer. N-3-protection of 6-Br and 7-Cl-imidazo[4,5-b]pyridines was also achieved with high
N-3 regioselectivity (62–92%). In another study [8], the alkylation reactions of 2-aryl-1(3)H-
imidazo[4,5-b]pyridines with alkoxymethyl chlorides and bromoacetonitril was reported and
attention was drawn to the fact that N-3 alkyl derivatives were produced exclusively in basic
(Et3N/NaH) nonpolar media. It was declared that the formation ratio of the regioisomers could be
changed with the reaction conditions and solvent effects were evident with the loss of N-3/N-1
selectivity when the polar aprotic solvent DMF was used [8]. Zeinyeh et al. [11] stated that
benzylation of imidazo[4,5-b] pyridine-4-oxide and 2-methylimidazo[4,5-b]pyridine-4-oxide with
benzyl bromide or benzyl iodide at RT using K2CO3 in DMF gave the corresponding regioisomers
with only a slight difference in
N-1/N-3 ratios.
There are more samples during the N-alkylation of imidazo[4,5-b]pyridine derivatives mainly
led to N-3 regioisomer [7,20,21]. Conversely, some studies have shown that N-1 regiosomers were
12

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obtained at a higher ratio during N-alkylation of imidazopyridines [22]. Moreover, the influence of
the electronic nature of the C-2 aryl substituent on the formation of regioisomers has been reported.
While this position of imidazopyridines was substituted with a 4-MeO-phenyl group, the N-3 ratio
was found to be higher [8], whereas when substituted with 4-Cl-phenyl, the N-1 ratio was higher
[23]. It has been reported that 4-chloro-1H-imidazo[4,5-c]pyridine alkylated with benzyl bromide or
2-fluorobenzyl bromide in the presence of potassium carbonate gave a mixture of N-1 and N-3
isomers in almost equal ratio [24], whereas treatment of 2-aryl-imidazo[4,5-c]pyridine with benzyl
chloride only produced the N3 regisiomer [25]. Furthermore, 2-phenyl-1(3)H-imidazo[4,5-
c]pyridines were mainly converted substituted 5-benzyl-2-phenyl-5H-imidazo[4,5-c] pyridines with
alkyl bromides and aqueous NaOH solution in DMF [26,27].
Finally, the most accurate result was published in obtaining 4-benzyl-6-bromo-2-(4-
methoxyphenyl)-4H-imidazo[4,5b]pyridine under basic conditions by X-ray data [28]. When we
attempted alkylation of compounds 2 – 4 with n-butyl bromide and 4-fluorobenzyl bromide under
basic conditions (K2CO3, DMF), alkylation occurred primarily at the N-4 position, so compounds
5 – 8 were obtained as the N-4 isomer, consistent with the literature [28]. Benzylation of 2 and 4 also
gave mainly 6 and 7 (N-4) with a small amount of 6b (N-3) and 7a (N-1) in the same reaction
medium (Scheme 2), which were isolated by chance using column chromatography. Both of the third
regioisomers 6a and 7b were followed in their MS chromatogram (Fig. 1) but could not be isolated
since their quantities were too low. In the case of alkylation of 4 with 4-fluorobenzyl bromide,
compound 7, which was present at a higher concentration (tR = 4.1 min) was separated by
crystallisation, then compound 7a (tR = 6.3 min) was isolated by column chromatography from the
supernatant liquid after the separation of 7. Characterisation of the individual isomeric products was
determined by observation of
2D-NOESY enhancements between the N-CH2 and H-7,6,5,4
protons. In the NOESY spectra of compounds 6a and 7a (Fig. 2), strong correlations were observed
between the benzylic and H-7 proton. For compounds 5, 6, 7 (Fig. 3), and 8, NOE cross peaks
between the
N-CH2 protons and H-5 were observed in the NOESY spectra. In the literature [11],
NOESY interaction was reported between the benzylic protons and H-5 for compound 3-benzyl-3H-
imidazo[4,5-b]pyridine 4-oxide. However, we do not believe this is possible, since the distance is too
far for NOE interaction. It is likely they obtained the N-4 isomer, instead of the N-3 isomer, as we
observed that none of our N-3 regioisomers gave similar NOE interactions at the same position.
N-oxide groups could not be accounted as a protection group; this nitrogen atom is easily alkylated
as shown in Schemes 2 and 4. In the LC-MS chromatogram of compound 6 (Fig. 1), the Rt values of
the N-1 (7.1 min) and N-3 (7.5 min) regioisomers are close to one another (i.e., this is why it is
difficult to separate them from each other), whereas the N-4 (3.95 min) regioisomer is far away from
13

ACCEPTED MANUSCRIPT
the N-1 and N-3 peaks and the peak area of N-4 is greater than the other isomers. Hence, the N-4
regioisomer could be easily separated from the others, as shown in our experiments. The NOE effect
is weaker when the nitrogen atoms have oxides, so a stronger NOE effect was observed in compound
6 compared with its N-oxide derivative 7.
For structural elucidation of the regioisomers, in addition to NOESY data, selective
magnetisation transfer experiments, namely the INAPT method [8], have been used for determining
the correlation of C-3a,7a,2 with N-CH2 protons. Since this application is known today as HMBC
(Heteronuclear Multiple Bond Correlation), we also carried out these experiments for all of our
synthesised compounds and obtained good profiles with expected correlations in the HMBC spectra.
It is possible to determine the chemical shift value (δ ppm) of C-3a and C-7a by their correlations
with pyridine hydrogens in their HMBC spectra. Thus, it should be possible to determine other
possible correlations (whether N-CH2: C-3a or N-CH2: C-7a) for the separation of regioisomers, as
observed in the gHMBC spectrum of 6b (Fig. 4).
Finally, to verify both NOESY and gHMBC data, we designed a regioselective synthesis
method for 6a, 6b, 7a, and 7b (Scheme 3). Reaction between 2,3-diaminopyridine and 4-
fluorobenzaldehyde gave the selective formation of a Schiff base 9. Reduction of 9 with NaBH4
formed 10. Cyclisation of 10 with the Na2S2O5 adduct of 3,4-dimethoxybenzaldehyde gave 6a with a
good yield. Oxidising 6a with H2O2 gave 7a. For the N-3 regioisomer, compounds 11 and 12 were
prepared according to the literature [16]. Cyclisation of 12 with the Na2S2O5 adduct of 3,4-
dimethoxybenzaldehyde gave 6b in good yield. Finally, 6b was converted to 7b with H2O2. The RT
values of 6a and 7b exactly correspond with the third unknown peaks obtained in the LC-MS
chromatograms of 6 and 7 (at the end point of the reaction).
Butylation and 4-fluorobenzylation of 13 and 14 (Scheme 4) in the same conditions gave
mainly 5H isomers (15–17) as recently reported in the literature [26,27], hence very strong NOE
interactions have been demonstrated between N-CH2 and H-4,6 in the NOESY spectra of 15, 16 (Fig.
5), and 17.
Actually, there are no important differences in the 1H-NMR spectra of 6 and 7, and 16 and 17.
However, in the carbon spectrum of 16, benzylic carbon was observed at 60.2 δ ppm, whereas in the
carbon spectrum of 17, the corresponding carbon was observed at 81.4 δ ppm because of the
paramagnetic effect of N-oxides. Similar results were shown for compounds 6 (54.9 δ ppm) and 7
(79 δ ppm). The complete assignments of all synthesised compounds were made using 1D and 2D
NMR experiments including COSY, NOESY, gHSQC, and gHMBC techniques.
14

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Compound 17 was totally converted to its C4-D form (17a) by keeping it at RT in CD3OD for
24 h. This was also observed with D2O in DMSO-d6 after 20 days. The C6-H signal appeared at 8.3 δ
1
ppm as dd (Jm,o = 2.4 and 7.2 Hz) in the H-NMR spectrum of 17, whereas the same proton was
1
observed at the same point, but only d (Jo = 7.2 Hz) coupling in the H-NMR spectrum of 17a as
C4-H was transformed to C4-D. Furthermore, in the 13C spectrum of 17a, the C-4 signal was
observed at 128.6 δ ppm as t (J = 29 Hz) due to the spin-spin coupling pattern rules [2NI+1]. For this
reason, the H-4 atoms of imidazo[4,5-c]pyridine 5-oxides are easily converted to chlorine by POCl3
[10, 22, 29].
Compound 6 was also prepared from 2, with dry NaH and p-fluorobenzylbromide in DMF, and
its LC-MS chromatogram (See Supp. Inf. for LC-MS pg 13) was similiar to our previous data
obtained with K2CO3. There were no important differences in the formation ratio of regioisomers.
Conclusions
It was found that the N-4 and N-5 regiosomers were formed at higher ratio during the N-alkylation
of 2-(3,4-dimethoxyphenyl)imidazo[4,5-b]pyridine (2) and 2-(3,4-dimethoxyphenyl)imidazo[4,5-c]
pyridine (13), respectively. Based on our experience and literature examples that support us, we
recognize that the previous tautomeric forms corresponding to these regioisomers are more stable.
NOESY was used for the structural elucidation of these regioisomers. The cross peaks for N-CH2
protons (at approximately around 5–6 ppm) and H-4,5,6,7 protons (at approximately 7–8 ppm)
confirms their spatial proximity as evidence for N-1,3,4,5-substitution. gHMBC analysis could be
effective as an alternative way for assignment of the regiosomers.
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Acknowledgement : Central Laboratory of Pharmacy Faculty of Ankara University provided
support for the acquisition of the NMR and mass spectrometer used in this work.
19

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Fig. 1. MS chromatogram (for 364 m/e) for 6, 6a & 6b at the endpoint of synthesis.

ACCEPTED MANUSCRIPT
Fig 2. Partial NOESY spectrum of compound 7a showing cross
peaks between N-CH2 and H-7, 2’,2’’,6’’ (in DMSO-d6).

ACCEPTED MANUSCRIPT
Fig 3. Partial NOESY spectrum of compound 7 showing cross peaks between
N-CH2 and H-5, 2’’,6’’ (in DMSO-d6).

ACCEPTED MANUSCRIPT
Fig. 4 gHMBC spectra of compound 6b, showing cross peaks
between N-CH2 / C-2, C-3a, C-1’’, C-2’’,6’’correlations (in CDCl3).

ACCEPTED MANUSCRIPT
Fig 5. Partial NOESY spectrum of compound 16 showing cross peaks between
N-CH2 and H-4, 6, 2’’,6’’ (in DMSO-d6).

ACCEPTED MANUSCRIPT
OMe
OMe
(4H)
N
OMe
(4H)
R
N
OMe
OMe
OMe
H
O
N
N
N
O
N
4
4
H
H
a
OMe
OMe
OMe
OMe
(3H)
(1H)
(3H)
(1H)
NaO3S
R
N
N
OMe
OMe
OH
R
NH2
NH2
N
H
3
N
H
3
N
N
O
1
b
N
R : H, Br
OMe
OMe
OMe
OMe
H
H
R
1
N
1
N
N
N
N
N
2
R : H
3
R : Br
4
O
Reagents : a) Na2S2O5 b) H2O2
Scheme 1. Synthesis of 2 – 4 and their tautomeric forms.

ACCEPTED MANUSCRIPT
OMe
OMe
N
N
N
a
OMe
OMe
N
N
N
H
HCl
5
2
OMe
OMe
**
OMe
F
N
N
2'
N
N
1'
OMe
N
OMe
OMe
b
2''
N
+
N
N
3''
2
6' 5'
+
6''
F
N
5''
F
6b (9.5 %)*
6a (2 %)*
6 (88 %)*
F
**
OMe
OMe
OMe
N
N
OMe
OMe
OMe
OMe
OMe
N
N
N
b
N
N
O
N
N
+
+
O
N
N
O
N
F
O
H
F
4
7a (3 %)*
7b (4 %)*
7 (93 %)*
OMe
OMe
OMe
Br
N
N
Br
N
N
OMe
OMe
OMe
b
Br
N
N
N
N
+
N
H
F
3
F
8 (70%)*
8a (12%)*
Reagents : a) K2CO3 / n-butyl bromide b) K2CO3 / 4-F-Benzyl bromide. *These values were calculated from MS
chromatograms ** Could not be isolated.
Scheme 2. N 1,3,4- Butylation and benzylation of imidazo[4,5-b]pyridines.

ACCEPTED MANUSCRIPT
F
F
c
NH2
NH2
N
NH
NH2
a
b
N
N
NH2
N
9
10
F
F
OMe
OMe
OMe
N
N
N
N
d
OMe
N
O
N
N
7a
6a
NH2
NH
NO2
NH
NO2
Cl
c
e
f
N
N
12
11
F
F
OMe
OMe
OMe
N
N
N
N
OMe
d
N
N
O
F
F
7b
6b
Reagents : a) 4-F-benzaldehyde b) NaBH4
c) Na2S2O5 adduct of 3,4-dimethoxybenzaldehyde (1)
d) H2O2 e) 4-F-Benzylamine f) H2 / Pd.C
Scheme 3. Selective synthesis of 6a - 6b and 7a - 7b.

ACCEPTED MANUSCRIPT
OMe
(5H)
OMe
OMe
(5H)
OMe
OMe
N
N
N
N
H
O
OMe
HN
5
HN
5
O
O
a
OMe
OMe
OMe
OMe
OMe
OMe
NaO3S
NH2
(3H)
(3H)
N
N
OH
b
N
N
N
N
1
N
H
N
H
3
3
N
NH2
(1H)
(1H)
OMe
OMe
OMe
OMe
H
N
H
N
1
1
N
N
O
14
13
OMe
OMe
N
a
OMe
N
N
N
OMe
HN
N
HCl
13
15
b
*
OMe
OMe
F
OMe
N
N
N
OMe
N
OMe
N
+
N
+
N
N
OMe
N
F
16
F
OMe
OMe
OMe
OMe
N
O
N
N
OMe
OMe
O
N
N
N
N
b
N
D
c
HN
O
17a
17
14
F
F
Reagents : a) K2CO3 / n-butyl bromide b) K2CO3 / 4-F-Benzyl bromide c) CD3OD
* Could not be observed in its LC-MS chromatogram
Scheme 4. N5- Butylation and benzylation of imidazo[4,5-c]pyridines.