Design, synthesis and anti-HBV activity evaluation of new substituted imidazo[4,5-b]pyridines

Article abstract of DOI:10.1016/j.bioorg.2020.103580

The design and synthesis of a number of new imidazo[4,5-b]pyridines is described. The heterocyclic scaffold possesses 6-chloro- or 5,6-dichloro-substitution and bears various 2-alkylamino-methyl or ethyl groups. The corresponding N¹ and N³-tosylates are also presented. The anti-HBV activity of the compounds was evaluated in HBV infectious system at the level of HBV rcDNA secretion and CC₅₀, EC₅₀ and selectivity index values were determined. The tosylates showed low antiviral potency and relatively high cytotoxicity, on the contrary, a number of 2,5 and/or-6-substituted imidazopyridines, mainly those belonging to the 6-chloroimidazo[4,5-b]pyridine series, were endowed with a very interesting profile and were further investigated. The most promising among them, along with the reduction of the secreted HBV rcDNA, also caused a reduction in HBV cccDNA and pgRNA levels, with a concomitant accumulation of the intracellular encapsidated rcDNA. Surprisingly, the most active 2-diethylaminoethyl-substituted derivative (21d), was highly competitive to interferon.

Full text of DOI:10.1016/j.bioorg.2020.103580

BioorganicChemistryxxx(xxxx)xxxx
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and anti-HBV activity evaluation of new substituted
imidazo[4,5-b]pyridines☆
Maria Gerasia,1, Efseveia Frakolakib,1, Georgios Papadakisa, Anna Chalarib, Nikolaos Lougiakisa,
⁎
⁎
Panagiotis Marakosa, Nicole Poulia, , Niki Vassilakib,
a Division of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
b Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias ave., 11521 Athens, Greece
A R T I C L E I N F O
A B S T R A C T
Keywords:
The design and synthesis of a number of new imidazo[4,5-b]pyridines is described. The heterocyclic scaffold
possesses 6-chloro- or 5,6-dichloro-substitution and bears various 2-alkylamino-methyl or ethyl groups. The
corresponding N1 and N3-tosylates are also presented. The anti-HBV activity of the compounds was evaluated in
HBV infectious system at the level of HBV rcDNA secretion and CC50, EC50 and selectivity index values were
determined. The tosylates showed low antiviral potency and relatively high cytotoxicity, on the contrary, a
number of 2,5 and/or-6-substituted imidazopyridines, mainly those belonging to the 6-chloroimidazo[4,5-b]
pyridine series, were endowed with a very interesting profile and were further investigated. The most promising
among them, along with the reduction of the secreted HBV rcDNA, also caused a reduction in HBV cccDNA and
pgRNA levels, with a concomitant accumulation of the intracellular encapsidated rcDNA. Surprisingly, the most
active 2-diethylaminoethyl-substituted derivative (21d), was highly competitive to interferon.
Purine
Imidazopyridine
Benzimidazole
hepatitis B virus
Viral replication
1. Introduction
dslDNA (10%) [5,6]. The rcDNA synthesis is initiated when the epsilon
(ɛ) stem-loop near the 5′ end of pgRNA binds to the reverse tran-
Hepatitis B virus (HBV) is the major causative factor of chronic
hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) [1]. De-
spite an efficient prophylactic vaccine, about 257 million people are
estimated to be chronically infected by HBV, and > 850,000 people die
annually due to HBV-related complications [2]. HBV belongs to He-
padnaviridae family and is classified into four serotypes (adw, adr, ayw,
and ayr) and at least 10 genotypes (A to J) and several subtypes with
specific geographical distributions [3].
scriptase (RT) domain of HBV polymerase in a manner depended on
host chaperone proteins, such as Hsp90 [7,8]. This binding is necessary
for the assembly of core protein into nucleocapsids and the initiation of
reverse transcription. The polymerase first synthesizes the minus-strand
DNA, concurrently degrading the template pgRNA, and then the plus-
strand DNA. This procedure is modulated by the capsid environment, as
phosphorylation of core protein in the immature nucleocapsids is re-
quired for the minus-strand DNA synthesis, while dephosphorylation
facilitates plus-strand DNA synthesis and particle maturation [9]. The
DNA-containing particles are directed for envelopment and secretion,
although the specific signal is not yet fully understood, or re-trans-
ported into the nucleus to maintain the cccDNA pool [10]. The exact
mechanism for this intracellular amplification of cccDNA is not known,
however it has been reported that it is regulated by the viral envelope
proteins [11], as well as host DNA polymerase alpha [12]. dslDNA can
also integrate into the host cell genome [6].
HBV is the smallest human DNA virus with a genome of 3200 bp
[4]. The HBV genome carries four open reading frames (ORFs), en-
coding seven different proteins: three surface proteins (L-, M-, and S-
HBsAg), core (HBcAg) and e (HBeAg) antigens, polymerase (P protein)
and the X protein (HBx). Upon infection, the viral genome, a relaxed
circular partially double-stranded DNA (rcDNA), is delivered in the
nucleus where it is converted into covalently closed circular DNA
(cccDNA), the transcriptional template for six viral RNAs. After trans-
port to the cytoplasm the 3.5 kb pre-genomic RNA (pgRNA) is en-
capsidated and reverse transcribed to rcDNA (90%) and occasionally
Current available antiviral therapy for chronic hepatitis B (CHB)
includes nucleos (t)ide analogues (NAs) and pegylated interferon (PEG-
☆ This work is dedicated to the memory of Professor George B. Foscolos
⁎ Corresponding authors.
E-mail addresses: pouli@pharm.uoa.gr (N. Pouli), nikiv@pasteur.gr (N. Vassilaki).
1 Equal contribution.
https://doi.org/10.1016/j.bioorg.2020.103580
Received 26 July 2019; Received in revised form 18 December 2019; Accepted 10 January 2020
0045-2068/©2020ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:MariaGerasi,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2020.103580

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
IFN) [13]. PEG-IFN, the first treatment approved for CHB, inhibits the
HBV lifecycle by inducing long-term immune control of the virus. It has
the benefit of finite treatment duration, absence of resistance and
higher rates of HBeAg and HBsAg seroconversion as compared to NAs.
However, PEG-IFN is not used widely because of low effectiveness
against viral replication, the inconvenience of frequent subcutaneous
injections and severe side effects [14].
target(s) of this class of compounds was also investigated, focusing on
the benzimidazole derivative BM601 [34]. This compound was found to
selectively reduce the secretion of hepatitis B virions and HBsAg,
probably resulting in a decrease of HBV DNA levels and interference
with HBsAg-mediated immunosuppression [35].
In continuation of our previous research on the discovery of
bioactive purine isosters [36–38] and in an effort to contribute to the
structure–activity relationship studies of the above mentioned benzi-
midazoles, we have designed and synthesized a number of novel imi-
dazo[4,5-b]pyridines in order to gain insight in their biological activity
as potential HBV inhibitors. With the aim to investigate the optimum
substitution pattern of this new central scaffold, we have introduced
alkyl linkers with variable length, bearing four different terminal
amines on 2-position of the bicyclic ring system, we have inserted one
or two chlorine atoms on the pyridine ring and we have prepared the
corresponding N1 and N3 tosylates as well.
Concerning NAs, the currently approved, recommended as first-line
therapy, are tenofovir disoproxil fumarate (TDF), tenofovir alafenamide
fumarate (TAF), and entecavir (ETV) [15,16]. They inhibit HBV re-
plication at three distinct phases: the HBV DNA polymerase priming,
the reverse transcription from the pgRNA, and the synthesis of the
positive-strand of rcDNA [17,18]. TDF, TAF and ETV are very effective
in suppressing viral replication and show a high barrier to resistance
[15]. However, treatment with NAs is not curative for the vast majority
of patients, with low rates of HBV seroclearance [19], and consequently
require lifelong therapy which is of high cost. Also, NAs have no direct
effect on viral transcription or the de novo formation and stability of the
cccDNA and the integrated HBV DNA, which persist in the nucleus of
the liver cell, even in people being treated [6,20]. Thus, the discovery of
novel antivirals that will eliminate or functionally inactivate cccDNA is
necessary for the 'definitive cure' of CHB.
2. Results and discussion
2.1. Chemistry
For the synthesis of the target compounds, we used as starting
materials the previously reported 5-chloro-3-nitropyridin-2-amine (1),
easily prepared upon nitration of the corresponding aminopyridine
[39], as well as 5,6-dichloro-3-nitropyridin-2-amine (6), which was
prepared according to the reactions presented in Scheme 1. Thus,
commercially pyridinamine 2 was converted to the pivaloyl derivative
3 [40], which was treated with N-chlorosuccinimide and the resulting
amide 4 [41] was successively hydrolyzed and nitrated to result in the
nitropyridine 6. This compound has already been reported and was
synthesized from the chlorination of 5- or, alternatively, 6-chloro-3-
nitropyridin-2-amine [42,43].
Due to the limitations of the available therapeutics for CHB, new
drugs are being developed to either directly target the viral life cycle or
modulate the immune responses to HBV [21]. Concerning direct-acting
antivirals that are in clinical trials, Tenofovir exalidex (TXL), a prodrug
of tenofovir, is a novel viral DNA polymerase inhibitor [22]. Myrcludex-
B prevents HBV entry via binding to the viral receptor sodium taur-
ocholate cotransporting polypeptide (NTCP) [23]. RNA interfering
(RNAi) gene silencers affect viral translation and genome encapsidation
[24]. HBV core protein allosteric modulators (CpAM) disturb nucleo-
capsid formation [25], while agents targeting HBsAg prevent its se-
cretion out of hepatocytes [26]. Immune response modulators include
toll-like receptor (TLR) agonists and immune checkpoint inhibitors,
which enhance innate immune responses, as well as therapeutic vac-
cines, that activate adaptive immunity [13].
Each nitropyridine 1 or 6 was treated with chloracetyl chloride or 3-
chloropropionyl chloride (Scheme 2) and the resulting chloracetamides
7–10, upon hydrogenation in the presence of Raney Ni as catalyst were
converted to the pyridinamines 11–14 respectively. These pyr-
idinamines were subjected to nucleophilic substitution by selected
amines, namely piperidine, N-methylpiperazine, morpholine and die-
thylamine to provide the amines 15a-d- 18a-d, which were ring-closed
upon heating in PPA, to give the target imidazopyridines 19a-d − 22a-
d.
In recent years the extensive list of miscellaneous classes of non-
nucleoside derivatives is being actively investigated [27], with the aim
to identify novel, safe and efficacious antiviral agents. These can in-
terfere with diverse drugable targets, other than HBV polymerase and
may be useful in overcoming problems associated with nucleoside
analogue cross-resistance [28]. Furthermore, they may open opportu-
nities for combination regiments in HBV infection management, re-
sulting in improvement of response to current therapies. Within this
context, a series of benzimidazole derivatives bearing a variety of
substitution patterns have been recently reported to possess interesting
anti-HBV activities [29]. The fundamental benzimidazole heterocyclic
scaffold has long drawn attention in medicinal chemistry [30]. Being
isosteric and functionally competitive to the privileged purine ring, it is
frequently present in several approved and investigational drugs [31].
In this case, the initial hit, the dichlorobenzimidazole derivative A
(Fig. 1), was initially identified from a random screening test of a small
library of compounds as moderate HBV inhibitor. This derivative was
subjected to lead optimization studies, to result in comparably active
and highly selective compounds B and C. The N1-substitution is gen-
erally important for HBV inhibitory activity, albeit several substituents,
with different lipophilic, electronic, and steric characters are well tol-
erated in this position. The presence of a flexible ethyl linker with a
terminal amide group was initially indicated as essential for HBV ac-
tivity, which increases with the replacement of the phthalimide group
with maleimide. However, further SAR and water solubility optimiza-
tion studies resulted in even more promising benzimidazole derivatives,
which possess a pyrrol-2-yl- or pyridin-2-yl ethyl group at position-2 of
the imidazole ring and lack the 5,6-dichlorosubstitution (for example
compound D) [32], or have instead a large 5- or 6-substituent (for ex-
ample compound E) [33]. In a parallel research, the possible molecular
From the ring-closure of 17b and 18b we have also isolated as by-
products the olefins 23 and 24 (Fig. 2), which obviously resulted from
the elimination of N-methylpiperazine.
The next step involved the introduction of a tosyl group to the
imidazopyridines 19a-d − 22a-d. The imidazopyridines were thus
treated with tosyl chloride in the presence of NaH and both possible
regio-isomers were formed from each one of the initial heterocycles
(Scheme 3). In each case, a substantial regio-selectivity was obtained,
concerning N1-substitution.
We have studied the tosylation reaction conditions, in an attempt to
improve the yield of the minor isomer. More specifically, we used a
variety of reaction temperatures for the conversion of 21a to 29a and
31a. The results, concerning the obtained yield for each isomer, are
presented in Table 1. We concluded that the addition of tosyl chloride
to the mixture of the imidazopyridine in DMF, in the presence of NaH,
at room temperature, provided the optimum percentage in N3-isomer
and in parallel the best overall yield.
The chromatographic purification of the isomeric mixtures proved,
in certain cases, not to be too simple. Concerning the tosylation of 22a
and 22b, only the major isomers 30a and 30b were isolated pure,
whereas the minor ones (32a and 32b) were obtained in mixture with
the corresponding N1-counterparts. In addition, attempts to isolate pure
the diethylamino-analogues of 26 and 28 were unsuccessful, even if
indication for their formation could be detected in the corresponding
1H NMR data, consequently these compounds are not considered
2

M. Gerasi, et al.
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Fig. 1. Representative benzimidazole derivatives with anti-HBV activity.
further in this study. Furthermore, from the tosylation of the diethy-
laminoimidazopyridines 21d and 22d we did not isolate the anticipated
tosylates, but the olefinic derivatives 33 and 34 corresponding to the
dominant N1-regio isomers (Fig. 3).
levels. Cells treated with the solvent DMSO were used as negative
control. The cytotoxic concentrations leading to death of 50% of the
cells (CC50) were calculated by regression analysis (Table 2). Next, we
performed an initial anti-HBV screening at concentration of 50 μΜ for
the safest compounds, whereas for the ones showing cytotoxicity at 50
μΜ, the maximum concentration used was 4–5 fold lower than the re-
spective CC50. HepG2-NTCP cells were cultured in the presence of the
compounds for 2 h before infection, during HBV inoculation (24 h) and
for an additional 6 days. DMSO-treated cells were used as negative
control. Virus titers were determined in cell supernatants by isolating
and quantifying HBV rcDNA of the released virions (qPCR). Moreover,
median effective concentration (EC50) values of the analogues against
HBV were determined using serial dilutions in dose-response analyses
and curve-fitting software (Table 2). Also, the selectivity index (SI) that
estimates possible future clinical potential, was determined as the ratio
A preliminary indication of the regio-selectivity of the tosylates can
be extracted from the 1H NMR spectral data, since, concerning the N1-
isomers, H-7 appears significantly deprotected, when compared to the
corresponding H-7 of the N3-isomers. However, the unambiguous de-
termination of the substitution pattern regarding the tosylates was ef-
fected using NOE spectral data. Thus, in the case of the N1-isomers we
observed a clear cross-peak between the ortho protons of the p-tosyl
group and H-7 in the scaffold. This correlation was not present in the
case of the N3-isomers (Fig. 4).
2.2. Biological evaluation
CC50/EC50
.
Based on the initial tests, we have concluded that the introduction
of a N-tosyl substituent on the imidazole ring is not in favor of the
activity, since derivatives 25a-d, 26a-c, 27a-d, 28a-c, 29a-c, 30a-c,
31a-c, 32c, 33 and 34 display low antiviral potency, and confer rela-
tively high cytotoxicity against host cells (Table 2, Fig. 5). The olefinic
derivatives 23 and 24 are highly cytotoxic, most probably because they
are easily subjected to nucleophilic attack on the double bond. We have
therefore decided to concentrate our efforts towards more extensive
The anti-HBV activity and cytotoxicity of the new compounds were
evaluated in an HBV infection system based on viral particles produced
from HepAD38 cells under the control of a tetracycline-regulated pro-
moter [44] and used to infect HepG2-NTCP cells, which express the
HBV receptor sodium taurocholate cotransporting polypeptide (NTCP)
[45]. First, we assessed the effect of compounds on the viability of
HepG2-NTCP cells, upon treating the cells with serial dilutions of each
compound (0.1–200 μM) for 7 days and quantifying intracellular ATP
Scheme 1. Reagents and conditions: (a) (CH3)3CCOCl, Et3N, dry DCM, r.t., 20 h, (b) NCS, dry DMF, 100 °C, 16 h, (c) c.HCl, EtOH/H2O, 60 °C, 2 h (d) c.HNO3,
c.H2SO4, r.t.,40 min.
3

M. Gerasi, et al.
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Scheme 2. Reagents and conditions: (a) chloroacetyl chloride, or 3-chloropropionyl chloride, dry DMF, r.t., 16 h, (b) H2, Raney Ni, EtOH, 34 or 57 psi, 8 h, (c)
appropriate amines, KI, EtOH, 60 0C, 2 h, (d) PPA, 95 0C, 2.5 h.
terminal amines shows that the diethylamine-substituted derivative
21d is about 2-fold more active than the piperidine- and N-methylpi-
perazine counterparts 21a and 21b respectively. It is noticeable that
none of the morpholine-bearing derivatives proved to possess clear
antiviral activity. Based on these data, for the most promising com-
pounds 19d, 20a, 21a,b and 21d, a dose-response curve analysis is
presented (Fig. 6).
Based on the above, we have identified potent imidazopyridines
showing anti-HBV activity in the low micromolar range, resulting in the
reduction of extracellular HBV rcDNA.
For defining the specific step of the viral life cycle targeted by the
imidazo[4,5-b]pyridines, the most active monochloro diethylami-
Fig. 2.
noethyl-substituted derivative 21d, in comparison to the respective
diethylaminomethyl analogue 19d, and the most active di-
chlorosubstituted derivative 20a were selected and their effect on the
levels of HBV cccDNA in the nucleus, pgRNA, as well as rcDNA of cy-
toplasmic and secreted virions were determined by RT-qPCR/qPCR
(Fig. 7A). Specifically, HepG2-NTCP cells were treated with 50 μM of
the compounds for 2 h before infection. HBV virions were subsequently
added for 24 h and cells were further cultured for 6 days in the presence
of the compounds. DMSO-treated cells were used as negative control.
Total RNA was isolated for pgRNA quantification by RT-qPCR. In ad-
dition, cell nuclei and cytoplasm were separated for HBV cccDNA and
rcDNA determination by qPCR, respectively. Secreted HBV rcDNA le-
vels in cell supernatants were quantified by qPCR.
evaluation of compounds 19a-d − 22a-d which present a more inter-
esting profile.
Concerning the series of imidazopyridines 19a-d − 22a-d, we can
assume that monochlorosubstitution (compounds 19a-d and 21a-d) of
the central scaffold is preferable over dichlorosubstitution (compounds
20a-d and 22a-d). Among dichlorosubstituted derivatives only the 2-
piperidinemethyl analogue 20a exhibits interesting antiviral activity
(EC50 = 14.08 µM), with an acceptable SI value exceeding 14.2. From a
structure activity point of view, it seems that, with the exception of
20a, the ethylene linker and the flexible diethylamine substitution are
best tolerated at position 2 of imidazole ring. Specifically, the 2-die-
thylaminoethyl- derivative 21d displays three times more potent anti-
HBV activity (EC50 = 13.53 µM) than the corresponding 2-diethyla-
minomethyl- analogue 19d, whereas low cytotoxicity is also noted,
resulting in a SI value of more than 14.8. In addition, comparison of the
We observed that, in addition to the reduction of the secreted
rcDNA, pgRNA levels were significantly decreased in the presence of
the compounds. Interestingly, the inhibitors also caused a reduction of
30–45% in the levels of cccDNA, which is important, as cccDNA
4

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
Scheme 3. Reagents and conditions: (a) NaH (60% in mineral oil), dry DMF, 0 °C, 10 min., then p-tosyl chloride, r.t., 20 min.
persistence is a major barrier to eradicating chronic HBV infection [20].
Surprisingly, we detected a concomitant accumulation of the rcDNA
contained in intracellular particles, suggesting that a later step of HBV
DNA replication/nucleocapsid maturation or particle secretion is tar-
geted by the inhibitors. This is in contrast to NAs, which target the early
steps of HBV DNA synthesis [18]. Moreover, the inhibition profile of
our compounds differs from the one reported for the structurally related
anti-HBV dichlorobenzimidazoles, which also reduce the extracellular
HBV DNA, but do not affect HBV transcription and DNA synthesis [34].
In agreement with these results, in vitro synthesis of HBV DNA was
not inhibited in an HBV endogenous polymerase assay (EPA), (Fig. 7B),
suggesting that 21d does not directly target viral polymerase. In addi-
tion, 21d did not reduce the levels of HBV cccDNA at an early time
point post infection (days 1–2) (Fig. 7C), when cccDNA is mainly pro-
duced from de novo HBV infection. However, an arrest in cccDNA
synthesis is observed only after 3 days of infection, favoring the pos-
sibility that 21d negatively affects the intracellular amplification of
HBV cccDNA, rather than blocking the incoming rcDNA.
Table 1
Correlation of the reaction temperature to the yield of N1- and N3-isomers 29a
και 31a respectively.
Temperature
Yield for N1-isomer (29a)
Yield for N3-isomer (31a)
Total yield
2–5 °C
25 °C
40 °C
80 °C
67%
68%
24%
traces
4%
7%
2%
–
71%
75%
26%
traces
Taken together, our results support the notion that the imidazo[4,5-
b]pyridines inhibit HBV secretion by targeting a later stage of HBV DNA
replication, not directly acting on the polymerase, but either on the
interaction of HBV DNA with the capsid, or the nucleocapsid trafficking
after maturation. Although they have a lower anti-HBV activity as
compared to the reported ones for the NAs Tenofovir (TFV) and its
approved bis-alkoxyester prodrug Tenofovir disoproxil fumarate (TDF),
they show a different mechanism of action. A direct comparison to NAs
cannot be made, as NAs activity (TFV EC50
= 1.1 μM, TDF
EC50 = 0.02 μM) has been previously determined by quantifying the
intracellular HBV rcDNA [46], which, in the case of our imidazopyr-
idine derivatives, is not inhibited. Moreover, the most effective imida-
zopyridines exhibit CC50 values higher than 200 μΜ, similarly to Te-
nofovir [47]. Based on the unique inhibition profile of 20a and 21d,
they could be used as lead compounds for further optimization.
To investigate the effect of the new compounds in combination with
other HBV treatments, HBV-infected cells were cultured in the presence
Fig. 3.
5

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
Fig. 4.
of the most active compound, 21d and/or pegylated interferon a2b
(Peg-IFN). Surprisingly, 21d was highly competitive to IFN (Fig. 7D
left), as shown by the high coefficient of drug interaction (CDI) values
(Fig. 7D right). A possible explanation for this competitive relationship
is that 21d could block the interferon signaling through the prevention
of particle maturation and the consequent excess of free HBsAg and
HBeAg that are not used in particle envelopment. It has been shown
that both viral proteins interfere with various steps of interferon sig-
naling and innate immune response [48]. Finally, this is in contrast to
the synergistic anti-HBV effect of NAs and interferon combination
[49,50].
Table 2
EC50, CC50 and SI of the new imidazopyridines against HBV particle production.
Compd
CC50 (μM)
EC50 (μΜ)
SI
19a
19b
19c
19d
20a
20b
20c
20d
21a
21b
21c
21d
22a
22b
22c
22d
23
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
50.54
49.88
0.96
> 4.0
> 50
–
> 50
–
34.79
4.66
0.60
> 5.7
14.08
> 14.2
> 50
–
> 50
–
> 50
–
21.14
1.01
1.45
> 9.5
25.31
> 7.9
> 50
–
3. Conclusions
13.53
0.92
> 14.8
0.30
(> 10)*
> 50
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
In conclusion, series of new 2-aminoalkylsubstituted 6-chloro- or
5,6-dichloro-1H-imidazo[4,5-b]pyridines were synthesized, along with
their corresponding N1 or N3-tosylates. Their anti-HBV activity was
evaluated in an efficient infectious system and for the most promising
compounds 19d, 20a, 21a,b and 21d, a dose-response curve analysis
was performed. The most active monochloro diethylaminoethyl-sub-
stituted derivative 21d was selected and underwent an in-depth study
together with its close diethylaminomethyl analogue 19d and a re-
presentative dichloro-derivative (20a), which was found to possess
comparable activity to 21d. Apart from their effect on HBV rcDNA
secretion, all compounds caused a reduction of HBV cccDNA and
pgRNA levels, with a concomitant accumulation of the intracellular
encapsidated rcDNA. This could imply intervention in a later step of
HBV DNA replication or the nucleocapsid maturation. Finally, in a
drug-drug interaction experiment, the most active compound 21d was
found to possess competitive anti-HBV effect to pegylated interferon
a2b, possibly suggesting blockage of the interferon signaling.
117.7
15.88
> 200
> 200
> 50
> 50
0.33
0.01
(> 0,1)*
(> 5)*
> 50
24
23.07
185.9
158.0
> 200
> 200
178.9
50.0
4.91
27.67
9.23
25a
25b
25c
25d
26a
26b
26c
27a
27b
27c
27d
28a
28b
28c
29a
29b
29c
30a
30b
30c
31a
31b
31c
32c
33
> 50
> 50
> 50
25.65
> 50
0.20
(> 10)*
29.04
192.0
> 200
162.7
> 200
> 200
16.83
47.06
42.98
45.65
46.0
7.14
2.33
> 50
31.64
> 50
> 50
> 50
0.41
0.79
0.56
1.19
(> 5)*
(> 10)*
(> 10)*
(> 5)*
(> 10)*
> 50
2.12
198.3
114.3
50.7
0.74
20.0
(> 20)*
(> 10)*
(> 5)*
(> 10)*
(> 10)*
(> 10)*
(> 10)*
(> 5)*
(> 10)*
4. Experimental section
3.38
26.66
46.52
36.65
49.66
43.58
26.61
44.5
0.15
1.93
2.57
7.26
1.98
0.74
4.1. Chemistry
Melting points were determined on a Büchi apparatus and are un-
corrected. 1H NMR spectra and 2D spectra were recorded on a Bruker
Avance III 600 or a Bruker Avance DRX 400 instrument, whereas 13C
NMR spectra were recorded on a Bruker Avance III 600 or a Bruker AC
200 spectrometer in deuterated solvents and were referenced to TMS (δ
scale). The signals of 1H and 13C spectra were unambiguously assigned
by using 2D NMR techniques: 1H1H COSY, NOESY, HMQC, and HMBC.
Mass spectra were recorded with a LTQ Orbitrap Discovery instrument,
possessing an Ionmax ionization source. Flash chromatography was
34
1.25
* In this case the EC50 value could not be determined; the cited value cor-
responds to the maximum concentration of the compound which could be used
based on its cytotoxicity.
6

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
Fig. 5. Screening of imidazo[4,5-b]pyr-
idines against HBV particle production.
HepG2-NTCP cells (6 × 104 cells/well) were
infected with HBV (multiplicity of infection
- MOI = 1000) in the presence of the max-
imum non-cytotoxic concentrations of 2-al-
kylamino-substituted imidazopyridines (A)
and their N-tosyl substituted derivatives (B).
Cells treated with the solvent DMSO were
used as control (C). Cell supernatants were
collected 7 days post infection (d.p.i) and
HBV DNA was isolated. HBV rcDNA levels
were determined with qPCR and normalized
to cell numbers by qPCR amplification of
cyclin A2. Values obtained with compound-
treated cells are expressed as percentage of
those obtained with control cells. The con-
centrations used are indicated under the
name of each compound. Bars represent
mean values obtained from three separate
experiments in triplicate. Error bars re-
present standard deviation (SD). *p < 0.01
vs control, **p
<
0.001 vs control
(Student’s t-test).
1H NMR and ΜS data previously reported [42].
4.1.2. N-(5-Chloro-3-nitropyridin-2-yl)chloracetamide (7)
Nitroaminopyridine 1 (1.00 g, 5.76 mmol) was suspended in an-
hydrous DMF (1 mL) and a solution of chloroacetyl chloride (1.83 mL,
23.04 mmol) in DMF (1 mL) was added dropwise under stirring and
cooling with an ice bath. After the addition was completed the reaction
mixture was allowed to stir at room temperature for 16 h and then
poured into ice and diluted with water. The resulting precipitate was
filtered, washed with water and dried to yield pure compound 7
(1.32 g, 92%). mp 111–112 °C (EtOAc/pentane); 1H NMR (600 MHz,
CDCl3) δ 4.35 (s, 2H, H-2), 8.52 (d, J = 2.2 Hz, 1H, H-4΄), 8.68 (d,
J = 2.2 Hz, 1H, H-6΄), 10.73 (br s, 1H, D2O exchangeable, NH); 13C
NMR (50 MHz, CDCl3) δ 43.6 (C-2), 127.50 (C-5΄), 133.6 (C-3΄), 134.3
(C-4΄), 143.0 (C-2΄), 152.7 (C-6΄), 164.6 (C-1); HR-MS (ESI) m/z: Calcd
for C7H4Cl2N3O3: [MeH]− = 247.9635, found 247.9632.
Fig. 6. Dose-response curve analysis for compounds 19d, 20a, 21a, 21b and
21d targeting HBV particle production. HepG2-NTCP cells (6x104 cells/well)
were infected with HBV (MOI = 1000) and treated with serial dilutions of the
compounds, or DMSO (control). Cell supernatants were collected 7 d.p.i and
HBV DNA was isolated. HBV rcDNA levels were determined with qPCR and
normalized to cell numbers by qPCR amplification of cyclin A2. Values obtained
with compound-treated cells are expressed as percentage of those obtained with
control cells. Markers represent mean values obtained from three separate ex-
periments in triplicate. Error bars represent standard deviation (SD).
4.1.3. N-(5,6-Dichloro-3-nitropyridin-2-yl)chloracetamide (8)
This compound was prepared by a procedure analogous to that of 7,
starting from nitroaminopyridine 6; yield 90%; mp 128–129 °C (EtOAc/
pentane); 1H NMR (200 MHz, CDCl3) δ 4.39 (s, 2H, H-2), 8.60 (s, 1H, H-
4΄), 10.66 (br s, 1H, D2O exchangeable, NH); 13C NMR (50 MHz, CDCl3)
δ 43.7 (C-2), 125.9 (C-5΄), 132.0 (C-3΄), 136.7 (C-4΄), 142.1 (C-2΄),
153.0 (C-6΄), 164.6 (C-1); HR-MS (ESI) m/z: Calcd for C7H3Cl3N3O3:
[MeH]− = 283.9216, found 283.9212.
performed on Merck silica gel 60 (0.040–0.063 mm). Analytical thin
layer chromatography (TLC) was carried out on precoated (0.25 mm)
Merck silica gel F-254 plates.
4.1.1. 2-Amino-5,6-dichloro-3-nitropyridine (6)
Aminopyridine 5 (1.20 g, 7.26 mmol) was dissolved in c.H2SO4
(11 mL) upon stirring in an ice/salt bath, followed by dropwise addition
of HNO3 (65%, 1.15 mL) at −10 °C. The reaction was subsequently
stirred at room temparature for 40 min. Then, the reaction mixture was
poured into ice and neutralized with an ammonium hydroxide solution,
adjusting the pH to 6. The precipitate was filtrered and the dry residue
was subjected to column chromatography (silica gel 60–200 μm) and
eluted with DCM/MeOH (99.5/0.5, v/v). Evaporation of the appro-
priate fractions afforded 951 mg (62%) of 6. mp 186–187 °C (EtOAc).
4.1.4. N-(5-Chloro-3-nitropyridin-2-yl)-3-chloropropanamide (9)
This compound was prepared by a procedure analogous to that of 7,
starting from nitroaminopyridine 1 using 3-chloropropionylchloride;
yield 82%; mp 130–131 °C (EtOAc/pentane); 1H NMR (600 MHz,
CDCl3) δ 3.21 (t, J = 6.6 Hz, 2H, H-2), 3.88 (t, J = 6.6 Hz, 2H, H-3),
8.50 (d, J = 2.4 Hz, 1H, H-4΄), 8.62 (d, J = 2.4 Hz, 1H, H-6΄), 9.90 (br
s, 1H, D2O exchangeable, NH); 13C NMR (151 MHz, CDCl3) δ 38.9 (C-
3), 41.4 (C-2), 126.7 (C-5΄), 133.0 (C-3΄), 134.4 (C-4΄), 143.5 (C-2΄),
152.6 (C-6΄), 169.1 (C-1); HR-MS (ESI) m/z: Calcd for C8H6Cl2N3O3:
7

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Fig. 7. A. Effect of compounds 19d, 20a and 21d on the levels of HBV replication intermediates. HepG2-NTCP cells (6x104 cells/well) were infected with HBV
(MOI = 1000) and treated with compounds at 50 μΜ, or DMSO (control - C) for 7 days. pgRNA levels were determined by RT-qPCR and normalized to YWHAZ
mRNA levels. Nuclear cccDNA, cytoplasmic encapsidated rcDNA and rcDNA in cell supernatants were quantified by qPCR and normalized to cell number by qPCR
amplification of cyclin A2. Values obtained with compound-treated cells are expressed as ratio of those obtained with control cells. B. 21d does not inhibit the
endogenous HBV DNA polymerase. HBV virion particles purified from supernatants of HepAD38 cells were subjected in endogenous polymerase assay (EPA) with
21d at the indicated concentrations. DMSO at 0.01% was used as a solvent control, and a reaction mixture without dNTPs was used as negative control of the
reaction. Following EPA, HBV DNA was extracted and quantified by qPCR. Values are expressed as ratio of those obtained from the reaction with DMSO. C. Effect of
compound 21d on cccDNA formation at different time-points post infection. HepG2-NTCP cells were infected with HBV and treated with 21d as described in A, for 1,
2, 3 and 7 days post virus inoculation (d.p.i.). Nuclear cccDNA, was quantified by qPCR and normalized to cyclin A2. Values are expressed as ratio of those obtained
with control cells. D. Activity of 21d and interferon combination against HBV particle production. HepG2-NTCP cells were infected with HBV and treated or not with
1000 or 2000 IU/ml interferon in the presence or absence of 21d at 10 μM. Cell supernatants were collected 7 d.p.i and HBV DNA was isolated. HBV rcDNA levels
were determined with qPCR and normalized to cell numbers by qPCR amplification of cyclin A2. Values are expressed as percentage of those obtained with cells
treated only with the solvent DMSO. Bars represent mean values obtained from three separate experiments in triplicate. Error bars represent standard deviation (SD).
*p < 0.01 vs control, **p < 0.001 vs control/DMSO (Student’s t-test).
[MeH]− = 261.9792, found 261.9793.
320 mg of 11 (60%); mp 284–285 °C (EtOAc, dec); 1H NMR (600 MHz,
CDCl3) δ 4.23 (s, 2H, H-2), 4.39 (br s, 2H, D2O exchangeable, NH2),
7.12 (d, J = 2.1 Hz, 1H, H-4΄), 7.80 (d, J = 2.1 Hz, 1H, H-6΄), 8.65 (br
s, 1H, D2O exchangeable, NH); 13C NMR (151 MHz, CDCl3) δ 42.7 (C-
2), 125.4 (C-4΄), 130.7 (C-5΄), 135.7 (C-3΄), 136.6 (C-6΄), 137.6 (C-2΄),
165.1 (C-1); HR-MS (ESI) m/z: Calcd for C7H6Cl2N3O:
[MeH]− = 217.9893, found 217.9892.
4.1.5. N-(5,6-Dichloro-3-nitropyridin-2-yl)-3-chloropropanamide (10)
This compound was prepared by a procedure analogous to that of 7,
starting from nitroaminopyridine 6 using 3-chloropropionylchloride.
Upon completion of the addition of 3-chloropropionylchloride the re-
action mixture was stirred at 70 °C for 6 h; yield 77%; mp 143–144 °C
(EtOAc/pentane); 1H NMR (600 MHz, CDCl3) δ 3.27 (t, J = 6.5 Hz, 2H,
H-2), 3.89 (t, J = 6.5 Hz, 2H, H-3), 8.59 (s, 1H, H-4΄), 9.96 (br s, 1H,
D2O exchangeable, NH); 13C NMR (151 MHz, CDCl3) δ 38.6 (C-3), 41.4
(C-2), 124.9 (C-5΄), 131.1 (C-3΄), 136.9 (C-4΄), 142.8 (C-2΄), 152.9 (C-
6΄), 169.2 (C-1); HR-MS (ESI) m/z: Calcd for C8H5Cl3N3O3:
[MeH]− = 295.9402, found 295.9391.
4.1.7. N-(3-Amino-5,6-dichloropyridin-2-yl)chloroacetamide (12)
This compound was prepared by a procedure analogous to that of
11, starting from 8. Compound 8 was hydrogenated at 34 psi for 8 h.
Purification was effected using a mixture of DCM/MeOH (99.5/0.5 to
99/1, v/v); yield 73%; amorphous solid; 1H NMR (600 MHz, CDCl3) δ
4.22 (s, 2H, H-2), 4.40 (br s, 2H, D2O exchangeable, NH2), 7.23 (s, 1H,
H-4΄), 8.63 (br s, 1H, D2O exchangeable, NH); 13C NMR (151 MHz,
CDCl3) δ 42.5 (C-2), 128.42 (C-4΄), 128.69 (C-5΄), 134.6 (C-6΄), 135.1
(C-3΄), 136.8 (C-2΄), 165.2 (C-1); HR-MS (ESI) m/z: Calcd for
C7H5Cl3N3O: [MeH]− = 251.9504, found 251.9505.
4.1.6. N-(3-Amino-5-chloropyridin-2-yl)chloroacetamide (11)
Nitroamide 7 (600 mg, 2.70 mmol) was dissolved in absolute EtOH
(80 mL) and was hydrogenated (34 psi) at room temperature for 8 h in
the presence of Raney Ni as catalyst. The reaction mixture was then
filtered through a celite pad and washed with a mixture of DCM/MeOH
(1/1). The combined filtrate and washings were evaporated. The re-
sidue was subjected to column chromatography (silica gel 60–200 μm)
and eluted with DCM/MeOH (99.5/0.5 to 98.5/1.5, v/v), providing
4.1.8. N-(3-Amino-5-chloropyridin-2-yl)-3-chloropropanamide (13)
This compound was prepared by a procedure analogous to that of
11, starting from 9. Compound 9 was hydrogenated at 57 psi for 8 h.
8

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Purification was effected using a mixture of DCM/MeOH (99/1 to 97/3,
v/v); yield 80%; mp: 277–278 °C (EtOAc, dec); 1H NMR (600 MHz,
CDCl3) δ 2.92 (t, J = 6.3 Hz, 2H, H-2), 3.88 (t, J = 6.3 Hz, 2H, H-3),
4.45 (br s, 2H, D2O exchangeable, NH2), 7.11 (d, J = 2.1 Hz, 1H, H-4΄),
7.76 (d, J = 2.1 Hz, 1H, H-6΄), 8.26 (br s, 1H, D2O exchangeable, NH);
13C NMR (151 MHz, CDCl3) δ 31.1 (C-3), 39.7 (C-2), 125.2 (C-4΄), 130.4
(C-5΄), 135.9 (C-6΄), 136.6 (C-3΄), 137.9 (C-2΄), 168.9 (C-1); HR-MS
(d, J = 2.0 Hz, 1H, H-6΄), 9.40 (br s, 1H, D2O exchangeable, NH); 13C
NMR (151 MHz, CDCl3) δ 53.9 (morpholine-C-3,5), 62.2 (C-2), 67.0
(morpholine-C-2,6), 125.0 (C-4΄), 129.9 (C-5΄), 136.3 (C-6΄), 136.8 (C-
3΄), 137.5 (C-2΄), 169.2 (C-1); HR-MS (ESI) m/z: Calcd for C11H14Cl
N4O2: [MeH]− = 269.0811, found 269.0811.
4.1.13. N-(3-Amino-5-chloropyridin-2-yl)-(diethylamino)acetamide (15d)
This compound was prepared by a procedure analogous to that of
15a, starting from 11 using diethylamine. Purification was effected
using a mixture of DCM/MeOH (99.5/0.5 to 98/2, v/v); yield 73%; mp
82–83 °C (Et2O); 1H NMR (600 MHz, CDCl3) δ 1.11 (t, J = 7.1 Hz, 6H,
2xCH3), 2.68 (q, J = 7.1 Hz, 4H, 2xCH2), 3.19 (s, 2H, H-2), 4.67 (br s,
2H, D2O exchangeable, NH2), 7.05 (d, J = 1.8 Hz, 1H, H-4΄), 7.75 (d,
J = 1.8 Hz, 1H, H-6΄), 9.74 (br s, 1H, D2O exchangeable, NH); 13C NMR
(151 MHz, CDCl3) δ 12.3 (2xCH3), 49.0 (2xCH2), 57.7 (C-2), 124.8 (C-
4΄), 129.5 (C-5΄), 136.2 (C-6΄), 137.16 (C-3΄), 137.33 (C-2΄), 171.6 (C-1);
HR-MS (ESI) m/z: Calcd for C11H16ClN4O: [MeH]− = 255.1018, found
255.1024.
(ESI) m/z: Calcd for C8H8Cl2N3O: [MeH]−
= 232.0050, found
232.0054.
4.1.9. N-(3-Amino-5,6-dichloropyridin-2-yl)-3-chloropropanamide (14)
This compound was prepared by a procedure analogous to that of
11, starting from 10. Compound 10 was hydrogenated at 57 psi for 8 h
Purification was effected using a mixture of DCM/MeOH (99/1 to 97/3,
v/v); mp 148–149 °C (CHCl3); 1H NMR (600 MHz, CDCl3) δ 2.90 (t,
J = 6.2 Hz, 2H, H-2), 3.86 (t, J = 6.2 Hz, 2H, H-3), 4.36 (br s, 2H, D2O
exchangeable, NH2), 7.22 (s, 1H, H-4΄), 8.07 (br s, 1H, D2O exchange-
able, NH); 13C NMR (151 MHz, CDCl3) δ 39.4, 39.7 (C-2,3) 128.13 (C-
5΄), 128.24 (C-4΄), 134.6 (C-6΄), 135.4 (C-3΄), 136.9 (C-2΄), 168.7 (C-1);
HR-MS (ESI) m/z: Calcd for C8H7Cl3N3O: [MeH]− = 265.9660, found
265.9653.
4.1.14. N-(3-Amino-5,6-dichloropyridin-2-yl)piperidin-1-ylacetamide
(16a)
This compound was prepared by a procedure analogous to that of
15a, starting from 12 using piperidine. Purification was effected using a
mixture of CHCl3/MeOH (99.5/0.5 to 94/6, v/v); yield 66%; mp
112–113 °C (EtOAc); 1H NMR (600 MHz, CDCl3) δ 1.47 (m, 2H, pi-
peridine-H-4), 1.65 (m, 4H, piperidine, H-3,5), 2.56 (m, 4H, piperidine-
H-2,6), 3.10 (s, 2H, H-2), 4.60 (br s, 2H, D2O exchangeable, NH2), 7.17
(s, 1H, H-4΄), 9.57 (br s, 1H, D2O exchangeable, NH); 13C NMR
(50 MHz, CDCl3) δ 23.7 (piperidine-C-4), 26.1 (piperidine-C-3,5), 55.2
(piperidine-C-2,6), 62.5 (C-2), 127.44 (C-4΄), 127.87 (C-5΄), 134.6 (C-
6΄), 136.2 (C-3΄), 136.7 (C-2΄), 170.4 (C-1); HR-MS (ESI) m/z: Calcd for
4.1.10. N-(3-Amino-5-chloropyridin-2-yl)piperidin-1-ylacetamide (15a)
Chloroamide 11 (400 mg, 1.82 mmol) and KI (180 mg, 1.09 mmol)
were dissolved in absolute EtOH (7 mL) under argon and a solution of
piperidine (1.80 mL, 18.2 mmol) in EtOH (2 mL) was added dropwise
under stirring and cooling with an ice bath. After the addition was
completed the reaction mixture was stirred at 60 °C for 2 h. EtOH and
the excess amount of piperidine were evaporated under reduced pres-
sure and the residue was dissolved in DCM followed by extraction with
H2O. The aqueous phase was extracted twice more with DCM, the or-
ganic layers were combined, dried (anhydrous Na2SO4) and evaporated
to dryness. The residue was subjected to column chromatography (silica
gel 60–200 μm) and eluted with DCM/MeOH (99/1 to 98/2, v/v); yield
86%; mp 108–109 °C (EtOAc/pentane); 1H NMR (600 MHz, CDCl3) δ
1.45 (m, 2H, piperidine-H-4), 1.63 (m, 4H, piperidine-H-3,5), 2.55 (m,
4H, piperidine-H-2,6), 3.10 (s, 2H, H-2), 4.61 (br s, 2H, D2O ex-
changeable, NH2), 7.05 (d, J = 2.1 Hz, 1H, H-4΄), 7.75 (d, J = 2.1 Hz,
1H, H-6΄), 9.57 (br s, 1H, D2O exchangeable, NH); 13C NMR (151 MHz,
CDCl3) δ 23.7 (piperidine-C-4), 26.2 (piperidine-C-3,5), 55.1 (piper-
idine-C-2,6), 62.5 (C-2), 124.8 (C-4΄), 129.6 (C-5΄), 136.1 (C-6΄), 137.0
(C-3΄), 137.5 (C-2΄), 170.3 (C-1); HR-MS (ESI) m/z: Calcd for
C
12H15Cl2N4O: [MeH]− = 301.0628, found 301.0622.
4.1.15. N-(3-Amino-5,6-dichloropyridin-2-yl)-(4-methylpiperazin-1-yl)
acetamide (16b)
This compound was prepared by a procedure analogous to that of
15a, starting from 12 using N-methylpiperazine. Purification was ef-
fected using a mixture of CHCl3/MeOH (97/3 to 90/10, v/v); yield
75%; mp 176–177 °C (EtOAc); 1H NMR (200 MHz, CDCl3) δ 2.31 (s, 3H,
CH3), 2.51 (m, 4H, piperazine-H), 2.67 (m, 4H, piperazine-H), 3.16 (s,
2H, H-2), 4.58 (br s, 2H, D2O exchangeable, NH2), 7.17 (s, 1H, H-4΄),
9.41 (br s, 1H, D2O exchangeable, NH); 13C NMR (50 MHz, CDCl3) δ
46.1 (CH3), 53.7 (piperazine-C-2,6), 55.1 (piperazine-C-3,5), 61.6 (C-
2), 127.56 (C-4΄), 127.94 (C-5΄), 134.6 (C-6΄), 136.0 (C-3΄), 136.7 (C-2΄),
C
12H16ClN4O: [MeH]− = 267.1018, found 267.1016.
4.1.11. N-(3-Amino-5-chloropyridin-2-yl)-(4-methylpiperazin-1-yl)
acetamide (15b)
169.7 (C-1); HR-MS (ESI) m/z: Calcd for
[MeH]− = 316.0737, found 316.0733.
C12H16Cl2N5O:
This compound was prepared by a procedure analogous to that of
15a, starting from 11, using N-methylpiperazine. Purification was ef-
fected using a mixture of DCM/MeOH (98/2 to 88/12, v/v); yield 78%;
mp 149–150 °C (EtOAc); 1H NMR (600 MHz, CDCl3) δ 2.32 (s, 3H,
CH3), 2.53 (m, 4H, piperazine-H), 2.69 (m, 4H, piperazine-H), 3.18 (s,
2H, H-2), 4.59 (br s, 2H, D2O exchangeable, NH2), 7.07 (d, J = 2.1 Hz,
1H, H-4΄), 7.76 (d, J = 2.1 Hz, 1H, H-6΄), 9.44 (br s, 1H, D2O ex-
changeable, NH); 13C NMR (151 MHz, CDCl3) δ 46.0 (CH3), 53.6 (pi-
perazine-C-2,6), 55.2 (piperazine-C-3,5), 61.6 (C-2), 125.0 (C-4΄), 129.8
(C-5΄), 136.26 (C-6΄), 136.94 (C-3΄), 137.5 (C-2΄), 169.6 (C-1); HR-MS
(ESI) m/z: Calcd for C12H17ClN5O: [MeH]− = 282.1127, found
282.1129.
4.1.16. N-(3-Amino-5,6-dichloropyridin-2-yl)-morpholin-4-ylacetamide
(16c)
This compound was prepared by a procedure analogous to that of
15a, starting from 12 using morpholine. Purification was effected using
a mixture of CHCl3/MeOH (99/1 to 98/2); yield 81%; mp 152–153 °C
(Et2O); 1H NMR (600 MHz, CDCl3) δ 2.65 (m, 4H, morpholine-H-3,5),
3.18 (s, 2H, H-2), 3.79 (m, 4H, morpholine-H-2,6), 4.57 (br s, 2H, D2O
exchangeable, NH2), 7.19 (d, J = 2.0 Hz, 1H, H-4΄), 9.38 (br s, 1H, D2O
exchangeable, NH); 13C NMR (151 MHz, CDCl3) δ 54.0 (morpholine-C-
3,5), 62.2 (C-2), 67.0 (morpholine-C-2,6), 127.7 (C-4΄), 128.0 (C-5΄),
134.7 (C-6΄), 135.9 (C-3΄), 136.7 (C-2΄), 169.3 (C-1); HR-MS (ESI) m/z:
Calcd for C11H13Cl2N4O2: [MeH]− = 303.0421, found 303.0419.
4.1.12. N-(3-Amino-5-chloropyridin-2-yl)morpholin-4-ylacetamide (15c)
This compound was prepared by a procedure analogous to that of
15a, starting from 11 using morpholine. Purification was effected using
a mixture of DCM/MeOH (98.5/1.5 to 96/4, v/v); yield 89%; mp
149–150 °C (EtOAc); 1H NMR (600 MHz, CDCl3) δ 2.66 (m, 4H, mor-
pholine-H-3,5), 3.19 (s, 2H, H-2), 3.78 (m, 4H, morpholine-H-2,6), 4.58
(br s, 2H, D2O exchangeable, NH2), 7.07 (d, J = 2.0 Hz, 1H, H-4΄), 7.76
4.1.17. N-(3-Amino-5,6-dichloropyridin-2-yl)-(diethylamino)acetamide
(16d)
This compound was prepared by a procedure analogous to that of
15a, starting from 12 using diethylamine. Purification was effected
using a mixture of CHCl3/MeOH (99/1, v/v); yield 88%; mp 91–92 °C
(Et2O); 1H NMR (600 MHz, CDCl3) δ 1.09 (t, J = 7.1 Hz, 6H, 2xCH3),
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2.66 (q, J = 7.1 Hz, 4H, 2xCH2), 3.17 (s, 2H, H-2), 4.67 (br s, 2H, D2O
exchangeable, NH2), 7.15 (s, 1H, H-4΄), 9.73 (br s, 1H, D2O exchange-
able, NH); 13C NMR (151 MHz, CDCl3) δ 12.2 (2xCH3), 49.0 (2xCH2),
57.7 (C-2), 127.26 (C-4΄), 127.76 (C-5΄), 134.4 (C-6΄), 136.19 (C-3΄),
136.56 (C-2΄), 171.7 (C-1), HR-MS (ESI) m/z: Calcd for C11H15Cl2N4O:
[MeH]− = 289.0628, found 289.0620.
4.1.22. N-(3-Amino-5,6-dichloropyridin-2-yl)-3-(piperidin-1-yl)
propanamide (18a)
This compound was prepared by a procedure analogous to that of
15a, starting from 14 using piperidine. Purification was effected using a
mixture of DCM/MeOH (97/3 to 90/10, v/v); yield 91%; mp
123–124 °C (EtOAc/pentane); 1H NMR (600 MHz, CDCl3) δ 1.51 (m,
2H, piperidine-H-4), 1.70 (m, 4H, piperidine-H-3,5), 2.55 (m, 6H, H-2,
piperidine-H-2,6), 2.71 (t, J = 6.0 Hz, 2H, H-3), 4.60 (br s, 2H, D2O
exchangeable, NH2), 7.14 (s, 1H, H-4΄), 12.08 (br s, 1H, D2O ex-
changeable, NH); 13C NMR (151 MHz, CDCl3) δ 24.32(piperidine-C-4),
25.9 (piperidine-C-3,5), 32.2 (C-2), 53.74 (piperidine-C-2,6), 54.07 (C-
3), 126.7 (C-5΄), 127.5 (C-4΄), 134.6 (C-6΄), 136.4 (C-3΄), 137.3 (C-2΄),
171.7 (C-1); HR-MS (ESI) m/z: Calcd for C13H17Cl2N4O:
[MeH]− = 315.0785, found 315.0788.
4.1.18. N-(3-Amino-5-chloropyridin-2-yl)-3-(piperidin-1-yl)propanamide
(17a)
This compound was prepared by a procedure analogous to that of
15a, starting from 13 using piperidine. Purification was effected using a
mixture of DCM/MeOH (99/1 to 94/6, v/v); yield 86%; mp 160–161 °C
(EtOAc); 1H NMR (400 MHz, CDCl3) δ 1.50 (m, 2H, piperidine-H-4),
1.69 (m, 4H, piperidine-H-3,5), 2.57 (m, 6H, H-2, piperidine-H-2,6),
2.73 (t, J = 6.0 Hz, 2H, H-3), 4.53 (br s, 2H, D2O exchangeable, NH2),
7.04 (d, J = 2.1 Hz, 1H, H-4΄), 7.83 (d, J = 2.1 Hz, 1H, H-6΄), 11.61 (br
s, 1H, D2O exchangeable, NH); 13C NMR (50 MHz, CDCl3) δ 24.3 (pi-
peridine-C-4), 25.9 (piperidine-C-3,5), 32.3 (C-2), 53.8 (piperidine-C-
2,6), 54.2 (C-3), 124.6 (C-4΄), 129.2 (C-5΄), 136.5 (C-6΄), 137.4 (C-3΄),
138.1 (C-2΄), 171.6 (C-1). HR-MS (ESI) m/z: Calcd for C13H18ClN4O:
[MeH]− = 281.1175, found 281.1172.
4.1.23. N-(3-Amino-5,6-dichloropyridin-2-yl)-3-(4-methylpiperazin-1-yl)
propanamide (18b)
This compound was prepared by a procedure analogous to that of
15a, starting from 14 using N-methylpiperazine. Purification was ef-
fected using a mixture of with DCM/MeOH (97/3 to 80/20, v/v); yield
93%; mp 132–133 °C (Et2O); 1H NMR (600 MHz, CDCl3) δ 2.36 (s, 3H,
CH3), 2.56 (t, J = 6.2 Hz, 2H, H-2), 2.68 (m, 8H, piperazine-H), 2.79 (t,
J = 6.2 Hz, 2H. H-3), 4.61 (br s, 2H, D2O exchangeble, NH2), 7.15 (s,
1H, H-4΄), 11.61 (br s, 1H, D2O exchangeable, NH); 13C NMR (50 MHz,
CDCl3) δ 32.2 (C-2), 45.7 (CH3), 51.8 (piperazine-C-2,6), 53.2 (C-3),
54.8 (piperazine-C-3,5), 126.9 (C-5΄), 127.6 (C-4΄), 134.6 (C-6΄), 136.4
(C-3΄), 137.0 (C-2΄), 171.2 (C-1); HR-MS (ESI) m/z: Calcd for
4.1.19. N-(3-Amino-5-chloropyridin-2-yl)-3-(4-methylpiperazin-1-yl)
propanamide (17b)
This compound was prepared by a procedure analogous to that of
15a, starting from 13 using N-methylpiperazine. Purification was ef-
fected using a mixture of CHCl3/MeOH (96/4 to 82/18, v/v); yield
70%; mp 154–155 °C (Et2O); 1H NMR (400 MHz, CDCl3) δ 2.30 (s, 3H,
CH3), 2.40–2.79 (m, 12H, H-2, H-3, piperazine-H), 4.52 (br s, 2H, D2O
C
13H18Cl2N5O: [MeH]− = 330.0894, found 330.0898.
4.1.24. N-(3-Amino-5,6-dichloropyridin-2-yl)-3-(morpholin-4-yl)
propanamide (18c)
exchangeable, NH2), 7.04 (d,
J = 2.0 Hz, 1H, H-4΄), 7.75 (d,
J = 2.0 Hz, 1H, H-6΄), 11.23 (br s, 1H, D2O exchangeable, NH); 13C
NMR (151 MHz, CDCl3) δ 32.3 (C-2), 46.0 (CH3), 52.4 (piperazine-C-
2,6), 53.6 (C-3), 55.1 (piperazine-C-3,5), 124.7 (C-4΄), 129.3 (C-5΄),
136.5 (C-6΄), 137.38 (C-3΄), 137.94 (C-2΄), 171.3 (C-1); HR-MS (ESI) m/
z: Calcd for C13H19ClN5O: [MeH]− = 296.1284, found 296.1276.
This compound was prepared by a procedure analogous to that of
15a, starting from 14 using morpholine. Purification was effected using
a mixture of with DCM/MeOH (99/1 to 94/6, v/v; yield 84%; mp
137–138 °C (Et2O); 1H NMR (600 MHz, CDCl3) δ 2.58 (t, J = 6.0 Hz,
2H, H-2), 2.65 (m, 4H, morpholine-H-3,5), 2.79 (t, J = 6.0 Hz, 2H, H-
3), 3.83 (m, 4H, morpholine-H-2,6), 4.59 (br s, 2H, D2O exchangeable,
NH2), 7.16 (s, 1H, H-4΄), 11.39 (br s, 1H, D2O exchangeable, NH); 13C
NMR (151 MHz, CDCl3) δ 31.9 (C-2), 52.9 (morpholine-C-3,5), 53.9 (C-
3), 66.8 (morpholine-C-2,6), 127.05 (C-5΄), 127.64 (C-4΄), 134.7 (C-6΄),
136.42 (C-3΄), 136.88 (C-2΄), 171.1 (C-1); HR-MS (ESI) m/z: Calcd for
4.1.20. N-(3-Amino-5-chloropyridin-2-yl)-3-(morpholin-4-yl)propanamide
(17c)
This compound was prepared by a procedure analogous to that of
15a, starting from 13 using morpholine. Purification was effected using
a mixture of CHCl3/MeOH (97/3 to 93/7, v/v); yield 82%; mp
147–148 °C (EtOAc); 1H NMR (400 MHz, CDCl3) δ 2.57 (t, J = 6.0 Hz,
2H, H-2), 2.62 (m, 4H, morpholine-H-3,5), 2.78 (t, J = 6.0 Hz, 2H, H-
3), 3.80 (m, 4H, morpholine-H-2,6), 4.54 (br s, 2H, D2O exchangeable,
NH2), 7.05 (d, J = 2.0 Hz, 1H, H-4΄), 7.76 (d, J = 2.0 Hz, 1H, H-6΄),
11.17 (br s, 1H, D2O exchangeable, NH); 13C NMR (151 MHz, CDCl3) δ
32.1 (C-2), 52.9 (morpholine-C-3,5), 54.1 (C-3), 66.9 (morpholine-C-
2,6), 124.7 (C-4΄), 129.4 (C-5΄), 136.5 (C-6΄), 137.34 (C-3΄), 137.83 (C-
C
12H15Cl2N4O2: [MeH]− = 317.0578, found 317.0584.
4.1.25. N-(3-Amino-5,6-dichloropyridin-2-yl)-3-(diethylamino)
propanamide (18d)
This compound was prepared by a procedure analogous to that of
15a, starting from 14 using diethylamine. Purification was effected
using a mixture of with CHCl3/MeOH (97/3 to 82/18, v/v; yield 73%;
mp 84–85 °C (EtOAc); 1H NMR (600 MHz, CDCl3) δ 1.18 (t, J = 7.1 Hz,
6H,2x CH3), 2.61 (m, 2H, H-2), 2.78 (q, J = 7.1 Hz, 4H, 2xCH2), 2.92
(m, 2H, H-3), 4.57 (br s, 2H, D2O exchangeable, NH2), 7.14 (s, 1H, H-
4΄), 11.45 (br s 1H, D2O exchangeable, NH); 13C NMR (151 MHz,
CDCl3) δ 10.9 (2xCH3), 32.7 (C-2), 46.3 (2x CH2), 48.8 (C-3), 126.98
(C-5΄), 127.42 (C-4΄), 134.6 (C-6΄), 136.69 (C-3΄), 136.83 (C-2΄), 171.4
2΄), 171.1 (C-1); HR-MS (ESI) m/z: Calcd for
[MeH]− = 283.0967, found 283.0969.
C12H16Cl N4O2:
4.1.21. N-(3-Amino-5-chloropyridin-2-yl)-3-(diethylamino)propanamide
(17d)
(C-1);
HR-MS
(ESI)
m/z:
Calcd
for
C12H17Cl2N4O:
This compound was prepared by a procedure analogous to that of
15a, starting from 13 using diethylamine. Purification was effected
using a mixture of DCM/MeOH (99/1 to 89/11, v/v); yield 87%; mp
79–80 °C (Et2O); 1H NMR (400 MHz, CDCl3) δ 1.15 (t, J = 7.1 Hz, 6H,
2xCH3), 2.62 (t, J = 5.7 Hz, 2H, H-2), 2.74 (q, J = 7.1 Hz, 4H, 2xCH2),
2.89 (t, J = 5.7 Hz, 2H, H-3), 4.54 (br s, 2H, D2O exchangeable, NH2),
7.04 (d, J = 2.1 Hz, 1H, H-4΄), 7.75 (d, J = 2.1 Hz, 1H, H-6΄), 11.34 (br
s, 1H, D2O exchangeable, NH); 13C NMR (151 MHz, CDCl3) δ 10.9
(2xCH3), 32.7 (C-2), 46.2 (C-3), 48.7 (2xCH2), 124.5 (C-4΄), 129.2 (C-
5΄), 136.3 (C-6΄), 137.48 (C-3΄), 137.75 (C-2΄), 171.4 (C-1); HR-MS (ESI)
m/z: Calcd for C12H18ClN4O: [MeH]− = 269.1175, found 269.1179.
[MeH]− = 303.0785, found 303.0794.
4.1.26. 6-Chloro-2-[(piperidin-1-yl)methyl]imidazo[4,5-b]pyridine (19a)
Aminoamide 15a (210 mg, 0.78 mmol) was suspended in PPA under
argon and the reaction mixture was stirred at 95 οC for 2.5 h. The re-
action mixture was then poured into ice and basified with an ammo-
nium hydroxide solution (12.5%) to a pH value of 10. The aqueous
phase was extracted three times with 100 mL of DCM and the combined
organic layers were dried (anhydrous Na2SO4) and evaporated to dry-
ness. The dry residue was then subjected to column chromatography
(silica gel 60–200 μm) and eluted with CHCl3/MeOH (98/2 to 96/4, v/
10

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
v), providing 140 mg of 19a; yield 71%; mp 233–234 °C (CHCl3); 1H
NMR (600 MHz, CDCl3) δ 1.50 (m, 2H, piperidine-H-4), 1.65 (m, 4H,
piperidine-H-3,5), 2.52 (m, 4H, piperidine-H-2,6), 3.81 (s, 2H, CH2),
7.96 (s, 1H, H-7), 8.41 (d, J = 2.1 Hz, 1H, H-5); 13C NMR (151 MHz,
CDCl3) δ 24.0 (piperidine-C-4), 26.1 (piperidine-C-3,5), 55.3 (piper-
idine-C-2,6), 57.8 (CH2), 125.96 (C-7), 126.7 (C-6), 136.2 (C-7a), 142.4
(C-5), 147.0 (C-3a), 156.7 (C-2); HR-MS (ESI) m/z: Calcd for
CDCl3) δ 46.0 (CH3), 53.5 (2x piperazine-C), 55.0 (2x piperazine-C),
56.6 (CH2), 124.0 (C-6), 128.6 (C-7), 134.6 (C-7a), 142.7 (C-5), 146.6
(C-3a), 156.2 (C-2); HR-MS (ESI) m/z: Calcd for
[MeH]− = 298.0632, found 298.0635.
C12H14Cl2N5:
4.1.32. 5,6-Dichoro-2-[(morpholin-4-yl)methyl]imidazo[4,5-b]pyridine
(20c)
C
12H14ClN4: [MeH]− = 249.0912, found 249.0907.
This compound was prepared by a procedure analogous to that of
19a, starting from 16c. Purification was effected using a mixture of
CHCl3/MeOH (99/1 to 96/4, v/v); yield 88%; mp 182–183 °C (EtOAc);
1H NMR (400 MHz, CDCl3) δ 2.59 (m, 4H, morpholine-H-3,5), 3.71 (m,
4H, morpholine-H-2,6), 3.87 (s, 2H, CH2), 8.09 (s, 1H, H-7), 10.74 (br s,
1H, D2O exchangeable, NH); 13C NMR (151 MHz, CDCl3) δ 54.0
(morpholine-C-3,5), 57.0 (CH2), 66.9 (morpholine-C-2,6), 124.2 (C-6),
129.5 (C-7), 135.4 (C-7a), 142.8 (C-5), 145.7 (C-3a), 155.7 (C-2); HR-
MS (ESI) m/z: Calcd for C11H11Cl2N4O: [MeH]− = 285.0315, found
285.0309.
4.1.27. 6-Chloro-2-[(4-methylpiperazin-1-yl)methyl]imidazo[4,5-b]
pyridine (19b)
This compound was prepared by a procedure analogous to that of
19a, starting from 15b. Purification was effected using a mixture of
DCM/MeOH (95/5 to 85/15, v/v) and DCM/MeOH/Et3N (84/15/1, v/
v); yield 84%; mp 217–218 °C (MeOH, dec); 1H NMR (600 MHz, CDCl3)
δ 2.32 (s, 3H, CH3), 2.44–2.73 (m, 8H, piperazine-H), 3.89 (s, 2H, CH2),
8.00 (s, 1H, H-7), 8.46 (d, J = 2.2 Hz, 1H, H-5), 12.32 (br s, 1H, D2O
exchangeable, NH); 13C NMR (151 MHz, CDCl3) δ 46.1 (CH3), 53.7 (2x
piperazine-C), 55.0 (2x piperazine-C), 57.2 (CH2), 125.9 (C-7), 127.1
(C-6), 136.5 (C-7a), 142.3 (C-5), 147.1 (C-3a), 156.2 (C-2); HR-MS (ESI)
m/z: Calcd for C12H15ClN5: [MeH]− = 264.1021, found 264.1020.
4.1.33. 5,6-Dichloro-2-[(diethylamino)methyl]imidazo[4,5-b]pyridine
(20d)
This compound was prepared by a procedure analogous to that of
19a, starting from 16d. Purification was effected using a mixture of
DCM/MeOH (97/3 to 92/8, v/v); yield 87%; mp 85–86 °C (Et2O); 1H
NMR (600 MHz, CDCl3) δ 1.06 (t, J = 7.1 Hz, 6H, 2x CH3), 2.64 (q,
J = 7.1 Hz, 4H, 2x CH2), 3.89 (s, 2H, CH2), 8.02 (s, 1H, H-7); 13C NMR
(151 MHz, CDCl3) δ 12.0 (2x CH3), 48.3 (2x CH2), 52.2 (CH2), 123.9 (C-
6), 127.7 (C-7), 134.2 (C-7a), 142.5 (C-5), 146.9 (C-3a), 158.3 (C-2);
HR-MS (ESI) m/z: Calcd for C11H13Cl2N4: [MeH]− = 271.0523, found
271.0531.
4.1.28. 6-Chloro-2-[(morpholin-4-yl)methyl]imidazo[4,5-b]pyridine
(19c)
This compound was prepared by a procedure analogous to that of
19a, starting from 15c. Purification was effected using a mixture of
DCM/MeOH (97/3 to 89/11, v/v); yield 96%; mp 249–250 °C (MeOH);
1H NMR (600 MHz, CDCl3) δ 2.61 (m, 4H, morpholineH-3,5), 3.77 (m,
4H, morpholine-H-2,6), 3.87 (s, 2H, CH2), 8.00 (s, 1H, H-7), 8.37 (s,
1H, H-5), 11.17 (br s, 1H, D2O exchangeable, NH); 13C NMR (151 MHz,
CDCl3) δ 54.1 (morpholine-C-3,5), 57.4 (CH2), 67.0 (morpholine-C-
2,6), 126.3 (C-7), 127.0 (C-6), 136.3 (C-7a), 142.6 (C-5), 146.6 (C-3a),
155.4 (C-2); HR-MS (ESI) m/z: Calcd for C11H12ClN4O:
[MeH]− = 251.0705, found 251.0707.
4.1.34. 6-Chloro-2-[2-(piperidin-1-yl)ethyl]imidazo[4,5-b]pyridine (21a)
This compound was prepared by a procedure analogous to that of
19a, starting from 17a. Purification was effected using a mixture of
DCM/MeOH (95/5 to 85/15, v/v); yield 76%; mp 184–185 °C (DCM);
1H NMR (600 MHz, CDCl3) δ 1.54 (m, 2H, piperidine-H-4), 1.72 (m, 4H,
piperidine-H-3,5), 2.59 (m, 4H, piperidine-H-2,6), 2.83 (t, J = 6.1 Hz,
2H, H-1΄), 3.15 (t, J = 6.1 Hz, 2H, H-2΄), 7.87 (d, J = 2.1 Hz, 1H, H-7),
8.26 (d, J = 2.1 Hz, 1H, H-5); 13C NMR (151 MHz, CDCl3) δ 24.2
(piperidine-C-4), 25.3 (C-1΄), 26.0 (piperidine-C-3,5), 54.2 (piperidine-
C-2,6), 55.8 (C-2΄), 124.9 (C-7), 125.8 (C-6), 134.3 (C-7a), 142.1 (C-5),
147.8 (C-3a), 158.3 (C-2); HR-MS (ESI) m/z: Calcd for C13H16ClN4:
[MeH]− = 263.1069, found 263.1065.
4.1.29. 6-Chloro-2-[(diethylamino)methyl]imidazo[4,5-b]pyridine (19d)
This compound was prepared by a procedure analogous to that of
19a, starting from 15d. Purification was effected using a mixture of
CHCl3/MeOH (99/1 to 97/3, v/v); yield 71%; mp 165–166 °C (EtOAc/
pentane); 1H NMR (600 MHz, CDCl3) δ 1.08 (t, J = 7.1 Hz, 6H, 2x CH3),
2.69 (q, J = 7.1 Hz, 4H, 2x CH2), 3.93 (s, 2H, CH2), 7.96 (s, 1H, H-7),
8.36 (d, J = 2.1 Hz, 1H, H-5); 13C NMR (151 MHz, CDCl3) δ 11.9 (2x
CH3), 48.3 (2x CH2), 52.6 (CH2), 125.7 (C-7), 126.5 (C-6), 136.7 (C-7a),
141.9 (C-5), 147.2 (C-3a), 158.5 (C-2); HR-MS (ESI) m/z: Calcd for
4.1.35. 6-Chloro-2-[2-(4-methylpiperazin-1-yl)ethyl]imidazo[4,5-b]
pyridine (21b)
C
11H14ClN4: [MeH]− = 237.0912, found 237.0910.
This compound was prepared by a procedure analogous to that of
19a, starting from 17b. Purification was effected using a mixture of
DCM/MeOH (95/5 to 80/20, v/v); yield 65%; mp 166–168 °C (EtOAc);
1H NMR (600 MHz, CDCl3) δ 2.32 (s, 3H, CH3), 2.42–2.73 (m, 8H, pi-
perazine-H), 2.83 (t, J = 6.3 Hz, 2H, H-1΄), 3.11(t, J = 6.3 Hz, 2H, H-
2΄), 7.86 (d, J = 2.2 Hz, 1H, H-7), 8.23 (d, J = 2.2 Hz, 1H, H-5); 13C
NMR (50 MHz, CDCl3) δ 25.6 (C-1΄), 45.9 (CH3), 52.8 (2x piperazine-C),
55.11 (2x piperazine-C), 55.24 (C-2΄), 125.0 (C-7), 125.8 (C-6), 134.3
(C-7a), 142.0 (C-5), 147.7 (C-3a), 157.9 (C-2); HR-MS (ESI) m/z: Calcd
for C13H17ClN5: [MeH]− = 278.1178, found 278.1169.
4.1.30. 5,6-Dichloro-2-[(piperidin-1-yl)methyl]imidazo[4,5-b]pyridine
(20a)
This compound was prepared by a procedure analogous to that of
19a, starting from 16a. Purification was effected using a mixture of
DCM/MeOH (99.5/0.5 to 96/4, v/v); yield 97%; mp 227–228 °C
(EtOAc); 1H NMR (600 MHz, CDCl3) δ 1.49 (m, 2H, piperidine-H-4),
1.61 (m, 4H, piperidine-H-3,5), 2.50 (m, 4H, piperidine-H-2,6), 3.78 (s,
2H, CH2), 8.02 (s, 1H, H-7); 13C NMR (151 MHz, CDCl3) δ 23.9 (pi-
peridine-C-4), 26.1 (piperidine-C-3,5), 55.2 (piperidine-C-2,6), 57.3
(CH2), 124.0 (C-6), 128.1 (C-7), 134.1 (C-7a), 142.7 (C-5), 146.5 (C-
3a), 157.0 (C-2); HR-MS (ESI) m/z: Calcd for
[MeH]− = 283.0523, found 283.0520.
C
12H13Cl2N4:
4.1.36. 6-Chloro-2-[2-(morpholin-4-yl)ethyl]imidazo[4,5-b]pyridine
(21c)
This compound was prepared by a procedure analogous to that of
19a, starting from 17c. Purification was effected using a mixture of
DCM/MeOH (97/3 to 90/10, v/v); yield 79%; mp: 170–171 °C (EtOAc);
1H NMR (600 MHz, CDCl3) δ 2.63 (m, 4H, morpholine-H-3,5), 2.85 (t,
J = 6.1 Hz, 2H, H-1΄), 3.15 (t, J = 6.1 Hz, 2H, H-2΄), 3.83 (m, 4H,
morpholine-H-2,6), 7.91 (d, J = 2.1 Hz, 1H, H-7), 8.28 (d, J = 2.1 Hz,
1H, H-5); 13C NMR (151 MHz, CDCl3) δ 25.2 (C-1΄), 53.5 (morpholine-
C-3,5), 55.8 (C-2΄), 67.0 (morpholine-C-2,6), 126.02 (C-7), 126.06 (C-
4.1.31. 5,6-Dichloro-2-[(4-methylpiperazin-1-yl)methyl]imidazo[4,5-b]
pyridine (20b)
This compound was prepared by a procedure analogous to that of
19a, starting from 16b. Purification was effected using a mixture of
DCM/MeOH (95/5 to 82/18, v/v); yield: 71%; mp 197–198 °C (EtOAc);
1H NMR (600 MHz, CDCl3) δ 2.30 (s, 3H, CH3), 2.43–2.67 (m, 8H, pi-
perazine-H), 3.85 (s, 2H, CH2), 8.04 (s, 1H, H-7); 13C NMR (151 MHz,
11

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
6), 135.4 (C-7a), 142.3 (C-5), 147.0 (C-3a), 157.7 (C-2); HR-MS (ESI)
C12H15Cl2N4: [MeH]− = 285.0679, found 285.0673.
m/z: Calcd for C12H14ClN4O: [MeH]− = 265.0862, found 265.0864.
4.1.42. 6-Chloro-2-vinylimidazo[4,5-b]pyridine (23)
4.1.37. 6-Chloro-2-[2-(diethylamino)ethyl]imidazo[4,5-b]pyridine (21d)
This compound was prepared by a procedure analogous to that of
19a, starting from 17d. Purification was effected using a mixture of
DCM/MeOH (98/2 to 85/15, v/v); yield 82%; mp 164–165 °C (EtOAc);
1H NMR (600 MHz, CDCl3) δ 1.08 (t, J = 7.2 Hz, 6H, 2x CH3), 2.67 (q,
J = 7.2 Hz, 4H, 2x CH2), 2.88 (t, J = 6.1 Hz, 2H, H-1΄), 3.09 (t,
J = 6.1 Hz, 2H, Η-2΄), 7.87 (d, J = 2.1 Hz, 1H, H-7), 8.25 (d,
J = 2.1 Hz, 1H, H-5); 13C NMR (151 MHz, CDCl3) δ 11.4 (2x CH3), 26.1
(C-1΄), 46.4 (2x CH2), 50.2 (C-2΄), 124.9 (C-7), 125.7 (C-6), 134.0 (C-
7a), 142.0 (C-5), 147.7 (C-3a), 158.6 (C-2); HR-MS (ESI) m/z: Calcd for
This compound was obtained as a by-product during the preparation
of 21b. Yield: 12%; mp 243–244 °C (dec) (EtOAc); 1H NMR (600 MHz,
DMSO‑d6) δ 5.81 (d, Jab = 11.5 Hz, 1H, Hb), 6.42 (d, Jac = 17.8 Hz, 1H,
Hc), 6.78 (dd, Jab = 11.5 Hz, Jac = 17.8 Hz, 1H, Ha), 8.09 (d,
J = 2.2 Hz, 1H, H-7), 8.32 (d, J = 2.2 Hz, 1H, H-5); 13C NMR
(151 MHz, DMSO) δ (ppm) 126.0 (C-2΄), 127.5 (C-1΄), 141.2 (C-7),
144.3 (C-6), 146.1 (C-7a), 154.0 (C-5), 156.3 (C-3a), 164.0 (C-2). HR-
MS (ESI) m/z: Calcd for C8H5ClN3: [MeH]− = 178.0177, found
178.0169.
C
12H16ClN4: [MeH]− = 251.1069, found 251.1068.
4.1.43. 5,6-Dichloro-2-vinylimidazo[4,5-b]pyridine (24)
This compound was obtained as a by-product during the preparation
of 22b. Yield: 7%; mp 211–212 °C (EtOAc); 1H NMR (600 MHz,
DMSO‑d6) δ 5.85 (d, Jab = 10.5 Hz, 1H, Hb), 6.43 (d, Jac = 17.8, 1H,
Hc), 6.80 (m, 1H, Ha), 8.11 (s, 1H, H-7); 13C NMR (151 MHz, DMSO) δ
(ppm) 122. (C-2΄), 124.2 (C-1΄), 126.2 (C-6), 140.9 (C-7), 146.2 (C-7a),
148.0 (C-5), 149.4 (C-3a), 150.0 (C-2). HR-MS (ESI) m/z: Calcd for
C8H4Cl2N3: [MeH]− = 211.9788, found 211.9792.
4.1.38. 5,6-Dichloro-2-[2-(piperidin-1-yl)ethyl]imidazo[4,5-b]pyridine
(22a)
This compound was prepared by a procedure analogous to that of
19a, starting from 18a. Purification was effected using a mixture of
CHCl3/MeOH (97/3 to 82/18, v/v); yield 72%; mp 152–153 °C
(EtOAc); 1H NMR (400 MHz, CDCl3) δ 1.55 (m, 2H, piperidine-H-4),
1.72 (m, 4H, piperidine-H-3,5), 2.59 (m, 4H, piperidine-H-2,6), 2.83 (t,
J = 6.0 Hz, 2H, H-1΄), 3.15 (t, J = 6.0 Hz, 2H, H-2΄), 7.98 (s, 1H, H-7);
13C NMR (151 MHz, CDCl3) δ 24.0 (piperidine-C-4), 25.2 (C-1΄), 25.8
(piperidine-C-3,5), 54.1 (piperidine-C-2,6), 55.6 (C-2΄), 123.4 (C-6),
127.1 (C-7), 133.2 (C-7a), 141.9 (C-5), 147.1 (C-3a), 158.7 (C-2); HR-
MS (ESI) m/z: Calcd for C13H15Cl2N4: [MeH]− = 297.0679, found
297.0688.
4.1.44. 1-(4-Methylbenzenesulfonyl)-6-chloro-2-[(piperidin-1-yl)methyl]-
1H-imidazo[4,5-b]pyridine (25a) and 3-(4-methylbenzenesulfonyl)-6-
chloro-2-[(piperidin-1-yl)methyl]-3H-imidazo[4,5-b]pyridine (27a)
Imidazopyridine 19a (140 mg, 0.56 mmol) was dissolved in anhy-
drous DMF (5 mL) under argon, NaH (60% in mineral oil, 56 mg,
1.39 mmol) was added at 0 °C and the mixture was stirred for 5 min.
Then, the ice bath was removed and p-tosylchloride was added and the
reaction mixture was stirred at room temperature for 20 min. Upon
completion of the reaction, DMF was evaporated and the residue was
treated with 100 mL saturated NaHCO3. The aqueous phase was ex-
tracted three times with 100 mL of DCM and the combined organic
layers were dried (anhydrous Na2SO4) and evaporated to dryness. The
dry residue was then subjected to column chromatography (silica gel
40–60 μm) and eluted with DCM/EtOAc (95/5 to 50/50, v/v) and
DCM/MeOH (99/1, v/v).
4.1.39. 5,6-Dichloro-2-[2-(4-methylpiperazin-1-yl)ethyl]imidazo[4,5-b]
pyridine (22b)
This compound was prepared by a procedure analogous to that of
19a, starting from 18b. Purification was effected using a mixture of
CHCl3/MeOH (97/3 to 82/18, v/v); yield 82%; mp 162–163 °C
(EtOAc); 1H NMR (400 MHz, CDCl3) δ 2.36 (s, 3H, CH3), 2.42–2.78 (m,
8H, piperazine-H), 2.85 (t, J = 6.1 Hz, 2H, H-1΄), 3.14 (t, J = 6.1 Hz,
2H, H-2΄), 8.00 (s, 1H, H-7); 13C NMR (151 MHz, CDCl3) δ 25.4 (C-1΄),
45.9 (CH3), 52.8 (2x piperazine-C), 55.06 (2x piperazine-C), 55.13 (C-
2΄), 123.6 (C-6), 127.6 (C-7), 133.7 (C-7a), 142.1 (C-5), 146.6 (C-3a),
Data for 25a: Yield 89%; mp 162–163 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 1.41–1.52 (m, 6H, piperidine-H-3,4,5), 2.42 (s, 3H,
CH3), 2.56 (m, 4H, piperidine-H-2,6), 4.03 (s, 2H, CH2), 7.33 (d,
J = 8.2 Hz, 2H, tosyl-H-3,5), 8.21 (d, J = 2.2 Hz, 1H, H-7), 8.23 (d,
J = 8.2 Hz, 2H, tosyl-H-2,6), 8.50 (d, J = 2.2 Hz, 1H, H-5); 13C NMR
(151 MHz, CDCl3) δ 21.9 (CH3), 24.2 (piperidine-C-4), 25.8 (piperidine-
C-3,5), 54.8 (piperidine-C-2,6), 56.3 (CH2), 121.6 (C-7), 126.5 (C-7a),
128.20 (tosyl-C-2,6), 128.32 (C-6), 130.1 (tosyl-C-3,5), 135.2 (tosyl-C-
1), 145.8 (C-5), 146.6 (tosyl-C-4), 152.5 (C-3a), 155.2 (C-2); HR-MS
(ESI) m/z: Calcd for C19H21ClN4O2SNa: [M+Na]+ = 427.0966, found
427.0975.
158.5 (C-2); HR-MS (ESI) m/z: Calcd for
[MeH]− = 312.0788, found 312.0782.
C13H16Cl2N5:
4.1.40. 5,6-Dichloro-2-[2-(morpholin-4-yl)ethyl]imidazo[4,5-b]pyridine
(22c)
This compound was prepared by a procedure analogous to that of
19a, starting from 18c. Purification was effected using a mixture of
CHCl3/MeOH (97/3 to 82/18, v/v); yield 81%; mp: 173–174 °C
(EtOAc/pentane); 1H NMR (600 MHz, CDCl3) δ 2.63 (m, 4H, morpho-
line-H-3,5), 2.86 (t, J = 6.0 Hz, 2H, H-1΄), 3.16 (t, J = 6.0 Hz, 2H, H-
2΄), 3.83 (m, 4H, morpholine-H-2,6), 8.00 (s, 1H, H-7); 13C NMR
(151 MHz, CDCl3) δ 25.1 (C-1΄), 53.4 (morpholine-C-3,5), 55.7 (C-2΄),
67.0 (morpholine-C-2,6), 123.8 (C-6), 127.9 (C-7), 134.0 (C-7a), 142.2
(C-5), 146.4 (C-3a), 158.2 (C-2); HR-MS (ESI) m/z: Calcd for
Data for 27a: Yield 8%; mp 145–146 °C (Et2O); 1H NMR (600 MHz,
CDCl3) δ 1.49 (m, 2H, piperidine-H-4), 1.57 (m, 4H, piperidine-H-3,5),
2.41 (s, 3H, CH3), 2.55 (m, 4H, piperidine-H-2,6), 4.03 (s, 2H, CH2),
7.31 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.92 (d, J = 2.2 Hz, 1H, H-7),
8.40 (d, J = 2.2 Hz, 1H, H-5), 8.45 (d, J = 8.2 Hz, 2H, tosyl-H-2,6); 13
C
C
12H13Cl2N4O: [MeH]− = 299.0472, found 299.0471.
NMR (151 MHz, CDCl3) δ 21.9 (CH3), 24.3 (piperidine-C-4), 26.0 (pi-
peridine-C-3,5), 54.9 (piperidine-C-2,6), 57.4 (CH2), 127.6 (C-7), 128.3
(C-6), 129.43 (tosyl-C-2,6), 129.58 (tosyl-C-3,5), 134.3 (C-7a), 135.2
(tosyl-C-1), 144.3 (C-5), 145.6 (C-3a), 146.2 (tosyl-C-4), 154.4 (C-2);
HR-MS (ESI) m/z: Calcd for C19H21ClN4O2SNa: [M+Na]+ = 427.0966,
found 427.0969.
4.1.41. 5,6-Dichloro-2-[2-(diethylamino)ethyl]imidazo[4,5-b]pyridine
(22d)
This compound was prepared by a procedure analogous to that of
19a, starting from 18d. Purification was effected using a mixture of
DCM/MeOH (95/5 to 84/16, v/v); yield 82%; mp: 154–155 °C (Et2O);
1H NMR (200 MHz, CDCl3) δ 1.11 (t, J = 7.2 Hz, 6H, 2x CH3), 2.72 (q,
J = 7.2 Hz, 4H, 2x CH2), 2.93 (t, J = 6.0 Hz, 2H, H-1΄), 3.14 (t,
J = 6.0 Hz, 2H, H-2΄), 7.95 (s, 1H, H-7), 8.10 (br s, 1H, D2O ex-
changeable, NH); 13C NMR (50 MHz, CDCl3) δ 11.1 (2x CH3), 25.8 (C-
1΄), 46.4 (2x CH2), 50.0 (C-2΄), 123.5 (C-6), 127.2 (C-7), 133.3 (C-7a),
142.0 (C-5), 147.0 (C-3a), 158.9 (C-2); HR-MS (ESI) m/z: Calcd for
4.1.45. 1-(4-Methylbenzenesulfonyl)-6-chloro-2-[(4-methylpiperazin-1-yl)
methyl]-1H-imidazo[4,5-b]pyridine
(25b)
and
3-(4-
methylbenzenesulfonyl)-6-chloro-2-[(4-methylpiperazin-1-yl)methyl]-3H-
imidazo[4,5-b]pyridine (27b)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 19b. Purification was effected using a
12

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
mixture of CHCl3/MeOH (99/1, v/v);
(C-2); HR-MS (ESI) m/z: Calcd for C18H22ClN4O2S: [M
Data for 25b: Yield 70%; mp 151–152 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 2.26 (s, 3H, piperazine-CH3), 2.34 (m, 4H, piper-
azine-H), 2.43 (s, 3H, C6H4-CH3), 2.67 (m, 4H, piperazine-H), 4.08 (s,
2H, CH2), 7.33 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 8.14 (d, J = 8.2 Hz,
2H, tosyl-H-2,6), 8.22 (d, J = 2.1 Hz, 1H, H-7), 8.51 (d, J = 2.1 Hz, 1H,
H-5); 13C NMR (151 MHz, CDCl3) δ 21.8 (C6H4-CH3), 46.2 (piperazine-
CH3), 53.3 (piperazine-C-2,6), 54.8 (piperazine-C-3,5), 55.4 (CH2),
121.6 (C-7), 126.4 (C-7a), 127.9 (tosyl-C-2,6), 128.4 (C-6), 130.2
(tosyl-C-3,5), 135.2 (tosyl-C-1), 145.9 (C-5), 146.5 (tosyl-C-4), 152.4
(C-3a), 154.6 (C-2); HR-MS (ESI) m/z: Calcd for C19H23ClN5O2S: [M
+H]+ = 420.1255, found 420.1249.
+H]+ = 393.1147, found 393.1141.
Data for 27d: Yield 9%; mp 130–131 °C (Et2O/pentane); 1H NMR
(600 MHz, CDCl3) δ 0.99 (t, J = 7.1 Hz, 6H, 2x CH3), 2.40 (s, 3H, C6H4-
CH3), 2.71 (q, J = 7.1 Hz, 4H, 2xCH2), 4.19 (s, 2H, CH2), 7.31 (d,
J = 8.2 Hz, 2H, tosyl-H-3,5), 7.92 (d, J = 2.2 Hz, 1H, H-7), 8.32 (d,
J = 8.2 Hz, 2H, tosyl-H-2,6) 8.41 (d, J = 2.2 Hz, 1H, H-5); 13C NMR
(151 MHz, CDCl3) δ 10.8 (2x CH3), 21.9 (C6H4-CH3), 46.5 (2x CH2),
52.9 (CH2), 127.6 (C-7), 128.3 (C-6), 129.12 (tosyl-C-2,6), 129.65
(tosyl-C-3,5), 134.4 (C-7a), 135.2 (tosyl-C-1), 144.2 (C-5), 145.7 (C-3a),
146.2 (tosyl-C-4), 155.6 (C-2); HR-MS (ESI) m/z: Calcd for
C
18H22ClN4O2S: [M+H]+ = 393.1147, found 393.1138.
Data for 27b: Yield 5%; mp 144–145 °C (EtOAc/pentane); 1H NMR
(600 MHz, CDCl3) δ 2.34 (s, 3H, piperazine-CH3), 2.42 (s, 3H, C6H4-
CH3), 2.51 (m, 4H, piperazine-H), 2.71 (m, 4H, piperazine-H), 4.10 (s,
2H, CH2), 7.31 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.92 (d, J = 2.1 Hz,
1H, H-7), 8.37 (d, J = 8.2 Hz, 2H, tosyl-H-2,6), 8.39 (d, J = 2.1 Hz, 1H,
H-5); 13C NMR (151 MHz, CDCl3) δ 21.9 (C6H4-CH3), 45.9 (piperazine-
CH3), 52.9 (piperazine-C-2,6), 54.8 (piperazine-C-3,5), 56.4 (CH2),
127.7 (C-7), 128.5 (C-6), 129.3 (tosyl-C-2,6), 129.7 (tosyl-C-3,5), 134.3
(C-7a), 135.1 (tosyl-C-1), 144.4 (C-5), 145.5 (C-3a), 146.4 (tosyl-C-4),
4.1.48. 1-(4-Methylbenzenesulfonyl)-5,6-dichloro-2-[(piperidin-1-yl)
methyl]-1H-imidazo[4,5-b]pyridine
(26a)
and
3-(4-
methylbenzenesulfonyl)-5,6-dichloro-2-[(piperidin-1-yl)methyl]-3H-
imidazo[4,5-b]pyridine (28a)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 20a. Purification was effected using a
mixture of cHex/EtOAc (90/10, v/v);
Data for 26a: Yield 56%; mp 147–148 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 1.44–1.51 (m, 6H, piperidine-H-3,4,5), 2.43 (s, 3H,
CH3), 2.56 (m, 4H, piperidine-H-2,6), 4.02 (s, 2H, CH2), 7.34 (d,
J = 8.2 Hz, 2H, tosyl-H-3,5), 8.20 (d, J = 8.2 Hz, 2H, tosyl-H-2,6), 8.31
(s, 1H, H-7). 13C NMR (151 MHz, CDCl3) δ 21.9 (CH3), 24.12 (piper-
idine-C-4), 25.8 (piperidine-C-3,5), 54.7 (piperidine-C-2,6), 56.2 (CH2),
124.1 (C-7), 125.7, 126.5 (C-6 and C-7a), 128.2 (tosyl-C-2,6), 130.2
(tosyl-C-3,5), 135.0 (tosyl-C-1), 145.7 (C-5), 146.8 (tosyl-C-4), 151.7
(C-3a), 156.1 (C-2); HR-MS (ESI) m/z: Calcd for C19H20Cl2N4O2SNa: [M
+Na]+ = 461,0576, found 461,0571.
153.7 (C-2); HR-MS (ESI) m/z: Calcd for
C19H23ClN5O2S: [M
+H]+ = 420.1255, found 420.1253.
4.1.46. 1-(4-Methylbenzenesulfonyl)-6-chloro-2-[(morpholin-4-yl)
methyl]-1H-imidazo[4,5-b]pyridine
(25c)
and
3-(4-
methylbenzenesulfonyl)-6-chloro-2-[(morpholin-4-yl)methyl]-3H-imidazo
[4,5-b]pyridine (27c)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 19c. Purification was effected using a
mixture of CHCl3/MeOH (99.5/0.5 to 96/4, v/v);
Data for 28a: Yield 5%; mp 158–159 °C (petroleum ether); 1H NMR
(600 MHz, CDCl3) δ 1.49 (m, 2H, piperidine-H-4), 1.58 (m, 4H, pi-
peridine-H-3,5), 2.43 (s, 3H, CH3), 2.55 (m, 4H, piperidine-H-2,6), 4.01
(s, 2H, CH2), 7.34 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.99 (s, 1H, H-7),
8.46 (d, J = 8.2 Hz, 2H, tosyl-H-2,6); 13C NMR (151 MHz, CDCl3) δ
21.9 (CH3), 24.3 (piperidine-C-4), 26.0 (piperidine-C-3,5), 54.9 (pi-
peridine-C-2,6), 57.4 (CH2), 126.6 (C-7), 129.65 (tosyl-C-2,6), 129.74
(tosyl-C-3,5), 129.87, 133.5 (C-6 and C-7a), 134.7 (tosyl-C-1), 144.3 (C-
5), 144.4 (C-3a), 146.6 (tosyl-C-4), 154.7 (C-2). HR-MS (ESI) m/z: Calcd
for C19H20Cl2N4O2SNa: [M+Na]+ = 461,0576, found 461,0579.
Data for 25c: Yield 65%; mp 167–168 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 2.43 (s, 3H, C6H4-CH3), 2.63 (m, 4H, morpholine-
H-3,5), 3.59 (m, 4H, morpholine-H-2,6), 4.08 (s, 2H, CH2), 7.34 (d,
J = 8.1 Hz, 2H, tosyl-H-3,5), 8.10 (d, J = 8.1 Hz, 2H, tosyl-H-2,6), 8.24
(d, J = 2.2 Hz, 1H, H-7), 8.51 (d, J = 2.2 Hz, 1H, H-5); 13C NMR
(151 MHz, CDCl3) δ 21.9 (CH3), 53.8 (morpholine-C-3,5), 55.9 (CH2),
66.7 (morpholine-C-2,6), 121.7 (C-7), 126.5 (C-7a), 127.8 (tosyl-C-2,6),
128.6 (C-6), 130.2 (tosyl-C-3,5), 135.2 (tosyl-C-1), 146.0 (C-5), 146.8
(tosyl-C-4), 152.4 (C-3a), 154.1 (C-2); HR-MS (ESI) m/z: Calcd for
C
18H20ClN4O3S: [M+H]+ = 407.0939, found 407.0944.
Data for 27c: Yield 7%; mp 159–160 °C (Et2O); 1H NMR (600 MHz,
4.1.49. 1-(4-Methylbenzenesulfonyl)-5,6-dichloro-2-[(4-methylpiperazin-
CDCl3) δ 2.43 (s, 3H, C6H4-CH3), 2.66 (m, 4H, morpholine-H-3,5), 3.71
(m, 4H, morpholine-H-2,6), 4.11 (s, 2H, CH2), 7.34 (d, J = 8.2 Hz, 2H,
tosyl-H-3,5), 7.93 (d, J = 2.2 Hz, 1H, H-7), 8.38 (d, J = 8.2 Hz, 2H,
tosyl-H-2,6), 8.42 (d, J = 2.2 Hz, 1H, H-5); 13C NMR (151 MHz, CDCl3)
δ 21.9 (CH3), 53.9 (morpholine-C-3,5), 57.0 (CH2), 66.9 (morpholine-C-
2,6), 127.7 (C-7), 128.5 (C-6), 129.22 (tosyl-C-2,6), 129.73 (tosyl-C-
3,5), 134.3 (C-7a), 135.1 (tosyl-C-1), 144.5 (C-5), 145.5 (C-3a), 146.5
(tosyl-C-4), 153.3 (C-2); HR-MS (ESI) m/z: Calcd for C18H20ClN4O3S:
[M+H]+ = 407.0939, found 407.0940.
1-yl)methyl]-1H-imidazo[4,5-b]pyridine
(26b)
and
3-(4-
methylbenzenesulfonyl)-5,6-dichloro-2-[(4-methylpiperazin-1-yl)methyl]-
3H-imidazo[4,5-b]pyridine (28b)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 20b. Purification was effected using a
mixture of DCM/MeOH (94/6 to 88/12, v/v);
Data for 26b: Yield 81%; mp 178–179 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 2.32–2.44 (m, 10H, piperazine-CH3, piperazine-H-
3,5, C6H4-CH3), 2.70 (m, 4H, piperazine-H-2,6), 4.06 (s, 2H, CH2), 7.34
(d, J = 8.2 Hz, 2H, tosyl-H-3,5), 8.08 (d, J = 8.2 Hz, 2H, tosyl-H-2,6),
8.33 (s, 1H, H-7); 13C NMR (151 MHz, CDCl3) δ 21.9 (C6H4-CH3), 46.1
(piperazine-CH3), 53.2 (piperazine-C-2,6), 54.8 (piperazine-C-3,5),
55.4 (CH2), 124.2 (C-7), 125.7, 126.8 (C-6 and C-7a), 127.9 (tosyl-C-
2,6), 130.4 (tosyl-C-3,5), 135.0 (tosyl-C-1), 145.9 (C-5), 146.9 (tosyl-C-
4), 151.6 (C-3a), 155.6 (C-2); HR-MS (ESI) m/z: Calcd for
4.1.47. 1-(4-Methylbenzenesulfonyl)-6-chloro-2-[(diethylamino)methyl]-
1H-imidazo[4,5-b]pyridine (25d) and 3-(4-methylbenzenesulfonyl)-6-
chloro-2-[(diethylamino)methy]l-3H-imidazo[4,5-b]pyridine (27d)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 19d. Purification was effected using a
mixture of DCM/EtOAc (90/10 to 50/50, v/v);
C
19H21Cl2N5O2SNa: [M+Na]+ = 476.0685, found 476.0696.
Data for 25d: Yield 72%; mp 142–143 °C (Et2O); 1H NMR (600 MHz,
CDCl3) δ 0.95 (t, J = 7.2 Hz, 6H, 2x CH3), 2.39 (s, 3H, C6H4-CH3), 2.72
(q, J = 7.2 Hz, 4H, 2x CH2), 4.21 (s, 2H, CH2), 7.32 (d, J = 8.2 Hz, 2H,
tosyl-H-3,5), 8.09 (d, J = 8.2 Hz, 2H, tosyl-H-2,6), 8.27 (d, J = 2.2 Hz,
1H, H-7), 8.46 (d, J = 2.2 Hz, 1H, H-5); 13C NMR (151 MHz, CDCl3) δ
10.7 (2x CH3), 21.9 (C6H4-CH3), 46.3 (2x CH2), 51.4 (CH2), 121.7 (C-7),
126.6 (C-7a), 128.0 (tosyl-C-2,6), 129.1 (C-6), 130.1 (tosyl-C-3,5),
135.2 (tosyl-C-1), 145.8 (C-5), 146.6 (tosyl-C-4), 152.6 (C-3a), 155.9
Data for 28b: Yield 3%; mp 156–157 °C (petroleum ether); 1H NMR
(600 MHz, CDCl3) δ 2.33–2.54 (m, 10H, piperazine-H-3,5, piperazine-
CH3, C6H4-CH3), 2.68 (m, 4H, piperazine-H-2,6), 4.09 (s, 2H, CH2),
7.35 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 8.00 (s, 1H, H-7), 8.40 (d,
J = 8.2 Hz, 2H, tosyl-H-2,6); 13C NMR (151 MHz, CDCl3) δ 21.9 (C6H4-
CH3), 46.3 (piperazine-CH3), 53.4 (piperazine-C-2,6), 55.1 (piperazine-
C-3,5), 56.5 (CH2), 126.7 (C-7), 129.7 (tosyl-C-2,6), 129.74 (tosyl-C-
3,5), 129.96, 133.4 (C-6 and C-7a), 134.6 (tosyl-C-1), 144.20 (C-5),
13

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
144.56 (C-3a), 146.7 (tosyl-C-4), 154.0 (C-2); HR-MS (ESI) m/z: Calcd
4.1.52. 1-(4-Methylbenzenesulfonyl)-6-chloro-2-[2-(4-methylpiperazin-1-
for C19H21Cl2N5O2SNa: [M+Na]+ = 476.0685, found 476.0689.
yl)ethyl]-1H-imidazo[4,5-b]pyridine
(29b)
and
3-(4-
methylbenzenesulfonyl)-6-chloro-2-[2-(4-methylpiperazin-1-yl)ethyl]-3H-
imidazo[4,5-b]pyridine (31b)
4.1.50. 1-(4-Methylbenzenesulfonyl)-5,6-dichloro-2-[(morpholin-4-yl)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 21b. Purification was effected using a
mixture of CHCl3/ MeOH (97/3, v/v);
methyl]-1H-imidazo[4,5-b]pyridine
(26c)
and
3-(4-
methylbenzenesulfonyl)-5,6-dichloro-2-[(morpholin-4-yl)methyl]-3H-
imidazo[4,5-b]pyridine (28c)
Data for 29b: Yield 56%; mp 136–137 °C (Et2O); 1H NMR (600 MHz,
CDCl3) δ 2.29 (s, 3H, piperazine-CH3), 2.37–2.50 (m, 7H, piperazine-H-
3,5, C6H4-CH3), 2.60 (m, 4H, piperazine-H-2,6), 3.01 (t, J = 7.60 Hz,
2H, CH2CH2-piperazine), 3.34 (t, J = 7.60 Hz, 2H, CH2CH2-piper-
azine), 7.34 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.78 (d, J = 8.2 Hz, 2H,
tosyl-H-2,6), 8.31 (d, J = 2.1 Hz, 1H, H-7), 8.49 (d, J = 2.1 Hz, 1H, H-
5); 13C NMR (151 MHz, CDCl3) δ 21.9 (C6H4-CH3), 27.3 (CH2CH2-pi-
perazine), 46.2 (piperazine-CH3), 53.0 (piperazine-C-2,6), 55.20 (pi-
perazine-C-3,5), 55.59 (CH2CH2-piperazine), 121.64 (C-7), 126.30 (C-
7a), 127.1 (tosyl-C-2,6), 128.1 (C-6), 130.7 (tosyl-C-3,5), 134.9 (tosyl-
C-1), 145.8 (C-5), 147.0 (tosyl-C-4), 153.0 (C-3a), 157.3 (C-2); HR-MS
(ESI) m/z: Calcd for C20H24ClN5O2SNa: [M+Na]+ = 456.1231, found
456.1231.
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 20c. Purification was effected using a
mixture of DCM/MeOH (99.5/0.5 to 98/2, v/v);
Data for 26c: Yield 69%; mp 176–177 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 2.44 (s, 3H, CH3), 2.63 (m, 4H, morpholine-H-3,5),
3.59 (m, 4H, morpholine-H-2,6), 4.07 (s, 2H, CH2), 7.36 (d, J = 8.2 Hz,
2H, tosyl-H-3,5), 8.08 (d, J = 8.2 Hz, 2H, tosyl-H-2,6), 8.35 (s, 1H, H-
7); 13C NMR (151 MHz, CDCl3) δ 21.9 (CH3), 53.8 (morpholine-C-3,5),
55.8 (CH2), 66.7 (morpholine-C-2,6), 124.2 (C-7), 125.7, 126.9 (C-6
and C-7a), 127.8 (tosyl-C-2,6), 130.4 (tosyl-C-3,5), 135.0 (tosyl-C-1),
145.9 (C-5), 147.1 (tosyl-C-4), 151.6 (C-3a), 155.1 (C-2); HR-MS (ESI)
m/z: Calcd for C18H18Cl2N4O3SNa: [M+Na]+ = 463.0369, found
463.0392.
Data for 31b: Yield 6%; mp 133–134 °C (petroleum ether); 1H NMR
(600 MHz, CDCl3) δ 2.31 (s, 3H, piperazine-CH3), 2.40 (s, 3H, C6H4-
CH3), 2.48 (m, 4H, piperazine-H-3,5), 2.66 (m, 4H, piperazine-H-2,6),
3.02 (t, J = 7.60 Hz, 2H, CH2CH2-piperazine), 3.50 (t, J = 7.60 Hz, 2H,
CH2CH2-piperazine), 7.32 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.87 (d,
J = 2.1 Hz, 1H, H-7), 8.10 (d, J = 8.2 Hz, 2H- tosyl, H-2,6), 8.36 (d,
J = 2.1 Hz, 1H, H-5). 13C NMR (151 MHz, CDCl3) δ 21.9 (C6H4-CH3),
28.4 (CH2CH2-piperazine), 46.1 (piperazine-CH3), 53.1 (piperazine-C-
2,6), 55.21 (piperazine-C-3,5), 55.86 (CH2CH2-piperazine), 127.1 (C-
7), 128.4 (C-6, tosyl-C-2,6), 130.1 (tosyl-C-3,5), 134.8 (C-7a), 135.1
(tosyl-C-1), 143.7 (C-5), 145.3 (C-3a), 146.5 (tosyl-C-4), 156.4 (C-2);
HR-MS (ESI) m/z: Calcd for C20H24ClN5O2SNa: [M+Na]+ = 456.1231,
found 456.1224.
Data for 28c: Yield 6%; mp 161–162 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 2.44 (s, 3H, CH3), 2.65 (m, 4H, morpholine-H-3,5),
3.71 (m,4H, morpholine-H-2,6), 4.09 (s, 2H, CH2), 7.37 (d, J = 8.2 Hz,
2H, tosyl-H-3,5), 8.01, (s, 1H, H-7), 8.40 (d, J = 8.2 Hz, 2H, tosyl-H-
2,6); 13C NMR (151 MHz, CDCl3) δ 22.0 (CH3), 53.9 (morpholine-C-
3,5), 57.0 (CH2), 67.0 (morpholine-C-2,6), 126.8 (C-7), 129.6 (tosyl-C-
2,6), 129.8 (tosyl-C-3,5), 130.0, 133.4 (C-6 and C-7a), 134.6 (tosyl-C-
1), 144.19 (C-5), 144.64 (C-3a), 146.9 (tosyl-C-4) 153.7 (C-2); HR-MS
(ESI) m/z: Calcd for C18H18Cl2N4O3SNa: [M+Na]+ = 463.0369, found
463.0377.
4.1.51. 1-(4-Methylbenzenesulfonyl)-6-chloro-2-[2-(piperidin-1-yl)ethyl]-
1H-imidazo[4,5-b]pyridine (29a) and 3-(4-methylbenzenesulfonyl)-6-
chloro-2-[2-(piperidin-1-yl)ethyl]-3H-imidazo[4,5-b]pyridine (31a)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 21a. Purification was effected using a
mixture of DCM/EtOAc (90/10 to 70/30, v/v);
4.1.53. 1-(4-Methylbenzenesulfonyl)-6-chloro-2-[2-(morpholin-4-yl)
ethyl]-1H-imidazo[4,5-b]pyridine (29c) and 3-(4-methylbenzenesulfonyl)-
6-chloro-2-[2-(morpholin-4-yl)ethyl]-3H-imidazo[4,5-b]pyridine (31c)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 21c. Purification was effected using a
mixture of CHCl3/ MeOH (99.5/0.5 to 97/3, v/v);
Data for 29a: Yield 75%; mp 128–129 °C (EtOAC); 1H NMR
(400 MHz, CDCl3) δ 1.45 (m, 2H, piperidine-H-4), 1.61 (m, 4H, pi-
peridine-H-3,5), 2.43 (s, 3H, CH3), 2.50 (m, 4H, piperidine-H-2,6), 3.00
(t, J = 8.1 Hz, 2H, CH2CH2-piperidine), 3.37 (t, J = 8.1 Hz, 2H,
CH2CH2-piperidine), 7.34 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.81 (d,
J = 8.2 Hz, 2H, tosyl-H-2,6), 8.30 (d, J = 2.2 Hz, 1H, H-7), 8.49 (d,
J = 2.2 Hz, 1H, H-5); 13C NMR (151 MHz, CDCl3) δ 21.8 (CH3), 24.2
(piperidine-C-4), 25.9 (piperidine-C-3,5), 27.2 (CH2CH2-piperidine),
54.5 (piperidine-C-2,6), 56.2 (CH2CH2-piperidine), 121.6 (C-7), 126.3
(C-7a), 127.1 (tosyl-C-2,6), 128.0 (C-6), 130.7 (tosyl-C-3,5), 134.8
(tosyl-C-1), 145.7 (C-5), 147.0 (tosyl-C-4), 152.9 (C-3a), 157.4 (C-2);
HR-MS (ESI) m/z: Calcd for C20H23ClN4O2SNa: [M+Na]+ = 441.1122,
found 441.1114.
Data for 29c: Yield 86%; mp 140–141 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 2.42 (s, 3H, CH3), 2.54 (m, 4H, morpholine-H-3,5),
2.99 (t, J = 7.5 Hz, 2H, CH2CH2-morpholine), 3.33 (t, J = 7.5 Hz, 2H,
CH2CH2-morpholine), 3.69 (m, 4H, morpholine-H-2,6), 7.34 (d,
J = 8.2 Hz, 2H, tosyl-H-3,5), 7.78 (d, J = 8.2 Hz, 2H, tosyl-H-2,6), 8.31
(d, J = 2.2 Hz, 1H, H-7), 8.49 (d, J = 2.2 Hz, 1H, H-5); 13C NMR
(151 MHz, CDCl3) δ 21.9 (CH3), 27.3 (CH2CH2-morpholine), 53.6
(morpholine-C-3,5), 56.0 (CH2CH2-morpholine), 67.0 (morpholine-C-
2,6), 121.6 (C-7), 126.3 (C-7a), 127.1 (tosyl-C-2,6), 128.1 (C-6), 130.8
(tosyl-C-3,5), 134.9 (tosyl-C-1), 145.8 (C-5), 147.1 (tosyl-C-4), 152.9
(C-3a), 157.1 (C-2); HR-MS (ESI) m/z: Calcd for C19H22ClN4O3S: [M
+H]+ = 421.1096, found 421.1101.
Data for 31a: Yield 7%; mp: 124–125 °C (petroleum ether); 1H NMR
(400 MHz, CDCl3) δ 1.47 (m, 2H, piperidine-H-4), 1.64 (m, 4H, pi-
peridine-H-3,5), 2.40 (s, 3H, CH3), 2.57 (m, 4H, piperidine-H-2,4), 2.99
(t, J = 8.1 Hz, 2H, CH2CH2-piperidine), 3.52 (t, J = 8.1 Hz, 2H,
CH2CH2-piperidine), 7.32 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.87 (d,
J = 2.1 Hz, 1H, H-7), 8.11 (d, J = 8.2 Hz, 2H, tosyl-H-2,6), 8.36 (d,
J = 2.1 Hz, 1H, H-5); 13C NMR (151 MHz, CDCl3) δ 21.9 (CH3), 24.3
(piperidine-C-4), 25.9 (piperidine-C-3,5), 29.8 (CH2CH2-piperidine),
54.5 (piperidine-C-2,6), 56.5 (CH2CH2-piperidine), 126.4 (C-7a), 127.1
(C-7), 128.5 (tosyl-C-2,6), 130.1 (tosyl-C-3,5), 134.8 (C-6), 135.1
(tosyl-C-1), 143.7 (C-5), 145.4 (C-3a), 146.5 (tosyl-C-4), 156.6 (C-2);
HR-MS (ESI) m/z: Calcd for C20H23ClN4O2SNa: [M+Na]+ = 441.1122,
found 441.1117.
Data for 31c: Yield 15%; mp 138–139 °C (Et2O); 1H NMR (600 MHz,
CDCl3) δ 2.41 (s, 3H, CH3), 2.61 (m, 4H, morpholine-H-3,5), 3.01 (t,
J = 7.6 Hz, 2H, CH2CH2-morpholine), 3.51 (t, J = 7.6 Hz, 2H, CH2CH2-
morpholine), 3.74 (m, 4H, morpholine-H-2,6), 7.33 (d, J = 8.2 Hz, 2H,
tosyl-H-3,5), 7.88 (d, J = 2.2 Hz, 1H, H-7), 8.10 (d, J = 8.2 Hz, 2H,
tosyl-H-2,6), 8.36 (d, J = 2.2 Hz, 1H, H-5); 13C NMR (151 MHz, CDCl3)
δ 21.9 (CH3), 28.3 (CH2CH2-morpholine), 53.7 (morpholine-C-3,5),
56.3 (CH2CH2-morpholine), 67.1 (morpholine-C-2,6), 127.1 (C-7),
128.45 (tosyl-C-2,6), 128.49 (C-6), 130.1 (tosyl-C-3,5), 134.8 (C-7a),
135.1 (tosyl-C-1), 143.8 (C-5), 145.3 (C-3a), 146.6 (tosyl-C-4), 156.2
(C-2); HR-MS (ESI) m/z: Calcd for
C19H22ClN4O3S: [M
+H]+ = 421.1096, found 421.1103.
14

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4.1.54. 1-(4-Methylbenzenesulfonyl)-5,6-dichloro-2-[2-(piperidin-1-yl)
ethyl]-1H-imidazo[4,5-b]pyridine (30a)
attempt to prepare the 2-diethylamino derivative of 21d. Oil; 1H NMR
(600 MHz, CDCl3) δ (ppm) 2.41 (s,3H, CH3), 5.88 (d, Jab = 11.5 Hz, 1H,
Hb), 6.76 (d, Jac = 17.8, 1H, Hc), 7.33 (d, 2H, J = 8.9 Hz, tosyl-Η-3,5),
7.44 (dd, Jab = 11.5, Jac = 17.8, 1H, Ha), 7.76 (d, 2H, J = 8.9 Hz,
tosyl-Η-2,6), 8.36 (d, J = 2.2 Hz, 1H, H-7), 8.52 (d, J = 2.2 Hz, 1H, H-
5), 13C NMR (151 MHz, CHCl3) δ (ppm) 21.8 (CH3), 122.0 (C-7), 123.2
(C-2΄), 126.5 (C-7a), 127.1 (tosyl-C-2,6), 128.3 (C-6), 129.1 (C-1΄),
130.7 (tosyl-C-3,5), 134.9 (tosyl-C-1), 146.59 (C-5), 147.05 (tosyl-C-4),
153.2 (C-3a), 153.7 (C-2); HR-MS (ESI) m/z: Calcd for
This compound was prepared by a procedure analogous to that of
25a and 27a, starting from 22a. Purification was effected using a
mixture of DCM/EtOAc (99/1 to 50/50, v/v); Yield 46%; oil; 1H NMR
(600 MHz, CDCl3) δ 1.44 (m, 2H, piperidine-H-4), 1.59 (m, 4H, pi-
peridine-H-3,5), 2.43 (s, 3H, CH3), 2.49 (m, 4H, piperidine-H-2,6), 2.97
(t, J = 7.6 Hz, 2H, CH2CH2-piperidine), 3.35 (t, J = 7.6 Hz, 2H,
CH2CH2-piperidine), 7.35 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.80 (d,
J = 8.2 Hz, 2H, tosyl-H-2,6), 8.41 (s, 1H, H-7); 13C NMR (151 MHz,
CDCl3) δ 21.9 (CH3), 24.3 (piperidine-C-4), 26.0 (piperidine-C-3,5),
27.4 (CH2CH2-piperidine), 54.6 (piperidine-C-2,6), 56.2 (CH2CH2-pi-
peridine), 124.0 (C-7), 125.4, 126.3 (C-6, C-7a), 127.2 (tosyl-C-2,6),
130.8 (tosyl-C-3,5), 134.7 (tosyl-C-1), 145.6 (C-5), 147.3 (tosyl-C-4),
152.2 (C-3a), 157.6 (C-2); HR-MS (ESI) m/z: Calcd for
C
15H12ClN3O2SNa: [M+Na]+ = 356.0231 found 356.0225.
4.1.58. 1-(4-Methylbenzenesulfonyl)-5,6-dichloro-2-vinyl-1H-imidazo
[4,5-b]pyridine (34)
This compound was obtained as the single product during our at-
tempt to prepare the 2-diethylamino derivative of 22d. mp 187–188 °C
(EtOAc); 1H NMR (600 MHz, CDCl3) δ (ppm) 2.42 (s,3H, CH3), 5.94 (d,
Jab = 11.5 Hz, 1H, Hb), 6.76 (d, Jac = 17.8, 1H, Hc), 7.33 (d, 2H,
J = 8.9 Hz, tosyl-Η-3,5), 7.41 (dd, Jab = 11.5, Jac = 17.8, 1H, Ha), 7.71
(d, 2H, J = 8.9 Hz, tosyl-Η-2,6), 8.45 (d, J = 2.2 Hz, 1H, H-7); 13C
NMR (151 MHz, CHCl3) δ (ppm) 21.9 (CH3), 122.9 (C-2΄), 124.3 (C-7),
125.6, 126.4 (C-6, C-7a), 127.1 (tosyl-C-2,6), 129.8 (C-1΄), 130.8 (tosyl-
C-3,5), 134.6 (tosyl-C-1) 146.4 (C-5), 147.3 (tosyl-C-4), 152.4 (C-3a),
154.4 (C-2); HR-MS (ESI) m/z: Calcd for C15H11Cl2N3O2SNa: [M
+Na]+ = 389.9841 found 389.9834.
C
20H22Cl2N4O2SNa: [M+Na]+ = 475.0733, found 475.0729.
4.1.55. 1-(4-Methylbenzenesulfonyl)-5,6-dichloro-2-[2-(4-
methylpiperazin-1-yl)ethyl]-1H-imidazo[4,5-b]pyridine (30b)
This compound was prepared by a procedure analogous to that of
25a and 27a, starting from 22b. Purification was effected using a
mixture of CHCl3/MeOH (99/1 to 85/15, v/v); Yield 26%; oil; 1H NMR
(600 MHz, CDCl3) δ 2.30 (s, 3H, piperazine-CH3), 2.37–2.69 (m, 11H,
CH3, piperazine-H), 2.98 (t, J = 7.60 Hz, 2H, CH2CH2-piperazine), 3.31
(t, J = 7.60 Hz, 2H, CH2CH2-piperazine), 7.34 (d, J = 8.2 Hz, 2H,
tosyl-H-3,5), 7.75 (d, J = 8.2 Hz, 2H, tosyl-H-2,6), 8.41 (s, 1H, H-7);
13C NMR (151 MHz, CDCl3) δ 21.6 (CH3), 27.5 (CH2CH2-piperazine),
44.1 (piperazine-CH3), 53.2 (piperazine-C-2,6), 55.6 (piperazine-C-
3,5), 57.1 (CH2CH2-piperazine), 121.3 (C-7), 126.2, 128.0 (C-7a, C-6),
129.1 (tosyl-C-2,6), 131.6 (tosyl-C-3,5), 134.9 (tosyl-C-1), 144.7 (C-5),
146.9 (tosyl-C-4), 152.9 (C-3a), 157.5 (C-2); HR-MS (ESI) m/z: Calcd for
4.2. Biology
4.2.1. Cell lines
HepAD38 and HepG2-NTCP cell lines are derived from HepG2 cells.
HepAD38 cells (kindly provided by Dr. Christoph Seeger, Fox Chase
Center, USA) contain the HBV genome (subtype ayw) under tetra-
cycline control [44]. They were maintained in Dulbecco modified Eagle
medium (DMEM)/F-12 (Gibco) supplemented 10% (v/v) fetal calf
serum (FCS), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 100
U/mL penicillin, 100 µg/mL streptomycin, 0.2 µg/ml hydrocortisone
(STEMCELL), and 5 μg/ml insulin (Sigma). HepG2-NTCP cells (kindly
provided by Prof. Stephan Urban, Heidelberg University, Germany)
stably overexpress human NTCP [45]. They were maintained in high
glucose (25 mM) DMEM (Gibco) supplemented with 2 mM L-glutamine,
0.1 mM non-essential amino acids, 100 U/mL penicillin, 100 µg/mL
streptomycin and 10% (v/v) FCS (referred to as complete DMEM).
C
20H24Cl2N5O2S: [M+H]+ = 468.1022 found 468.1011.
4.1.56. 1-(4-Methylbenzenesulfonyl)-5,6-dichloro-2-[2-(morpholin-4-yl)
ethyl]-1H-imidazo[4,5-b]pyridine (30c) and 3-(4-methylbenzenesulfonyl)-
5,6-dichloro-2-[2-(morpholin-4-yl)ethyl]–3H-imidazo[4,5-b]pyridine
(32c)
These compounds were prepared by a procedure analogous to that
of 25a and 27a, starting from 22c. Purification was effected using a
mixture of CHCl3/ MeOH (99.5/0.5 to 98.5/1.5, v/v);
Data for 30c: Yield 89%; mp 179–180 °C (EtOAc); 1H NMR
(600 MHz, CDCl3) δ 2.41 (s, 3H, CH3), 2.54 (m, 4H, morpholine-H-3,5),
2.98 (t, J = 7.2 Hz, 2H, CH2CH2-morpholine), 3.33 (t, J = 7.2 Hz, 2H,
CH2CH2-morpholine), 3.69 (m, 4H, morpholine-H-2,6), 7.34 (d,
J = 8.2 Hz, 2H, tosyl-H-3,5), 7.78 (d, J = 8.2 Hz, 2H, tosyl-H-2,6), 8.40
(s, 1H, H-7); 13C NMR (151 MHz, CDCl3) δ 21.8 (CH3), 27.0 (CH2CH2-
morpholine), 53.3 (morpholine-C-3,5), 55.4 (CH2CH2-morpholine),
66.6 (morpholine-C-2,6), 124.0 (C-7), 125.4, 126.4 (C-6, C-7a), 127.2
(tosyl-C-2,6), 130.9 (tosyl-C-3,5), 132.2 (tosyl-C-1), 145.7 (C-5), 147.4
(tosyl-C-4), 152.0 (C-3a), 157.1 (C-2); HR-MS (ESI) m/z: Calcd for
4.2.2. Virus production
Virus stocks were produced on HepAD38 cells as described pre-
viously [51]. Briefly, cells were cultured in Williams’ medium E (Gibco)
with 5% FCS, 35 µg/ml hydrocortisone (STEMCELL), and 5 μg/ml in-
sulin (Sigma), and 2% dimethyl sulfoxide (DMSO). Cell supernatants
were collected every third to fourth day and filtered through a 0.45-μm
filter. HBV particles were concentrated from the clarified supernatant
by overnight precipitation with 5% PEG 8000 and centrifugation at 4 °C
for 60 min at 4,000 rpm. HBV titers were determined after DNA ex-
traction using QIAamp DNA blood mini kit (Qiagen), followed by
quantification of HBV rcDNA with quantitative real-time polymerase
chain reaction (qPCR), using primers HBV-rcDNA-F (5′-GACTCTCTCG
TCCCCTTCTC-3′) and HBV-rcDNA-R (5′-ATGGTGAGGTGAACAAT
GCT-3′). Standard curves were generated from an HBV genome-con-
taining plasmid (payw1.2) [52].
C
19H21Cl2N4O3S: [M+H]+ = 455.0706 found 455.0710.
Data for 32c: Yield 5%; mp 162–163 °C (Et2O); 1H NMR (600 MHz,
CDCl3) δ 2.43 (s, 3H, CH3), 2.61 (m, 4H, morpholine-H-3,5), 3.02 (~t,
2H, CH2CH2-morpholine), 3.51 (~t, 2H, CH2CH2-morpholine), 3.74 (m,
4H, morpholine-H-2,6), 7.36 (d, J = 8.2 Hz, 2H, tosyl-H-3,5), 7.95 (s,
1H, H-7), 8.13 (d, J = 8.2 Hz, 2H, tosyl-H-2,6); 13C NMR (151 MHz,
CDCl3) δ 21.9 (CH3), 28.3 (CH2CH2-morpholine), 53.7 (morpholine-C-
3,5), 56.3 (CH2CH2-morpholine), 67.0 (morpholine-C-2,6), 126.7 (C-7),
129.0 (tosyl-C-2,6), 129.4 (C-6), 130.1 (tosyl-C-3,5), 133.78 (C-7a),
133.88 (tosyl-C-1), 134.5 (C-5), 143.9 (tosyl-C-4), 146.9 (C-3a), 156.4
4.2.3. Cytotoxicity assay
Cytotoxicity of the compounds was determined by measuring in-
tracellular ATP levels. Specifically, 104 HepG2-NTCP cells per well were
seeded in 96-well flat bottom plates in total volume of 100 μL complete
DMEM. After 24 h, cells were incubated with the compounds for 7 days
at 37 °C (5% CO2). Cells treated with the solvent DMSO were used as
control. ATP was measured using the ViaLight HS BioAssay kit (Lonza)
according to the manufacturer’s protocol in a GloMax 20/20 single-tube
(C-2); HR-MS (ESI) m/z: Calcd for
C19H21Cl2N4O3S: [M
+H]+ = 455.0706 found 455.0715.
4.1.57. 1-(4-Methylbenzenesulfonyl)-6-chloro-2-vinyl-1H-imidazo[4,5-b]
pyridine (33)
This compound was obtained as the single product during our
15

M. Gerasi, et al.
BioorganicChemistryxxx(xxxx)xxxx
luminometer (Promega) for 1 s. ATP levels were normalized to total
protein amounts and used for the calculation of the compound con-
centration causing 50% cell death (CC50). CC50 values were determined
by nonlinear regression analysis after converting the drug concentra-
tions into log-X using the Prism 5.0 software (GraphPad Software Inc.).
were determined after DNA extraction using QIAamp DNA blood mini
kit (Qiagen), followed by qPCR as described in Paragraph 4.2.2.
4.2.7. Statistical analysis
Statistical calculations were carried out using Excel Microsoft
Office® or GraphPad Prism (GraphPad Software, USA). Only results
subjected to statistical analysis using Student’s t-test with p ≤ 0.05
were considered as statistically significant and presented.
4.2.4. Infection and antiviral assay
HepG2-NTCP cells were seeded in 24-well plate (60,000 cells/well).
After 16 h, cells were inoculated with HBV at 1000 genome equivalents
(GEq) per cell in the presence of 2% DMSO and 4% PEG 8000. After
24 h, cells were washed 3 times and incubated with complete DMEM
containing 2% DMSO for an additional 6 days. Then, cells were lysed or
their supernatants were collected for quantification of intracellular and
secreted, respectively, HBV nucleic acids. For antiviral assays, the in-
hibitors were used to pre-incubate the cells 2 h before virus inoculation
and were also added during infection.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
4.2.5. HBV DNA/RNA isolation and quantification
We are grateful to Dr. Christoph Seeger (Fox Chase Center, USA)
and Prof. Stephan Urban, Heidelberg University, Germany) for pro-
viding HepAD38 and HepG2-NTCP cell lines, respectively, and Dr.
Christine Neuveut (Institut Pasteur Paris, France) for scientific advice
on HBV infectious system and viral life cycle. G. Papadakis was funded
by a Scholarship through the Onassis Foundation, Liechtenstein.
HBV rcDNA in cell supernatants was extracted and quantified as
described above for virus stocks. HBV rcDNA values from cells treated
with serial dilutions of the most active inhibitors were used for the
calculation of their median effective concentrations (EC50), by non-
linear regression analysis after converting the drug concentrations into
log-X using the Prism 5.0 software (GraphPad Software Inc.). Total RNA
was extracted from cells lyzed with Trizol (Invitrogen), according to the
manufacturer’s instructions. HBV pgRNA was quantified with reverse-
transcription (RT) and qPCR. RT was performed using Moloney Murine
Leukemia Virus (MMLV) reverse transcriptase (Promega) with oligo
(dT) primers (NEB). qPCR was performed using KAPA SYBR FAST qPCR
Master Mix (Kapa Biosystems) and primers HBV-pgRNA-F (5′-TGTCA
ACACTAATATGGGCCTAA-3′) and HBV-pgRNA-R (5′-AGGGGCATTTG
GTGGTCTAT-3′). The housekeeping gene YWHAZ was used for nor-
malization (primers 5′-GCTGGTGATGACAAGAAAGG-3′ and 5′- GGAT
GTGTTGGTTGCATTTCCT-3′). For HBV cccDNA and intracellular en-
capsidated rcDNA quantification, cells were lysed with 10 mM Tris-HCl
pH 7.5, 1 mM EDTA, 50 mM NaCl, 8% sucrose and 0.375% NP-40. After
3 min of centrifugation at 12000 g, the pellet (nuclei) and the super-
natant (cytoplasm) were recovered. DNA was extracted from the pellet
using QIAamp DNA blood mini kit (Qiagen) and HBV cccDNA was
quantified with qPCR using primers HBV-cccDNA-F (5′-GTGCACTTCG
CTTCACCTCT-3′) and HBV-cccDNA-R 5′-AGCTTGGAGGCTTGAAC
AGT-3′. The supernatant was treated with 10 U RQ1 DNase (Promega)
and 50 μg RNase Α (Invitrogen) for 30 min at 37 °C. Then, viral capsids
were precipitated by adding 350 mΜ NaCl, 6.25 mM EDTA and 6.5%
PEG 8000 for 30 min on ice. After centrifugation (4 min at 12000 g) the
pellet was resuspended in 10 mM Tris-HCl pH 7.5 and 6 mM MgCl2 and
treated with 10 U RQ1 DNase, for 15 min at 37 °C. After proteinase K
treatment, phenol-chloroform extraction and isopropanol precipitation,
HBV rcDNA was quantified by qPCR using primers HBV-rcDNA-F and
HBV-rcDNA-R. Data were normalized to cell number by qPCR ampli-
fication of Cycline A2 gene (CCNA2) using primers CCNA2-F (5′-CCT
GCTCAGTTTCCTTTGGT-3′) and CCNA2-R (5′-AGACGCCCAGAGATG
CAG-3′). Mean values were calculated according to the ΔCT quantifi-
cation method.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103580.
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