Synthesis and antiviral activity of new phenylimidazopyridines and N-benzylidenequinolinamines derived by molecular simplification of phenylimidazo[4,5-g]quinolines

Article abstract of DOI:10.1016/j.ejmech.2014.07.011

Continuing our program of research concerning the antiviral activity of a wide series of new angular and linear azolo bicyclic and tricyclic derivatives, now we have simplified and modified the 4-chloro-2-(4-nitrophenyl)-3H-imidazo[4, 5-g]quinoline 1, which previously resulted the most active derivative, through either the elimination of the central ring or the opening of the imidazole ring, obtaining various imidazopyridines and N-benzylidenequinolinamines respectively. Title compounds were tested in cell-based assays for cytotoxicity and antiviral activity against representatives of two DNA virus families as wells as against representatives of RNA virus families containing single-stranded, either positive-sense (ssRNA⁺) or negative-sense (ssRNA^-), and double-stranded genomes (dsRNA). Some imidazo[4,5-b]pyridines emerged as new derivatives endowed with antiviral activity against Vaccinia Virus (VV) at concentrations ranging from 2 to 16 μM. In particular, compound 2b demonstrate to be about 10 times more potent than Cidofovir, used as reference drug. Similarly, the imidazo[4,5-c]pyridines and N-benzylidenequinolinamines derivatives resulted active against Bovine Viral Diarrhoea virus (BVDV), at concentrations ranging from 1.2 to 28 μM. Above all compounds 1, 3a and 3f showed an EC₅₀ of the same order of magnitude of the reference drug, the 2′-C-methyl-guanosine. Moreover, several N-benzylidenequinolinamines showed an interesting activity against Respiratory Syncytial Virus (RSV) at concentrations between 12 and 26 μM.

Full text of DOI:10.1016/j.ejmech.2014.07.011

European Journal of Medicinal Chemistry 84 (2014) 8e16
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiviral activity of new phenylimidazopyridines and
N-benzylidenequinolinamines derived by molecular simplification of
phenylimidazo[4,5-g]quinolines
,
*
,
,
Roberta Loddo b 1, Irene Briguglio a 1, Paola Corona a, Sandra Piras a, Mario Loriga a,
Giuseppe Paglietti a, Antonio Carta a *, Giuseppina Sanna b, Gabriele Giliberti b,
,
Cristina Ibba b, Pamela Farci b, Paolo La Colla b
a
ꢀ
Dipartimento di Chimica e Farmacia, Universita degli Studi di Sassari, Via Muroni 23/A, 07100 Sassari, Italy
Dipartimento di Scienze Biomediche, Sezione di Microbiologia e Virologia, Universita degli Studi di Cagliari, Cittadella Universitaria s.p.8, Km 0,700, 09042
b
ꢀ
Monserrato, Ca, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Continuing our program of research concerning the antiviral activity of a wide series of new angular and
linear azolo bicyclic and tricyclic derivatives, now we have simplified and modified the 4-chloro-2-(4-
nitrophenyl)-3H-imidazo[4,5-g]quinoline 1, which previously resulted the most active derivative,
through either the elimination of the central ring or the opening of the imidazole ring, obtaining various
imidazopyridines and N-benzylidenequinolinamines respectively.
Received 5 February 2014
Received in revised form
9 June 2014
Accepted 3 July 2014
Available online 4 July 2014
Title compounds were tested in cell-based assays for cytotoxicity and antiviral activity against rep-
resentatives of two DNA virus families as wells as against representatives of RNA virus families con-
taining single-stranded, either positive-sense (ssRNAþ) or negative-sense (ssRNAꢀ), and double-stranded
genomes (dsRNA). Some imidazo[4,5-b]pyridines emerged as new derivatives endowed with antiviral
Keywords:
Phenylimidazopiridines
N-benzylidenequinolinamines
Antiviral activity
activity against Vaccinia Virus (VV) at concentrations ranging from 2 to 16 mM. In particular, compound
2b demonstrate to be about 10 times more potent than Cidofovir, used as reference drug. Similarly, the
imidazo[4,5-c]pyridines and N-benzylidenequinolinamines derivatives resulted active against Bovine
Bovine viral diarrhoea virus (BVDV)
Vaccinia virus (VV)
Respiratory syncytial virus (RSV)
Coxsackie virus type B5 (CVB-5)
Herpes simplex virus type 1 (HSV-1)
Viral Diarrhoea virus (BVDV), at concentrations ranging from 1.2 to 28 mM. Above all compounds 1, 3a
and 3f showed an EC50 of the same order of magnitude of the reference drug, the 20-C-methyl-guanosine.
Moreover, several N-benzylidenequinolinamines showed an interesting activity against Respiratory
Syncytial Virus (RSV) at concentrations between 12 and 26
m
M.
© 2014 Published by Elsevier Masson SAS.
1. Introduction
Particularly we focused our attention on HSV-1, the most com-
mon etiologic agent of sporadic fatal encephalitis worldwide [4].
The clinical syndrome is often characterized by the rapid inception
of fever, headache and nausea, followed by acute or subacute onset
of an encephalopathy whose symptoms include lethargy, confu-
sion, and delirium [5]. It is estimated to affect at least 1 in 500,000
per year [6]. Nucleoside analogues acyclovir (ACV) and its de-
rivatives with better bioavailability such as famciclovir, valacyclovir
and penciclovir have become the first line drugs for prophylaxis
and treatment of HSV infections. However, their wide use to treat
Herpes Simplex infections and their long-term administration for
the treatment of chronic infections in the immunocompromised
host (such as the organ transplant patient or patient with AIDS) can
lead to the development of ACV-resistant mutant strain [7]. As
result, the efficacy of these nucleoside analogues has been
compromised.
Over 200 species of DNA and RNA viruses are known to be able
to infect and cause significant diseases in humans [1,2]. Examples of
DNA pathogen viruses are Herpesvirus and Poxvirus [3]. Herpes
viruses belongs to the Herpesviridae family, subdivided into
numerous genera, comprehending Herpes simplex virus type 1
(HSV-1), that causes facial cold-sores, Herpes simplex virus type 2
(HSV-2), also called genital herpes, and Varicella-Zoster virus
(VZV), which causes chickenpox in children and shingles in adults.
* Corresponding authors.
E-mail addresses: rloddo@unica.it (R. Loddo), acarta@uniss.it (A. Carta).
The first two authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.07.011
0223-5234/© 2014 Published by Elsevier Masson SAS.

R. Loddo et al. / European Journal of Medicinal Chemistry 84 (2014) 8e16
9
Poxviruses (members of the Poxviridae family) are viruses that
can infect both vertebrate and invertebrate animals. They are
subdivided in four genera, among which the Orthopoxvirus genus
include Variola virus, Vaccinia virus (VV), Cowpox virus and Mon-
keypox virus [8]. Before the introduction of vaccination and erad-
ication of Variola virus's infection (smallpox) in 1970s, it caused
significant morbidity in human populations with anywhere from 1
to 25 percent of mortality [9]. In recent years, however, the po-
tential of the use of Variola virus or another Orthopoxvirus, as a
bioterrorism weapon, has heightened our awareness as to our
vulnerability to this disease since vaccination for smallpox was
discontinued in 1980 [10e14]. This has stimulated efforts to
develop new drugs for treatment of Poxvirus infections. This po-
tential threat has resulted in a resurgent effect to identify and
develop more agents that can be used in an emergency situation to
treat these viral diseases.
RNA viruses include more than 350 different human pathogens
and most of the etiological agents of emerging diseases. According
to their type of genetic material, RNA viruses are classified as single-
stranded RNA (ssRNA) or double-stranded RNA (dsRNA). Further
classification accounts for the ssRNA viruses polarity into negative-
sense and positive-sense, or ambisense. Unfortunately, most of
these human pathogens have no available vaccine, and only a few
selective antiviral drugs are utilizable in the clinic to prevent and/or
treat their infections. Between the ssRNA, viruses belonging to the
Flaviviridae family cause clinically significant diseases in humans
and animals. This virus family includes three genera: Pestiviruses
(i.e., Bovine Viral Diarrhoea virus [BVDV]), Flaviviruses (i.e., Yellow
Fever [YFV], Dengue [DENV], and West Nile [WNV]), and Hep-
aciviruses (Hepatitis C virus [HCV]). Actually, with the exception of
YFV, there is no vaccine and some new drugs are actually in study
[15e23].
have took in account the simplification of imidazo[4,5-g]quinoline
nucleus through the elimination of the central ring or molecular
modification by the opening of the imidazole ring, obtaining imi-
dazopyridines (2) and N-benzylidenequinolinamines (3) de-
rivatives respectively (Fig. 1).
Here we report the chemical synthesis and the biological
assessment of their antiviral activity.
2. Chemistry
All chemical structures of the synthesized compounds are
depicted in Figs. 2 and 3. The nitrogen atom positions in imidazo-
pyridines and the substituents of all compounds were chosen with
the aim to evaluate the influence of electron-withdrawing groups,
lipophilicity and/or steric hindrance, on the antiviral activities.
The synthetic route to obtain the designed imidazopyridines
2ael (Fig. 2) and their intermediates is described in Scheme 1. The
diamines 4aec underwent nucleophilic attack by the correspond-
ing bisulphite compounds 5aee, to give the imidazo[4,5-b] (2aej)
and [4,5-c]pyridine (2k,l).
In turn N-benzylidenequinolinamines 3a,b,feh were obtained
by condensation of diaminoquinolines 6a,b with bisulphite com-
pounds 5a,b,f,g, operating by soft condition in refluxed ethanol
(Scheme 2). Whereas more extreme conditions, DMF at 130 ꢁC, in
past principally afforded the corresponding imidazo[4,5-g]quino-
lines [36].
Bisulphites 5aeg were obtained in high yields from the
commercially available corresponding aldehydes (Aldrich) with
Na2S2O5 in ethanol, according to the procedure used by Shriner and
Land [37].
3. Results, discussion and conclusion
Other important ssRNA viruses pathogens are those belonging
to the Picornaviridae (Coxsackievirus, Poliovirus) and Para-
mixoviridae (Respiratory syncytial virus) and Rhabdoviridae (Ve-
sicular Stomatitis Virus, VSV) families. These viruses cause a variety
of illnesses, and at present, no specific antiviral therapy is available
for the treatment [24,25].
Hence the need to identify new lead compounds targeting a
distinct stages of replication cycle in a virus-specific way. In this
context, according to an antiviral research multiannual program,
our group described the series of 2-[4-substituted naphtyl] [26], 2-
[4-substituted biphenyl] [27], substituted-2-styryl [28] and 5-
acetyl-2-[2,3,4-substituted aryl] benzimidazoles [29]. After bio-
logical evaluation, most of these compounds emerged for their
selective activity against Respiratory Syncytial virus (RSV), Yellow
Fever virus (YFV) and Coxsackie virus type B5 (CVB-5). Afterwards,
with the aim to increase the antiviral behaviour, we operated a
bioisosteric modification replacing the benzimidazole ring with a
benzotriazole nucleus to obtain the series of N-[4-(1H(2H)-benzo-
triazol-1(2)-yl)phenyl]alkylcarboxamides [30] and N,N0-bis[4-
Title compounds 2ael and 3a,b,feh were evaluated in cell based
assays for their cytotoxicity and antiviral activity against a panel of
RNA and DNA viruses. Among single-stranded, positive RNA viruses
(ssRNAþ), we considered a Retrovirus (Human Immunodeficiency
Virus type 1, HIV-1), two Picornaviruses (Coxsackie Virus type-5,
CVB-5, and Poliovirus type-1, Sabin strain, Sb-1), and viruses
representative of two of the three genera of the Flaviviridae family,
i.e., a Flavivirus (Yellow Fever Virus, YFV), and a Pestivirus (Bovine
Viral Diarrhoea Virus, BVDV). Among single-stranded, negative RNA
viruses (ssRNA-) a Paramyxoviridae (Respiratory Syncytial Virus,
RSV) and a Rhabdoviridae (Vesicular Stomatitis Virus, VSV) were
selected as representatives. Among double-stranded RNA (dsRNA)
viruses, a Reoviridae family member (Reo-1) was included. Finally,
(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamides
[31].
Meantime, to provide molecular diversity of benzotriazole and
benzimidazole nucleus for further structure activity relationship
(SAR) analysis, we synthesized and tested for antiviral activity three
new classes of angular [32] and linear N-tricyclic compounds
[33e35].
Recently we have reported new linear azolo tricyclic derivatives
as selective inhibitors of the RNA-dependent RNA polymerases
(RdRps) of various RNA virus [36]. Between them, imidazo[4,5-g]
quinoline derivatives showed an interesting activity against some
ssRNAþ (BVDV, HCV and CVB), and dsRNA (Reo) viruses. Notably
the 4-chloro-2-(4-nitrophenyl)-3H-imidazo[4,5-g]quinoline (1),
resulted endowed with high and selective activity against BVDV
and HCV. Now, with the purpose to extend the SAR analysis we
Fig. 1. Molecular modification of the lead compound 1.

10
R. Loddo et al. / European Journal of Medicinal Chemistry 84 (2014) 8e16
Fig. 2. Imidazo[4,5-b] and [4,5-c]pyridines 2ael synthesized.
that various benzimidazole derivatives are endowed with the same
mechanism of action [38,39], is acceptable to suppose that our
compounds act similarly. In this direction actually are going more
biological and in silico essays.
In all the active derivatives is present a bromine atom in 6 po-
sition, whereas their counterpart without Br 2a,c,i are completely
inactive. Surprisingly in the copies 2e/2f and 2g/2h also the 6-Br-
derivatives resulted inactive. Probably the cause of this behaviour is
the higher cytotoxicity of 2f and 2h, in comparison with 2b,d,j,
associated with the decrease of biological activity due to either 4-
CH3CO-phenyl or furan-3-yl substituents in position 2. Consid-
ering the virtual chemical reactivity paired with the bromine sub-
stitution, we investigated the chemical and enzymatic stability of
this class of compounds. In view of the high activity of 2b against
VV, we chose this compound as an example of the entire class.
Compound was then incubated in rat plasma and in a pH 7.4
buffered solution, and results showed a good stability in buffer
(t > 4) h and plasma (t ¼ 2) h.
Fig. 3. N-benzylidenequinolinamines 3a,b,feh synthesized.
½
½
While, among the imidazo[4,5-c]pyridines, only 2k resulted
active against VV even if at high concentration (EC50 ¼ 56
mM). In
two representatives of DNA virus families were also included:
Herpes Simplex Virus type-1, HSV-1 (Herpesviridae) and Vaccinia
Virus, VV (Poxviridae).
Results are reported in Table 1. In general, they exhibited low
cytotoxicity, with the exception of compounds 2f,h and 3b,g that
resulted be slightly cytotoxic against some cell lines used in this
conclusion this preliminary SAR study suggests that the presence of
both a 6-Br substituent and 40eCNephenyl (2b), 40eCF3ephenyl
(2d) or cyclohexyl (2j) substituents in position 2 is associated to a
selective antiviral activity against VV of the imidazo[4,5-b]pyridine
series.
Furthermore, the imidazo[4,5-b]pyridine 2b turned out to be
active against the second representative DNA-virus tested, the
study (CC50 between 20 and 40
76).
As far as the antiviral activity is concerned, the imidazopyridines
synthesized can be divided into derivatives connected by b (2aej)
or c (2k,l) sides on the pyridine ring and both the adjacent carbon
atoms of the [1,2,3]triazole ring.
mM against MT-4, MDBK and Vero-
Herpes Simplex type-1 (HSV-1), at the concentration of 15
mM.
About the others virus tested only the derivatives 2c and 2j showed
a moderate activity against CVB-5 in the range 18e20 mM but not
against Sb-1.
Among the second series, both the imidazo[4,5-c]pyridine de-
rivatives 2k,l turned out to be selective inhibitors of BVDV in the
range 7e9 mM, this highlighting that the c side connection of the
Three derivatives 2b,d,j belonging to the first series are specif-
ically active against Vaccinia Virus (VV) at concentrations ranging
from 2 to 16 mM. In particular, compound 2b showed an activity
pyridine ring is determining for the anti-BVDV behaviour of this
class of compounds.
about 10 times more potent than the reference drug Cidofovir.
Being Cidofovir molecular target a polymerase, and being known
Scheme 1. Synthesis of compounds 2ael. Reagents and conditions: (i) DMF, 130 ꢁC for 2 h. (ii) EtOH, reflux for 8 h.

R. Loddo et al. / European Journal of Medicinal Chemistry 84 (2014) 8e16
11
Scheme 2. Synthesis of compounds 3a,b,feh.
About the antiviral activity of the N-benzylidenequinolinamines
3a,b,feh, all the compounds resulted active against BVDV and re-
combinant BVDV RNA-dependent RNA polymerase (RdRp) [40]. In
4. Experimental
4.1. Chemistry
particular 3a and 3f resulted the most potent (EC50 ¼ 1.7 and 2
mM
respectively), showing an activity comparable with the reference
4.1.1. General remarks
drug 20-C-methyl-guanosine (EC50 ¼ 1.1
mM).
Melting points were carried out with a Kofler hot stage or Digital
Electrothermal melting point apparatus and are uncorrected.
Infrared spectra were recorded as nujol mulls on NaCl plates with a
€
Moreover, compound 3f can be identified as the opened
analogue of 4-chloro-2-(4-nitrophenyl)-3H-imidazo[4,5g]quino-
line, one of the lead compounds from the previously synthesized
series of RpRd inhibitors [36]. This activity confirms the relevance
of an electron withdrawing substituent on the 4 position of the
phenyl moiety, as previously observed. At the same time the
opening of the imidazole ring seem do not lead to an anti-BVDV
activity loss, while causing a broader antiviral spectrum. In fact,
with the sole exception of 3h, all tested compounds exhibited an
interesting activity against CVB-5, Sb-1 and RSV. In particular 3g
PerkinElmer 781 IR spectrophotometer and are expressed in
n
(cmꢀ1). Uv spectra are qualitative and were recorded in nm for
solutions in EtOH with a PerkinElmer Lambda 5 spectrophotom-
eter. Nuclear magnetic resonance (1HNMR) spectra were deter-
mined in CDCl3, DMSO-d6, CDCl3/DMSO-d6 (1:3 ratio) and were
recorded with a Varian XL-200 (200 MHz) spectrometer. Chemical
shifts (d scale) are reported in parts per million (ppm) downfield
from tetramethylsilane (TMS) used as internal standard. Splitting
patterns are designated, as follows: s, singlet; d, doublet; t, triplet;
q, quadruplet; m, multiplet; br s, broad singlet; dd, double doublet.
The assignment of exchangeable protons (OH and NH) was
confirmed by the addition of D2O. 13C NMR were determined in
DMSO-d6 and were recorded at 100 MHz with Brucher 400 Avance
Nanobay.
turned out as the most active against CVB-5 (EC50 ¼ 16
against Sb-1 (EC50 22 M) and finally 3f versus RSV
(EC50 ¼ 12 M).
mM), 3g
¼
m
m
On the contrary of the imidazo[4,5-c]pyridines, the N-benzyli-
denequinolinamines resulted completely inactive towards VV.
None of the tested compounds, however, resulted active against
HIV-1, YFV, Reo-1 and VSV.
Chromatographic separations were performed using a 1090L
Liquid Chromatograph (HewlettPackard, Palo Alto, USA) equipped
with a diode array detector HP 1040A. All separations were
accomplished on a Phenomenex Luna C18 (200 ꢂ 4.6 mm, particle
In the light of the above mentioned results, we can conclude
that imidazo[4,5-b]pyridine derivatives showed, in general, to be
endowed with good activity against VV, whereas N-benzylidene-
quinolinamines exhibited an interesting activity versus BVDV
comparable to those of both reference drug and lead compound 1
size 5 mm).
Ms spectra were performed on combined HP 5790-HP 5970 GC/
MS apparatus or with a combined Liquid Chromatograph-Agilent
1100 series Mass Selective Detector (MSD). All exact mass data
(HRMS)were obtained with a “Thermo Scientific Quantitative Plus
Orbitrap LC/MS/MS System” apparatus. Analytical thin-layer chro-
matography (TLC) was performed on Merck silica gel F-254 plates.
Pure compounds showed a single spot in TLC. For flash chroma-
tography, Merck silica gel 60 was used with a particle size
0.040e0.063 mm (230e400 mesh ASTM). Elemental analysis were
performed on a Perkin-Elmer 2400 instrument at Laboratorio di
(EC50 ¼ 1.1 and 1.2
mM respectively). Owing to their good ratio of
activity/cytotoxicity imidazo[4,5-b]pyridines might represent new
interesting leads that can be further developed as new agents
against poxvirus infections, through the introduction of alternative
substituents on the positions 2 and 6, and submitted to in vivo
experiments.
On the other hand, N-benzylidenequinolinamines turned out as
attractive leads for the development of new derivatives endowed
with an improved activity against BVDV and RSV, towards the
introduction of either further electron withdrawing or electron
donor substituents on the phenyl moiety and on the pyridine ring.
Finally we can affirm that the molecular simplification and
opening ring of the lead compound 1 results favourable for differ-
entiation and address the antiviral activity against either VV in the
case of imidazo[4,5-b]pyridines or BVDV and RSV in the case of N-
benzylidenequinolinamines.
ꢀ
Microanalisi, Dipartimento di Chimica e Farmacia, Universita di
Sassari, Italy, and the results were within 0.4% of theoretical
values.
4.1.2. Intermediates
The diamines (4aec) were commercially available while bisul-
phite compounds (5aeg) were obtained in high yields from the

12
R. Loddo et al. / European Journal of Medicinal Chemistry 84 (2014) 8e16
Table 1
Cytotoxicity and antiviral activity of imidazo[4,5-b] and [4,5-c]pyridine 2ael and N-benzylidenequinolinamines 3a,b,feh against ssRNAþ (HIV-1, BVDV, YFV, CV-5, Sb-1),
ssRNAꢀ (RSV, VSV), dsRNA (Reo-1) and DNA (VV, HSV-1) viruses.
Compd
MT-4a
CC50
HIV-1b
EC50
MDBKc
CC50
BVDVd
EC50
BHK-21e
CC50
YFVf
EC50
Reo-1g
Vero-76h
CC50
CVB-5i
EC50
Sb-1j
RSVk
VSVl
VVm
HSV-1n
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
3a
3b
3f
>100
>100
53
>100
>100
>100
28
>100
>100
>53
>100
>100
>100
>28
>100
>100
>100
>100
>100
>100
>100
>100
40
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.2
>100
>100
>100
>100
65
>100
>100
>40
>100
>100
9
7
2
14
1.7
11
28
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
>100
>100
ꢃ100
>100
>100
>100
>100
>100
>25
>100
>100
>100
>100
>100
>100
>100
>100
ꢃ100
>100
>100
>100
>100
>100
>25
>100
>100
73
>100
>100
>100
50
>100
>100
>73
18
>100
>100
>50
>100
>40
86
>100
>100
>73
>100
>100
>100
>50
>100
>100
>73
>100
>100
>100
>50
>100
>100
>73
>100
>100
>100
>50
>100
>100
2
>100
4
>100
>50
>100
>40
>100
16
44
>100
15
>100
>100
>100
>50
>100
20
>100
>20
>100
40
>100
>40
>100
>40
>100
>40
>100
>40
>100
>100
>100
>100
46
49
53
20
>100
40
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
90
>100
90
>100
>100
>100
>100
>100
>100
57
>100
22
>100
>100
>100
>100
>100
26
12
14
>90
>100
>100
>100
>100
>100
>100
>90
>100
>90
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
20
>100
>100
46
29
>100
16
56
>100
>100
>90
88
>90
>100
>100
>100
>100
>100
>75
>87
>100
>100
>100
3g
3h
>100
EFV
Met-Gua
Met-Cyt
Eth-Cyt
Aza
CDF
ACG
0.002
>100
1.1
>100
1.9
>100
16
>100
ꢃ12.5
>100
>100
27
23
1.2
19
3
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%.
ND ¼ Not determined.
EFV ¼ Efavirenz.
MetGua ¼ 20-C-methyl-guanosine.
Met-Cyt ¼ 20-C-methyl-cytidine.
Eth-Cyt ¼ 20-C-ethynyl-cytidine.
Aza ¼ 6-aza-uridine.
CDF ¼ Cidofovir.
ACG ¼ Acycloguanosine.
a
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
m
m
m
m
M) required to reduce the proliferation of mock-infected MT-4 cells by 50%, as determined by the MTT method.
M) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, as determined by the MTT method.
М) required to reduce the viability of mock-infected MDBK (Bovine normal kidney) cells by 50%, as determined by the MTT method.
M) required to achieve 50% protection of MDBK cells from BVDV-induced cytopathogenicity, as determined by the MTT method.
М) required to reduce the viability of mock-infected BHK (Hamster normal kidney fibroblast) monolayers by 50%, as determined by the MTT
b
c
d
e
Compound concentration (
m
method.
f
Compound concentration (
m
m
m
m
m
m
m
m
m
M) required to achieve 50% protection of BHK cells from YFV-induced cytopathogenicity, as determined by the MTT method.
M) required to achieve 50% protection of BHK cells from Reo-1-induced cytopathogenicity, as determined by the MTT method.
M) required to reduce the viability of mock-infected VERO-76 cells by 50% as determined by the MTT method.
M) required to reduce the plaque number of CVB-5 by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of Sb-1 by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of RSV by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of VSV by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of VV by 50% in VERO-76 monolayers.
M) required to reduce the plaque number of HSV-1 by 50% in VERO-76 monolayers.
g
h
i
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
Compound concentration (
j
k
l
m
n
commercially available corresponding aldehydes (Aldrich) with
Na2S2O5 in ethanol, according to the procedure used by Shriner and
Land [37]. 6,7-Diamine-8-chloroquinolines (6a,b) was prepared
according to procedure previously reported by us [41].
the general procedure starting from 4a (218 mg, 2 mmol) and so-
dium (4-cyanophenyl)(hydroxy)methanesulfonate (5a) (470 mg,
2 mmol); m.p. 285e288 ꢁC (from EtOH); TLC (chloroform/methanol
9:1): Rf 0.44; IR (nujol): n
3554, 2224, 1597, cmꢀ1; 1H NMR (CDCl3):
d
13.82 (1H, br s, NH), 8.40 (3H, J ¼ 8.8 Hz, H-30, 50 and H-5 partially
obscured), 8.06 (3H, d, J ¼ 8.6 Hz, H-20, 60 and H-7 partially
4.1.3. General procedure for the preparation of the imidazo[4,5-b]
pyridines (2aeh) and imidazo[4,5-c]pyridines (2k,l)
obscured), 7.31(1H, dd, J ¼ 8.0 Hz and J ¼ 4.6 Hz, H-6); 13C-NMR
(CDCl3):
d 162.09 (s), 149.19 (s), 146.11 (d), 139.22 (s), 132.89 (d, 2
A mixture of diamine (4a, 4b or 4c) (2) mmol and either sodium
hydroxy(R-substituted-phenyl-1-yl)methanesulfonate (5aec) or
sodium furan-3-yl(hydroxy)methanesulfonate (5d) (2) mmol was
stirred for 2 h in 4 mL of DMF at 130 ꢁC (for 2aeh, and 2k,l). On
cooling, the mixture was diluted with water and the formed pre-
cipitate was collected and dried on oven. The solid residues were
purified by recrystallization from EtOH.
CH), 129.75 (s), 128.71 (d, 2 CH), 123.23 (d), 123.06 (d), 119.21 (s),
112.66 (s). LC/MS: m/z 221 [M þ 1]. HRMS: 220.0741. Anal. Calcd. for
C13H8N4: C,70.90; H, 3.66; N, 25.44; found C, 70.70; H, 3.56; N,
25.42.
4.1.3.2. 4-(6-Bromo-3H-imidazo[4,5-b]pyridin-2-yl)benzonitrile
(2b). This compound was obtained in 79% yield by the protocol
described in the general procedure starting from 4b (376 mg,
2 mmol) and sodium (4-cyanophenyl)(hydroxy)methanesulfonate
4.1.3.1. 4-(3H-imidazo[4,5-b]pyridin-2-yl)benzonitrile
(2a). This
compound was obtained in 90% yield by the protocol described in

R. Loddo et al. / European Journal of Medicinal Chemistry 84 (2014) 8e16
13
(5a) (470 mg, 2 mmol); m.p. 239e242 ꢁC (from EtOH); TLC (chlo-
HRMS: 315.0017. Anal. Calcd. for C14H10BrN3O: C, 53.19; H, 3.19; N,
13.29; found C, 53.00; H, 3.16; N, 3.26.
roform/methanol 9:1): Rf 0.49; IR (nujol): n ;
3554, 2231, 1597, cmꢀ1
1H NMR (CDCl3):
d
13.99 (1H, br s, NH), 8.40 (2H, J ¼ 8.4 Hz, H-30, 50),
8.39 (1H, s, H-5), 8.25 (1H, s, H-7), 7.94 (2H, d, J ¼ 8.2 Hz, H-20, 60);
4.1.3.7. 2-(Furan-3-yl)-3H-imidazo[4,5-b]pyridine (2g). This com-
pound was obtained in 18% yield by the protocol described in the
general procedure starting from 4a (218 mg, 2 mmol) and sodium
furan-3-yl(hydroxy)methanesulfonate (5e) (400 mg, 2 mmol); m.p.
203e206 ꢁC (from EtOH); TLC (chloroform/methanol 9:1): Rf 0.29;
13C-NMR (CDCl3):
d 161.91 (s), 151.06 (s), 148.79 (d), 140.46 (s),
139.51 (d), 133.19 (d, 2 CH), 132.05 (s), 127.85 (d, 2 CH), 119.44 (d),
118.81 (s), 113.41 (s). LC/MS: m/z 299 [M þ 1]. HRMS: 297.9849.
Anal. Calcd. for C13H7BrN4: C,52.20; H, 2.36; N,18.73; found C, 52.16;
H, 2.29; N, 18.69.
IR (nujol):
n ; d 13.90 (1H, br s,
3558, 1631 cm-1 1H NMR (CDCl3):
NH), 8.35 (1H, s, H-20), 8.30 (1H, dd, J ¼ 7.6 Hz and J ¼ 1.8 Hz, H-5),
7.96 (1H, dd, J ¼ 8.0 Hz and J ¼ 1.8 Hz, H-7), 7.55 (1H, d, J ¼ 1.6 Hz, H-
40), 7.18 (1H, dd, J ¼ 8.0 Hz and J ¼ 4.8 Hz, H-6), 7.14 (1H, d,
4.1.3.3. 2-(4-(trifluoromethyl)phenyl)-3H-imidazo[4,5-b]pyridine
(2c). This compound was obtained in 67% yield by the protocol
described in the general procedure starting from 4a (218 mg,
2 mmol) and sodium hydroxy(4-(trifluoromethyl)phenyl)meth-
anesulfonate (5b) (556 mg, 2 mmol); m.p. 272e274 ꢁC (from EtOH);
J ¼ 1.6 Hz, H-50); 13C-NMR (CDCl3):
d 150.33 (s), 147.41 (s), 145.41
(d), 144.57 (d), 138.65 (d), 130.09 (s), 129.81 (s), 108.77 (d), 123.36
(d), 121.96 (d). LC/MS: m/z 186 [M þ 1]. HRMS: 185.0601. Anal.
Calcd. for C14H7N3O: C, 64.86; H, 3.81; N, 22.69; found C, 64.76; H,
3.78; N, 22.64.
TLC (chloroform/methanol 9:1): Rf 0.44; IR (nujol):
n 3558, 1588,
cmꢀ1; 1H NMR (CDCl3):
d
13.99 (1H, br s, NH), 8.45 (2H, J ¼ 8.4 Hz,
H-30, 50), 8.38 (1H, d, J ¼ 4.2 Hz, H-5), 8.01 (1H, d, J ¼ 8.2 Hz, H-7),
7.83 (2H, d, J ¼ 8.8 Hz, H-20, 60), 7.25 (1H, dd, J ¼ 8.2 Hz and
4.1.3.8. 6-Bromo-2-(furan-3-yl)-3H-imidazo[4,5-b]pyridine
(2h).
J ¼ 4.8 Hz, H-6); 13C-NMR (CDCl3):
d 162.88 (s), 149.63 (s), 146.33
This compound was obtained in 14% yield by the protocol described
in the general procedure starting from 4b (376 mg, 2 mmol) and
sodium furan-3-yl(hydroxy)methanesulfonate (5e) (400 mg,
2 mmol); m.p. 274e277 ꢁC (from EtOH); TLC (chloroform/methanol
(d), 139.12 (s), 131.79 (s), 130.89 (s), 126.68 (d, 2 CH), 125.44 (d, 2
CH), 124.91 (q), 124.01 (s), 123.71 (d). LC/MS: m/z 264 [M þ 1].
HRMS: 263.0645 Anal. Calcd. for C13H8F3N3: C, 59.32; H, 3.06; N,
15.96; found C, 59.29; H, 2.29; N, 15.90.
95:0.5): Rf 0.31; IR (nujol):
8.38 (1H, s, H-5), 8.31 (1H, s, H-20), 8.03 (1H, br s, NH), 7.96 (1H, s,
H-7), 7.66 (1H, s, H-40), 7.10 (1H, s, H-50); 13C-NMR (CDCl3):
150.69
n ;
3558, 1623 cm-1 1H NMR (CDCl3):
d
d
4.1.3.4. 6-Bromo-2-(4-(trifluoromethyl)phenyl)-3H-imidazo[4,5-b]
pyridine (2d). This compound was obtained in 72% yield by the
protocol described in the general procedure starting from 4b
(376 mg, 2 mmol) and sodium hydroxy(4-(trifluoromethyl)phenyl)
methanesulfonate (5b) (556 mg, 2 mmol); m.p. 288e291 ꢁC (from
(s), 149.03 (d), 147.71 (s), 144.26 (d), 140.09 (d), 139.41 (d), 132.39
(s), 129.32 (s), 119.63 (s), 108.91 (d). LC/MS: m/z 265 [M þ 1]. HRMS:
262.9711. Anal. Calcd. for C10H6BrN3O: C, 45.48; H, 2.29; N, 15.91;
found C, 45.38; H, 2.20; N, 15.88.
EtOH); TLC (chloroform/methanol 9:1): Rf 0.45; IR (nujol):
n 3556,
4.1.3.9. 4-(3H-imidazo[4,5-c]pyridin-2-yl)benzonitrile
(2k).
1615, cmꢀ1 1H NMR (CDCl3):
; d 13.93 (1H, br s, NH), 8.44 (2H,
This compound, previously synthesized by D. W. Robertson et al.
[42], was obtained in 51% yield by the protocol described in the
general procedure starting from 4c (218 mg, 2 mmol) and sodium
J ¼ 7.8 Hz, H-30, 50), 8.42 (1H, s, H-5), 8.23 (1H, s, H-7), 7.84 (2H, d,
J ¼ 7.6 Hz, H-20, 60); 13C-NMR (CDCl3):
d 161.48 (s), 150.93 (s), 148.57
(d), 140.07 (d), 138.21 (s), 131.65 (s), 130.77 (s), 126.94 (d, 2 CH),
124.36 (d, 2 CH), 123.71 (d), 120.34 (s). LC/MS: m/z 342 [M þ 1].
HRMS: 340.9769. Anal. Calcd. for C13H7BrF3N3: C, 45.64; H, 2.06; N,
12.28; found C, 45.54; H, 2.00; N, 12.21.
(4-cyanophenyl)(hydroxy)methanesulfonate
(5a)
(470
mg,
2 mmol); m.p. >300 ꢁC (from EtOH); TLC (chloroform/methanol
9:1): Rf 0.32. LC/MS: m/z 145 [M þ 1]. HRMS: 220.0753. Anal. Calcd.
for C7H4N4: C, 58.33; H, 2.80; N, 38.87; found C, 58.29; H, 2.60; N,
38.70.
4.1.3.5. 1-(4-(3H-imidazo[4,5-b]pyridin-2-yl)phenyl)ethanone (2e).
This compound was obtained in 72% yield by the protocol described
in the general procedure starting from 4a (218 mg, 2 mmol) and
sodium (4-acetylphenyl)(hydroxy)methanesulfonate (5c) (504 mg,
2 mmol); m.p. >300 ꢁC (from EtOH); TLC (chloroform/methanol
4.1.3.10. 1-(4-(3H-imidazo[4,5-c]pyridin-2-yl)phenyl)ethanone (2l).
This compound was obtained in 55% yield by the protocol described
in the general procedure starting from 4c (218 mg, 2 mmol) and
sodium (4-acetylphenyl)(hydroxy)methanesulfonate (5c) (470 mg,
2 mmol); m.p. 214e217 ꢁC (from EtOH); TLC (chloroform/methanol
9:1): Rf 0.35; IR (nujol): n
3556, 1672, 1615, cmꢀ1; 1H NMR (CDCl3):
13.96 (1H, br s, NH), 8.38 (2H, J ¼ 8.8 Hz, H-30, 50 and H-7), 8.10
9:1): Rf 0.30; IR (nujol): n d 9.08
1672, 1600 cm-1; 1H NMR (CDCl3):
d
(3H, d, J ¼ 8.8 Hz, H-20, 60), 7.24 (1H, dd, J ¼ 8.2 and J ¼ 4.8 Hz, H-6),
(1H, s, H-4), 8.38 (3H, d, J ¼ 7.2 Hz, H-30, 50 and 1H, partially
2.65 (3H, s, CH3); 13C-NMR (CDCl3):
d 196.67 (CO), 162.08 (s), 151.68
obscured, H-6), 8.12 (2H, d, J ¼ 7.2 Hz, H-20, 60) 7.72 (1H, d,
J ¼ 5.0 Hz, H-4), 2.62 (3H, s, CH3); 13C-NMR (CDCl3):
d 196.77 (CO),
(s), 144.12 (d), 140.83 (s), 137.01 (s), 130.86 (s), 130.56 (d, 2 CH),
127.88 (d, 2 CH), 124.51 (d), 121.44 (d), 27.09 (q). LC/MS: m/z 238
[M þ 1]. HRMS: 237.0912. Anal. Calcd. for C14H11N3O: C, 70.87; H,
4.67; N, 17.71; found C, 70.80; H,4.58; N, 17.68.
176.81 (s), 147.66 (s), 143.37 (d), 141.89 (d), 140.91 (s), 139.34 (s),
138.02 (s), 129.34 (d, 2 CH), 128.01 (d, 2 CH), 113.21 (d), 27.19 (q).LC/
MS: m/z 238 [M þ 1]. HRMS: 237.1005. Anal. Calcd. for C14H11N3O:
C, 70.87; H, 4.67; N, 17.71; found C, 70.77; H,4.58; N, 17.62.
4.1.3.6. 1-(4-(6-bromo-3H-imidazo[4,5-b]pyridin-2-yl)phenyl)etha-
none (2f). This compound was obtained in 74% yield by the pro-
tocol described in the general procedure starting from 4b (376 mg,
2 mmol) and sodium (4-acetylphenyl)(hydroxy)methanesulfonate
(5c) (504 mg, 2 mmol); m.p. >300 ꢁC (from EtOH); TLC (chloroform/
4.1.4. General procedure for the preparation of the imidazo[4,5-b]
pyridines (2i,j)
A mixture of diamine 4a or 4b (2) mmol and sodium cyclo-
hexyl(hydroxy)methanesulfonate (5d) (2) mmol was refluxed in
ethanol (10 mL) for 8 h. After cooling, an excess of the sodium salt
was filtered off through filter paper and the mother liquors were
evaporated to dryness under reduced pressure. The solid residues
were purified by silica gel flash column chromatography, eluting
with a mixture of chloroform-methanol in the ratio indicated under
the Rf of each compound as described below.
methanol 9:1): Rf 0.45; IR (nujol): n
3558,1680,1612, cmꢀ1; 1H NMR
(CDCl3):
d
13.90 (1H, br s, NH), 8.38 (3H, J ¼ 8.6 Hz, H-30, 50and H-5),
8.19 (1H, s, H-7), 8.11 (2H, d, J ¼ 8.6 Hz, H-20, 60), 2.65 (3H, s, CH3);
13C-NMR (CDCl3):
d 198.29 (CO), 161.43 (s), 150.89 (s), 148.54 (d),
141.07 (s), 140.01 (d), 137.51 (s), 132.17 (s), 131.04 (d, 2 CH), 130.11
(s), 128.43 (d, 2 CH), 120.07 (s), 24.91 (q). LC/MS: m/z 317 [M þ 1].

14
R. Loddo et al. / European Journal of Medicinal Chemistry 84 (2014) 8e16
4.1.4.1. 2-Cyclohexyl-3H-imidazo[4,5-b]pyridine (2i). This com-
pound was obtained in 54% yield by the protocol described in the
general procedure starting from 4a (218 mg, 2 mmol) and sodium
cyclohexyl(hydroxy)methanesulfonate (5d) (585 mg, 2 mmol); m.p.
197e198 ꢁC (from EtOH); TLC (chloroform/methanol 9:1): Rf 0.60;
138.54 (s), 136.14 (d), 133. 11 (s), 129.34 (d, 2 CH), 125.90 (d, 2 CH),
122.30 (s), 121.05 (s), 118.58 (d), 112.13 (d), 111.91 (s). LC/MS: m/z
352 [M þ 1]. HRMS: 349.1002. Anal. Calcd. for C17H11ClF3N3: C,
58.38; H, 3.17; N, 12.01; found C, 58.07; H, 3.32; N, 11.75.
IR (nujol):
n
1539 cm-1; 1H NMR (CDCl3):
d
13.47 (1H, s a, NH), 8.33
4.1.5.3. 8-Chloro-N6-(4-nitrobenzylidene)quinoline-6,7-diamine (3f).
This compound was obtained in 30% yield by the protocol described
in the general procedure starting from 6a (387 mg, 2 mmol) and
sodium hydroxy(4-nitrophenyl)methanesulfonate (5f) (510 mg,
2 mmol); m.p. 161e163 ꢁC (from EtOH); TLC (chloroform/methanol
(1H, d, J ¼ 5.0 Hz, H-5), 8.06 (1H, d, J ¼ 7.8 Hz, H-7) 7.26 (1H, dd,
J ¼ 7.8 Hz and J ¼ 5.2 Hz, H-6), 3.12e3.00 (1H, m, CH), 2.30e1.35
(10H, m, H-cyclohexyl); 13C-NMR (CDCl3):
d 151.89 (s), 147.56 (s),
145.49 (d), 132.19 (s), 127.21 (d), 123.01 (d), 40.41 (d), 33.27 (t, 2
CH2), 26.89 (t), 25.78 (t, 2 CH2). LC/MS: m/z 201 [M þ 1]. HRMS:
201,1204. Anal. Calcd. for C12H15N3: C, 71.61; H, 7.51; N, 20.88;
found C, 71.55; H, 7.42; N, 20.77.
9:1): Rf 0.78; IR (nujol): n d 8.78
3375, 1603, cmꢀ1; 1H NMR (CDCl3):
(1H, dd, J ¼ 4.4 and 1.6 Hz, H-2), 8.68 (1H, s, N]CH), 8.27 (2H, d,
J ¼ 8.2 Hz, H-30, 50), 8.12 (1H, dd, J ¼ 8.4 and 1.6 Hz, H-4), 7.70 (2H, d,
J ¼ 8.2 Hz, H-20, 60), 7.35 (1H, s, H-5), 7.29 (1H, dd, J ¼ 8.4 and 4.4 Hz,
4.1.4.2. 6-Bromo-2-cyclohexyl-3H-imidazo[4,5-b]pyridine
(2j).
H-3), 5.01 (2H, s, NH2); 13C-NMR (CDCl3):
d 160.13 (d), 150.22 (s),
This compound was obtained in 78% yield by the protocol described
in the general procedure starting from 4b (376 mg, 2 mmol) and
sodium cyclohexyl(hydroxy)methanesulfonate (5d) (585 mg,
2 mmol); m.p. 226e227 ꢁC (from EtOH); TLC (chloroform/methanol
149.72 (d), 142.5 (s), 141.78 (s), 139.59 (s), 138.60 (s), 135.07 (d),
127.92 (d, 2 CH), 123.87 (d, 2 CH), 124.20 (s), 120.69 (d), 119.93 (d),
114.14 (s). LC/MS: m/z 329 [M þ 1]. HRMS: 326.0511. Anal. Calcd. for
C
16H11ClN4O2: C, 58.82; H, 3.39; N, 17.15; found C, 59.21; H, 3.22; N,
9:1): Rf 0.50; IR (nujol):
J ¼ 1.8 Hz, H-5), 7.97 (1H, d, J ¼ 1.8 Hz, H-7), 2.60e2.83 (1H, m, CH),
2.10e1.36 (10H, m, H-cyclohexyl); 13C-NMR (CDCl3):
151.42 (s),
n
1535 cm-1; 1H NMR (CDCl3):
d
8.29 (1H, d,
17.35.
d
4.1.5.4. 8-Chloro-N6-(3-nitro-4-(trifluoromethyl)benzylidene)quino-
line-6,7-diamine (3g). This compound was obtained in 34% yield by
the protocol described in the general procedure starting from 6a
(387 mg, 2 mmol) and sodium hydroxy(3-nitro-4-(trifluoromethyl)
phenyl)methanesulfonate (5g) (626 mg, 2 mmol); m.p. 170e172 ꢁC
147.33 (s), 148.29 (d), 137.87 (d), 130.94 (s), 118.61 (s), 38.78 (d),
33.45 (t, 2 CH2), 27.07 (t), 25.43 (t, 2 CH2). LC/MS: m/z 280 [M þ 1].
HRMS: 279.0348. Anal. Calcd. for C12H14BrN3: C, 51.44; H, 5.04; N,
15.00; found C, 51.34; H, 5.00; N, 14.99.
(from EtOH); TLC (chloroform/methanol 9:1): Rf 0.60; IR (nujol):
n
4.1.5. General procedure for the preparation of the N-
benzylidenequinolinamines (3a,b,feh)
3389, 1624, cmꢀ1; 1H NMR (CDCl3):
d 9.17 (1H, s, N]CH), 8.91 (1H,
dd, J ¼ 4.4 and 1.8 Hz, H-2), 8.50e8.37 (3H, m, H-20, 50, 60), 8.08 (1H,
dd, J ¼ 8.4 and 1.8 Hz, H-4), 7.39 (1H, s, H-5), 7.27 (1H, dd, J ¼ 8.4 and
A mixture of diaminoquinolines 6a, or 6b (2) mmol and sodium
hydroxy(R-substituted-phenyl-1-yl)methanesulfonate
(5a,b,f,g)
4.4 Hz, H-3), 5.11 (2H, s, NH2); 13C-NMR (CDCl3):
d 160.19 (d), 149.50
(2) mmol was stirred for 8 h in 15 mL of refluxed EtOH. On cooling,
the mixture was diluted with water and the formed precipitate was
collected and dried on oven. The solid residue was flash-
chromatographed on silica gel column eluting with a mixture of
chloroform/methanol in the ratio of 9:1, obtaining the N-benzyli-
denequinolinamines (3a,b,feh) in 30-40% yield.
(d),144.94 (s), 142.23 (s), 140.55 (s), 138.89 (s), 138.41 (s), 135.88 (d),
135.41 (d), 126. 07 (s), 126.62 (d), 124.22 (s), 122.98 (d), 122.89 (s),
114.12 (s), 120.33 (d), 120.22 (d). LC/MS: m/z 397 [M þ 1]. HRMS:
394.0409. Anal. Calcd. for C17H10ClF3N4O2: C, 51.73; H, 2.55; N,
14.19; found C, 51.49; H, 2.68; N, 14.01.
4.1.5.5. 4-((7-Amino-8-chloro-4-oxo-1,4-dihydroquinolin-6-ylimino)
methyl)benzonitrile (3h). This compound was obtained in 31% yield
by the protocol described in the general procedure starting from 6b
(419 mg, 2 mmol) and sodium (4-cyanophenyl)(hydroxy)meth-
anesulfonate (5a) (470 mg, 2 mmol); m.p. > 300 ꢁC (from EtOH);
4.1.5.1. 4-((7-Amino-8-chloroquinolin-6-ylimino)methyl)benzonitrile
(3a). This compound was obtained in 36% yield by the protocol
described in the general procedure starting from 6a (387 mg,
2 mmol) and sodium (4-cyanophenyl)(hydroxy)methanesulfonate
(5a) (470 mg, 2 mmol); m.p. > 300 ꢁC (from EtOH); TLC (chloro-
TLC (chloroform/methanol 9:1): Rf 0.48; IR (nujol):
n 3359, 2225,
form/methanol 9:1): Rf 0.80; IR (nujol):
n
3358, 2228, 1598, cmꢀ1
;
1605, cmꢀ1
;
1H NMR (CDCl3):
d
10.95 (1H, d, J ¼ 3.8 Hz, NH), 8.90
1H NMR (CDCl3):
d
8.88 (1H, dd, J ¼ 4.2 and 1.6 Hz, H-2), 8.70 (1H, s,
(1H, s, N]CH), 8.23 (2H, d, J ¼ 8.4 Hz, H-30, 50), 7.94 (1H, s, H-5), 7.85
N]CH), 8.09 (2H, d, J ¼ 8.4 Hz, H-30, 50), 8.04 (1H, dd, J ¼ 8.2 and
1.6 Hz, H-4), 7.82 (2H, d, J ¼ 8.4 Hz, H-20, 60), 7.31 (1H, s, H-5), 7.25
(1H, dd, J ¼ 8.2 and 4.2 Hz, H-3), 5.10 (2H, s, NH2); 13C-NMR (CDCl3):
(2H, d, J ¼ 8.4 Hz, H-20, 60). 8.04 (1H, d, J ¼ 7.2 Hz, H-3), 6.03 (1H, dd,
J ¼ 7.2 and 3.8 Hz, H-2), 5.92 (2H, s, NH2); 13C_NMR (CDCl3):
d 177.
31 (s), 159.94 (d), 140.88 (s), 147.98 (s), 146.95 (s), 132.98 (s), 129.89
(s), 139.21 (d), 132.45 (d, 2 CH), 126.88 (d, 2 CH), 123.08 (s), 122.03
(d), 118.71 (s) 114.96 (s), 108.55 (d). LC/MS: m/z 325 [M þ 1]. HRMS:
323.1021. Anal. Calcd. for C17H11ClN4O: C, 63.26; H, 3.44; N, 17.36;
found C, 63.03; H, 3.58; N, 17.19.
d
159.81 (d), 149.72 (d), 142.36 (s), 141.97 (s), 139.82 (s), 138.41 (s),
134.79 (d), 128.11 (d, 2 CH), 124.22 (d, 2 CH), 124.06 (s), 120.83 (d),
120.13 (d), 118.93 (s) 114.42 (s), 114.31 (s). LC/MS: m/z 309 [M þ 1].
HRMS: 306.1064. Anal. Calcd. for C17H11ClN4: C, 66.56; H, 3.61; N,
18.26; found C, 66.91; H, 3.44; N, 18.03.
4.1.6. Chemical and enzymatic stability
4.1.5.2. 8-Chloro-N6-(4-(trifluoromethyl)benzylidene)quinoline-6,7-
diamine (3b). This compound was obtained in 40% yield by the
protocol described in the general procedure starting from 6a
(387 mg, 2 mmol) and hydroxy(4-(trifluoromethyl)phenyl)meth-
anesulfonate (5b) (556 mg, 2 mmol); m.p. 150-152 ꢁC (from EtOH);
Aliquots of 2b were dissolved in pH 7.4 phosphate buffer
(chemical stability) or in plasma (enzymatic stability). The solu-
tions were maintained at 37 ꢁC and aliquots were withdrawn every
1 h. Plasma samples were extracted with acetonitrile (1:2) and
centrifuged at 3000 RPM (1000 g) for 10 min. The supernatant and
buffer solutions were analysed by HPLC.
Chromatographic separations were performed using a Liquid
Chromatograph, accomplished on a reversed phase HPLC column.
The selected wavelength was 250 nm. The mobile phase used in the
separation consisted of methanol/water [80:20]. The flow-rate was
TLC (chloroform/methanol 9:1): Rf 0.85; IR (nujol):
n 3383, 1627,
cmꢀ1; 1H NMR (CDCl3):
d
8.89 (1H, dd, J ¼ 4.2 and 1.8 Hz, H-2), 8.71
(1H, s, N]CH), 8.10 (2H, d, J ¼ 8.4 Hz, H-30, 50), 8.04 (1H, dd, J ¼ 8.4
and 1.8 Hz, H-4), 7.62 (2H, d, J ¼ 8.4 Hz, H-20, 60), 7.30 (1H, s, H-5),
7.24 (1H, dd, J ¼ 8.4 and 4.2 Hz, H-3), 5.11 (2H, s, NH2); 13C-NMR
(CDCl3):
d
159.31 (d), 150.42 (d), 144.76 (s), 141.24 (s), 139.67 (s),
1 mL/min with an injection volume of 20 ml.

R. Loddo et al. / European Journal of Medicinal Chemistry 84 (2014) 8e16
15
Pseudo-first-order half-times (t½) determining the chemical
and enzymatic stability were calculated from the linear slopes of
plots of the logarithm of remaining compound against time. In
results compound 2b appeared quite stable in a pH 7.4 buffered
solution (t > 4 h), slightly reduced in plasma (t ¼ 2 h).
37 ꢁC in a humidified, 5% CO2 atmosphere in the absence or pres-
ence of serial dilutions of test compounds. Cell viability was
determined after 48-96 h at 37 ꢁC by the MTT method.
½
½
4.2.4. Antiviral assays
Antiviral activity against HIV-1 was based on inhibition of virus-
induced cytopathogenicity in MT-4 cells acutely infected with a
4.2. Biology
multiplicity of infection (m.o.i.) of 0.01. Briefly, 50
mL of RPMI
containing 1 ꢂ 104 MT-4 cells were added to each well of flat bot-
4.2.1. Test compounds
Compounds were dissolved in DMSO at 100 mM, and then
diluted in culture medium.
tom microtitre trays, containing 50
mL of RPMI without or with
serial dilutions of test compounds. Then, 20 mL of a HIV-1 suspen-
sion containing 100 CCID50 were added. After a 4-day incubation at
37 ꢁC, cell viability was determined by the MTT method. Antiviral
activity against YFV and Reo-1 was based on inhibition of virus-
induced cytopathogenicity in BHK-21 cells acutely infected with a
m.o.i. of 0.01. Activity of compounds activity against BVDV was
based on inhibition of virus-induced cytopathogenicity in MDBK
cells acutely infected with a m.o.i. of 0.01. Briefly, BHK and MDBK
cells were seeded in 96-well plates at a density of 5 ꢂ 104 and
3 ꢂ 104 cells/well, respectively, and were allowed to form confluent
monolayers by incubating overnight in growth medium at 37 ꢁC in
a humidified CO2 (5%) atmosphere. Cell monolayers were then
4.2.2. Cells and viruses
Cell lines were purchased from American Type Culture Collec-
tion (ATCC). The absence of mycoplasma contamination was
checked periodically by the Hoechst staining method. Cell lines
supporting the multiplication of RNA and DNA viruses were the
following: CD4þ human T-cells containing an integrated HTLV-1
genome (MT-4); Madin Darby Bovine Kidney (MDBK) [ATCC
CCL22 (NBL-1) Bos Taurus], Baby Hamster Kidney (BHK-21) [ATCC
CCL10 (C-13) Mesocricetus auratus] and Monkey kidney (Vero 76)
[ATCCCRL 1587 Cercopithecus Aethiops]. Viruses were purchased
from American Type Culture Collection (ATCC), with the exception
of Yellow Fever Virus (YFV) and Human Immunodeficiency Virus
type-1 (HIV-1). Viruses representative of positive-sense single
stranded RNAs (ssRNAþ) were: (i) Retroviridae: the IIIB laboratory
strain of HIV-1, obtained from the supernatant of the persistently
infected H9/IIIB cells (NIH 1983); (ii) Flaviviridae: yellow fever virus
(YFV) [strain 17-D vaccine (Stamaril Pasteur J07B01)] and bovine
viral diarrhoea virus (BVDV) [strain NADL (ATCC VR-534)]; (iii)
Picornaviridae: human enterovirus B [coxsackie type B5 (CV-B5),
strain Faulkner, (ATCC VR-185)], and human enterovirus C [polio-
virus type-1 (Sb-1), Sabin strain Chat (ATCC VR-1562)]. Viruses
representative of negative-sense, single-stranded RNAs (ssRNA-)
were: (iv) Paramyxoviridae: human respiratory syncytial virus (RSV)
[strain A2 (ATCC VR-1540)]; (v) Rhabdoviridae: vesicular stomatitis
virus (VSV) [lab strain Indiana (ATCC VR 158)]. The virus repre-
sentative of double-stranded RNAs (dsRNA) Reoviridae was reovirus
type-1 (Reo-1) [simian virus 12, strain 3651 (ATCC VR- 214)]. DNA
virus representatives were: (vi) Poxviridae: vaccinia virus (VV)
[strain Elstree (Lister Vaccine) (ATCC VR-1549)]; and (vii) Herpes-
viridae: human herpesvirus 1 (HSV-1) [strain KOS (ATCC VR- 1493)].
infected with 50 mL of a proper virus dilution in maintenance me-
dium [MEM-Earl with L-glutamine, 1 mM sodium pyruvate and
0.025 g/L kanamycin, supplemented with 0.5% inactivated FBS] to
give an m.o.i of 0.01. After 2 h, 50
mL of maintenance medium,
without or with serial dilutions of test compounds, were added.
After a 3-/4-day incubation at 37 ꢁC, cell viability was determined
by the MTT method.
Antiviral activity against CV-B5, Sb-1, VV, HSV-1, VSV and RSV
was determined by plaque reduction assays in infected cell mono-
layers. To this end, Vero 76-cells were seeded in 24-well plates at a
density of 2 ꢂ 105 cells/well and were allowed to form confluent
monolayers by incubating overnight in growth medium [Dulbecco's
Modified Eagle Medium (D-MEM) with
-glucose and 0.025 g/L kanamycin, supplemented with 10% FBS] at
37 ꢁC in a humidified CO2 (5%) atmosphere. Then, monolayers were
infected for 2 h with 250 L of proper virus dilutions to give 50 to
100 PFU/well. Following removal of unadsorbed virus, 500 L of
L-glutamine and 4500 mg/L
D
m
m
maintenance medium [D-MEM with L-glutamine and 4500 mg/L
Dglucose, supplemented with 1% inactivated FBS] containing 0.75%
methylecellulose, without or with serial dilutions of test com-
pounds, were added. Cultures were incubated at 37 ꢁC for 2 (Sb-1
and VSV), 3 (CVB-5, VV and HSV-1) or 5 days (RSV) and then fixed
with PBS containing 50% ethanol and 0.8% crystal violet, washed
and air-dried. Plaques were then counted.
4.2.3. Cytotoxicity assays
Cytotoxicity assays were run in parallel with antiviral assays.
Exponentially growing MT-4 cells were seeded at an initial density
of 1 ꢂ 105 cells/mL in 96-well plates in RPMI-1640 medium, sup-
plemented with 10% foetal bovine serum (FBS), 100 units/mL
penicillin G and 100 mg/mL streptomycin. Cell cultures were then
incubated at 37 ꢁC in a humidified, 5% CO2 atmosphere, in the
absence or presence of serial dilutions of test compounds. Cell
viability was determined after 96 h at 37 ꢁC by the 3-(4,5- dime-
thylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method
[43]. MDBK and BHK cells were seeded in 96-well plates at an initial
density of 6 ꢂ 105 and 1 ꢂ 106 cells/mL, respectively, in Minimum
Essential Medium with Earle's salts (MEM-E), Lglutamine, 1 mM
sodium pyruvate and 25 mg/L kanamycin, supplemented with 10%
horse serum (MDBK) or 10% foetal bovine serum (FBS) (BHK). Cell
cultures were then incubated at 37 ꢁC in a humidified, 5% CO2 at-
mosphere in the absence or presence of serial dilutions of test
compounds. Cell viability was determined after 72 h at 37 ꢁC by the
MTT method. Vero-76 cells were seeded in 96-well plates at an
initial density of 4 ꢂ 105 cells/mL, in Dulbecco's Modified Eagle
Medium (D-MEM) with L-glutamine and 25 mg/L kanamycin,
supplemented with 10% FBS. Cell cultures were then incubated at
4.2.5. Linear regression analysis
The extent of cell growth/viability and viral multiplication, at
each drug concentration tested, were expressed as percentage of
untreated controls. Concentrations resulting in 50% inhibition (CC50
or EC50) were determined by linear regression analysis.
References
[1] S.J. Flint, L.W. Enquist, R.M. Krug, V.R. Racaniello, A.M. Skalka, Principles of
Virology: Molecular Biology, Pathogenesis, and Control, 2000. Washington,
D.C.
[2] M. Woolhouse, F. Scott, Z. Hudson, R. Howey, M. Chase-Topping, Human vi-
ruses: discovery and emergence, Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 367
(2012) 2864e2871.
[3] D.M. Knipe, P.M. Howley, D.E. Griffin, Virus-host cell interactions, in:
W.W. Lippincott (Ed.), Fields Virology, 2001, pp. 133e170. Philadelphia.
[4] P.G. Kennedy, Viral encephalitis: causes, differential diagnosis, and manage-
ment, J. Neurol. Neurosurg. Psychiatry 75 (Suppl. 1) (2004) i10e15.
[5] R.B. Domingues, F.D. Lakeman, C.S. Pannuti, M.C. Fink, A.M. Tsanaclis,
Advantage of polymerase chain reaction in the diagnosis of herpes simplex

16
R. Loddo et al. / European Journal of Medicinal Chemistry 84 (2014) 8e16
encephalitis: presentation of 5 atypical cases, Scand. J. Infect. Dis. 29 (1997)
229e231.
[6] R.J. Whitley, Herpes simplex encephalitis: adolescents and adults, Antivir. Res.
71 (2006) 141e148.
[26] G. Vitale, A. Carta, M. Loriga, G. Paglietti, R. Loddo, B. Busonera, D. Collu, P. La
Colla, 2-Arylbenzimidazoles as antiviral and antiproliferative agents. Part 1,
Med. Chem. 4 (2008) 605e615.
[27] G. Vitale, P. Corona, M. Loriga, A. Carta, G. Paglietti, P. La Colla, B. Busonera,
E. Marongiu, D. Collu, R. Loddo, 2-Arylbenzimidazoles as antiviral and anti-
proliferative agents. Part 2, Med. Chem. (2009) 507e516.
[28] G. Vitale, P. Corona, M. Loriga, A. Carta, G. Paglietti, C. Ibba, G. Giliberti,
R. Loddo, E. Marongiu, P. La Colla, Styrylbenzimidazoles. Synthesis and bio-
logical activity e part 3, Med. Chem. 6 (2010) 70e78.
[29] G. Vitale, P. Corona, M. Loriga, A. Carta, G. Paglietti, G. Giliberti, G. Sanna,
P. Farci, M.E. Marongiu, P. La Colla, 5-acetyl-2-arylbenzimidazoles as antiviral
agents. Part 4, Eur. J. Med. Chem. 53 (2012) 83e97.
[30] A. Carta, G. Loriga, S. Piras, G. Paglietti, M. Ferrone, M. Fermeglia, S. Pricl, P. La
Colla, B. Secci, G. Collu, R. Loddo, Synthesis and in vitro evaluation of the anti-
viral activity of N-[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides,
Med. Chem. 2 (2006) 577e589.
[31] A. Carta, M. Loriga, S. Piras, G. Paglietti, M. Ferrone, M. Fermeglia, S. Pricl, P. La
Colla, G. Collu, T. Sanna, R. Loddo, Synthesis and anti-picornaviridae in vitro
activity of a new class of helicase inhibitors the N,N'-bis[4-(1H(2H)-benzo-
triazol-1(2)-yl)phenyl] alkyldicarboxamides, Med. Chem. 3 (2007) 520e532.
[32] A. Carta, M. Loriga, G. Paglietti, M. Ferrone, M. Fermeglia, S. Pricl, T. Sanna,
C. Ibba, P. La Colla, R. Loddo, Design, synthesis, and preliminary in vitro and in
silico antiviral activity of [4,7]phenantrolines and 1-oxo-1,4-dihydro-[4,7]
phenantrolines against single-stranded positive-sense RNA genome viruses,
Bioorg. Med. Chem. 15 (2007) 1914e1927.
[33] P. Sanna, A. Carta, G. Paglietti, Synthesis of two novel tricyclic rings: triazolo
[4,5-g]quinolines and pyrido[2,3-g]quinoxalines derived from 6,7-
diaminoquinolines, Heterocycles 53 (2000) 423e432.
[34] A. Carta, P. Sanna, L. Gherardini, D. Usai, S. Zanetti, Novel functionalized pyrido
[2,3-g]quinoxalinones as antibacterial, antifungal and anticancer agents,
Farmaco 56 (2001) 933e938.
[35] A. Carta, G. Boatto, G. Paglietti, G. Poni, M.G. Setzu, P. Caredda, Synthesis and
biological evaluation of triazolo[4,5-g]quinolines, imidazo[4,5-g]quinolines
and pyrido[2,3-g]quinoxaline. Part II, Heterocycles 60 (2003) 833e842.
[36] A. Carta, I. Briguglio, S. Piras, P. Corona, G. Boatto, M. Nieddu, P. Giunchedi,
M.E. Marongiu, G. Giliberti, F. Iuliano, S. Blois, C. Ibba, B. Busonera, P. La Colla,
Quinoline tricyclic derivatives. Design, synthesis and evaluation of the anti-
viral activity of three new classes of RNA-dependent RNA polymerase in-
hibitors, Bioorg. Med. Chem. 19 (2011) 7070e7084.
[37] R.L. Shriner, A. Land, The structure of the bisulfite compound of acetaldehyde,
J. Org. Chem. 6 (6) (1941) 888e894.
[38] F. Sparatore, M.I. La Rotonda, G. Caliendo, E. Novellino, C. Silipo, A. Vittoria,
Structure-activity relationships for the auxin-like activity of benzotriazole
derivatives, Farm. Ed. Sci. 43 (1988) 141e151.
[39] A. Boido, I. Vazzana, F. Sparatore, B. Cappello, V. Diurno, C. Matera,
R. Marrazzo, S. Russo, M.L. Cenicola, E. Marmo, Synthesis and biological
evaluation of N-oxides of 40-(benzotriazol-2-yl)-phenyl and phenoxy alkanoic
acids, Farm. Ed. Sci. 44 (1989) 257e277.
[40] Unpublished data, currently under study to evaluate, in enzyme assays and
putative binding mode, the mechanism of action of all the title compounds in
comparison with the tricyclic derivatives previously reported.
[7] W.C. Tan, I.B. Jaganath, R. Manikam, S.D. Sekaran, Evaluation of antiviral ac-
tivities of four local Malaysian Phyllanthus species against herpes simplex
viruses and possible antiviral target, Int. J. Med. Sci. 10 (2013) 1817e1829.
[8] P. Chayavichitsilp, J.V. Buckwalter, A.C. Krakowski, S.F. Friedlander, Herpes
simplex, Pediatr. Rev./Am. Acad. Pediatr. 30 (2009) 119e129 quiz 130.
[9] D.A. Henderson, Bioterrorism as a public health threat, Emerg. Infect. Dis. 4
(1998) 488e492.
[10] D.L. Heymann, M. Szczeniowski, K. Esteves, Re-emergence of monkeypox in
Africa: a review of the past six years, Br. Med. Bull. 54 (1998) 693e702.
[11] Y.J. Hutin, R.J. Williams, P. Malfait, R. Pebody, V.N. Loparev, S.L. Ropp,
M. Rodriguez, J.C. Knight, F.K. Tshioko, A.S. Khan, M.V. Szczeniowski,
J.J. Esposito, Outbreak of human monkeypox, Democratic Republic of Congo,
1996 to 1997, Emerg. Infect. Dis. 7 (2001) 434e438.
[12] T. O'Toole, Smallpox: an attack scenario, Emerg. Infect. Dis. 5 (1999) 540e546.
[13] J.G. Breman, D.A. Henderson, Poxvirus dilemmasemonkeypox, smallpox, and
biologic terrorism, N. Engl. J. Med. 339 (1998) 556e559.
[14] Russia, Iraq, and other potential sources of anthrax, smallpox, and other
bioterrorist weapons: hearing before the Committee on International Re-
lations, in: House of Representatives, One Hundred Seventh Congress, First
Session, United States Congress, Washington, DC, 2001, p. 40.
[15] A.M. Di Bisceglie, J.H. Hoofnagle, Optimal therapy of hepatitis C, Hepatology
36 (2002) S121eS127.
[16] M.W. Fried, M.L. Shiffman, K.R. Reddy, C. Smith, G. Marinos, F.L. Goncales Jr.,
D. Haussinger, M. Diago, G. Carosi, D. Dhumeaux, A. Craxi, A. Lin, J. Hoffman,
J. Yu, Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infec-
tion, N. Engl. J. Med. 347 (2002) 975e982.
[17] F. Poordad, J. McCone Jr., B.R. Bacon, S. Bruno, M.P. Manns, M.S. Sulkowski,
I.M. Jacobson, K.R. Reddy, Z.D. Goodman, N. Boparai, M.J. DiNubile,
V. Sniukiene, C.A. Brass, J.K. Albrecht, J.P. Bronowicki, S.-Investigators, Boce-
previr for untreated chronic HCV genotype 1 infection, N. Engl. J. Med. 364
(2011) 1195e1206.
[18] B.R. Bacon, S.C. Gordon, E. Lawitz, P. Marcellin, J.M. Vierling, S. Zeuzem,
F. Poordad, Z.D. Goodman, H.L. Sings, N. Boparai, M. Burroughs, C.A. Brass,
J.K. Albrecht, R. Esteban, H.R.-Investigators, Boceprevir for previously treated
chronic HCV genotype 1 infection, N. Engl. J. Med. 364 (2011) 1207e1217.
[19] I.M. Jacobson, J.G. McHutchison, G. Dusheiko, A.M. Di Bisceglie, K.R. Reddy,
N.H. Bzowej, P. Marcellin, A.J. Muir, P. Ferenci, R. Flisiak, J. George, M. Rizzetto,
D. Shouval, R. Sola, R.A. Terg, E.M. Yoshida, N. Adda, L. Bengtsson, A.J. Sankoh,
T.L. Kieffer, S. George, R.S. Kauffman, S. Zeuzem, A.S. Team, Telaprevir for
previously untreated chronic hepatitis C virus infection, N. Engl. J. Med. 364
(2011) 2405e2416.
[20] S. Zeuzem, P. Andreone, S. Pol, E. Lawitz, M. Diago, S. Roberts, R. Focaccia,
Z. Younossi, G.R. Foster, A. Horban, P. Ferenci, F. Nevens, B. Mullhaupt,
P. Pockros, R. Terg, D. Shouval, B. van Hoek, O. Weiland, R. Van Heeswijk, S. De
Meyer, D. Luo, G. Boogaerts, R. Polo, G. Picchio, M. Beumont, R.S. Team,
Telaprevir for retreatment of HCV infection, N. Engl. J. Med. 364 (2011)
2417e2428.
[21] E.A. Gould, T. Solomon, Pathogenic flaviviruses, Lancet 371 (2008) 500e509.
[22] A.M. O'Connor, S.D. Sorden, M.D. Apley, Association between the existence of
calves persistently infected with bovine viral diarrhea virus and commingling
on pen morbidity in feedlot cattle, Am. J. Vet. Res. 66 (2005) 2130e2134.
[23] C.C. Chase, G. Elmowalid, A.A. Yousif, The immune response to bovine viral
diarrhea virus: a constantly changing picture, the Veterinary clinics of North
America, Food Anim. Pract. 20 (2004) 95e114.
[24] C. Centers for Disease, Prevention, Enterovirus surveillanceeUnited States,
2002-2004, Morb. Mortal. Wkly. Rep. 55 (2006) 153e156.
[25] A.T. Borchers, C. Chang, M.E. Gershwin, L.J. Gershwin, Respiratory syncytial
virusea comprehensive review, Clin. Rev. Allergy Immunol. 45 (2013)
331e379.
[41] A. Carta, M. Palomba, P. Corona, Synthesis of substituted aminoquinolines as
useful intermediates for preparation of aromatic N-tricyclic systems, Het-
erocycles 68 (2006) 1715e1722.
[42] D.W. Robertson, E.E. Beedle, J.H. Krushinski, G.D. Pollock, H. Wilson, V.L. Wyss,
J.S. Hayes, Structure-activity relationships of arylimidazopyridine cardio-
tonics: discovery and inotropic activity of 2-[2-methoxy-4-(methylsulfinyl)
phenyl]-1H-imidazo[4,5-c]pyridine, J. Med. Chem. 28 (1985) 717e727.
[43] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn,
J. Desmyter, E. De Clercq, Rapid and automated tetrazolium-based colori-
metric assay for the detection of anti-HIV compounds, J. Virol. Methods 20
(1988) 309e321.