The Versatile Nature of the 6-Aminoquinolone Scaffold: Identification of Submicromolar Hepatitis C Virus NS5B Inhibitors

Article abstract of DOI:10.1021/jm401362f

We have previously reported that the 6-aminoquinolone chemotype is a privileged scaffold to obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, and enzymatic and cellular characterization of new 6-aminoquinolone derivatives as potent inhibitors of NS5B polymerase, an attractive and viable therapeutic target to develop safe anti-HCV agents. The 6-amino-7-[4-(2-pyridinyl)-1-piperazinyl]quinolone derivative 8 proved to be the best compound of this series, exhibiting an IC₅₀ value of 0.069 μM against NS5B polymerase and selective antiviral effect (EC₅₀ = 3.03 μM) coupled with the absence of any cytostatic effect (CC₅₀ > 163 μM; SI > 54) in Huh 9-13 cells carrying a HCV genotype 1b, as measured by MTS assay. These results indicate that the 6-aminoquinolone scaffold is worthy of further investigation in the context of NS5B-targeted HCV drug discovery programs.

Full text of DOI:10.1021/jm401362f

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Article
The versatile nature of the 6-aminoquinolone scaffold:
identification of submicromolar hepatitis C virus NS5B inhibitors
Giuseppe Manfroni, Rolando Cannalire, Maria Letizia Barreca, Neerja Kaushik-Basu, Pieter LEYSSEN,
Johan Winquist, Nunzio Iraci, Dinesh Manvar, Jan Paeshuyse, Rupa Guhamazumder, Amartya
Basu, Stefano Sabatini, Oriana Tabarrini, U. Helena Danielson, Johan Neyts, and Violetta Cecchetti
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 16 Oct 2013
Downloaded from http://pubs.acs.org on October 19, 2013
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The Versatile Nature of the 6ꢀAminoquinolone
Scaffold: Identification of Submicromolar Hepatitis
C Virus NS5B Inhibitors
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Giuseppe Manfroni,*, † Rolando Cannalire,† Maria Letizia Barreca,*, † Neerja KaushikꢀBasu,§
Pieter Leyssen,‡ Johan Winquist,# Nunzio Iraci,† Dinesh Manvar,§ Jan Paeshuyse, ‡ Rupa
Guhamazumder,§ Amartya Basu,§ Stefano Sabatini,† Oriana Tabarrini,† U. Helena Danielson,#
Johan Neyts,‡ and Violetta Cecchetti†
†Dipartimento di Chimica e Tecnologia del Farmaco, Università degli Studi di Perugia, Via del
Liceo 1, 06123 Perugia, Italy
§Department of Biochemistry and Molecular Biology, RutgersꢀThe State University of New
Jersey, 185 South Orange Avenue, New Jersey 07103, USA
‡Rega Institute for Medical Research, Katholieke Universiteit Leuven, Bꢀ3000 Leuven, Belgium
#Department of Chemistry – BMC, Uppsala University, Box 576, SEꢀ751 23 Uppsala, Sweden.
KEYWORDS. Hepatitis C virus, RNAꢀdependent RNA polymerase, quinolones, NS5B
inhibitors, inducedꢀfit docking
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ABSTRACT.
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We have previously reported that the 6ꢀaminoquinolone chemotype is a privileged scaffold to
obtain antibacterial and antiviral agents. Herein we describe the design, synthesis, enzymatic and
cellular characterization of new 6ꢀaminoquinolone derivatives as potent inhibitors of NS5B
polymerase, an attractive and viable therapeutic target to develop safe antiꢀHCV agents. The 6ꢀ
aminoꢀ7ꢀ[4ꢀ(2ꢀpyridinyl)ꢀ1ꢀpiperazinyl] quinolone derivative 8 proved to be the best compound
of this series, exhibiting IC50 value of 0.069 ꢁM against NS5B polymerase and selective antiviral
effect (EC50 = 3.03 ꢁM, EC90 =13.5 ꢁM) coupled with the absence of any cytostatic effect (CC50
>163 ꢁM, SI >54) in Huhꢀ9ꢀ13 cells carrying a HCV genotype 1b, as measured by MTS assay.
These results indicate that the 6ꢀaminoquinolone scaffold is worthy of further investigation in the
context of NS5Bꢀtargeted HCV drug discovery programs.
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Introduction
Hepatitis C virus (HCV), the causative agent of chronic hepatitis C, infects nearly 170 million
people worldwide.1 Chronic HCV infection results in major liver disorders such as cirrhosis and
hepatocellular carcinoma ultimately necessitating liver transplantation.1,2 Among the six major
genotypes, HCV genotype 1b causes the majority of HCV infections in industrialized countries
and is the most refractory to therapy.2 To date there is no vaccine available.3 The current
combination therapy consists of ribavirin (RBV), pegylated interferon α (pegIFNꢀα) and for
genotype 1 since midꢀ2011 a NS3/4A protease inhibitor, i.e. telaprevir or boceprevir.4 Although
the triple combination therapy induces an improved sustained virological response (SVR), it
suffers from longꢀterm toxic effects, high costs, and increased pillꢀburden.5 Therefore an
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alternative pegIFNꢀα/RBVꢀfree treatment, based on a combination of several directꢀacting
antivirals (DAAs) with different mode of action, is still a need of primary importance.
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HCV is a small, enveloped, singleꢀstranded RNAꢀ(+) virus that belongs to the genus
Hepacivirus, of the Flaviviridae family.6 Its RNA genome encodes a polyprotein precursor of
about 3000 aminoacids, which is processed by cellular and viral proteases to yield four structural
(S) and six nonꢀstructural (NS) proteins.6 Among the NS proteins, NS5B is a key enzyme for
HCV replication with a RNAꢀdependent RNA polymerase (RdRp) function, thus representing an
attractive target for the development of selective antiviral agents.7 NS5B inhibitors are divided
into nucleoside inhibitors (NIs) that bind to the active site and nonꢀnucleoside inhibitors (NNIs)
that bind to one of the five identified allosteric sites.8 The allosteric sites are currently classified
as follows: (i) thumb site I (TSI), (ii) thumb site II (TSII), (iii) palm site I (PSI), (iv) palm site II
(PSII), and (v) palm site III (PSIII).8a In the need to identify new antiꢀHCV drugs targeting
NS5B, pharmaceutical companies and academic groups are making tremendous efforts to
discover potent and selective inhibitors8b to be combined in a pegIFNꢀα/RBVꢀfree therapy based
on the use of different DAAs. Anyway, although a broad range of NIs and NNIs have been
reported in literature, drugs active against NS5B polymerase have not yet been approved by
FDA. Regarding the NS5B NIs, sofosbuvir (PSIꢀ7977) and mericitabine (RG7128) are the most
advanced drug candidates in clinical trials, whilst filibuvir (PFꢀ00868554) and setrobuvir
(ANA598) are the most advanced NNIs.9 In particular, the combination of sofosbuvir with an
NS5A inhibitor is the most promising interferon free regimen reaching a SVR >90%.10
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During the last few years our research group has been involved in HCV drug discovery, and in
particular in the identification of novel chemotypes endowed with antiꢀNS5B and/or antiꢀHCV
activity.11 As a continuation of this endeavor, we herein report the design and synthesis of new 6ꢀ
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aminoquinolone derivatives as potential antiꢀHCV compounds. Since many years, we have
focused on the 6ꢀaminoquinolone chemotype as a privileged scaffold to obtain novel
chemotherapeutic agents. This research work started off by replacing for the first time, the usual
fluorine atom at the Cꢀ6 position of fluoroquinolone antimicrobial drugs with an amino group.12
This chemical modification produced potent antibacterial agents which were less phototoxic
than the corresponding fluorinated compounds.13 Subsequently, we demonstrated that suitable
derivatization of the 6ꢀaminoquinolone scaffold resulted in numerous derivatives endowed with a
wide range of antiviral activities, such as antiꢀHIV,14 antiꢀHCMV15 and antiꢀHPV.16
Given this background, our attention was caught by a recent publication by Kumar et
al.,17describing the discovery of quinolone benzylesters as a new class of NS5B TSIIꢀNNIs (e.g.,
compounds 1 and 2, Figure 1A).
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Figure 1. A) Chemical structures of 1 and 2 as representative quinolones TSIIꢀNNIs. B)
Schematic representation of the interaction between NS5B polymerase and compound 1 (PDB
ID 3PHE). NS5B residues lying within a distance of 4 Å from the bound ligand are shown and
color coded as follows: greenꢀhydrophobic; purpleꢀbasic; cyanꢀpolar. The TSII pocket is
displayed with a line, exhibiting the color of the nearest protein residue. The gap in the line
shows the opening of the pocket. Specific interactions between ligand atoms and protein residues
are marked with lines: pink, Hꢀbonds to protein backbone; green, πꢀπ stacking interactions.
Ligand atoms that are exposed to solvent are marked with gray spheres.
The reported quinolones showed potent NS5B inhibitory activity in the submicromolar range,
binding to the NS5B TSII, as confirmed by the coꢀcrystal structure of ligand 1 bound to NS5B
polymerase (PDB ID 3PHE)17 (Figure 1B). This structure revealed the formation of two
hydrogen bonds between the ligand carbonyl oxygen atoms and the backbone amide NH atoms
of Ser476 and Tyr477. The benzyl ester occupied a hydrophobic pocket mainly lined by Leu419,
Met423 and Trp528, whereas the Nꢀ1 substituted benzyl group filled a pocket defined by
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Leu419, Val485, Ala486, Leu489 and Leu497, establishing hydrogenꢀbond interaction with
Leu497 through the sulfone moiety. Finally, the methylpiperazine substituent was largely solvent
exposed (Figure 1B). The compounds reported by Kumar et al. also demonstrated the ability to
inhibit HCV replication in subgenomic replicon system at micromolar concentrations, with
derivative 2 showing the best biological profile (IC50 (NS5B) = 0.008 ꢁM; EC50 (replicon) = 0.2
ꢁM; CC50 = 20 ꢁM).17
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Based on these observations and given our longꢀstanding experience with the 6ꢀ
aminoquinolone chemotype, we explored the design of a series of new 6ꢀaminoquinolone
derivatives, in an effort to obtain more potent antiꢀNS5B inhibitors and/or HCV replication
inhibitors. In particular, keeping the Cꢀ6 amino substituent constant, we designed compounds 3ꢀ5
(Scheme 1 and Table 1) in which i) the Cꢀ7 position of quinolone scaffold was occupied by a
chlorine atom as a suitable replacement of the methoxyl group present in compound 2, and ii) the
Nꢀ1 and Cꢀ3 positions were functionalized with some benzyl substituents already reported as the
best fragments in the known antiꢀNS5B quinolone series. Compounds 6 and 7 were instead
designed by keeping the 4ꢀchlorobenzyl moiety constant at both Nꢀ1 and Cꢀ3 positions, and by
replacing the chlorine atom with a piperazine or a methylpiperazine, respectively. These
modifications were pursued considering that the latter substituents granted a better solubility in
known antiꢀHCV quinolones such as compound 1. Finally, to better explore the role of the Cꢀ7
substituent in this new series of 6ꢀaminoquinolones, we designed derivatives 8ꢀ10, where the 1ꢀ
(2ꢀpyridinyl)piperazine,
2ꢀ(1ꢀpiperazinyl)ꢀ1,3ꢀbenzothiazole,
and
1ꢀ[3ꢀ
(trifluoromethyl)phenyl]piperazine fragments were placed at the Cꢀ7 position, while maintaining
the 4ꢀchlorobenzyl substituent at Nꢀ1 and Cꢀ3 positions (Scheme 1 and Table 1). The three
arylpiperazines were used as Cꢀ7 substituents in this series of compounds for two main reasons:
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the Cꢀ7 substituents resulted in potent antiꢀHIV activity,14 and the synthetic pathway to obtain
arylpiperazinyl quinolones was well known to us.
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Results and discussion
Inducedꢀfit docking studies
Before initiating chemical synthesis of the described quinoloneꢀbased compounds, we
performed inducedꢀfit docking (IFD) studies of derivatives 3ꢀ10 using Prime and Glide
programs.18 Here, receptor flexibility upon ligand binding was taken into account in an attempt
to describe the inhibitor binding mode (see Experimental Section). The compounds were
prepared using the LigPrep utility19 and docked by the IFD procedure against the crystal
structure of NS5B in complex with inhibitor 1 (PDB ID 3PHE).17 In order to validate the IFD
performance, test calculations using 1 were carried out, extracting the ligand from the
corresponding NS5B complex and then docking it back into the allosteric pocket of the enzyme
crystal structure. The top IFD conformation of 1 agreed well with its experimental binding
conformation, showing a rootꢀmean square deviation (RMSD) value of 0.7 Å. Moreover, IFD of
the known TSIIꢀNNI 2 was carried out as well, since this inhibitor was later used as reference
compound in our biological assays, being the quinolone derivative with the highest antiꢀNS5B
potency reported in literature.17 The best IFD structure of 2 showed a ligand binding
conformation resembling the conformation observed for compound 1.
IFD of compounds 3ꢀ10 into TSII generated a number of NS5B/ligand complexes, and only
the best scoring pose for each ligand was retained. This revealed that all the 6ꢀaminoquinolones
exhibited similar ligand orientation within the binding pocket, and that they may potentially
interact with the NS5B residues of TSII in a comparable fashion to the known TSIIꢀNNIs 1 and
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2, as discussed below. For instance, the topꢀranked IFD orientation of 6ꢀaminoquinolone 8 is
shown in Figure 2, together with the experimental position of compound 1. Beside the ligandꢀ
NS5B interactions already highlighted for compound 1 involving the two benzyl groups and the
carbonyl group of the quinolone scaffold (Figure 1B), our derivative was able to establish two
additional hydrogenꢀbond interactions; in particular, the Cꢀ6 amino group interacted with the
backbone carbonyl of Tyr477, whereas the pyridinyl moiety showed hydrogenꢀbonding to
Asn483 (Figure 2). Furthermore, different from 1, derivative 8 was not able to interact with the
Ser476 residue through its carbonyl ester group.
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Figure 2. IFD pose of the designed 6ꢀaminoquinolone 8 (green) overlaid with the Xꢀray
conformation of the known quinolone TSIIꢀNNI 1 (magenta). Key NS5B residues are
represented as sticks, whereas hydrogen bonds are represented as yellow dashed lines.
Subsequently, inhibition constant (Ki) values were estimated for each compound
employing the corresponding top IFD complex and the “epdb” function of AutoDock20 (Table 1).
The predicted Ki values suggested that the 6ꢀaminoquinolones harbored high binding affinity for
NS5B. In particular, the piperazineꢀbased fragments at the Cꢀ7 position of the quinolone scaffold
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were preferred relative to the chlorine atom (compounds 3ꢀ5 vs 6ꢀ10), with compounds 7 and 8
predicted to be the most potent derivatives within the series (Table 1). Paradoxically though, the
AutoDock estimated inhibitory activity of 2, did not agree well with its experimentally reported
IC50 value (estimated Ki = 0.652 ꢁM vs experimental IC50 = 0.008 ꢁM). Taken together, the in
silico data suggested that, in analogy with the known quinolones, the newly designed compounds
3ꢀ10 may act as potent TSIIꢀNNIs, thus providing an incentive to synthesize and test them as
potential inhibitors of NS5B polymerase and HCV replication.
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Synthesis of the target 6ꢀaminoquinolones
The 6ꢀaminoquinolones 3ꢀ10 were prepared through the canonical cycloaracylation procedure,
as shown in Scheme 1. In particular, acrylate 1112 was reacted with the appropriate benzylamine
in a 4:1 Et2O/EtOH mixture at room temperature (rt) to give the intermediates 12 and 13, which
were then cyclized to quinolone esters 14 and 15, using K2CO3 in dry DMF at 80 °C, and
subsequently hydrolyzed in aqueous 8 N HCl to the corresponding acids 16 and 17 by using
microwave (MW) irradiation at 120 °C for 130 min. The MW irradiation showed some
advantages than conventional heating: i) reduction of the reaction time from days to few hours;
ii) improvement of yield reaching values close to 90%. Quinolone acids 16 and 17 so obtained
were reacted with the appropriate benzylchloride in DMF in presence of K2CO3 at 60 °C,
obtaining the key nitro benzylester synthons 18ꢀ20 which were converted into their amino target
derivatives 3ꢀ5 using Feꢀpowder in a mixture of DMF/aqueous 3.5% NaCl at reflux.21 Although
the yields of this reduction were very low (from 10% to 40%), the neutral reaction conditions
were necessary to grant the reduction of the amino group avoiding the hydrolysis of the Cꢀ3
benzyl ester. The key intermediate 20 was further functionalized with the appropriate piperazine
side chains, in dry MeCN in presence of Et3N at 80 °C, to give the derivatives 21ꢀ25. This step
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suffers from low yield (35ꢀ50%) when applied to the preparation of intermediates 23ꢀ25. Finally
the key nitro derivatives 21ꢀ25 were reduced to amino target compounds 6ꢀ10, using the same
conditions as used for the preparation of compounds 3ꢀ5.
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The low yields obtained for the preparation of intermediates 23ꢀ25 prompted us to explore an
alternative synthetic procedure. In particular, the planned alternative synthetic pathway reported
in Scheme 2 was conveniently applied, selecting compound 8 as synthetic probe. Thus, the ethyl
ester intermediate 15 was initially reacted with the 1ꢀ(2ꢀpyridinyl)piperazine in dry DMF at 80
°C obtaining intermediate 26 in good yield (85%); it was then hydrogenated using RaneyꢀNi as
catalyst to the corresponding amino derivative 27, which was hydrolyzed under basic conditions
into the corresponding acid intermediate 28 and successively benzylated to the target compound
8 by employing the same conditions as previously used. The benzylation reaction of intermediate
28 afforded only the Cꢀ3 benzyl ester in fairly good yield, without any trace of Cꢀ6 amino
benzylation. This alternative synthetic route permitted the avoidance of neutral reduction
condition, which suffers from low yields as described above.
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Scheme 1. Synthesis of Target Compounds 3ꢀ10a
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aReagents and conditions: i) BnNH2, Et2O/EtOH 4:1, rt; ii) K2CO3, dry DMF, 80 °C; iii) 8 N
HCl, MW, 120 °C, 15 bar; iv) BnCl, K2CO3, DMF, 60 °C; v) piperazines, Et3N, MeCN, 80 °C;
vi) aqueous NaCl 3.5%, Feꢀpowder, DMF, reflux.
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Scheme 2. Alternative Synthesis of Target Compound 8a
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aReagents and conditions: i) Arylpiperazines, DMF, 80 °C; ii) H2, RaneyꢀNi, DMF, rt, atm
pressure; iii) aqueous NaOH 5%, EtOH 1:1, reflux; iv) BnCl, K2CO3, DMF, 60 °C.
Biochemical and Biological Studies.
The target compounds 3ꢀ10 were evaluated by two methods to determine the antiꢀNS5B and
the antiꢀHCV activities (Table 1). In addition, intermediates 27 and 28 were also evaluated in
order to assess the importance of the Cꢀ3 benzyl fragment on NS5B inhibition. The cytotoxicity
of all ten compounds was also investigated.
NS5B inhibition assay. The antiꢀNS5B activity was evaluated by the standard primerꢀ
dependent elongation reaction employing poly rA/U12 templateꢀprimer and recombinant HCV
NS5BCꢂ21 as previously described.22 Compound 2 was reꢀsynthesized23 and included for
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comparison as a reference, yielding an IC50 value of 0.211 ꢃM under the assay conditions used,
in contrast to the reported value of 0.008 ꢃM.17
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Table 1. Estimated Ki values, inhibitory activity on NS5B, antiꢀHCV activity and cytotoxicity of the
studied compounds .
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31
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O
O
R3
H2N
R7
O
N
R1
R4
R2
AutoDock
Estimated Ki
Ki
NS5B
Replicon assay on Huh5ꢀ2
functional assay
Cpds
R1
R2
R3
R4
R7
IC50
EC50
(ꢁM)c
1.5
EC90
(ꢁM)d
NDg
CC50
(ꢁM)e
10.5
SIf
6.8
(ꢁM)a
(ꢁM)b
3
4
5
F
F
CF3
CF3
Cl
CH3
H
H
CH3
Cl
Cl
Cl
Cl
0.340
1.41±0.2
0.154±0.015
NDg
0.255
0.249
72.3
>192.3
>200
>192.3
>200
>2.5
NDg
H
H
>200
6
7
H
H
Cl
Cl
H
H
Cl
Cl
0.096
0.070
1.7
2.7
NDg
NDg
3.7
2.8
2.3
1.2
0.067±0.02
N
N CH3
0.040±0.004
8
9
H
H
Cl
Cl
H
H
Cl
Cl
0.043
0.093
2.2
8.6
13.5
NDg
>244
>149
>54
>17
0.069±0.002
0.138 ± 0.023
10
H
Cl
H
Cl
0.178
23.8
94.6
NDg
>147
>6
6.132 ± 1.6
27
2.49
31.3
>193
3.06
3.06± 0.6
28
NCh
7.3
NDg
>204
42.3
>28
23.7 ± 6
2
0.211± 0.01
(0.008)i
2.02
4.7
21
0.652
aAutoDockꢀpredicted inhibition constant (Ki) for each selected IFD complex bIC50
concentration of compound that inhibits 50% enzyme activity in vitro. The IC50 values of the
compounds were determined from dose–response curves employing 8ꢀ12 concentrations of the
indicated compounds in duplicate in two independent experiments ± SD. EC50 = the effective
concentration required to inhibit virus replication by 50%. The reported values represent the means
=
c
d
of data derived at least from three independent experiments. EC90 = the effective concentration
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required to inhibit virus replication by 90%. The reported value represents the means of data
derived at least from three independent experiments. CC50 = is the concentration of compound
e
exhibiting 50% antimetabolic effect as evaluated by the MTS assay. The reported value represents
6
7
8
9
the means of data derived at least from three independent experiments. fSI = selectivity index (ratio
g
h
of CC50 to EC50). ND = not determined. NC= not calculated: the IFD top pose of this 6ꢀ
i
aminoquinolone derivative showed that the compound was unable to properly occupy TSI. IC50
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value reported by Kumar et al.17
This discrepancy between the two IC50 values may be due to several reasons such as
differences in the type of assay, reaction conditions employed, and the different strains of the
HCV NS5B genotype 1b isolate.
For preliminary screening of antiꢀNS5B candidates, all compounds were first investigated at 25
ꢃM concentration and compounds exhibiting ≥50% inhibition of NS5B RdRp activity at this
concentration were subjected to IC50 determination. With the exception of compound 5, all the
molecules satisfied this criterion. Thus, compounds 3, 4, 6ꢀ10 were further investigated to
evaluate their potency, yielding IC50 values ranging from 0.04 to 6.13 ꢁM (Table 1). Compounds
6ꢀ8 turned out to be the most potent derivatives within this 6ꢀaminoquinolone series, exhibiting
IC50 values of 0.067 ꢁM, 0.040 ꢁM, and 0.069 ꢁM, respectively. These three compounds were
characterized by a 4ꢀchlorobenzyl fragment at both Nꢀ1 and Cꢀ3 positions, and a 1ꢀpiperazinyl
(compound 6), a 4ꢀmethylꢀ1ꢀpiperazinyl (compound 7) or a 4ꢀ(2ꢀpyridinyl)ꢀ1ꢀpiperazinyl
(compound 8) group at Cꢀ7 position. With the exception of the inactive compound 5,
replacement of the Cꢀ7 piperazineꢀbased substituents with a chlorine atom, still exhibited NS5B
inhibition in the low micromolar range, although there was a 9ꢀfold decrease in activity
(compound 3, IC50 = 1.4 ꢁM; compound 4, IC50 = 0.15 ꢁM). It was clearly evident that the
chemical nature of the Nꢀ4 piperazine substituent significantly influenced the antiꢀNS5B activity.
In particular, insertion of the benzothiazole fragment at Nꢀ4 position of the piperazine ring
yielded compound 9 (IC50 = 0.138ꢁM), with a slight decrease in its activity by 2 to 3.5ꢀfold
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compared to derivatives 6ꢀ8. By contrast, introduction of a 3ꢀtrifluoromethylphenyl group at the
same position of the piperazine moiety proved to be highly detrimental as is evident from
compound 10 (IC50 = 6.13 ꢁM), which exhibited ~89 to153ꢀfold decrease in its activity relative
to compounds 6ꢀ8. In line with the SAR reported for known antiꢀHCV quinolones,17 replacement
of the benzyl ester at the Cꢀ3 position in the most potent derivative 8 with an ethyl ester group
(compound 27) or a free carboxylic acid (compound 28) produced a significant reduction in
potency against NS5B by ~45ꢀfold and 345ꢀfold, respectively. These results strongly suggest that
the 6ꢀaminoquinolones exert their antiꢀHCV activity through binding to the TSIIꢀNNIs pocket,
and once more highlight the importance of the Cꢀ3 benzyl ester in the quinolones series for
NS5B inhibition.
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In a head to head comparison, compound 7, the most potent NS5B inhibitor among the 6ꢀ
aminoquinolone series, exhibited 5ꢀfold higher inhibition than the reference compound 2 under
our assay conditions. In order to validate the binding of 6ꢀaminoquinolones to NS5B, we next
investigated the dissociation constant (KD) for compound 7 as a representative. We reasoned that
since KD value is independent of the enzyme and inhibitor amounts used in the assay, it
represents the intrinsic affinity of the ligand for its biological target. The KD for compound 7 was
evaluated both by Surface Plasmon Resonance (SPR) and fluorescence quenching assays, and
yielded values of 14 ꢁM and 1.2 ꢁM, respectively, confirming the binding of our 6ꢀ
aminoquinolones to NS5B.
We also compared the experimental IC50 values with the predicted Ki values (Table 1) and
found that with the exception of compound 10, a high correlation existed between these two data
sets (R2 = 0.83). Of note, AutoDock correctly predicted the piperazineꢀbased 6ꢀaminoquinolones
6ꢀ9 as the most potent NS5B inhibitors within this series. Interestingly, we also noticed that,
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even though the inhibition constant predicted by AutoDock for the reference compound 2 (Ki=
0.652 ꢁM) showed low agreement with the inhibitory activity reported in literature (IC50 = 0.008
ꢁM),17 it agreed well with the antiꢀNS5B potency obtained in our assay (IC50 = 0.211ꢁM).
Replicon system assay. Compounds 3ꢀ10 and intermediates 27 and 28 were investigated for
their ability to inhibit HCV replication in Huhꢀ5ꢀ2 cells carrying a subgenomic genotype 1b
replicon. In parallel, we also investigated the cytostatic effect of the compounds on the same
cells type. Towards this goal, we evaluated the compound concentration that inhibits virus
replication by 50% (EC50) and the concentration that reduces host cell metabolism by 50%
(CC50). These values allowed us to calculate the selectivity index (SI = CC50/EC50), as a measure
of the therapeutic potential of each compound in the assay system. All values are reported in
Table 1. A preliminary analysis showed that most compounds inhibited HCV replication in the
micromolar concentration range. 6ꢀAminoquinolone derivatives 6, 7, and 8, the most potent
compounds in the antiꢀNS5B assay, inhibited HCV replication with EC50 values between 1.7 ꢁM
to 2.7 ꢁM. Compound 9 (EC50 = 8.6 ꢁM) exhibited ~3 to 5ꢀfold decrease in antiꢀHCV activity
compared to derivatives 6ꢀ8, while quinolones 10 (EC50 = 23.8 ꢁM) and 27 (EC50 = 31.3 ꢁM)
displayed ~12 to 18ꢀfold decrease. Compound 3 also displayed a low micromolar antiꢀHCV
activity (EC50 = 1.5 ꢁM), consistent with its low micromolar antiꢀNS5B activity; moreover,
compound 28 (EC50 = 7.3 ꢁM) was more promising than compound 27 (EC50 = 31.3 ꢁM) in the
antiviral assay. Finally, the Cꢀ7 chloro derivatives 4 and 5 were found to be neither active nor
toxic.
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It is important to underline that only compounds that produce significant inhibition of viral
replication (>70% inhibition) at concentrations that do not elicit an antimetabolic effect on the
host cells (cell viability >90%) can be considered selective inhibitors of HCV replication in the
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replicon system (hit selection criteria). Thus, derivatives 3, 6 and 7 were less promising because
of their antimetabolic effects on the cells, as confirmed by their low CC50 values and selectivity
index (SI). By contrast, compounds 8 (EC50 = 2.2 ꢁM, CC50 >244 ꢁM), 9 (EC50 = 8.6 ꢁM, EC50
>169 ꢁM) and 28 (EC50 = 7.3 ꢁM, CC50 >204 ꢁM) exhibited reasonable efficacy as deduced
from their low EC50 and high SI values. Of these, derivative 8 was the most promising, as it
inhibited HCV replication without any cytostatic effects at all concentrations employed.
Furthermore, the 6ꢀaminoquinolone 8 achieved a reasonably low EC90 value of 13.5 ꢁM.
As reported in Table 1, the known quinolone 2 was also assayed in parallel in order to compare
our molecules with a structurally related reference compound. Compound 2 (EC50 = 2.02 ꢁM)
inhibited HCV replication, although with an EC50 different from that reported by Kumar et al.
(i.e., EC50 = 0.23 ꢁM),17 thus exhibiting 10ꢀfold less antiꢀHCV activity under our assay
conditions. However, analysis of the doseꢀresponse curve (data not shown) unexpectedly
underlined that reference 2 was a nonꢀselective antiꢀHCV agent. In the context of its
antimetabolic effect, our data was comparable to that reported previously. Thus, it was gratifying
to find that the presence of the Cꢀ6 amino group in association with a Cꢀ7 4ꢀ(2ꢀpyridinyl)ꢀ1ꢀ
piperazinyl substituent as in compound 8 allowed us to obtain a more selective quinolone
derivative than the reference compound.
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Considering the promising results obtained for derivative 8, it was selected for further studies
with the aim to quantitatively validate its antiꢀHCV activity. In particular, the compound was
tested in Huhꢀ9ꢀ13 cells by realꢀtime RTꢀPCR. The data thus obtained were consistent with the
data acquired using Huhꢀ5ꢀ2 cells. In fact, 6ꢀaminoquinolone 8 showed a selective antiviral
effect (EC50 = 3.03 ꢁM) coupled with the absence of any cytostatic effect as measured by the 3ꢀ
(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium
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(MTS) assay (CC50 >163 ꢁM). However, an inꢀdepth microscopic analysis of the cells
highlighted that quinolone 8 had an impact on the cellular morphology as apparent from the
observed cell aggregation. The morphological alterations of the treated cells induced by
derivative 8 might suggest that its antiꢀHCV activity could be in part due to potential pleiotropic
effects on the cells.
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Conclusions
Given our longꢀstanding experience in design, synthesis and biological characterization of 6ꢀ
aminoquinolone derivatives, and the antiꢀHCV activity reported for quinoloneꢀbased derivatives
by Kumar et al., we have herein described new 6ꢀaminoquinolones functionalized at Nꢀ1, Cꢀ3
and Cꢀ7 positions as potential antiꢀHCV agents. Biochemical and biological data confirmed that
the strategy of arylpiperazine moieties insertion at the Cꢀ7 position of the quinolone scaffold was
successful in developing potent new antiꢀHCV NS5B quinolones. In particular, compound 8
emerged as one of the most potent NS5B inhibitors and the most selective antiꢀHCV derivative
within this series.
The knowledge acquired from this study will guide the future structural functionalization of
the 6ꢀaminoquinolone scaffold, in search of optimized derivatives endowed with enhanced
pharmacological profiles.
Experimental section
Inducedꢀfit docking methodology. The crystal structure of NS5B RdRP complexed with
inhibitor 1 (PDB ID 3PHE)17 was retrieved from the RCSB Protein Data Bank and used as target
structure for our modeling studies. Water molecules were deleted. Schrodinger Protein
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Preparation Wizard,24 a workflow designed to ensure chemical correctness and to optimize a
protein structure for further analysis, was then used to obtain a satisfactory starting structure. In
particular, hydrogen atoms were added and bond orders and charges were assigned; the
orientation of hydroxyl groups on Ser, Thr, and Tyr, the side chains of Asn and Gln residues, and
the protonation state of His residues were optimized. Steric clashes were relieved by performing
a small number of minimization steps, not intended to minimize the system completely. In our
study, the minimization (OPLS 2005 force field) was stopped when the RMSD of the nonꢀ
hydrogen atoms reached 0.30 Å.
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Prior to IFD experiments, compounds 2ꢀ10 were built using the Schrodinger Maestro
interface25 and then submitted to the LigPrep utility,19 which rapidly produces low energy 3D
structures taking into account ionization states, tautomers, sterochemistries, and ring
conformations at the desired pH. For our study, a pH range of 6−8 was set. The ionization state
of all the ligands was double checked using MoKa,26 a software able to accurately compute pKa
values. In all the compounds, the 6ꢀamino group was considered in the neutral form (average
pKa = 2.0). The distal nitrogen atom of the piperazine (pKa = 8.41) and methylpiperazine rings
(pKa = 8.0) of compounds 6 and 7 were both modeled as protonated, whereas the arylpiperazines
at the Cꢀ7 position of the quinolone scaffold in derivatives 8ꢀ10 were considered in the neutral
form. The methylpiperazine moiety of compound 1 was also considered as protonated
(pKa=8.01).
The IFD protocol developed by Schrödinger18 was then employed for accurate prediction of
ligand binding modes and concomitant structural changes in the NS5B receptor. Briefly, this
methodology was used for the inducedꢀfit docking of compounds 1ꢀ10 using the protein prepared
as described above and the following steps (the description below is from the IFD manual):
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1. Initial Glide docking of each ligand using a softened potential (van der Waals radii scaling).
By default, a maximum 20 poses per ligand are retained, and by default poses to be retained must
have a CoulombꢀvdW score less than 100 and an Hꢀbond score less than –0.05;
2. Prime sideꢀchain prediction for each proteinꢀligand complex, on residues within a given
distance of any ligand pose (6 Å in this study);
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3. Prime minimization of the same set of residues and the ligand for each protein/ligand
complex pose. The receptor structure in each pose now reflects an induced fit to the ligand
structure and conformation.
4. Glide redocking of each proteinꢀligand complex structure within a specified energy of the
lowestꢀenergy structure (default 30 kcal/mol). The ligand is now rigorously docked, using default
Glide settings, into the inducedꢀfit receptor structure.
5. Estimation of the binding energy (IFD Score) for each output pose.
In our study, all docking calculation were run in the “Standard Precision” mode of Glide, and
the grid box was centered on the crystallographic position of TSIIꢀNNI 1. All of the docked
structures were automatically ranked according to the IFD score. RMSD value for 1 was
computed by all nonꢀ hydrogens atoms. The image representing the best IFD pose of 8 (Figure 2)
was prepared using PyMOL.27 Figure 1B was instead generated using the Ligand Interaction
Diagram tool of Maestro GUI.25
Computation of ligand inhibition constants. AutoDock’s epdb function20 was then used to
estimate the inhibition costant (Ki) of derivatives 2ꢀ10. The IFD top pose for each ligand was
processed by AutodockTools version 1.5.6, that merged nonpolar hydrogens and assigned
Gasteiger charges to each of the eight NS5B/ligand complexes. For each of the NS5B proteins,
AutoGrid version 4.2.5 was used to generate separate grid maps for all atom types of the
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corresponding ligand structure, plus one for the electrostatic interactions. The grid was centered
on the ligand, and its dimensions were 50Åx50Åx50Å with a spacing of 0.375Å. Finally,
AutoDock version 4.2.5.1 was used in nonꢀdocking mode to calculate the ligand Ki value; the
calculations were performed selecting the “epdb” utility.
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Chemistry. All starting materials were commercially available, unless otherwise indicated.
Reagents and solvents were purchased from common commercial suppliers and were used as
such. Organic solutions were dried over anhydrous Na2SO4 and concentrated with a rotary
evaporator at low pressure. All reactions were routinely checked by thinꢀlayer chromatography
(TLC) on silica gel 60F254 (Merck) and visualized by using UV or iodine. Column
chromatography separations were carried out on Merck silica gel 60 (mesh 70ꢀ230), flash
chromatography on Merck silica gel 60 (mesh 230ꢀ400). Melting points were determined in
capillary tubes (Büchi Electrotermal model 9100) and are uncorrected. Yields were of purified
products and were not optimized. The reactions conducted under microwaves irradiation were
1
carried out employing a microwave oven Biotage InitiatorTM 2.0 version 2.3 build 6250. H
NMR spectra were recorded at 200 or 400 MHz (BrukerAvance DRXꢀ200 or 400, respectively)
13
while C NMR spectra were recorded at 100 MHz (BrukerAvance DRXꢀ400). Chemical shifts
are given in ppm (δ) relative to TMS. Spectra were acquired at 298 K. Data processing was
performed with standard Bruker software XwinNMR and the spectral data are consistent with the
assigned structures. The purity of the compounds was determined by combustion analysis
employing a Fisons elemental analyzer, model EA1108CHN, and data for C, H, and N are within
0.4% of the theoretical values (purity ≥95%).
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Ethyl
2ꢀ(2,4ꢀdichloroꢀ5ꢀnitrobenzoyl)ꢀ3ꢀ{[2ꢀfluoroꢀ4ꢀ
5
6
7
8
(trifluoromethyl)benzyl]amino}acrylate (12). To a solution of ethyl 2ꢀ(2,4ꢀdiclhoroꢀ5ꢀ
nitrobenzoyl)ꢀ3ꢀ(dimethylamino)acrylate 1112 (2.00 g, 5.5 mmol) in a mixture of Et2O/EtOH 4:1
(30 mL), 2ꢀfluoroꢀ4ꢀtrifluoromethylbenzylamine (1.28 mg, 6.6 mmol) was added dropwise and
the solution was stirred at room temperature for 5 min. The formed precipitate was filtered under
vacuum and washed with Et2O to give the title compound 12 (1.98 g, 70%) as pale yellow solid,
pure by TLC in two eluents (CHCl3/MeOH 95:5 and cyclohexane/EtOAc 50:50): mp 134ꢀ135
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1
°C. H NMR (200 MHz, DMSOꢀd6): δ 1.00 (t, J = 7.0 Hz, 3H, CH3), 3.90 (q, J = 7.0 Hz, 2H,
OCH2), 4.90 (d, J = 6.2 Hz, 2H, NCH2), 7.50ꢀ7.80 (m, 3H, benzylꢀH), 8.00 (s, 1H, Hꢀ3), 8.05 (s,
1H, Hꢀ6), 8.40 (d, J = 14.3 Hz, 1H, acrylicꢀH), 11.00ꢀ11.10 (m, 1H, NH).
Ethyl 3ꢀ[(4ꢀchlorobenzyl)amino]ꢀ2ꢀ(2,4ꢀdichloroꢀ5ꢀnitrobenzoyl)acrylate (13). Following
the procedure described to prepare compound 12 and using the 4ꢀchlorobenzylamine, the title
compound 13 was obtained as pale yellow solid, pure by TLC in two eluents (CHCl3/MeOH 95:5
and cyclohexane/EtOAc 50:50), in 98% yield: mp 145ꢀ146 °C. 1H NMR (200 MHz, DMSOꢀd6):
δ 1.00 (t, J = 7.0 Hz, 3H, CH3), 3.90 (q, J = 7.0 Hz, 2H, OCH2), 4.75 (d, J = 6.2 Hz, 2H, NCH2),
7.35ꢀ7.50 (m, 4H, benzylꢀH), 8.00 (s, 1H, Hꢀ3), 8.05 (s, 1H, Hꢀ6), 8.30 (d, J = 14.6 Hz, 1H,
acrylicꢀH), 11.10 (m, 1H, NH).
Ethyl
7ꢀchloroꢀ1ꢀ[2ꢀfluoroꢀ4ꢀ(trifluoromethyl)benzyl]ꢀ6ꢀnitroꢀ4ꢀoxoꢀ1,4ꢀdihydroꢀ3ꢀ
quinolinecarboxylate (14). A mixture of compound 12 (1.95 g, 3.8 mmol) in dry DMF (25 mL)
and K2CO3 (1.59 g, 11.5 mmol) was heated under stirring at 80 °C for 3 h. The solvent was
concentrated and the mixture was poured into ice/water and acidified with 2N HCl (pH~2). The
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precipitate so obtained was filtered under vacuum to give the title compound 14 (1.68 g, 93%),
pure by TLC in two eluents (CHCl3/MeOH 95:5 and CHCl3/acetone 70:30), as pale yellow solid:
mp 273ꢀ275 °C. 1H NMR (200 MHz, DMSOꢀd6): δ 1.30 (t, J = 7.0 Hz, 3H, CH3), 4.30 (q, J = 7.0
Hz, 2H, OCH2), 5.90 (s, 2H, NCH2), 7.35ꢀ7.45 (m, 1H, benzylꢀHꢀ6), 7.60 (d, J = 8.0 Hz, 1H,
benzylꢀHꢀ5), 7.80 (d, JHꢀF= 10.0 Hz, 1H, benzylꢀHꢀ3), 8.15 (s, 1H, Hꢀ8), 8.80 (s, 1H, Hꢀ2), 8.95
(s, 1H, Hꢀ5).
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Ethyl 7ꢀchloroꢀ1ꢀ(4ꢀchlorobenzyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ1,4ꢀdihydroꢀ3ꢀquinolinecarboxylate (15).
Following the procedure described to prepare compound 14 and starting from intermediate 13,
the title compound 15 was obtained as brown solid, pure by TLC in two eluents (CHCl3/MeOH
1
95:5 and CHCl3/acetone 70:30), in 95% yield: mp 240ꢀ242 °C. H NMR (200 MHz, DMSOꢀd6):
δ 1.25 (t, J = 7.0 Hz, 3H, CH3), 4.25 (q, J = 7.0 Hz, 2H, OCH2), 5.70 (s, 2H, NCH2), 7.25ꢀ7.35
(m, 2H, benzylꢀHꢀ2 and ꢀHꢀ6), 7.35ꢀ7.45 (m, 2H, benzylꢀHꢀ3 and ꢀHꢀ5), 8.00 (s, 1H, Hꢀ8), 8.75
(s, 1H, Hꢀ2), 8.90 (s, 1H, Hꢀ5).
7ꢀChloroꢀ1ꢀ[2ꢀfluoroꢀ4ꢀ(trifluoromethyl)benzyl]ꢀ6ꢀnitroꢀ4ꢀoxoꢀ1,4ꢀdihydroꢀ3ꢀ
quinolinecarboxylic acid (16). In a microwave oven tube, compound 14 (0.50 g, 1.07 mmol)
was suspended in 8N HCl (10 mL).The mixture was irradiated at 120 °C for 130 min employing
the following experimental parameters: pressure 15 bar, cooling on, FHT on, preꢀstirring 30 s,
very high absorption. The mixture was diluted with water and the solid obtained was filtered
under vacuum to give the title compound 16 (0.39 g, 93%), pure by TLC in two eluents
(CHCl3/MeOH 90:10 and CHCl3/MeOH/AcOH 85:14.5:0.5), as pale yellow solid: mp 259ꢀ260
°C. 1H NMR (200 MHz, DMSOꢀd6): δ 6.00 (s, 2H, NCH2), 7.35ꢀ7.40 (m, 1H, benzylꢀHꢀ6), 7.50
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(d, J = 8.0 Hz, 1H, benzylꢀHꢀ5), 7.80 (d, JHꢀF = 10.0 Hz, 1H, benzylꢀHꢀ3) 8.30 (s, 1H, Hꢀ8), 8.10
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(s, 1H, Hꢀ2), 9.00 (s, 1H, Hꢀ5), 14.00 (bs, 1H, COOH).
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7ꢀChloroꢀ1ꢀ(4ꢀchlorobenzyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ1,4ꢀdihydroꢀ3ꢀquinolinecarboxilic acid (17).
Following the procedure described to prepare the compound 16 and starting from intermediate
15, the title compound 17 was obtained as pale brown solid, pure by TLC in two eluents
1
(CHCl3/MeOH 90:10 and CHCl3/MeOH/AcOH 85:14.5:0.5), in 90% yield: mp 276ꢀ278 °C. H
NMR (400 MHz, DMSOꢀd6): δ 5.90 (s, 2H, NCH2), 7.30ꢀ7.40 (m, 2H, benzylꢀHꢀ2 and ꢀHꢀ6),
7.40ꢀ7.50 (m, 2H, benzylꢀHꢀ3 and ꢀHꢀ5),8.25 (s, 1H, Hꢀ8), 8.90 (s, 1H, Hꢀ2), 9.30 (s, 1H, Hꢀ5),
14.10 (bs, 1H, COOH).
General procedure of esterification (method A). To a solution of the quinolone 3ꢀcarboxylic
acids 16 or 17 (1 mmol) in DMF (30 ml), K2CO3 (2 mmol) and the appropriate benzylchloride
(1.5 mmol) were added and the mixture was heated at 60 °C for 24 h. The solvent was
concentrated, and the mixture was poured into ice/water and acidified with 2N HCl (pH~1ꢀ2) to
give a precipitated which was filtered under vacuum. The solid obtained was triturated with
EtOH affording compounds 18ꢀ20.
2ꢀMethylbenzyl
7ꢀchloroꢀ1ꢀ[2ꢀfluoroꢀ4ꢀ(trifluoromethyl)benzyl]ꢀ6ꢀnitroꢀ4ꢀoxoꢀ1,4ꢀ
dihydroꢀ3ꢀquinolinecarboxylate (18). Following the general procedure method A and using the
2ꢀmethylbenzylchloride, compound 18 was obtained from 16, in 89% yield as yellow solid: mp
1
183ꢀ185 °C. H NMR (200 MHz, DMSOꢀd6): δ 2.30 (s, 3H, CH3), 5.25 (s, 2H, OCH2), 5.80 (s,
2H, NCH2), 7.10ꢀ7.25 (m, 3H, OꢀbenzylꢀH), 7.30ꢀ7.35 (m, 1H, NꢀbenzylꢀHꢀ6), 7.40ꢀ7.60 (m, 2H,
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NꢀbenzylꢀHꢀ5 and OꢀbenzylꢀH), 7.75 (d, JHꢀF= 10.0 Hz, 1H, NꢀbenzylꢀHꢀ3), 8.10 (s, 1H, Hꢀ8),
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8.75 (s, 1H, Hꢀ2), 8.90 (s, 1H, Hꢀ5).
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4ꢀMethylbenzyl
7ꢀchloroꢀ1ꢀ[2ꢀfluoroꢀ4ꢀ(trifluoromethyl)benzyl]ꢀ6ꢀnitroꢀ4ꢀoxoꢀ1,4ꢀ
dihydroꢀ3ꢀquinolinecarboxylate (19). Following the general procedure method A and using the
4ꢀmethylbenzylchloride, compound 19 was obtained from 16, in 75% yield as yellow solid: mp
1
223ꢀ224 °C. H NMR (200 MHz, DMSOꢀd6): δ 2.30 (s, 3H, CH3), 5.20 (s, 2H, OCH2), 5.90 (s,
2H, NCH2), 7.10ꢀ7.20 (m, 2H, OꢀbenzylꢀHꢀ3 and ꢀHꢀ5), 7.30ꢀ7.45 (m, 3H, OꢀbenzylꢀHꢀ2, ꢀHꢀ6
and NꢀbenzylꢀHꢀ6), 7.55 (d, J = 7.0 Hz, 1H, NꢀbenzylꢀHꢀ5), 7.75 (d, JHꢀF = 10.0 Hz, 1H, Nꢀ
benzyl Hꢀ3), 8.10 (s, 1H, Hꢀ8), 8.75 (s, 1H, Hꢀ2), 8.95 (s, 1H, Hꢀ5).
4ꢀChlorobenzyl
7ꢀchloroꢀ1ꢀ(4ꢀchlorobenzyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ1,4ꢀdihydroꢀ3ꢀ
quinolinecarboxylate (20). Following the general procedure method A and using the 4ꢀ
chlorobenzylchloride, compound 20 was obtained from 17, in 90% yield as pale brown solid: mp
1
220ꢀ221 °C. H NMR (200 MHz, DMSOꢀd6): δ 5.30 (s, 2H, OCH2), 5.80 (s, 2H, NCH2), 7.30ꢀ
7.60 (m, 8H, N,OꢀbenzylꢀH), 8.10 (s, 1H, Hꢀ8), 8.80 (s, 1H, Hꢀ2), 9.00 (s, 1H, Hꢀ5).
General procedure of coupling with piperazines (method B). A mixture of compound 20 (1
mmol) in dry CH3CN (20 mL), Et3N (3.0 mmol) and the appropriate piperazine (1.5 mmol) was
stirred at reflux for 3ꢀ20h. The solvent was then concentrated to half volume and after cooling
the precipitated so obtained was collected by filtration under vacuum and purified, if necessary,
by the method below reported, affording compounds 21ꢀ25.
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4ꢀChlorobenzyl
1ꢀ(4ꢀchlorobenzyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ7ꢀ(1ꢀpiperazinyl)ꢀ1,4ꢀdihydroꢀ3ꢀ
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quinolinecarboxylate (21). Following the general procedure method B and using piperazine
(reaction time 3h), after treatment with Et2O, compound 21 was obtained in 90% yield as a
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yellow solid: mp 140ꢀ143 °C. H NMR (200 MHz, DMSOꢀd6): δ 2.75ꢀ3.00 and 3.25ꢀ3.50 (m,
each 4H, piperazineꢀCH2), 5.30 (s, 2H, OCH2), 5.70 (s, 2H, NCH2), 7.10 (s, 1H, Hꢀ8), 7.30ꢀ7.60
(m, 8H, N,OꢀbenzylꢀH), 8.65 (s, 1H, Hꢀ2), 9.00 (s, 1H, Hꢀ5).
4ꢀChlorobenzyl 1ꢀ(4ꢀchlorobenzyl)ꢀ7ꢀ(4ꢀmethylꢀ1ꢀpiperazinyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ1,4ꢀdihydroꢀ
3ꢀquinolinecarboxylate (22). Following the general procedure method B and using 4ꢀ
methylpiperazine (reaction time 4h), compound 22 was obtained in 71% yield as a yellow
1
crystalline solid: mp 230ꢀ232 °C. H NMR (200 MHz, CDCl3): δ 2.40 (s, 3H, CH3), 2.45ꢀ2.55
and 2.95ꢀ3.05 (m, each 4H, piperazineꢀCH2), 5.30 (s, 2H, OCH2), 5.40 (s, 2H, NCH2), 6.60 (s,
1H, Hꢀ8), 7.10ꢀ7.20 (m, 2H, NꢀbenzylꢀHꢀ2 and ꢀHꢀ6), 7.35ꢀ7.45 (m, 4H, OꢀbenzylꢀH), 7.45ꢀ7.55
(m, 2H, NꢀbenzylꢀHꢀ3 and ꢀHꢀ5), 8.55 (s, 1H, Hꢀ2), 8.80 (s, 1H, Hꢀ5).
4ꢀChlorobenzyl
1ꢀ(4ꢀchlorobenzyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ7ꢀ[4ꢀ(2ꢀpyridinyl)ꢀ1ꢀpiperazinyl]ꢀ1,4ꢀ
dihydroꢀ3ꢀquinolinecarboxylate (23). Following the general procedure method B and using 1ꢀ
(2ꢀpyridinyl)piperazine (reaction time 20h), after purification by flash chromatography eluting
with CHCl3/MeOH (97:3), compound 23 was obtained in 50% yield as a yellow solid: mp 199ꢀ
1
201 °C. H NMR (200 MHz, DMSOꢀd6): δ 3.10ꢀ3.25 and 3.50ꢀ3.70 (m, each 4H, piperazineꢀ
CH2), 5.30 (s, 2H, OCH2), 5.75 (s, 2H, NCH2), 6.70 (dd, J = 5.0 and 6.8 Hz, 1H, pyridineꢀH),
6.85 (d, J = 9.0 Hz, 1H, pyridineꢀH), 7.10 (s, 1H, Hꢀ8), 7.30ꢀ7.60 (m, 9H, N,OꢀbenzylꢀH and
pyridineꢀH), 8.20 (dd, J = 2.8 and 5.0 Hz, 1H, pyridineꢀH), 8.60 (s, 1H, Hꢀ2), 9.00 (s, 1H, Hꢀ5).
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4ꢀChlorobenzyl 7ꢀ[4ꢀ(1,3ꢀbenzothiazolꢀ2ꢀyl)ꢀ1ꢀpiperazinyl]ꢀ1ꢀ(4ꢀchlorobenzyl)ꢀ6ꢀnitroꢀ4ꢀ
oxoꢀ1,4ꢀdihydroꢀ3ꢀquinolinecarboxylate (24). Following the general procedure method B, and
using the 2ꢀ(1ꢀpiperazinyl)ꢀ1,3ꢀbenzothiazole (reaction time 18h), after purification by flash
chromatography eluting with CHCl3/MeOH (97:3), compound 24 was obtained in 35% yield as
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yellow solid: mp 200ꢀ202 °C. H NMR (400 MHz, DMSOꢀd6): δ 3.15ꢀ3.25 and 3.65ꢀ3.75 (m,
each 4H, piperazineꢀCH2), 5.30 (s, 2H, OCH2), 5.70 (s, 2H, NCH2), 7.10ꢀ7.15 (m, 2H, Hꢀ8 and
benzothiazoleꢀH), 7.25ꢀ7.30 (m, 1H, benzothiazoleꢀH), 7.35ꢀ7.55 (m, 9H, benzothiazoleꢀH and
N,OꢀbenzylꢀH), 7.75ꢀ7.80 (m, 1H, benzothiazoleꢀH), 8.60 (s, 1H, Hꢀ2), 9.00 (s, 1H, Hꢀ5).
4ꢀChlorobenzyl
1ꢀ(4ꢀchlorobenzyl)ꢀ6ꢀnitroꢀ4ꢀoxoꢀ7ꢀ{4ꢀ[3ꢀ(trifluoromethyl)phenyl]ꢀ1ꢀ
piperazinyl}ꢀ1,4ꢀdihydroꢀ3ꢀquinolinecarboxylate (25). Following the general procedure
method B, and using 1ꢀ[3ꢀ(trifluoromethyl)phenyl]piperazine (reaction time 16 h), after
purification by flash chromatography eluting with CHCl3/MeOH (97:3), compound 25 was
obtained in 45% yield as yellow solid: mp 213ꢀ216 °C. 1H NMR (400 MHz, DMSOꢀd6): δ 3.15ꢀ
3.25 and 3.30ꢀ3.40 (m, each 4H, piperazineꢀCH2), 5.30 (s, 2H, OCH2), 5.75 (s, 2H, NCH2), 7.05
(s, 1H, Hꢀ8), 7.10 (d, J = 8.0 Hz, 1H, phenylꢀH), 7.20ꢀ7.25 (m, 2H, phenylꢀH), 7.35ꢀ7.40 (m, 2H,
benzylꢀH), 7.35ꢀ7.45 (m, 5H, benzylꢀH and phenylꢀH), 7.55ꢀ7.60 (m, 2H, benzylꢀH), 8.55 (s, 1H,
Hꢀ2), 9.00 (s, 1H, Hꢀ5).
General procedure for reduction of nitro group (method C). To a solution of the nitro
derivatives 18ꢀ25 (1.0 mmol) in DMF (30 mL), aqueous NaCl 3.5% w/w (150 mL) and Fe
powder (55 mmol) were added; the mixture was then heated at 150 °C for 24h. The hot mixture
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was filtered over Celite and the residue washed with warm DMF (50 mL). The filtrate was
evaporate to dryness and the residue was further workedꢀup as follows. For compounds 3ꢀ5 the
residue was poured into ice/water, acidified with 2N HCl (pH = 4) and the formed precipitated
filtered under vacuum, collected and crystallized by EtOH/DMF. In the case of compounds 6ꢀ10,
the residue was treated with CHCl3 and the inorganic component was removed by filtration. The
organic layer was evaporated to dryness and the residue was purified as indicated.
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2ꢀMethylbenzyl
6ꢀaminoꢀ7ꢀchloroꢀ1ꢀ[2ꢀfluoroꢀ4ꢀ(trifluoromethyl)benzyl]ꢀ4ꢀoxoꢀ1,4ꢀ
dihydroꢀ3ꢀquinolinecarboxylate (3). Following the general procedure method C and starting
from intermediate 18, the target compound 3 was obtained in 36% yield as pale yellow solid: mp
1
259ꢀ260 °C. H NMR (400 MHz, DMSOꢀd6): δ 2.30 (s, 3H, CH3), 5.25 (s, 2H, OCH2), 5.75 (s,
2H, NH2), 5.80 (s, 2H, NCH2), 7.20ꢀ7.30 (m, 4H, OꢀbenzylꢀH), 7.50 (d, J = 7.0 Hz, 1H, Nꢀ
benzylꢀHꢀ6), 7.60 (d, J = 7.0 Hz, 1H, NꢀbenzylꢀHꢀ5), 7.65 (s, 1H, Hꢀ8), 7.70 (s, 1H, Hꢀ5), 7.75
(d, JHꢀF = 10.0 Hz, 1H, NꢀbenzylꢀHꢀ3), 8.75 (s, 1H, Hꢀ2). 13C NMR (100 MHz, DMSOꢀd6): δ
19.00, 50.59, 64.25, 108.82, 110.00, 113.64ꢀ113.88 (m, NꢀbenzylꢀCꢀ3), 118.30, 122.30 (m, Nꢀ
benzylꢀCꢀ5), 123.65 (q, JCꢀF = 268.0 Hz, CF3), 124.21, 126.16, 128.38, 128.54 (d, JCꢀF = 14.0 Hz,
NꢀBenzylꢀCꢀ1), 128.96, 129.17, 129.98 (d, JCꢀF = 5.0 Hz, NꢀBenzylꢀCꢀ6), 130.33, 130.40, 130.80
(dd, JCꢀF = 9.0 and 33.0 Hz, NꢀBenzylꢀCꢀ4), 135.00, 136.91, 143.47, 149.18, 160.08 (d, JCꢀF
=
250.0 Hz, NꢀBenzylꢀCꢀ2), 165.23, 172.60. Anal. Calcd. for C26H19ClF4N2O3: C, 60.18; H, 3.69;
N, 5.40. Found: C, 59.96; H, 3.51; N, 5.80.
4ꢀMethylbenzyl
6ꢀaminoꢀ7ꢀchloroꢀ1ꢀ[2ꢀfluoroꢀ4ꢀ(trifluoromethyl)benzyl]ꢀ4ꢀoxoꢀ1,4ꢀ
dihydroꢀ3ꢀquinolinecarboxylate (4). Following the general procedure method C and starting
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6
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280ꢀ281 °C. H NMR (400 MHz, DMSOꢀd6): δ 2.30 (s, 3H, CH3), 5.20 (s, 2H, OCH2), 5.75 (s,
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9
2H, NH2), 5.80 (s, 2H, NCH2 ), 7.15ꢀ7.20 (m, 2H, OꢀbenzylꢀHꢀ3 and ꢀHꢀ5), 7.25 (t, J = 7.7 Hz,
1H, NꢀbenzylꢀHꢀ6), 7.35ꢀ7.40 (m, 2H, OꢀbenzylꢀHꢀ2 and ꢀHꢀ6), 7.50 (d, J = 8.1 Hz, 1H, Nꢀ
benzylꢀHꢀ5), 7.60 (s, 1H, Hꢀ8), 7.65 (s, 1H, Hꢀ5), 7.75 (d, JHꢀF = 10.0 Hz, 1H, NꢀbenzylꢀHꢀ6),
8.75 (s, 1H, Hꢀ2). 13C NMR (100 MHz, DMSOꢀd6): δ 21.19, 50.55, 65.46, 108.85, 109.98,
113.75 (d, JCꢀF = 21.0 Hz, NꢀbenzylꢀCꢀ3), 118.29, 121.5ꢀ123.00 (m, NꢀbenzylꢀCꢀ5), 123.60 (q,
JCꢀF= 270.0 Hz, CF3), 124.22, 128.21, 128.57 (d, JCꢀF = 10.0 Hz, NꢀbenzylꢀCꢀ1), 129.13, 129.32,
129.89 (d, JCꢀF = 4.0 Hz, NꢀbenzylꢀCꢀ6), 130.33, 130.78 (dd, JCꢀF = 8.0 and 30.0 Hz, NꢀbenzylꢀCꢀ
4), 134.13, 137.40, 143.47, 149.16, 160.05 (d, JCꢀF = 250.0 Hz, NꢀbenzylꢀCꢀ2), 165.03, 172.62.
Anal. Calcd. for C26H19ClF4N2O3: C, 60.18; H 3.69; N, 5.40. Found:C, 60.30; H, 3.84; N, 5.13.
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4ꢀChlorobenzyl
6ꢀaminoꢀ7ꢀchloroꢀ1ꢀ(4ꢀchlorobenzyl)ꢀ4ꢀoxoꢀ1,4ꢀdihydroꢀ3ꢀ
quinolinecarboxylate (5). Following the general procedure method C and starting from
intermediate 20, the target compound 5 was obtainedin 34% yield as pale yellow solid: mp 274ꢀ
1
275 °C. H NMR (400 MHz, DMSOꢀd6): δ 5.25 (s, 2H, OCH2), 5.50 (s, 2H, NH2), 5.75 (s, 2H,
NCH2), 7.20ꢀ7.30 (m, 2H, NꢀbenzylꢀHꢀ2 and ꢀHꢀ6), 7.30ꢀ7.45 (m, 4H, NꢀbenzylꢀHꢀ3, and –Hꢀ5,
OꢀbenzylꢀHꢀ2, and ꢀHꢀ6), 7.50ꢀ7.60 (m, 3H, Hꢀ5, and NꢀbenzylꢀHꢀ3, and ꢀHꢀ5), 7.65 (s, 1H, Hꢀ
13
8), 8.75 (s, 1H, Hꢀ2). C NMR (100 MHz, DMSOꢀd6): δ 55.20, 64.74, 108.26, 109.86, 118.82,
124.07, 128.77, 128.88, 129.36, 129.79, 130.23, 130.70, 132.91, 135.46, 136.29, 143.40, 149.00,
165.19, 172.61. Anal. Calcd. for C24H17Cl3N2O3: C, 59.10; H, 3.51; N, 5.74. Found: C, 59.19; H,
3.64; N, 5.52.
29
ACS Paragon Plus Environment