Synthesis, cytotoxicity and antiviral evaluation of new series of imidazo[4,5-g[quinoline and pyrido[2,3-g[quinoxalinone derivatives

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Synthesis, cytotoxicity and antiviral evaluation of new series of

imidazo[4,5-g]quinoline and pyrido[2,3-g]quinoxalinone derivatives

a§

b*§

c§

c

a

Irene Briguglio , Roberta Loddo , Erik Laurini , Maurizio Fermeglia , Sandra Piras , Paola Corona , Paolo

a

b

Giunchedi , Elisabetta Gavini , Giuseppina Sanna , Gabriele Giliberti , Cristina Ibba , Pamela Farci , Paolo

La Colla , Sabrina Pricl , Antonio Carta

b+

cd+

a*+

a

Dipartimento di Chimica e Farmacia, Università degli Studi di Sassari, Via Muroni 23/A, 07100 Sassari,

Italy

b

Dipartimento di Scienze Biomediche, Sezione di Microbiologia e Virologia, Università degli Studi di

Cagliari, Cittadella Universitaria s.p.8, Km 0,700, 09042 Monserrato (Ca), Italy

c

Molecular Simulation Engineering (MOSE) Laboratory, Department of Engineering and Architecture

(DEA), University of Trieste, 34127 Trieste, Italy

d

National Interuniversity Consortium for Material Science and Technology (INSTM), Research Unit MOSE-

DEA, University of Trieste, 34127 Trieste, Italy

§

These authors contributed equally to this work

+

Senior co-authors

Keywords: imidazo[4,5-g]quinolines, pyrido[2,3-g]quinoxalines, antiviral activity, RNA viruses,

DNA viruses.

*

Corresponding Authors:

Tel.: +39 079 228722; fax: +39 079 228720. E.mail address acarta@unisss.it;

Tel.: +39 070 6754202; fax: +39 070 6754210. E.mail address rloddo@unica.it

Abstract

Linear aromatic N-tricyclic compounds with promising antiviral activity and minimal cytotoxicity

were prepared and analyzed in the last years. Specifically, the pyrido[2,3-g]quinoxalinone nucleus

was found endowed with high potency against several pathogenic RNA viruses as etiological agents

of important veterinary and human pathologies. Following our research program on new antiviral

agents we have designed, synthesized and assayed new series of imidazo[4,5-g]quinoline and

pyrido[2,3-g]quinoxalinone derivatives. Lead compounds 1-4 were further modified to enhance

their antiviral activity and reduce their cytotoxicity. Thus, different substituents were introduced on

N atom at position 1 or the O atom at position 2 of the leads; contextually, several groups were

inserted on the nitrogen atom at position 7 of diaminoquinoline intermediates. Title compounds

were tested in cell-based assays for cytotoxicity and antiviral activity against RNA virus families

containing single-stranded (either positive-sense (ssRNA+) or negative-sense (ssRNA-)), and

double-stranded genomes (dsRNA), and against two representatives of DNA virus families. Some

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derivatives emerged as potential leads for further development as antiviral agents against some

viruses of public health significance, such as RSV, Reo, BVDV and HCV. Particularly, compounds

4

, 11b, 11c, 13c, 15a, 18 and 21 resulted active against BVDV at concentrations ranging from 1.3

to 5 µM. Compound 21 was also evaluated for its activity on the BVDV RdRp. Compound 4 was

also tested as potential anti-HCV compound in a subgenomic replication assay. Molecular

simulation results provided a molecular rationale for the anti-BVDV activity of these compounds.

1. Introduction

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 Herpesviruses, e.g., Herpes

simplex virus type 1 (HSV-1) and Poxviruses [3], which can infect both vertebrate and invertebrate

animals, or, from Orthopoxvirus genus the Vaccinia virus (VV). The latter emerged in recent years

for its potential use as an attractive bioterrorist weapon [4]. This menace to public health increased

our awareness around human vulnerability to this infection, especially since smallpox vaccination

was discontinued in 1980 [5-9]. RNA viruses include many highly pathogens affecting humans,

several of them showing the ability to cross the species barrier and resulting in zoonotic-human

epidemics [10]. Examples of RNA pathogen viruses are Flaviviruses, which can cause life-

threatening diseases in both humans and animals. This virus family includes three genera:

Pestiviruses, Flaviviruses and Hepaciviruses. The Pestivirus genus includes animal pathogens of

major economic impact for the livestock industry, such as the Bovine viral diarrhea virus (BVDV).

Hepatitis C virus (HCV) is the sole representative of the Hepacivirus genus and constitutes the

major cause of human hepatitis per se [11]. Other important RNA viruses belong to the

Picornaviridae family. These viruses induce a variety of maladies including meningitis, cold, heart

infection, conjunctivitis, and hepatitis. This family includes nine genera, some of which comprise

major human pathogens (e.g., Poliovirus and Coxsackievirus) [12]. At present, no speciﬁc antiviral

therapy is available for the treatment of Picornaviridae infections.

In this scenario, there is an urgent need for new lead compounds that either target distinct stages of

the viral replication cycle in a virus-specific way, have a broader spectrum of activity or act through

novel mechanisms of action. In this context, according to a long-lasting antiviral research program

our group designed and synthesized three new classes of linear aromatic N-tricyclic compounds

endowed with promising antiviral activity and selectivity index against several pathogenic RNA

viruses: triazolo[4,5-g]quinolines, imidazo[4,5-g]quinolones, and pyrido[2,3-g]quinoxalines [13].

All three molecular classes share the same key intermediate: a 6,7-diaminoquinoline scaffold

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variously substituted on the nitrogen at position 7. Specifically, the pyrido[2,3-g]quinoxalinone

nucleus was found particularly active against the Pestivirus BVDV and partly against the Human

respiratory syncytial virus (RSV). Both these two viruses are of major interest for livestock and

human therapies, respectively. In antiviral drug discovery, BVDV is commonly used as a surrogate

model for Hepatitis C virus [14]. Thus, given their interesting activity against BVDV, the

pyrido[2,3-g]quinoxalinones were also tested against HCV in a subgenomic replication assay that

allows viral replication in a human hepatoma cell line (GS4.1). Furthermore, the lead compounds of

that series were evaluated in enzyme assays performed with the recombinant BVDV and HCV

RNA-dependent RNA polymerases (RdRps) [13]. Unfortunately, both the pyrido[2,3-

g]quinoxalines and pyrido[2,3-g]quinoxalinones resulted more active against the BVDV than the

HCV enzyme. Moreover, they showed slight cytotoxicity against several cell lines.

In the light of these results, and with the aim of enhancing the antiviral activity and reducing the

cytotoxicity of these molecules, in this work we selected lead compounds 1 and 2 from the previous

molecular series (Fig. 1), which bear a phenyl and a benzyl substituent at position 3 of the

pyrazinone moiety, respectively, for further development. Furthermore, we adopted derivative 3,

which contains an isopropyl side chain at the same position and is totally devoid of activity on all

tested viruses and cell lines, as a negative benchmark. Finally, as shown in Scheme 1, we

synthesized and assayed the thienyl derivative 4 since no derivative bearing a heterocycle moiety at

position 3 was previously reported and its intermediate 4b is readily available on the market.

This study is divided in three parts. In the first part we modified the structure of lead compound 1

by inserting a propyl, cyclohexyl or methylcyclohexyl group on the nitrogen at position 7 of the

diaminoquinoline intermediates 8a-c, as reported in Scheme 2.

Unexpectedly, the subsequent cyclization reaction with 2-oxo-2-phenylacetic acid provided, in

addition to the desired pyrido[2,3-g]quinoxalinone derivatives with the phenyl ring at position 2

(11a-c), the imidazo[4,5-g]quinolines 9a,b. Interestingly, the same molecular scaffold was obtained

in the past by another chemical route [13]. To verify the reproducibility of this mechanism the

diaminoquinolines 8b,c were submitted to cyclization reaction with 2-oxo-2-(thiophen-2-yl) acetic

and 2-oxo-3-phenylpropanoic acids, respectively. In the first case both the pyrido[2,3-

g]quinoxalinone bearing the thiophene ring at position 2 (12b) and the corresponding imidazo[4,5-

g]quinoline 10b were obtained. In the second case the reaction afforded the pyrido[2,3-

g]quinoxalinone 13c and only non-isolable traces of the corresponding imidazo[4,5-g]quinoline 9c.

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In the second part of this study we introduced several substituents - methyl, ethyl, acetonitrile,

benzyl and 4-nitrobenzyl group - either on the nitrogen atom at position 1 or on the oxygen atom at

position 2, as shown in Scheme 3. Through direct alkylation of compound 1, N-alkyl derivatives

(14a-e) were always obtained, while the isomeric O-derivatives were not always obtained.

Antiviral evaluation of the N and O alkyl derivatives 14 and 15 highlighted N-methyl (14a) and O-

methyl (15a) derivatives as the only compounds endowed of antiviral activity against BVDV, with

EC50 values for compound 15a comparable to that of lead compound 1. Moreover, these compounds

showed activity also against CVB-5 and Sb-1 (14a), RSV and Reo-1 (15a) viruses.

Consequently, we submitted the other three lead compounds (2, 3 and 4) only to a methylation

reaction to dissect the relevance of the different substituents at position 3, as shown in Scheme 4.

All synthesized compounds were tested for cytotoxicity and for activity in cell-based assays against

+

ssRNA viruses [Human immunodeﬁciency virus (HIV-1), Coxsackie virus type B5 (CVB-5), Polio

-

virus (Sb-1), Yellow Fever virus (YFV), Bovine Viral Diarrhea virus (BVDV)], ssRNA viruses

[

Human respiratory syncytial virus (RSV), Vescicular stomatitis virus (VSV)], dsRNA viruses

Reovirus (Reo-1)], and DNA viruses [(Herpes simplex virus type 1 (HSV-1), Vaccinia virus (VV)],

with the aim of comparing their antiviral activity with that of the former molecular series. In

addition, compound 4 was submitted to the HCV subgenomic replication assay to assess the activity

of this new lead compound against this critical human pathogen. Finally, a molecular-base rationale

for the anti-BVDV activity of these compounds is proposed based on the results of computer-based

simulations.

All viruses selected were among those most harmful to human health. With the exception of YFV,

there is no vaccine available against the above-mentioned Flaviviruses to date, and only a few new

drugs are currently under successful pre-clinical development in this field [12, 15-19]. This

perspective indeed emphasizes the compelling need for the identification of new molecules with

therapeutic potential against RNA viruses.

2. Results and discussion

2.1 Chemistry

The synthetic route to obtain compound 4 is illustrated in Scheme 1. The key intermediate 8-

chloroquinoline-6,7-diamine 4a, obtained as reported in [20], was submitted to cyclization reaction

with 2-oxo-2-(thiophen-2-yl)acetic acid (4b) in 10% sulfuric acid to give the expected 5-chloro-3-

(thiophen-2-yl)pyrido[2,3-g]quinoxalin-2(1H)-one (4) (yield 43%) but not the 2-thiophen-2-yl

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isomer (4c). As shown in Scheme 1 and previously reported in [21], the reaction conditions

determine its regioselectivity. In fact, the simultaneous presence of the acidic environment and an

electron-withdrawing group on the α-carbonyl of 4b leads to the exclusive formation of the desired

isomer 4.

The diamine intermediates 8a-c, crucial for the preparation of quinoxalines 11a-c, 12b and 13c,

were generally obtained in good yield through nucleophilic substitution of the chlorine at position 7

of the known 7,8-dichloro-6-nitroquinoline (5) [22] with the amines 6a-c. The nitro derivatives

intermediates (7a-c) were subsequently submitted to reduction reaction with 10% Pd/C in ethanol,

as depicted in Scheme 2.

The succeeding condensation reaction of the diamines 8a-c with the appropriate α-ketoacids in

refluxed ethanol led not only to the expected pyrido[2,3-g]quinoxalinones (11a-c, 12b and 13c), but

also to the imidazo[4,5-g]quinolines derivatives 9a-c and 10b. This behavior is somewhat different

from that previously discussed for compound 4. In fact, the lack of an acidic environment and the

presence of an alkyl group on the nitrogen at position 7 of the diaminoquinolines 8a-c - which

increase the steric hindrance around that site - both reverse the reactivity providing only the 2-aryl

isomers 11a-c, 12b and 13c. On the other hand, this reaction is in competition with the formation of

the imidazo[4,5-g]quinoline nucleus, as previously reported by our group [21].

Scheme 3 reports the synthesis of the pyrido[2,3-g]quinoxalinones 14a-e and pyrido[2,3-

g]quinoxalines 15a,b,e. The lead compound 1 was subjected to alkylation reaction with methyl and

ethyl sulfate, acetonitrile, benzyl bromide and 4-nitrobenzyl chloride in dimethylformamide (DMF)

2 3

and Cs CO at 60 °C for 1-16 hours. From these reactions the expected N alkyl derivatives (14a-e)

were isolated, with yields ranging from 67% to 95%. When the alkylating agents were methyl and

ethyl sulfate or 4-nitrobenzyl chloride, even O-alkyl derivatives (15a,b,e) were isolated, even if the

yields in this case were rather low (7% - 23%).

Finally, as confirmation of the observed behavior, the methylation of the remaining lead compounds

(2-4) provided both products of N-alkylation and O-alkylation with yields of 26-30% (16-18) and

13-23% (19-21), respectively (Scheme 4).

2.2 Biology

Title compounds were evaluated in cell-based assays for cytotoxicity and antiviral activity against

ssRNA+ viruses representative of the Flaviviridae and Picornaviridae families. In particular,

representative viruses of two of the three genera of the Flaviviridae family, YFV (Flaviviruses) and

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BVDV (Pestiviruses), and one genus of the Picornaviridae family, CVB-5 and Sb-1

+

(

Enteroviruses), were tested. Compounds were also tested against other representatives of ssRNA ,

-

ssRNA and DNA viruses.

Table 1 shows the overall results of the cell-based assays obtained for imidazo[4,5-g]quinolines

(9a-c and 10b), pyrido[2,3-g]quinoxalinones (4, 11a-c, 12b, 13c, 14a-e, 16-18) and pyrido[2,3-

g]quinoxalines (15a,b,e, 19-21). Efavirenz (EFV), 2′-C-methyl-guanosine (Met-Gua), 2′-C-methyl-

cytidine (Met-Cyt), 2′-C-ethynyl-cytidine (Eth-Cyt), 6-azauridine (Aza), cidofovir (CDF) and

acyclovir (ACG) were used as reference compounds. As concerns the antiviral activity, none of title

compounds turned out to be active against HIV-1, YFV, VSV, VV and HSV-1, although some (9a,

11a, 14a) show a modest and scattered activity against CVB-5 and Sb-1. On the contrary,

compounds belonging to all classes proved to be endowed with an interesting anti-BVDV activity.

Among these compounds (bold in Table 1), only 9c, 11a, 11c, and 13c exerted cytotoxic effects on

exponentially growing MT-4 cell lines, and on MDBK and BHK cells in stationary growth, at

concentrations ≤ 40 µM. However, as expected, the imidazo[4,5-g]quinolines derivatives showed

only moderate activity against BVDV. Indeed, in a previous paper our research group demonstrated

the need of the presence of a chlorine atom on the quinoline ring for activity against this virus [13].

Conversely, pyrido[2,3-g]quinoxalines derivatives exhibited moderate to good activity towards

BVDV, especially when an aryl substituent at position 3 is present in addition to the chlorine atom

at position 5.

Among lead compounds, thienyl derivative 4 showed BVDV selectivity, good potency and

selectivity index. For this reason, compound 4 was also tested as potential anti-HCV compound in a

subgenomic replication assay. Experimental results (Table 2) showed that 4 was indeed able to

inhibit HCV replication (EC50 = 7.5 ±0.5 µM), but was also cytotoxic for GS4.1 cells (CC50 = 21 ±

2.0 µM), overall resulting in poor selectivity index (S.I. ≈ 3).

Biological analysis further confirmed that N-alkylated and O-alkylated derivatives were in general

devoid of cytotoxicity. Among them, compounds 18 and 21 not only show activity greater than the

corresponding unmethylated lead 4 but also are the most potent and selective molecules against

BVDV with EC50 values of 2 and 1.3 µM, respectively.

Compound 21 was finally evaluated for its activity on the BVDV RdRp (Figure 2). In vitro

enzymatic assay demonstrated that this compound inhibits the BVDV RNA-dependent RNA

polymerase activity with an IC value of 0.64 µM, thus confirming both compound potency and

50

viral target protein.

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2.3 Modeling results

Previous enzymatic assays clearly showed that the lead compounds 1 and 2 [13] and 21 target one

of the most important enzymes involved in the BVDV replication-cycle, namely the RNA-

dependent RNA-polymerase (RdRp). Molecular simulation studies were then performed on a subset

of 12 present compounds including the leads 1-4 with two specific purposes: i) to dissect the effect

of the substitution of the quinoxalin-2(1H)-one scaffold in the corresponding N-methylated

compounds 14a and 16-18 or the 2-methoxy-quinoxaline moiety in compounds 15a and 19-21; and

ii) the role of a bulky aromatic substituent at position 3 of the double-nitrogen ring. For this

purpose, a preliminary search for a putative binding site for our compounds on the BVDV RdRp

was first conducted [23-28]. In line with previous findings [26-28], the resulting portion of the

enzyme making up the binding pocket for our molecular set is located between the RdRp Fingers

domains 1 and 2 (residues 139–313 and 351–410, Figure 3A). Accordingly, all molecules were

docked within the selected protein cavity according to a well-validated procedure, and the

corresponding protein/drug free energies of binding (ꢀGbind) were scored by applying molecular

dynamics (MD) simulations based on the MM/PBSA approach [23-34]. Table 3 shows the result of

the MM/PBSA analysis, from which a direct correlation between enzyme/drug affinity and in vitro

EC50 values is apparent. As seen in this Table, computational data confirm that lead compounds 1, 2

and 4 all bind the BVDV polymerase with highly comparable affinity, while compound 3, chosen as

negative reference, does not. The critical presence of a bulky aromatic substituent at position 3 of

all compounds for their optimal BVDV RdRp binding is also confirmed by the other data in Table

3. Indeed, for both series of compounds 14a,16-18 and 15a,19-21 a marked decrease in the value of

ꢀG

bind is predicted when an isopropyl group is found at that position.

To investigate in more detail the BVDV RdRp binding mode of these selected compounds, we

proceeded and analyzed the main interactions involved in the stabilization of the relevant

enzyme/drug complexes. Taking the quinoxaline derivative 21 as proof-of-concept, the analysis of

the corresponding equilibrated MD trajectory reveals that 21 is nestled in the detected RdRp

binding pocket, where it occupies two cavities lined by the side chains of residues A221, I261, I287,

and Y289 and residues V216, Y303, V306, K307, P408 and A412, respectively (Figure 3B).

Contextually, three stabilizing hydrogen bonds (HBs) are identified between donor atoms on the

side chain of protein residues R295, S411 and Y674 are the corresponding counterparts in the

compound structure represented by the N-pyrido atom, the nitrogen atom at position 1 of the

quinozaline moiety, and the sulfur atom of the thienyl ring, respectively (Figure 3B).

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A quantitative analysis of the drug/protein interaction was performed via a per-residue

deconvolution of the enthalpic contribution to binding afforded by each single residue involved [32-

38], as reported in Table 4. To facilitate reading, the values of ꢀHbind,res for the critical RdRp

residues were clustered according to a specific underlying interaction type as follows: A221, I261,

I287, and Y289 belong to the first hydrophobic cavity (HC1), and were considered for hydrophobic

(HI) and π-interactions (π). Analogously, amino acids V216, Y303, V306, K307, P408 and A412

were clustered in the other hydrophobic cavity (HC2) and considered for analogous interaction

types. On the other hand, R295, Y674 and S411, as responsible for H-bond interactions, were

classified as HB1, HB2, and HB3, respectively.

The permanent hydrogen bond between the N-pyrido atom of 21 and the side chain of R295 (HB1,

average dynamic length (ADL) = 1.96 ± 0.05 Å) reflects in a stabilizing contribution of -2.03

kcal/mol. Concomitantly, the second stable H-bond interaction detected between the hydroxyl

group of Y674 and the quinoxaline nitrogen atom (HB2, average dynamic length (ADL) = 1.94 ±

0.03 Å) provides a favorable contribution to binding of -1.98 kcal/mol. The third polar interaction

HB3, involving the –OH on the side chain of S411 and the S atom of the thiophene ring of 21 is

decidedly weaker, as confirmed by its ꢀHbind,res value of -0.58 and the corresponding longer ADL

(2.15 ± 0.05 Å). Finally, the network of HI and π interactions, originated among the aromatic

portions of 21 and the two identified receptor cavities HC1 and HC2, considerably concur in

stabilizing the protein/drug binding with an overall contribution of -5.95 kcal/mol.

With the aim of finding a molecular rationale for the points i) and ii) above, the same computational

analysis was then applied to all remaining 11 compounds in Table 3. As can be seen by

reconsidering all ꢀGbind values listed in this Table, the modification of the amide group in lead

compounds 1-4 did not substantially affect their binding affinity towards the target protein. Taking

the thienyl derivatives 4, 18, and 21 as proof-of-concept, the relevant ꢀGbind values are all very

similar, being in the range -8.00 ÷ -8.19 kcal/mol (Table 3). In keeping with these results, the same

compound trio exhibits an utterly comparable interactions profile against the RdRp of BVDV, as

confirmed by their interaction spectra (Figure 4A) and their binding pose (Figure 4B).

It is worth noting how the interactions involving the second hydrophobic cavity (HC2) are not

negligible, as they afford a stabilization to compound binding comparable to those originating from

HC1. Further, taking the quinoxaline series as a meaningful example, the corresponding interaction

spectra in Figure 5A well illustrate the importance of the presence of a bulky, aromatic substituent

able to fulfill the HC2 HI and π network. As indeed illustrated in Figure 5A, both the phenyl and

benzyl derivatives 15a and 19 respectively show a similar interactions spectra compared to the best

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compound 21; this, in turn, reflects in a good estimated affinity against the target RdRp (Table 3).

Notably, the presence of a further polar interaction in the thienyl derivative 21 (HB3) is not a

pivotal requirement for BVDV RdRp binding, but it can be eventually exploited to optimize it. On

the other hand, the isopropyl group in compound 20 is too small to aptly fit the second hydrophobic

cavity: this is reflected in a plummet of both its RdRp binding affinity (Table 3) and (even more so)

in the relevant interactions with each residue involved in the binding site (Figure 5A). Furthermore,

since the small aliphatic substituent cannot establish the required network of hydrophobic and π

interactions with the side chains of the residues belonging to the HC2, the compound assumes an

ineffective pose, which, in turn, exerts a negative influence also on the remaining portion of the

BVDV RdRp binding site (Figure 5A-B).

2.4 Computer Aided Drug Design

Based on the computational results reported in Section 2.3, we designed 4 new potential BVDV

RdRp inhibitors. Focusing on the position 3 of the molecular scaffold, we propose the replacement

of the thienyl ring of lead compound 21 with other (hetero)cyclic entities theoretically able to fulfill

the determined network of intermolecular interactions. For this purpose, the following moieties

were selected: 2-Furanyl (23), 2-Naphtyl (24), and 2-Benzofuranyl (25). Compound 22, featuring a

cyclohexyl as the substituent, was also included in the design as a sort of negative control. In fact,

based on the results obtained for 20, we surmised that the presence of a flexible moiety with no

heteroatoms available for hydrogen bonding with the enzyme and with a non-aromatic character

should ultimately result in a drop of the corresponding enzyme/drug affinity. Applying the same

computational protocol, we docked compounds 22-25 in the enzyme binding site and calculated the

corresponding enzyme affinity (∆G_b_i_n_d). As seen from Table 5, the new derivatives 23-25 are

endowed with excellent BVDV affinities (-8.43, -8.14, and -8.72 kcal/mol, respectively), whereas

compound 22 shows a remarkable lower ∆Gbind value (-6.36 kcal/mol), as expected.

From the deconvolution of the enthalpic component of the binding free energy (∆Hbind, Figure 6A),

the higher affinity of the furanyl derivatives 23 compared to 21 is justified by a shorter and stronger

hydrogen bond detected between the oxygen atom and the hydroxyl group of S411 (ADL = 2.08 ±

0.04 Å). while all other interactions within the protein binding site remain unperturbed.

Contextually, in the case of compound 24, the lack of HB3 pharmacophoric requirement is

compensated by an improvement of the hydrophobic and p interaction network, ultimately resulting

in the preservation of the drug affinity towards BVDV RdRp (Figure 6A). Compound 25 represents

the best structural combination in that the oxygen atom of the furanyl group is engaged in a strong

H-bond with S411 (ADL = 2.06 ± 0.03 Å) whereas the aromatic portion of the moiety is nicely

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encased in the HC2, thereby increasing the strength of the specific hydrophobic interactions (Figure

6A). Figure 6B shows a comparison of the binding mode of compounds 21 and 25 within the

BVDV RdRP binding pocket.

3. Conclusions

In this work, starting from a set of lead compounds 1-4, different series of molecules based on the

-chloro-pyrido[2,3-g]quinoxalin-2(1H)-one nuclei have been synthesized, tested as antiviral agents

5

on a series of RNA- and DNA-viruses. Overall, the results suggest that this tricyclic nuclei appear

to be interesting scaffolds in the drug discovery and development processes of new antiviral agents

actives against important animal and human pathogens such as BVDV and HCV.

Specifically, biological evaluation of N-methylated (14a, 16-18) and O-methylated (15a, 19-21)

derivatives demonstrated that these compounds are endowed with good anti-BVDV activity only

when they bear a phenyl or a thienyl group at position 3. Molecular modeling studies confirmed that

some modifications in the structure of the synthesized quinoxalinones 1-4 are beneficial in

decreasing the lead compound toxicity profiles without affecting their affinity against their target

polymerase. Additionally, and maybe more important from drug development point of view, the

computational approach allowed to identify important molecular determinants for the BVDV RdRp

binding that could be exploited to further optimize the structure of new derivatives provided with a

more potent antiviral activity. Accordingly, further developments could interest position 3 either

through the introduction of different electron donor or electron withdrawing substituents on the

phenyl moiety or its replacement with other heterocycles able to fulfill the determined network of

intermolecular interactions. In this perspective, we designed and tested in silico 4 new derivatives

(22-25) featuring different (hetero)cyclic substituents at position 3 of the general molecular

scaffold. The results confirmed that compounds 23-25, which can preserve or ameliorate the

network of protein/drug intermolecular interactions, are endowed with a high affinity towards the

viral enzyme. On the other hand, the free energy of binding for compound 22 plummets in the

presence of a flexible, cycloaliphatic group as a result of a global detriment of the corresponding

interaction network. The synthesis and the subsequent evaluation of the antiviral activity of

compounds 23-25 are in progress in order to develop and characterize a new series of a more potent

BVDV RdRp inhibitors. More, compound 21 is currently undergoing assays to test its druggability

profile.

Acknowledgements

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The authors thank the “Assessorato della Programmazione, Bilancio, Credito e Assetto del

territorio, della Regione Autonoma della Sardegna (Italia)”, LEGGE

ﬁnancial support.

REGIONALE 7 AGOSTO 2007, for

4

. Experimental

.1. Chemistry

.1.1. General remarks

Melting points were carried out with a Köfler hot stage or Digital Electrothermal melting point

apparatus and are uncorrected. Infrared spectra were recorded as nujol mulls with a Perkin-Elmer

-1

1

7

81 IR spectrophotometer and are expressed in  (cm ). Nuclear magnetic resonance ( H-NMR)

spectra were determined in CDCl , DMSO-d , CDCl /DMSO-d (1:3 ratio) and were recorded with a

3

6

3

6

Bruker Avance III 400 NanoBay. Chemical shifts (



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

2

D O.

1

3

6

C NMR were determined in DMSO-d and were recorded at 100 MHz with Bruker Avance III 400

NanoBay. Ms spectra were performed on combined Liquid Chromatograph-Agilent 1100 series

Mass Selective Detector (MSD). Analytical thin-layer chromatography (TLC) was performed on

Merck silica gel F-254 plates. Pure compounds showed a single spot in TLC. For flash

chromatography, Merck silica gel 60 was used with a particle size 0.040-0.063 mm (230-400 mesh

ASTM). Elemental analysis were performed on a Perkin-Elmer 2400 instrument at Laboratorio di

Microanalisi, Dipartimento di Chimica e Farmacia, Università degli studi di Sassari, Italy, and the

results were within ±0.4% of theoretical values.

4.1.2. Starting material and known intermediates

The intermediates 8-Chloro-N-cyclohexylquinoline-6,7-diamine (7b), 8-chloroquinoline-6,7-

diamine (4a) and 7,8-dichloro-6-nitroquinoline (5) were prepared according to procedure previously

reported by us [13, 20, 22]. 2-Oxo-2-(thiophen-2-yl)acetic acid (4b), propan-1-amine (6a),

cyclohexanamine (6b), cyclohexylmethanamine (6c), were commercially available.

ACCEPTED MANUSCRIPT

4.1.3. 5-chloro-3-(thiophen-2-yl)pyrido[2,3-g]quinoxalin-2(1H)-one (4)

The intermediate, 6,7-diamino-8-chloroquinoline (4a) (2.06 mmol) of scheme 1, was dissolved in 8

mL of H SO 10% followed by the addition of 2.06 mmol of 2-oxo-2-(thiophen-2-yl)acetic acid

2

4

(4b). The mixture was heated at 60°C under stirring for 1h. After cooling, the orange crude

precipitate was filtered off and purified by flash chromatography over silica gel eluting with a 9:1

mixture of chloroform/methanol (43% yield); m.p. >300°C (from EtOH); IR (nujol):  3300, 1672

-1 1

cm ; H NMR (CDCl

3 6

+ DMSO-d ):  12.90 (1H, br s, NH), 9.00 (1H, d, J = 4.4 Hz, H-7), 8.58

(1H, d, J = 4.8 Hz, H-5’), 8.43 (1H, d, J = 8.4 Hz, H-9), 7.82 (1H, d, J = 3.6 Hz, H-3’), 7.73 (1H, s,

13

H-10), 7.62 (1H, dd, J = 8.4 and 4.4 Hz, H-8), 7.26 (1H, dd, J = 4.8 Hz and 3.6 Hz, H-4’). C-NMR

DMSO-d ): δ 121.2 (CH), 122.3 (CH), 123.3 (C),124.3 (CH), 125.3 (C), 126.4 (CH), 126.2 (CH),

27.3 (C),132.2 (CH), 133.8 (C),135.2 (C), 139.8 (C),144.8 (CH), 154.2 (C), 156.3 (C). LC/MS: m/z

16 [M + 1]. Anal. Calcd. for C15 ClN OS: C, 57.42; H, 2.57; N, 13.39; found C, 57.28; H, 2.49;

(

6

1

3

H

8

3

N, 13.28.

4.1.4. General procedure for the preparation of the intermediates 7a,c.

To a solution of 7,8-dichloro-6-nitroquinoline (5) (1 g, 4.1 mmol) in DMF (10 mL) an excess of the

required substituted amines 6a,c (16 mmol) was added dropwise with stirring. The solution was then

stirred for further 8 h at 100°C. After cooling, water was added (10 mL) and the solution was

3

extracted with CHCl . The organic extracts were dried on sodium sulfate, filtered, and the

chloroform solution concentrated under reduced pressure. The oil residues were purified by flash

chromatography eluting with a 8:2 mixture of petroleum ether/ethyl acetate.

4.1.4. 1. 8-Chloro-6-nitro-N-propylquinolin-7-amine (7a) This compound was obtained in 25% yield

by the protocol above described; m.p. 91-93 °C (from EtOH); IR (nujol):  3388, 1612, 1525, 1345,

-1 1

cm ; H NMR (CDCl

3

):  9.05 (1H, dd, J = 4.2 and 1.6 Hz, H-2), 8.37 (1H, s, H-5), 8.14 (1H, dd, J

=

8.2 and 1.6 Hz, H-4), 7.35 (1H, dd, J = 8.2 and 4.2 Hz, H-3), 5.94 (1H, br s, NH), 3.36 (2H, m,

1

3

NH-CH

2

-CH

): δ 11.0 (CH

CH), 137.8 (C), 139.5 (C), 142.5 (C), 154.2 (CH). LC/MS: m/z 268 [M + 1]. Anal. Calcd. for

: C, 54.25; H, 4.55; N, 15.82; found C, 54.05; H, 4.68; N, 15.69.

2

-CH

3

), 1.78-1.60 (2H, m, NH-CH

2 2 3 3

-CH -CH ), 0.99 (3H, t, J = 7.4 Hz, CH ). C-NMR

(CDCl

3

), 22.2 (CH ), 46.0 (CH ), 114.8 (C), 121.5 (C), 122.5 (CH), 124.0 (CH), 137.6

2

(

12 3 2

C H12ClN O

ACCEPTED MANUSCRIPT

4.1.4. 2. 8-Chloro-N-(cyclohexylmethyl)-6-nitroquinolin-7-amine (7c)

This compound was obtained in 58% yield by the protocol above described; m.p. 87-89 °C (from

-1 1

EtOH); IR (nujol):  3404, 1613, 1525, 1345, cm ; H NMR (CDCl

3

):  9.04 (1H, dd, J = 4.2 and

1.6 Hz, H-2), 8.34 (1H, s, H-5), 8.13 (1H, dd, J = 8.2 and 1.6 Hz, H-4), 7.34 (1H, dd, J = 8.2 and 4.2

1

3

Hz, H-3), 5.97 (1H, br s, NH), 3.20 (2H, t, CH

δ 24.8 (2 x CH ), 26.2 (CH ), 30.8 (2 x CH ), 37.7 (CH), 54.0 (CH

CH), 123.1 (CH), 136.9 (CH), 137.0 (C), 138.7 (C), 142.3 (C), 153.6 (CH). LC/MS: m/z 322 [M +

2

), 1.76-0.9 (11H, m, cyclohexyl). C-NMR (CDCl

3

):

2

), 113.8 (C), 120.8 (C), 121.70

(

3 2

1]. Anal. Calcd. for C16H18ClN O : C, 60.09; H, 5.67; N, 13.14; found C, 60.32; H, 5.38; N, 13.40.

4.1.5. General procedure for the preparation of the diamines 8a,c.

The 8-chloro-N-(substituted)-6-nitroquinolin-7-amines 7a,c (2.07 mmol) were dissolved in 70 mL of

ethanol followed by the addition of 10% Palladium on activated charcoal. This mixture was

hydrogenated in Parr at 20-25°C at 3 atm. After filtering and washing the catalyst thoroughly with

ethanol, the filtrates were concentrated in vacuo to give the desired intermediates 8a,c.

7

4

.1.5.1. 8-Chloro-N -propylquinoline-6,7-diamine (8a)

This compound was obtained, as an oil, in 90% yield by the protocol described in the general

-1 1

procedure starting from 7a (550 mg, 2.07 mmol); IR (nujol):  3450, 3330, 3240 cm ; H NMR

CDCl ):  8.73 (1H, dd, J = 4.2 and 1.6 Hz, H-2), 7.86 (1H, dd, J = 8.2 and 1.6 Hz, H-4), 7.25 (1H,

dd, J = 8.2 and 4.2 Hz, H-3), 6.87 (1H, s, H-5), 4.25 (3H, br s, NH), 3.13 (2H, t, J = 7.2 Hz, N-CH ),

), 23.2

CH ), 46.3 (CH ), 111.8 (C), 117.8 (CH), 118.9 (CH), 122.5 (C), 130.6 (CH), 132.9 (C), 133.6 (C),

(

3

2

13

1

2 3 3 3 3

.73-1.60 (2H, m, CH -CH ), 1.03 (3H, t, J = 7.2 Hz, CH ). C-NMR (CDCl ): δ 11.0 (CH

(

2

1

36.2 (C), 144.0 (CH). LC/MS: m/z 238 [M + 1]. Anal. Calcd. for C12

7.83; found C, 61.01; H, 5.88; N, 17.66.

3

H14ClN : C, 61.15; H, 5.99; N,

1

7

4

.1.5.2. 8-chloro-N -(cyclohexylmethyl)quinoline-6,7-diamine (8c)

This compound was obtained in 42% yield by the protocol described in the general procedure

starting from 7c (662 mg, 2.07 mmol) after flash chromatography, eluting with a 8:2 mixture of

-1 1

petroleum ether/ethyl acetate.M.p. 88-90 °C (from EtOH); IR (nujol):  3425, 3298, 3250 cm ; H

NMR (CDCl ):  8.74 (1H, dd, J = 4.4 and 1.6 Hz, H-2), 7.87 (1H, dd, J = 8.2 and 1.6 Hz, H-4),

3

ACCEPTED MANUSCRIPT

7.24 (1H, dd, J = 8.0 and 4.4 Hz, H-3), 6.86 (1H, s, H-5), 4.24 (3H, br s, NH), 2.94 (2H, d, J = 7.4

13

Hz, CH

2

) 1.95-1.04 (11H, m, cyclohexyl). C-NMR (CDCl

), 111.0 (C), 117.3 (CH), 118.0 (CH), 121.8 (C), 130.5 (CH), 132.8

C), 132.0 (C), 136.2 (C), 144.2 (CH). LC/MS: m/z 292 [M + 1]. Anal. Calcd. for C16 : C,

3 2 2

): δ 24.6 (2 x CH ), 25.8 (CH ), 30.0 (2

2 2

x CH ), 37.0 (CH), 54.7 (CH

(

H20ClN

3

66.31; H, 6.96; N, 14.50; found C, 66.17; H, 7.16; N, 14.22.

4.1.6. General procedure for the preparation of 2,3-substituted 3H-imidazo[4,5-g]quinolines 9a-c,

10b and the 5-chloro-2,4-substituted-pyrido[2,3-g]quinoxalin-3(4H)-ones 11a-c, 12b and 13c.

A solution of the quinolines (8a-c) (1.38 mmol) in ethanol (6 mL) was obtained by heating under

reflux, then an excess of either required acids (2-oxo-2-phenylacetic acid, 2-oxo-2-(thiophen-2-yl)

acetic acid or 2-oxo-3-phenylpropanoic acid) was added. Heating was maintained for different times

as reported below. After cooling, the solution was evaporated and the crude products were purified

by flash chromatography using as eluent a mixture of petroleum ether/ethyl acetate or acetone in

different ratios, as reported below. The imidazo[4,5-g]quinolines 9a, 9b, 9c, and 10b were obtained

associated with the pyrido[2,3-g]quinoxalines 11a, 11b, 13c, and 12b, respectively.

4.1.6.1.

2-Phenyl-3-propyl-3H-imidazo[4,5-g]quinoline

(9a)

and

5-chloro-2-phenyl-4-

propylpyrido[2,3-g]quinoxalin-3(4H)-one (11a)

9a was obtained in 66% yield by the protocol described in the general procedure starting from 8a

(367 mg, 1.38 mmol) and 2-oxo-2-phenylacetic acid (228 mg, 1.52 mmol) for 12h. The reaction

mixture was purified by silica gel column chromatography eluting with a 7:3 mixture of petroleum

ether/ethyl acetate. M.p. 128-130 °C (from EtOH); TLC (petroleum ether/ethyl acetate 7:3): R 0.10;

f

-

1 1

IR (nujol):  1652 cm ; H NMR (CDCl

8.1 and 1.6 Hz, H-8), 8.24 (1H, s, H-4), 8.13 (1H, s, H-9), 7.82-7.77 (2H, m, H-2’, 6’), 7.59-7.55

3H, m, H-3’, 4’, 5’), 7.33 (1H, dd, J = 8.1 and 3.8 Hz, H-7), 4.32 (2H, t, J = 7.6 Hz, N-CH ), 2.01-

.90 (2H, m, CH -CH ), 0.91 (3H, t, J = 7.4 Hz, CH ). C-NMR (CDCl ): δ 11.4 (CH ), 22.3 (CH ),

3

):  8.91 (1H, dd, J = 3.8 and 1.6 Hz, H-6), 8.36 (1H, dd, J

=

(

2

13

1

5

2

3

2

1.7 (CH ), 109.7 (CH), 116.9 (CH), 118.4 (CH), 124.3 (C), 128.7 (2 x CH), 129.6 (2 x CH), 130.3

(CH), 130.9 (C), 136.3(CH), 136.9 (C), 143.6 (C), 144.6 (C), 150.0 (CH), 158.4 (C). LC/MS: m/z

288 [M + 1]. Anal. Calcd. for C H N : C, 79.41; H, 5.96; N, 14.62; found C, 79.19; H, 5.86; N,

19 17 3

14.96.

ACCEPTED MANUSCRIPT

11a was obtained from 8a in 4% yield by the protocol above described. M.p. 100-102 °C (from

-1 1

EtOH); TLC (petroleum ether/ethyl acetate 7:3): R 0.24; IR (nujol):  1742, 1627 cm ; H NMR

f

(

CDCl ):  9.06 (1H, dd, J = 4.4 and 2.0 Hz, H-7), 8.36 (1H, dd, J = 8.2 and 2.0 Hz, H-9), 8.19 (1H,

3

s, H-10), 7.79-7.74 (2H, m, H-2’, 6’), 7.60-7.57 (3H, m, H-3’, 4’, 5’), 7.44 (1H, dd, J = 8.2 Hz and J

=

4.4 Hz, H-8), 4.66 (2H, t, J = 7.4 Hz, N-CH ), 1.93-1.80 (2H, m, CH -CH ), 0.79 (3H, t, J = 7.4

2

3

13

Hz, CH

3

). C-NMR (CDCl

3

): δ 11.2 (CH

3

2 2

), 19.90 (CH ), 44.1 (CH ), 113.0 (C), 109.9 (CH), 123.0

(CH), 128.2 (C), 130.1 (2 x CH), 129.7 (2 x CH), 129.4 (C), 130.8 (CH), 134.8 (CH), 138.2 (C),

139.0 (C), 143.0 (C), 151.8 (CH), 155.4 (C), 156.0 (C). LC/MS: m/z 352 [M + 1]. Anal. Calcd. for

20 3

C H16ClN

O: C, 68.67; H, 4.61; N, 12.01; found C, 68.39; H, 4.49; N, 11.79.

4.1.6.2. 3-Cyclohexyl-2-phenyl-3H-imidazo[4,5-g]quinoline (9b) and 5-chloro-4-cyclohexyl-2-

phenylpyrido[2,3-g]quinoxalin-3(4H)-one (11b)

9

b was obtained in 57% yield by the protocol described in the general procedure starting from 8b

380 mg, 1.38 mmol) and 2-oxo-2-phenylacetic acid (228 mg, 1.52 mmol) for 12h. The reaction

mixture was purified by silica gel column chromatography eluting with a 7:3 mixture of petroleum

ether/ethyl acetate;m.p. 183-185 °C (from EtOH); TLC (petroleum ether/ethyl acetate 7:3): R 0.11;

):  8.90 (1H, dd, J = 4.2 and 1.6 Hz, H-6),8.45 (1H, s, H-

), 8.36 (1H, dd, J = 8.8 and 1.6 Hz, H-8), 8.26 (1H, s, H-4), 7.74-7.70 (2H, m, H-2’, 6’), 7.60-7.56

3H, m, H-3’, 4’, 5’), 7.36 (1H, dd, J = 8.8 and 4.2 Hz, H-7), 4.42 (1H, m, cyclohexyl), 2.60-1.25

(

f

-1 1

IR (nujol):  1654 cm ; H NMR (CDCl

3

9

(

13

10H, m, cyclohexyl). C-NMR (CDCl

10.2 (CH), 116.3 (CH), 118.8 (CH), 124.50 (C), 128.8 (2 x CH), 129.4 (2 x CH), 130.3 (CH), 130.4

C), 136.7 (CH), 136.9 (C), 143.7 (C), 144.1 (C), 149.3 (CH), 158.8 (C). LC/MS: m/z 328 [M + 1].

Anal. Calcd. for C22 : C, 80.70; H, 6.46; N, 12.83; found C, 80.50; H, 6.30; N, 12.67.

3 2 2 2

): δ 25.1 (CH ), 25.9 (2 x CH ), 30.8 (2 x CH ), 57.4 (CH),

1

(

21 3

H N

11b was obtained from 8b in 8% yield by the protocol above described. M.p. 169-170 °C (from

-1 1

EtOH); TLC (petroleum ether/ethyl acetate 7:3): R 0.21; IR (nujol):  1716, 1633 cm ; H NMR

f

(CDCl

3

):  9.07 (1H, dd, J = 4.4 and 1.6 Hz, H-7), 8.35 (1H, dd, J = 8.2 and 1.6 Hz, H-9), 8.17 (1H,

s, H-10), 7.79-7.60 (2H, m, H-2’, 6’), 7.59-7.45 (3H, m, H-3’, 4’, 5’), 7.42 (1H, dd, J = 8.2 and 4.4

13

Hz, H-8), 4.23 (1H, m, cyclohexyl), 2.20-1.10 (10H, m, cyclohexyl). C-NMR (CDCl

3

): δ 24.9

(

CH ), 26.4 (2 x CH ), 33.5 (2 x CH ), 58.7 (CH), 109.4 (CH), 123.6 (CH), 127.4 (C), 128.5 (2 x

2

CH), 129.4 (2 x CH), 129.8 (C), 130.3 (CH), 130.8 (C), 135.7 (C), 136.8 (CH), 137.4 (C), 141.4 (C),

1

49.8 (CH), 153.0 (C), 160.8 (C). LC/MS: m/z 392 [M + 1]. Anal. Calcd. for C23

0.85; H, 5.17; N, 10.78; found C, 70.62; H, 5.02; N, 10.56.

3

H20ClN O: C,

7

ACCEPTED MANUSCRIPT

4.1.6.3. 2-Benzyl-3-(cyclohexylmethyl)-3H-imidazo[4,5-g]quinoline (9c) and 2-benzyl-5-chloro-4-

cyclohexylpyrido[2,3-g]quinoxalin-3(4H)-one (13c)

9c was obtained in 9 % yield by the protocol described in the general procedure starting from 8c

(399 mg, 1.38 mmol) and 2-oxo-3-phenylpropanoic acid (249 mg, 1.52 mmol) for 6h. The reaction

mixture was purified by silica gel column chromatography eluting with a 8.5:1.5 mixture of

petroleum ether/aceton; m.p. 67-69 °C (from EtOH); TLC (petroleum ether/aceton 8.5:1.5): R 0.16;

f

-1 1

IR (nujol):  3451, 2360, 1632, 1600 cm ; H NMR (CDCl

3

):  8.87 (1H, dd, J = 4.2 and 1.8 Hz, H-

), 8.33 (1H, dd, J = 8.4 and 1.8 Hz, H-8), 8.17 (1H, s, H-9), 8.00 (1H, s, H-4), 7.37-7.20 (6H, m, H-

and H-Ph), 4.41 (2H, s, CH -Ph), 3.94 (2H, d, J = 7.6 Hz, N-CH -cyclohexyl), 2.00-0.85 (11H, m,

6

7

2

1

3

cyclohexyl). C-NMR (CDCl ): δ 25.6 (2 x CH ), 26.0 (CH ), 31.1 (2 x CH ), 38.0 (CH), 31.1

3

2

2 2

(CH ), 50.6 (CH ), 107.2 (CH), 115.8 (CH), 118.6 (CH), 127.2 (CH), 128.5 (2 x CH), 128.9 (2 x

CH), 124.6 (C), 135.6 (C), 136.81 (CH), 139.1 (C), 142. 7 (C), 144.5 (C), 149.2 (CH), 158.7 (C).

LC/MS: m/z 356 [M + 1]. Anal. Calcd. for C24

H, 7.06; N, 11.64.

25 3

H N : C, 81.09; H, 7.09; N, 11.82; found C, 79.90;

13c was obtained from 8c in 33 % yield by the protocol above described. M.p. 53-55 °C (from

-1

1

EtOH); TLC (petroleum ether/aceton 8.5:1.5): R

CDCl ):  9.05 (1H, dd, J = 4.4 and 1.6 Hz, H-7), 8.38 (1H, dd, J = 8.2 and 1.6 Hz, H-9), 8.19 (1H,

s, H-10), 7.37-7.20 (6H, m, H-8 and H-Ph), 4.41 (2H, s, N-CH ), 3.94 (2H, s, CH -Ph), 2.00-0.85

), 26.1 (2 x CH ), 33.80 (2 x CH ), 52.6

CH ), 57.5 (CH ), 58.8 (CH), 109.9 (CH), 123.1 (CH), 126.8 (C), 128.0 (2 x CH), 129.7 (2 x CH),

f

0.25; IR (nujol):  1740, 1604 cm ; H NMR

(

3

2

13

(

11H, m, cyclohexyl). C-NMR (CDCl

3

): δ 24.9 (CH

2

(

2

129.3 (C), 130.4 (C), 130.9 (CH), 136.1 (C), 136.3 (CH), 137.7 (C), 141.0 (C), 150.4 (CH), 153.6

(C), 155.2 (C). LC/MS: m/z 420 [M + 1]. Anal. Calcd. for C25

3

H24ClN O: C, 71.85; H, 5.79; N,

10.05; found C, 71.59; H, 5.97; N, 10.29.

4.1.6.4. 3-Cyclohexyl-2-(thiophen-2-yl)-3H-imidazo[4,5-g]quinoline (10b) and 5-chloro-4-

cyclohexyl-2-(thiophen-2-yl)pyrido[2,3-g]quinoxalin-3(4H)-one (12b)

10b was obtained in 8% yield by the protocol described in the general procedure starting from 8b

(380 mg, 1.38 mmol) and 2-oxo-2-(thiophen-2-yl)acetic acid (237 mg, 1.52 mmol) for 12h. The

reaction mixture was purified by silica gel column chromatography eluting with a 6:4 mixture of

petroleum ether/ethyl acetate; m.p. 152-154 °C (from EtOH); TLC (petroleum ether/ethyl acetate

ACCEPTED MANUSCRIPT

-

1 1

7

:3): R

H-6), 8.39 (1H, s, H-4), 8.32 (1H, dd, J = 8.0 and 1.6 Hz, H-8), 8.22 (1H, s, H-9), 7.63 (1H, d, J =

.2 Hz, H-5’), 7.48 (1H, d, J = 3.8 Hz, H-3’), 7.32 (1H, dd, J = 8.0 and 4.0 Hz, H-7), 7.25 (1H, dd, J

5.2 and 3.8 Hz, H-4’), 4.79 (1H, m, cyclohexyl), 2.72-2.50 (2H, m, cyclohexyl), 2.06-1.42 (8H, m,

f

0.15; IR (nujol):  2360, 1671 cm ; H NMR (CDCl

3

):  8.89 (1H, dd, J = 4.0 and 1.6 Hz,

5

=

1

3

cyclohexyl). C-NMR (CDCl

3

): δ 25.6 (CH

CH), 121.3 (CH), 126.70 (C), 127.1 (CH), 128.5 (CH), 128.70 (C), 128.6 (CH), 129.2 (CH), 129.10

C), 136.0 (CH), 138.1 (C), 143.7 (C), 149.8 (CH), 158.8 (C). LC/MS: m/z 334 [M + 1]. Anal.

S: C, 72.04; H, 5.74; N, 12.60; found C, 71.88; H, 5.52; N, 12.85.

2 2 2

), 26.0 (2 x CH ), 32.0 (2 x CH ), 57.9 (CH), 105.0

(

19 3

Calcd. for C20H N

12b was obtained from 8b in 30 % yield by the protocol above described. M.p. 215-217 °C (from

-1 1

EtOH); TLC (petroleum ether/ethyl acetate 7:3): R

NMR (CDCl ):  9.07 (1H, dd, J = 4.4 and 1.6 Hz, H-7), 8.34 (1H, dd, J = 8.2 and 1.6 Hz, H-9),

.16 (1H, s, H-10), 7.63 (1H, dd, J = 5.0 and 1.0 Hz, H-5’), 7.57 (1H, dd, J = 4.0 and 1.0 Hz, H-3’),

.43 (1H, dd, J = 8.2 and 4.4 Hz, H-8), 7.24 (1H, dd, J = 5.0 and 4.0 Hz, H-4’), 4.28 (1H, m,

f

0.26; IR (nujol):  2368, 1744, 1679 cm ; H

3

8

7

13

3 2 2

cyclohexyl), 2.72-1.32 (10H, m, cyclohexyl). C-NMR (CDCl ): δ 24.9 (2 x CH ), 25.6 (CH ), 31.1

(

2 x CH ), 60.9 (CH), 113.0 (C), 109.2 (CH), 120.3 (CH), 123.8 (C), 124.1 (C), 125.3 (CH), 127.1

2

(CH), 127.3 (CH), 134.9 (CH),137.6 (C), 138.3 (C),139.4 (C),149.3 (CH), 152.4 (C), 159.0 (C).

3

LC/MS: m/z 398 [M + 1]. Anal. Calcd. for C21H18ClN OS: C, 63.71; H, 4.58; N, 10.61; found C,

63.98; H, 4.26; N, 10.85.

4.1.6.5. 5-chloro-4-(cyclohexylmethyl)-2-phenylpyrido[2,3-g]quinoxalin-3(4H)-one (11c)

This compound was obtained in 68 % yield by the protocol described in the general procedure

starting from 8c (399 mg, 1.38 mmol) and 2-oxo-2-phenylacetic acid (228 mg, 1.52 mmol) for 2h

and 45’; the compound was purified by silica gel column chromatography eluting with a 6:4 mixture

-1

of petroleum ether/ethyl acetate; m.p. 129-131 °C (from EtOH); IR (nujol):  1732, 1627, 1604 cm ;

1

H NMR (CDCl

.19 (1H, s, H-10), 7.80-7.72 (2H, m, H-2’, 6’), 7.62-7.50 (3H, m, H-3’, 4’, 5’), 7.41 (1H, dd, J = 8.2

and 4.4 Hz, H-8), 4.59 (2H, d, J = 7.2 Hz, CH ), 4.12 (1H, m, cyclohexyl), 1.95-0.85 (10H, m,

), 25.9 (CH ), 30.9 (2 x CH ), 45.1 (CH), 48.3

3

):  9.05 (1H, dd, J = 4.4 and 1.6 Hz, H-7), 8.38 (1H, dd, J = 8.2 and 1.6 Hz, H-9),

8

2

1

3

cyclohexyl). C-NMR (CDCl

3

): δ 25.3 (2 x CH

2

(

CH ), 115.9 (CH), 119.0 (CH), 126.1 (C), 128.2 (2 x CH), 129.9 (2 x CH), 130.4 (C), 131.1 (CH),

2

130.2 (C), 136.3 (C), 136.8 (CH), 138.4 (C), 142.1 (C), 148.9 (CH), 154.2 (C), 159.5 (C). LC/MS:

m/z 406 [M + 1]. Anal. Calcd. for C24

.68; N, 10.14.

3

H22ClN O: C, 71.37; H, 5.49; N, 10.40; found C, 71.06; H,

5

ACCEPTED MANUSCRIPT

4.1.7. General procedure for the preparation of 5-chloro-1-substituted-3-phenylpyrido[2,3-

g]quinoxalin-2(1H)-ones 14a-e and the 5-chloro-2-substituted-3-phenylpyrido[2,3-g]quinoxalines

5 a,b,e.

1

To a mixture of the 5-chloro-3-phenylpyrido[2,3-g]quinoxalin-2(1H)-one (1) [13] (492 mg, 1.60

mmol) and Cs CO (521 mg, 1.60 mmol)) in anhydrous N,N-dimethylformammide (DMF) (10 mL)

2

3

an excess of the required alkylant agents (dimethyl sulfate, diethyl sulfate, bromomethylbenzene, 1-

chloromethyl-4-nitrobenzene) (1.60-7.07 mmol) in N,N-dimethylformammide (5-10 mL) was added

dropwise. The mixture was stirred at 60°C for 1-16h, then allowed to cool to room temperature and

water (50 mL) was added. The resulting precipitates were collected by filtration, washed with water

and the crude products were purified by flash chromatography using a mixture of ethyl

ether/methanol as eluent in different ratios as reported below. The 1-substituted-pyrido[2,3-

g]quinoxalin-2(1H)-ones 14a,b,ewere obtained associated with the 2-substituted-pyrido[2,3-

g]quinoxalines 15a,b,e respectively.

4.1.7.1. 5-Chloro-1-methyl-3-phenylpyrido[2,3-g]quinoxalin-2(1H)-one (14a) and 5-chloro-2-

methoxy-3-phenylpyrido[2,3-g]quinoxaline (15a)

14a was obtained in 77 % yield by the protocol described in the general procedure starting from 1

(

492 mg, 1.60 mmol), Cs CO (521 mg, 1.60 mmol) and dimethyl sulfate (393 mg, 3.12 mmol) in

2

3

DMF (10 mL) for 16h; the mixture was purified by silica gel column chromatography eluting with a

9.5:0.5 mixture of ethyl ether/methanol; m.p. 256-258 °C (from EtOH); TLC (petroleum

-1 1

ether/aceton 8.5:1.5): R

f

0.65; IR (nujol):  1651, 1624 cm ; H NMR (CDCl ):  9.00 (1H, d, J =

3

4

7

1

.4 Hz, H-7), 8.52 (1H, d, J = 8.6 Hz, H-9), 8.17 (1H, d, J = 8.6 Hz, H-8), 7.60-7.45 (5H, m, H-Ph),

1

3

3 3 3

.42 (1H, s, H-10), 3.78 (3H, s, CH ). C-NMR (CDCl ): δ 35.3 (CH ), 109.1 (CH), 122.9 (CH),

28.1 (2 x CH), 128.7 (C), 129.2 (2 x CH), 130.8 (C), 131.5 (CH), 133.4 (C), 135.8 (C), 136.9 (CH),

37.9 (C), 139.2 (C), 150.0 (CH), 152.8 (C), 153.7 (C). LC/MS: m/z 324 [M + 1]. Anal. Calcd. for

C

18

H

12ClN

3

O: C, 67.19; H, 3.76; N, 13.06; found C, 67.03; H, 3.45; N, 12.96.

15a was obtained from 1in 19 % yield by the protocol above described. M.p. 148-150 °C (from

-1

1

EtOH); TLC (petroleum ether/aceton 8.5:1.5): R 0.46; IR (nujol):  1732, 1665 cm ; H NMR

f

1

(DMSO-d

6

):  H NMR (DMSO-d

6

):  9.13 (1H, d, J = 4.2 Hz, H-7), 8.35 (1H, d, J = 8.2 Hz, H-9),

13

8

3

.26 (1H, s, H-10), 8.10 (2H, m, H-2’, 6’), 7.53 (4H, m, H-8, H-3’, 4’, 5’), 4.23 (3H, s, CH ). C-

ACCEPTED MANUSCRIPT

NMR (DMSO-d

6

3

): δ 54.3 (CH ), 120.5 (CH), 123.9 (CH), 126.1 (C), 127.0 (2 x CH), 128.3 (CH),

1

6

29.0 (2 x CH), 130.1 (CH), 130.8 (CH), 131.2 (C), 131.9 (C), 134.8 (C), 136.0 (CH), 140.1 (C),

50.1 (CH), 151.9 (C), 154.0 (C). LC/MS: m/z 324 [M + 1]. Anal. Calcd. for C18

7.19; H, 3.76; N, 13.06; found C, 67.06; H, 3.54; N, 12.84.

3

H12ClN O: C,

4

.1.7.2. 5-chloro-1-ethyl-3-phenylpyrido[2,3-g]quinoxalin-2(1H)-one (14b) and 5-chloro-2-ethoxy-

-phenylpyrido[2,3-g]quinoxaline (15b)

3

14b was obtained in 71 % yield by the protocol described in the general procedure starting from 1

(492 mg, 1.60 mmol), Cs

2 3

CO (521 mg, 1.60 mmol) and diethyl sulfate(284 mg, 1.84 mmol) in

DMF (10 mL) for 4h; the mixture was purified by silica gel column chromatography eluting with a

9:1 mixture of ethyl ether/methanol; m.p. 252-253 °C (from EtOH); TLC (petroleum ether/aceton

-1

1

8.5:1.5): R

f

0.70; IR (nujol):  1664, 1597 cm ; H NMR (CDCl

3

):  H NMR (DMSO-d

6

):  9.00

(1H, s, H-7), 8.51 (1H, d, J = 7.6 Hz, H-9), 8.39 (2H, m, H-2’, 6’), 8.09 (1H, s, H-10), 7.60 (4H, m,

13

H-8, H-3’, 4’, 5’), 4.38 (2H, q, J = 6 Hz, CH ), 1.48 (3H, s, t, J = 7.2 Hz, CH ). C-NMR (DMSO-

2

3

d

6

): δ 11.9 (CH

30.9 (C), 131.0 (CH), 131.2 (C), 132.6 (C), 135.5 (C), 136.2 (CH), 139.9 (C), 151.0 (CH), 153.1

O: C, 67.96; H, 4.20; N,

3 2

), 37.6 (CH ), 109.3 (CH), 123.1 (CH), 127.9 (2 x CH), 128.2 (C), 129.0 (2 x CH),

1

(C), 154.8 (C). LC/MS: m/z 338 [M + 1]. Anal. Calcd. for C19

H

14ClN

3

12.51; found C, 68.31; H, 4.08; N, 12.88.

15b was obtained from 1 in 23 % yield by the protocol above described. M.p. 160-162 °C (from

-1

1

EtOH); TLC (petroleum ether/aceton 8.5:1.5): R 0.38; IR (nujol):  1733, 1669 cm ; H NMR

f

(

DMSO-d

6

):  9.04 (1H, d, J = 4.2 Hz, H-7), 8.51 (1H, d, J = 8.4 Hz, H-9), 8.27 (1H, s, H-10), 8.17

), 1.33 (3H, t, J = 6.8

), 122.2 (CH), 122.7 (CH), 127.8 (C),

(2H, m, H-2’, 6’), 7.48 (4H, m, H-8, H-3’, 4’, 5’), 4.55 (2H, q, J = 6.8 Hz, CH

2

1

3

Hz, CH

3

). C-NMR (DMSO-d

6

): δ 13.9 (CH

3

), 63.3 (CH

2

1

28.18 (2 x CH), 128.8 (C), 129.9 (2 x CH), 130.5 (CH), 131.0 (CH), 131.2 (C), 132.6 (C), 135.5

C), 136.2 (CH), 139.9 (C), 151.0 (CH), 153.1 (C), 154.8 (C). LC/MS: m/z 338 [M + 1]. Anal.

Calcd. for C19 O: C, 67.96; H, 4.20; N, 12.51; found C, 67.63; H, 4.08; N, 12.22.

(

H

14ClN

3

4.1.7.3. 5-chloro-1-(4-nitrobenzyl)-3-phenylpyrido[2,3-g]quinoxalin-2(1H)-one (14e) and 5-chloro-

2-(4-nitrobenzyloxy)-3-phenylpyrido[2,3-g]quinoxaline (15e)

14e was obtained in 67 % yield by the protocol described in the general procedure starting from 1

2 3

(492 mg, 1.60 mmol), Cs CO (521 mg, 1.60 mmol) and 1-(chloromethyl)-4-nitrobenzene (806 mg,

ACCEPTED MANUSCRIPT

4.70 mmol) in DMF (10 mL) for 4h; the mixture was purified by silica gel column chromatography

eluting with a 9:1 mixture of ethyl ether/methanol; m.p. 268-270 °C (from EtOH); TLC (petroleum

-

1

ether/aceton 8.5:1.5): R

DMSO-d

Ph), 8.22 (2H, d, J = 8.0 Hz, H-3’, H-5’ benzyl), 8.10 (1H, s, H-10),7.69 (2H, d, J = 8.4 Hz, H-2’,

f

0.61; IR (nujol):  1652, 1602 cm ; H NMR (CDCl

3

):  H NMR

(

6

):  9.09 (1H, d, J = 4.4 Hz, H-7), 8.51 (1H, d, J = 7.2 Hz, H-9), 8.61 (2H, m, H-2’, H-6’

13

H-6’ benzyl), 7.60 (4H, m, H-8, H-3’, H-4’, H-5’ Ph), 7.28, 5.75 (2H, s, CH

2

). C-NMR (DMSO-

d

6

): δ 45.5 (CH ), 109.6 (CH), 123.2 (CH), 123.6 (2 x CH), 128.0 (2 x CH), 128.1 (2 x CH), 128.8

2

(

C), 130.0 (2 x CH), 131,1 (C), 131.15 (CH), 131.7 (C), 132.9 (C), 135.9 (C), 136.1 (CH), 140.1

C), 143.4 (C), 146.7 (C), 151,1 (CH), 153.8 (C), 154.9 (C). LC/MS: m/z 445 [M + 1]. Anal. Calcd.

(

for C24

H

15ClN

4

O

3

: C, 65.09; H, 3.41; N, 12.65; found C, 64.91; H, 3.28; N, 12.39.

15e was obtained from 1 in 7 % yield by the protocol above described. M.p. 221-223 °C (from

-1

1

EtOH); TLC (petroleum ether/aceton 8.5:1.5): R

DMSO-d ):  9.19 (1H, d, J = 4.4 Hz, H-7), 8.73 (2H, m, H-2’, H-6’ Ph), 8.45 (2H, d, J = 8.6 Hz,

H-3’, H-5’ benzyl), 8.10 (1H, d, = 8.2 Hz, H-9), 8.02-7.85 (5H, m, H-8, H-10, H-3’, H-4’, H-5’ Ph),

f

0.25; IR (nujol):  1698, 1642 cm ; H NMR

(

6

1

3

7

1

2 6 2

.36 (2H, d, J = 8.6 Hz, H-2’, H-6’ benzyl), 5.91 (2H, s, CH ). C-NMR (DMSO-d ): δ 51.9 (CH ),

21.8 (CH), 124.1 (CH), 124.0 (2 x CH), 126.8 (C), 127.6 (2 x CH), 128.6 (2 x CH), 128.6 (CH),

29.1 (2 x CH), 131.4 (C), 131.8 (C), 135.0 (C), 137.9 (C), 136.4 (CH), 138.6 (C), 142.6 (C), 144.1

4 3

(C), 146.6 (C), 150.3 (CH), 153.3 (C). LC/MS: m/z 445 [M + 1]. Anal. Calcd. for C24H15ClN O : C,

65.09; H, 3.41; N, 12.65; found C, 64.91; H, 3.23; N, 12.88.

4.1.7.4. 2-(5-chloro-2-oxo-3-phenylpyrido[2,3-g]quinoxalin-1(2H)-yl)acetonitrile (14c)

To a solution of the 5-chloro-3-phenylpyrido[2,3-g]quinoxalin-2(1H)-one (1) (492 mg, 1.60 mmol)

in 2-chloroacetonitrile (12.63 mL) was added Cs CO (658 mg, 2.02 mmol). The reaction was

2

3

stirred at 60°C for 1h and then allowed to cool to room temperature. Water (50 mL) was added and

the resulting precipitate was collected by filtration, washed with water and dried in a oven to give a

-1 1

crystalline solid. Yield: 95 %; m.p. 293-295 °C (from EtOH); IR (nujol):  1653, 1602 cm ; H

1

NMR (DMSO-d

6

):  H NMR (DMSO-d

6

):  9.09 (1H, d, J = 4.4 Hz, H-7), 8.55 (1H, d, J = 8.6

Hz, H-9), 8.39-8.26 (2H, m, H-2’, H-6’), 8.24 (1H, s, H-10), 7.75 (1H, dd, J = 8.6 Hz and J = 4.4

13

Hz, H-8), 7.60-7.44 (3H, m, H-3’, 4’, 5’), 4.08 (2H, s, CH

09.2 (CH), 115.2 (C), 123.6 (CH), 128.11 (2 x CH), 128.8 (C), 129.9 (2 x CH), 130.8 (C), 131.3

CH), 133.1 (C), 135.0 (C), 136.14 (CH), 140.4 (C), 151.4 (CH), 153.0 (C), 154.5 (C). LC/MS: m/z

49 [M + 1]. Anal. Calcd. for C19 O: C, 65.81; H, 3.20; N, 16.16; found C, 65.54; H, 3.42;

N, 16.02.

2 6 2

). C-NMR (DMSO-d ): δ 30.8 (CH ),

1

(

3

H11ClN

4

ACCEPTED MANUSCRIPT

4.1.7.5. 1-benzyl-5-chloro-3-phenylpyrido[2,3-g]quinoxalin-2(1H)-one (14d)

This compound was obtained in 30 % yield by the protocol described in the general procedure

starting from 1 (492 mg, 1.60 mmol), Cs CO (521 mg, 1.60 mmol) and (bromomethyl)benzene

2

3

(1209 mg, 0.84 mL, 7.07 mmol) in DMF (10 mL) for 4h; m.p. 228-230 °C (from EtOH); IR (nujol):

-1 1



1663, 1597 cm ; H NMR (DMSO-d ):  9.06 (1H, d, J = 4.4 Hz, H-7), 8.62 (2H, m, H-Ph), 8.12

6

1

3

(

1H, d, J = 8.2 Hz, H-9), 7.50-7.30 (10H, m, H-8, H-10, H-Ph + benzyl), 5.56 (2H, s, CH

2

). C-

NMR (DMSO-d ): δ 45.7 (CH ), 109.8 (CH), 123.1 (CH), 126.9 (2 x CH), 127.3 (CH), 128.0 (2 x

6

2

CH), 128.4 (2 x CH), 128.6 (CH), 129.8 (2 x CH), 131,0 (C), 131.6 (C), 132.8 (C), 135.4 (C), 136.1

C), 139.9 (C), 151,08 (CH), 153.7 (C), 154.9 (C). LC/MS: m/z 400 [M + 1]. Anal. Calcd. for

C H ClN O: C, 72.45; H, 4.05; N, 10.56; found C, 72.15; H, 3.87; N, 10.26.

(

24

16

3

4

.1.8. General procedure for the preparation of 5-chloro-1-methyl-3-phenylpyrido[2,3-

g]quinoxalin-2(1H)-ones (16-18) and the 5-chloro-2-methoxy-3-phenylpyrido[2,3-g]quinoxalines

19-21).

(

To a mixture of the opportune 5-chloro-3-(thiophen-2-yl)pyrido[2,3-g]quinoxalin-2(1H)-one (4),

prepared as above reported, (502 mg, 1.60 mmol), 3-benzyl-5-chloropyrido[2,3-g]quinoxalin-2(1H)-

one (2) [1] (515 mg, 1.60 mmol), and 5-chloro-3-isopropylpyrido[2,3-g]quinoxalin-2(1H)-one (3)

[

13] (438 mg, 1.60 mmol) and one mole equivalent of Cs

2

CO

3

in anhydrous N,N-

dimethylformammide (10 mL) was dropwise added an excess of dimethyl sulfate (393 mg, 3.12

mmol) in N,N-dimethylformammide (10 mL). The mixture was stirred at 60°C for 16h, then allowed

to cool to room temperature and water (50 mL) was added. The resulting precipitates were collected

by filtration, washed with water and the crude products were purified by flash chromatography using

a mixture of ethyl ether/methanol 95:5 as eluent. The 1-methyl-pyrido[2,3-g]quinoxalin-2(1H)-

ones(16-18) were obtained associated with the 2-methoxy-pyrido[2,3-g]quinoxalines(19-21)

respectively.

4

.1.8.1. 3-benzyl-5-chloro-1-methylpyrido[2,3-g]quinoxalin-2(1H)-one (16) and 3-benzyl-5-chloro-

-methoxypyrido[2,3-g]quinoxaline (19)

2

ACCEPTED MANUSCRIPT

16 was obtained in 26 % yield by the protocol described in the general procedure starting from 2

-1 1

[

13]. M.p. 275-277 °C (from EtOH); IR (nujol):  3315, 1652, 1607 cm ; H NMR (DMSO-d ): 

6

9.56 (1H, d, J = 4.6 Hz, H-7), 9.35 (1H, d, J = 8.6 Hz, H-9), 9.01 (1H, s, H-10), 8.61 (2H, m, H-2’,

6

’), 7.89 (1H, dd, J = 8.6 and 4.6 Hz, H-8), 7.71 (3H, m, H-3’, 4’, 5’), 3.84 (2H, s, CH ), 3.60 (3H, s,

2

13

3 6 3 2

CH ). C-NMR (DMSO-d ): δ 37.5 (CH ), 44.8 (CH ), 109.3 (CH), 123.5 (CH), 125.3 (CH), 126.1

(C), 127.8 (C), 128.4 (2 x CH), 128.9 (2 x CH), 131.7 (C), 133.5 (CH), 134.1 (C), 136.1 (C), 140.6

(

C), 145.8 (CH), 154.6 (C), 155.3 (C). LC/MS: m/z 338 [M + 1]. Anal. Calcd. for C H ClN O: C,

1

9

14

3

67.96; H, 4.20; N, 12.51; found C, 67.67; H, 4.37; N, 12.27.

19 was obtained from 2in 23 % yield by the protocol above described. M.p. 158-160 °C (from

-1 1

EtOH); IR (nujol):  3306, 1643 cm ; H NMR (DMSO-d

6

):  9.71 (1H, d, J = 4.6 Hz, H-7), 9.38

(1H, d, J = 8.6 Hz, H-9), 8.95 (1H, s, H-10), 8.61 (2H, m, H-2’, 6’), 7.96 (1H, dd, J = 8.6 and 4.6 Hz,

1

3

H-8), 7.71 (3H, m, H-3’, 4’, 5’), 4.18 (3H, s, CH

3

), 3.82 (2H, s, CH

), 109.6 (CH), 123.7 (CH), 125.6 (CH), 126.8 (C), 128.1 (2 x CH), 128.9 (C), 130.2

2 x CH), 132.4 (C), 135.3 (C), 136.6 (C), 135.8 (CH), 139.5 (C), 147.5 (CH), 150.6 (C), 181.5 (C).

O: C, 67.96; H, 4.20; N, 12.51; found C,

2 6

). C-NMR (DMSO-d ): δ 44.6

2 3

(CH ), 49.8 (CH

(

LC/MS: m/z 338 [M + 1]. Anal. Calcd. for C19H14ClN

3

68.25; H, 4.07; N, 12.81.

4.1.8.2. 5-chloro-3-isopropyl-1-methylpyrido[2,3-g]quinoxalin-2(1H)-one (17) and 5-chloro-3-

isopropyl-2-methoxypyrido[2,3-g]quinoxaline (20)

17 was obtained, in 27 % yield by the protocol described in the general procedure starting from 3

-1 1

[

13]. M.p. 261-263 °C (from EtOH); IR (nujol):  3319, 1632 cm ; H NMR (DMSO-d

6

):  8.71

(1H, d, J = 4.0 Hz, H-7), 8.38 (1H, d, J = 7.2 Hz, H-9), 7.91 (1H, s, H-10), 7.74 (1H, dd, J = 7.2 and

1

3

4

3 3

.0 Hz, H-8), 3.61 (1H, m, CH), 3.48 (3H, s, N-CH ), 1.32 (6H, d, J = 6.4 Hz, 2 x CH ). C-NMR

(

CDCl ): δ 18.2 (2 x CH ), 37.8 (CH ), 39.6 (CH), 109.3 (CH), 123.3 (CH), 125.1 (C), 127.7 (C),

3 3 3

133.2 (CH), 134.3 (C), 135.2 (C), 140.9 (C), 145.3 (CH), 154.6 (C), 155.2 (C). LC/MS: m/z 290 [M

+

1]. Anal. Calcd. for C15 O: C, 62.61; H, 4.90; N, 14.60; found C, 62.38; H, 4.80; N, 14.34.

H14ClN

3

20 was obtained from 3 in 18 % yield by the protocol above described. M.p 153-155 °C (from

-1 1

EtOH); IR (nujol):  3342, 1629 cm ; H NMR (DMSO-d

6

):  8.64 (1H, d, J = 4.0 Hz, H-7), 8.60

(1H, d, J = 7.2 Hz, H-9), 8.04 (1H, s, H-10), 7.82 (1H, dd, J = 7.2 Hz and J = 4.0, H-8), 4.21 (s, 3H,

1

3

OCH

3 3 6

), 3.61 (1H, m, CH), 1.32 (6H, d, J = 6.4 Hz, 2 x CH ). C-NMR (DMSO-d ): δ 19.2 (2 x

CH ), 39.7 (CH), 54.1 (CH ), 123.0 (CH), 124.9 (C), 126.9 (CH), 128.3 (C), 132.6 (CH), 135.2 (C),

3

ACCEPTED MANUSCRIPT

136.1 (C), 142.0 (C), 147.6 (C), 149.3 (CH), 181.8 (C). LC/MS: m/z 290 [M + 1]. Anal. Calcd. for

15 3

C H14ClN O: C, 62.61; H, 4.90; N, 14.60; found C, 62.57; H, 5.12; N, 14.91.

4

.1.8.3. 5-chloro-1-methyl-3-(thiophen-2-yl)pyrido[2,3-g]quinoxalin-2(1H)-one (18) and 5-chloro-

-methoxy-3-(thiophen-2-yl)pyrido[2,3-g]quinoxaline (21)

2

18 was obtained in 30 % yield by the protocol described in the general procedure starting from 4.

-1 1

M.p. 247-249 °C (from EtOH); IR (nujol):  3300, 1652 cm ; H NMR (CDCl

.0 Hz, H-7), 8.50 (1H, d, J = 7.0 Hz, H-5’), 8.47 (1H, d, J = 8.0 Hz, H-9), 8.04 (1H, s, H-10), 7.96

1H, d, J = 4.2 Hz, H-3’), 7.67 (1H, dd, J = 8.0 and 4.0 Hz, H-8), 7.31 (1H, dd, J = 7.0 and 4.2 Hz,

3

):  9.01 (1H, d, J =

4

(

13

3 3 3

H-4’), 3.77 (3H, s, CH ). C-NMR (CDCl ): δ 41.58 (CH ), 109.4 (CH), 122.6 (CH), 124.0 (C),

125.1 (CH), 125.7 (C), 127.2 (CH), 127.5 (CH), 127.9 (C), 132.7 (CH), 134.4 (C), 135.7 (C), 140.2

(

C), 145.6 (CH), 153.8 (C), 157.6 (C). LC/MS: m/z 330 [M + 1]. Anal. Calcd. for C16 OS: C,

H10ClN

3

58.63; H, 3.07; N, 12.82; found C, 58.96; H, 2.91; N, 12.68.

21was obtained from 4 in 13 % yield by the protocol above described. M.p. 148-150 °C (from

-

1 1

EtOH); IR (nujol):  3321, 1663 cm ; H NMR (CDCl

d, J = 7.0 Hz, H-5’), 8.32 (1H, d, J = 8.0 Hz, H-9), 8.07 (1H, s, H-10), 7.61 (1H, d, J = 4.2 Hz, H-3’),

.41 (1H, dd, J = 8.0 and 4.4 Hz, H-8), 7.15 (1H, dd, J = 7.0 and 4.2 Hz, H-4’), 4.23 (3H, s, CH ).

), 109.1 (CH), 124.0 (CH), 125.8 (C), 126.3 (CH), 126.8 (C), 128.3

CH), 128.6 (CH), 129.1 (C), 134.2 (CH), 136.6 (C), 138.2 (C), 139.8 (C), 144.9 (C), 149.4 (CH),

3

):  9.07 (1H, d, J = 4.4 Hz, H-7), 8.34 (1H,

7

3

1

3

3 3

C-NMR (CDCl ): δ 54.26 (CH

(

1

58.2 (C). LC/MS: m/z 330 [M + 1]. Anal. Calcd. for C H ClN OS: C, 58.63; H, 3.07; N, 12.82;

16 10 3

found C, 59.01; H, 2.86; N, 12.94.

4.2. Biology

4.2.1 Test compounds

Compounds were dissolved in DMSO at 100 mM, and then diluted in culture medium.

.2.2. Cells and Viruses

4

Cell lines were purchased from American Type Culture Collection (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: CD4 human T-cells

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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)

Mesocricetusauratus] and Monkey kidney (Vero 76) [ATCCCRL 1587 CercopithecusAethiops].

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 (ssRNA ) were: (i) Retroviridae: the III

B

laboratory strain of HIV-1, obtained from the supernatant of the persistently infected H9/III

B

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 [poliovirus 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

representative 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) Herpesviridae: human

herpesvirus 1 (HSV-1) [strain KOS (ATCC VR- 1493)].

4.2.3. Cytotoxicity Assays

Cytotoxicity assays were run in parallel with antiviral assays. Exponentially growing MT-4 cells

5

were seeded at an initial density of 1x10 cells/mL in 96-well plates in RPMI-1640 medium,

supplemented 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% CO atmosphere, in

2

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- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method

5

6

[

39]. MDBK and BHK cells were seeded in 96-well plates at an initial density of 6x10 and 1x10

cells/mL, respectively, in Minimum Essential Medium with Earle’s salts (MEM-E), L-glutamine, 1

mM sodium pyruvate and 25 mg/L kanamycin, supplemented with 10% horse serum (MDBK) or

1

0% foetal bovine serum (FBS) (BHK). Cell cultures were then incubated at 37 °C in a humidified,

% CO atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability

5

2

was determined after 72 hrs at 37 °C by the MTT method [39]. Vero-76 cells were seeded in 96-

5

well plates at an initial density of 4x10 cells/mL, in Dulbecco’s Modified Eagle Medium (D-

ACCEPTED MANUSCRIPT

MEM) with L-glutamine and 25 mg/L kanamycin, supplemented with 10% FBS. Cell cultures were

2

then incubated at 37 °C in a humidified, 5% CO atmosphere in the absence or presence of serial

dilutions of test compounds. Cell viability was determined after 48-96 h at 37 °C by the MTT

method.

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 multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 µL of RPMI

4

containing 1x10 MT-4 cells were added to each well of flat bottom microtitre trays, containing 50

µL of RPMI without or with serial dilutions of test compounds. Then, 20 µL of a HIV-1 suspension

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 am.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 am.o.i. of 0.01. Briefly, BHK and MDBK cells were seeded in

4

9

6-well plates at a density of 5x10 and 3x10 cells/well, respectively, and were allowed to form

confluent monolayers by incubating overnight in growth medium at 37 °C in a humidified CO

5%) atmosphere. Cell monolayers were then infected with 50 µL of a proper virus dilution in

2

(

maintenance medium [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 hrs, 50 µL

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 [39].

Antiviral activity against CVB-5, Sb-1, VV, HSV-1, VSV and RSV was determined by plaque

reduction assays in infected cell monolayers. To this end, Vero 76-cells were seeded in 24-well

5

plates at a density of 2x10 cells/well and were allowed to form confluent monolayers by incubating

overnight in growth medium [Dulbecco’s Modified Eagle Medium (D-MEM) with L-glutamine and

4500 mg/L D-glucose and 0.025 g/L kanamycin, supplemented with 10% FBS] at 37 °C in a

humidified CO (5%) atmosphere. Then, monolayers were infected for 2 h with 250 µL of proper

2

virus dilutions to give 50 to 100 PFU/well. Following removal of unadsorbed virus, 500 µL of

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

compounds, were added. Cultures were incubated at 37 °C for 2 (Sb-1 and VSV), 3 (CVB-5, VV

ACCEPTED MANUSCRIPT

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.

4.2.5. 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

EC ) were determined by linear regression analysis.

50

4.2.6. HCV replicon assay

A human hepatoma cell line (Huh-7) bearing the HCV genotype 1b replicon (GS4.1 cells), kindly

provided by C. Seeger (Fox Chase University, Philadelphia, PA, USA) through Idenix

Pharmaceuticals, was grown in D-MEM supplemented with 10% FBS, 2 mM L-glutamine, 110

mg/L sodium pyruvate, 0.1 mM non-essential amino acids, 100 U/mL penicillin, 100 µg/mL

streptomycin and 0.5 mg/mL G418 (Invitrogen). For dose-response testing, GS4.1 cells were seeded

3

in 96-well plates at a density of 7.5 x 10 cells/well in 50 µL medium containing twofold serial

dilutions of test compounds (highest concentration, 75 µM). Huh-7 cells lacking the HCV replicon

served as negative controls. Plates were then incubated for 72 h at 37°C in a humidified, 5% CO

2

incubator. Inhibition of HCV replication was measured by quantification of the viral NS4A protein

using an enzyme-linked immunosorbent assay (ELISA) as follows: plates were fixed for 1 min with

1:1 acetone–methanol, washed twice with PBS containing 0.1% Tween 20, left for 1 h at room

temperature with TNE buffer containing 10% FBS, and then incubated for 2 h at 37°C with the anti-

NS4A mouse monoclonal antibody A-236 (ViroGen, Watertown, MA, USA) diluted in the same

buffer. After three washes with PBS containing 0.1% Tween 20, plates were incubated for 1 h at

37°C with anti-mouse immunoglobulin G-peroxidase conjugate in TNE buffer containing 10%

FBS. After further washing as above, the reaction was developed with phenylenediamine (Zymed,

San Francisco, CA, USA). The reaction was stopped after 30 min with 2 N H2SO4 and absorbance

was determined at 492 nm using a Sunrise Tecan (Durham, NC, USA) spectrophotometer. EC50

values were determined from % inhibition vs compound concentration data, using a sigmoidal non-

linear regression analysis based on four parameters, with a Tecan Magellan software. For

cytotoxicity evaluation, Huh-7 and GS4.1 cells were treated with compounds as described above,

and cellular viability was monitored using the Cell Titer 96 Aqueous One Solution cell proliferation

ACCEPTED MANUSCRIPT

assay (Promega). CC50 values were determined from the % cytotoxicity versus compound

concentration data with Tecan Magellan software, as described above.

4.2.7. Expression of the BVDV-NS5BD24 polymerase

Expression and purification of BVDV-NS5B∆24 polymerase were performed as described in details

in our previous work [13]. In brief, the expression plasmid encoding the His-tagged C-terminal 24-

aminoacid-deleted BVDV-NS5B was introduced into the Escherichia coli strain Rosetta™

2(DE3)pLysS (Novagene) by chemical transformation. Transformant bacteria were cultured

overnight at 30 C in 5 mL of lysogeny broth (LB) supplemented with 25 µg/mL kanamycin and 30

µg/mL chloramphenicol. Cultures were then diluted into 1 L of LB medium additivated by the same

quantities of the two antibiotics, and incubated at 30 C until the A600 reached 0.6–0.7. The culture

was then induced overnight with 1 mM isopropyl-β-D-thiogalactopyranoside, after which cells were

harvested by centrifugation and stored at 80 C until purification.

4.2.8. Purification of NS5B proteins

Cell pellets were thawed and immediately lysed by the addition of 10 mL of CelLytic B (Sigma).

Insoluble material was removed by centrifugation at 11,000 rpm for 60 min at 4 C. The soluble

extract was applied to a 5-mL column of nickel–nitrilotriacetic acid–agarose (Qiagen), previously

equilibrated with the lysis buffer (50 mM NaH

column was washed extensively with the wash buffer (50 mM NaH

imidazole, pH 8.0) and the protein was eluted stepwise with the elution buffer containing increasing

concentration of imidazole (50 mM NaH PO , 300 mM NaCl, 50–250 mM imidazole, pH 8.0). The

2

PO

4

, 300 mM NaCl, 10 mM imidazole, pH 8.0). The

2

PO , 300 mM NaCl, 20 mM

4

2

4

polypeptide composition of each column fraction was monitored via Coomassie-stained SDS–

PAGE analysis. Fractions enriched in pure 6xHis-tagged NS5B protein, recovered in the 130–250

mM imidazole eluates, were pooled and dialyzed against a buffer containing 25 mM Tris–HCl, pH

7

.5, 2.5 mM MgCl , 1 mM dithiothreitol, and 50% glycerol. Protein concentration was determined

2

by the micro-Bradford method (Bio-Rad) using Bovine Serum Albumin (BSA) as standard.

Following dialysis, the purified 6xHis-tagged BVDV-NS5B∆24 protein was divided into aliquots

and stored at -80 C.

4.2.9 RNA-dependent RNA polymerase assay

Enzyme assays were performed in 96-well plates using 10 µg/mL poly(rC) (GE Healthcare,

formerly Amersham Biosciences) as template, 0.1 µg/mL oligo(rG)12 (Invitrogen) as primer, and

80 µM GTP (Invitrogen) as substrate, in a 20 µL reaction mixture containing 20 mM Tris/HCl, pH

ACCEPTED MANUSCRIPT

2

7.0, 1 mM dithiothreitol, 25 mM NaCl, 20 U/mL RNasin (Promega), 5 mM MgCl , 5% DMSO, 5%

glycerol, and 500 ng of the purified protein. After enzyme/drug pre-incubation for 30 min at room

temperature, reactions were started by the addition of GTP. One microliter of three-fold serial

dilutions of test compounds in DMSO 0.5% were added, and the samples were incubated for 120

min at 37 C (BVDV-NS5B∆24). DMSO alone or the nucleotide analog 30-deoxyguanosine-50-

triphosphate (30-dGTP) (tebu-bio) were used as negative and positive controls, respectively.

Reactions were stopped by adding 2 µL of 200 mM EDTA. 138 microliters of the PicoGreen

Quantitation Reagent Molecular Probes diluted 1:345 in TE (Tris/EDTA) buffer were added to each

sample, followed by incubation for 5 min at room temperature in the dark. After excitation at 480

nm, fluorescence was measured at 520 nm in a fluorescence microplate reader (Infinite F200,

Tecan). Effective fluorescence was calculated by subtracting the mean fluorescence of the blank

samples and by converting it into % of activity. Percent of residual activity was then plotted vs

compound concentrations. Dose–response curves were fit with GraphPad Prism (v. 6.00 for

Windows, GraphPad Software, La Jolla California US) to obtain the drug concentration providing

50% inhibition (IC50).

4.3. Computational details

All simulations were carried out using the Pmemd modules of Amber 14 [40], running on own

CPU/GPU calculation cluster. The entire MD simulation and data analysis procedure was optimized

by integrating Amber 14 in modeFRONTIER, a multidisciplinary and multiobjective optimization

and design environment [41]. Molecular graphics images were produced using the UCSF Chimera

package (v.1.10) [42]. The interaction spectra in Figure 2A and 3A were obtained using GraphPad

Prism version 6.00 for Mac OS X Yosemite, GraphPad Software, La Jolla California USA.

4.3.1. Docking procedure

The optimized structures of selected compounds 1-4, 14a, 15a, 16-18 and 19-21 were docked into

the BVDV RdRp binding pockets using the optimized structure of the RdRp of BVDV taken from

our previous work [23-29]. All docking experiments were performed with Autodock 4.3/Autodock

Tools 1.4.6 [43] on a win64 platform. The resulting docked conformations were clustered and

visualized; then, in the absence of any relevant crystallographic information, the structure of each

resulting complex characterized by the lowest Autodock interaction energy in the prevailing cluster

was selected for further modeling.

4.3.2. Molecular Dynamics simulation

Article Doi

Synthesis, cytotoxicity and antiviral evaluation of new series of imidazo[4,5-g[quinoline and pyrido[2,3-g[quinoxalinone derivatives

DOI: 10.1016/j.ejmech.2015.10.002

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2015.10.002

Full text of DOI:10.1016/j.ejmech.2015.10.002

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