The Open Medicinal Chemistry Journal




ISSN: 1874-1045 ― Volume 15, 2021
LETTER

Anti-BVDV Activity Evaluation of Naphthoimidazole Derivatives Compared with Parental Imidazoquinoline Compounds



Roberta Ibba*, 1, Sandra Piras1, Ilenia Delogu2, Roberta Loddo2, Antonio Carta1
1 Department of Chemistry and Pharmacy, University of Sassari, Via Muroni, 23A, 07100 Sassari, Italy
2 Department of Biomedical Sciences, Section of Microbiology and Virology, University of Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy

Abstract

Background:

Pestivirus genus includes animal pathogens which are involved in economic impact for the livestock industry. Among others, Bovine Viral Diarrhoea Virus (BVDV) establish a persistent infection in cattle causing a long list of symptoms and a high mortality rate. In the last decades, we synthesised and reported a certain number of anti-BVDV compounds.

Methods:

In them, imidazoquinoline derivatives turned out as the most active. Their mechanism of actions has been deeply investigated, BVDV RNA-dependent RNA polymerase (RpRd) resulted as target and the way of binding was predicted in silico through three main H-bond interaction with the target.

The prediction could be confirmed by target or ligand mutation. The first approach has already been performed and published confirming the in silico prediction.

Results:

Here, we present how the ligand chemical modification affects the anti-BVDV activity. The designed compounds were synthesised and tested against BVDV as in silico assay negative control.

Conclusion:

The antiviral results confirmed the predicted mechanism of action, as the newly synthesised compounds resulted not active in the in vitro BVDV infection inhibition.

Keywords: BVDV, RdRp, Naphthoimidazoles, Antiviral activity, Pestivirus, Synthesis.


Article Information


Identifiers and Pagination:

Year: 2020
Volume: 14
First Page: 65
Last Page: 70
Publisher Id: TOMCJ-14-65
DOI: 10.2174/1874104502014010065

Article History:

Received Date: 6/5/2020
Revision Received Date: 21/7/2020
Acceptance Date: 22/7/2020
Electronic publication date: 22/09/2020
Collection year: 2020

© 2020 Ibba et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Chemistry and Pharmacy, University of Sassari, Via Muroni 23/A, 07100 Sassari, Italy; E-mail: ribba@uniss.it.





1. INTRODUCTION

Bovine Viral Diarrhoea Virus (BVDV) is the prototype of the Pestivirus genus of the Flaviviridae family. The Pestivirus genus also contains other animal pathogens such as the Bovine Viral Diarrhoea Virus (BVDV) [1Tautz, N.; Thiel, H-J. Cytopathogenicity of pestiviruses: cleavage of bovine viral diarrhea virus NS2-3 has to occur at a defined position to allow viral replication. Arch. Virol., 2003, 148(7), 1405-1412.
[http://dx.doi.org/10.1007/s00705-003-0106-9] [PMID: 12827468]
], the Border Disease Virus (BDV) and the Classical Swine Fever Virus (CSFV). BVDV causes infertility [2Oguejiofor, C.F.; Thomas, C.; Cheng, Z.; Wathes, D.C. Mechanisms linking bovine viral diarrhea virus (BVDV) infection with infertility in cattle. Anim. Health Res. Rev., 2019, 20(1), 72-85.
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[http://dx.doi.org/10.1590/s1984-29612019032] [PMID: 31215610]
] in cattle but also teratogenesis, early embryonic death, abortion, respiratory problems and immune system disorder on cows which results in acute infections of immunocompetent cattle causing a mortality rate of 17 to 32% [4Ellis, J.A.; West, K.H.; Cortese, V.S.; Myers, S.L.; Carman, S.; Martin, K.M.; Haines, D.M. Lesions and distribution of viral antigen following an experimental infection of young seronegative calves with virulent bovine virus diarrhea virus-type II. Can. J. Vet. Res., 1998, 62(3), 161-169.
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] and an estimated loss between 10 and 40 million $ per million calvings [5Gunn, G.J.; Saatkamp, H.W.; Humphry, R.W.; Stott, A.W. Assessing economic and social pressure for the control of bovine viral diarrhoea virus. Prev. Vet. Med., 2005, 72(1-2), 149-162.
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]. The virus was also considered to be a valuable surrogate for the Hepatitis C Virus (HCV) in antiviral drug studies [7Baginski, S.G.; Pevear, D.C.; Seipel, M.; Sun, S.C.; Benetatos, C.A.; Chunduru, S.K.; Rice, C.M.; Collett, M.S. Mechanism of action of a pestivirus antiviral compound. Proc. Natl. Acad. Sci. USA, 2000, 97(14), 7981-7986.
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]. During the years, several mammalians from different species have been infected by BVDV [8Passler, T.; Walz, P.H. Bovine viral diarrhea virus infections in heterologous species. Anim. Health Res. Rev., 2010, 11(2), 191-205.
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], which was not host-specific. BVDV can also be a problematic contaminant in the laboratory, BVDV strains have been identified in commercially available lots of foetal bovine serum and cell lines [9Harasawa, R.; Mizusawa, H. Demonstration and genotyping of pestivirus RNA from mammalian cell lines. Microbiol. Immunol., 1995, 39(12), 979-985.
[http://dx.doi.org/10.1111/j.1348-0421.1995.tb03301.x] [PMID: 8789057]
], and therefore in interferons and vaccines for medical use [10Harasawa, R.; Sasaki, T. Sequence analysis of the 5′ untranslated region of pestivirus RNA demonstrated in interferons for human use. Biologicals, 1995, 23(4), 263-269.
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, 11Harasawa, R.; Tomiyama, T. Evidence of pestivirus RNA in human virus vaccines. J. Clin. Microbiol., 1994, 32(6), 1604-1605.
[http://dx.doi.org/10.1128/JCM.32.6.1604-1605.1994] [PMID: 8077414]
]. Several conventional live and killed vaccines in the years have been developed, but they showed non-efficacy or safety issues [12Oirschot, J.T.V.; Bruschke, C.J.M.; Rijn, P.A.V. Vaccination of Cattle against Bovine Viral Diarrhoea. Veterinary Microbiology., 1999, 64, 169-183.
[http://dx.doi.org/10.1016/S0378-1135(98)00268-5]
-14Zemke, J.; König, P.; Mischkale, K.; Reimann, I.; Beer, M. Novel BVDV-2 mutants as new candidates for modified-live vaccines. Vet. Microbiol., 2010, 142(1-2), 69-80.
[http://dx.doi.org/10.1016/j.vetmic.2009.09.045] [PMID: 19875253]
]. No antivirals are currently available for controlling BVDV infections in laboratories or farms, only voluntary BVDV control programmes [15van Duijn, L.; Veldhuis, A.M.B.; Mars, M.H.; de Roo, B.; Lam, T.J.G.M. Efficacy of a voluntary BVDV control programme: Experiences from the Netherlands. Vet. J., 2019, 245, 55-60.
[http://dx.doi.org/10.1016/j.tvjl.2018.12.016] [PMID: 30819426]
] seemed to be successful, but BVDV remains an agronomical burden, hence the antiviral research on molecules that can specifically inhibit the virus replication could be useful to control outbreaks in farms. In the last decades, a certain number of selective anti-BVDV compounds have been reported, this includes virus targeting and host targeting derivatives, e.g. polymerase inhibitors [7Baginski, S.G.; Pevear, D.C.; Seipel, M.; Sun, S.C.; Benetatos, C.A.; Chunduru, S.K.; Rice, C.M.; Collett, M.S. Mechanism of action of a pestivirus antiviral compound. Proc. Natl. Acad. Sci. USA, 2000, 97(14), 7981-7986.
[http://dx.doi.org/10.1073/pnas.140220397] [PMID: 10869440]
, 16King, R.W.; Scarnati, H.T.; Priestley, E.S.; De Lucca, I.; Bansal, A.; Williams, J.K. Selection of a thiazole urea-resistant variant of bovine viral diarrhoea virus that maps to the RNA-dependent RNA polymerase. Antivir. Chem. Chemother., 2002, 13(5), 315-323.
[http://dx.doi.org/10.1177/095632020201300507] [PMID: 12630680]
-18Tabarrini, O.; Manfroni, G.; Fravolini, A.; Cecchetti, V.; Sabatini, S.; De Clercq, E.; Rozenski, J.; Canard, B.; Dutartre, H.; Paeshuyse, J.; Neyts, J. Synthesis and anti-BVDV activity of acridones as new potential antiviral agents. J. Med. Chem., 2006, 49(8), 2621-2627.
[http://dx.doi.org/10.1021/jm051250z] [PMID: 16610805]
], protease inhibitors [19Bukhtiyarova, M.; Rizzo, C.J.; Kettner, C.A.; Korant, B.D.; Scarnati, H.T.; King, R.W. Inhibition of the bovine viral diarrhoea virus NS3 serine protease by a boron-modified peptidyl mimetic of its natural substrate. Antivir. Chem. Chemother., 2001, 12(6), 367-373.
[http://dx.doi.org/10.1177/095632020101200607] [PMID: 12018682]
] and human cellular enzymes inhibitors [20Branza-Nichita, N.; Durantel, D.; Carrouée-Durantel, S.; Dwek, R.A.; Zitzmann, N. Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers. J. Virol., 2001, 75(8), 3527-3536.
[http://dx.doi.org/10.1128/JVI.75.8.3527-3536.2001] [PMID: 11264342]
-22Zitzmann, N.; Mehta, A.S.; Carrouée, S.; Butters, T.D.; Platt, F.M.; McCauley, J.; Blumberg, B.S.; Dwek, R.A.; Block, T.M. Imino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: implications for the development of broad spectrum anti-hepatitis virus agents. Proc. Natl. Acad. Sci. USA, 1999, 96(21), 11878-11882.
[http://dx.doi.org/10.1073/pnas.96.21.11878] [PMID: 10518544]
]. The antiviral research against animal pathogens of the Pestivirus genus is still a challenge. In the framework of a long-lasting antiviral research program, our group designed and synthesised a series of angular and linear N-polycyclic derivatives active against BVDV, YFV, HCV and other related viruses [23Vitale, G.; Corona, P.; Loriga, M.; Carta, A.; Paglietti, G.; Giliberti, G.; Sanna, G.; Farci, P.; Marongiu, M.E.; La Colla, P. 5-acetyl-2-arylbenzimidazoles as antiviral agents. Part 4. Eur. J. Med. Chem., 2012, 53, 83-97.
[http://dx.doi.org/10.1016/j.ejmech.2012.03.038] [PMID: 22513121]
-30Sanna, G.; Piras, S.; Madeddu, S.; Busonera, B.; Klempa, B.; Corona, P.; Ibba, R.; Murineddu, G.; Carta, A.; Loddo, R. 5,6-Dichloro-2-phenyl-benzotriazoles: New potent inhibitors of orthohantavirus. Viruses, 2020, 12(1), 122.
[http://dx.doi.org/10.3390/v12010122] [PMID: 31968537]
]. The molecular target for BVDV inhibition was identified in the viral RNA dependent RNA polymerase (RdRp) [24Carta, A.; Briguglio, I.; Piras, S.; Corona, P.; Boatto, G.; Nieddu, M.; Giunchedi, P.; Marongiu, M.E.; Giliberti, G.; Iuliano, F.; Blois, S.; Ibba, C.; Busonera, B.; La Colla, P. Quinoline tricyclic derivatives. Design, synthesis and evaluation of the antiviral activity of three new classes of RNA-dependent RNA polymerase inhibitors. Bioorg. Med. Chem., 2011, 19(23), 7070-7084.
[http://dx.doi.org/10.1016/j.bmc.2011.10.009] [PMID: 22047799]
, 28Briguglio, I.; Loddo, R.; Laurini, E.; Fermeglia, M.; Piras, S.; Corona, P.; Giunchedi, P.; Gavini, E.; Sanna, G.; Giliberti, G.; Ibba, C.; Farci, P.; La Colla, P.; Pricl, S.; Carta, A. Synthesis, cytotoxicity and antiviral evaluation of new series of imidazo[4,5-g]quinoline and pyrido[2,3-g]quinoxalinone derivatives. Eur. J. Med. Chem., 2015, 105, 63-79.
[http://dx.doi.org/10.1016/j.ejmech.2015.10.002] [PMID: 26479028]
, 31Carta, A.; Loriga, M.; Paglietti, G.; Ferrone, M.; Fermeglia, M.; Pricl, S.; Sanna, T.; Ibba, C.; La Colla, P.; Loddo, R. Design, synthesis, and preliminary in vitro and in silico antiviral activity of [4,7]phenantrolines and 1-oxo-1,4-dihydro-[4,7]phenantrolines against single-stranded positive-sense RNA genome viruses. Bioorg. Med. Chem., 2007, 15(5), 1914-1927.
[http://dx.doi.org/10.1016/j.bmc.2007.01.005] [PMID: 17251029]
, 32Carta, A.; Briguglio, I.; Piras, S.; Corona, P.; Ibba, R.; Laurini, E.; Fermeglia, M.; Pricl, S.; Desideri, N.; Atzori, E.M.M.; La Colla, P.; Collu, G.; Delogu, I.; Loddo, R. A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems. Eur. J. Med. Chem., 2016, 117, 321-334.
[http://dx.doi.org/10.1016/j.ejmech.2016.03.080] [PMID: 27161176]
]. Imidazo[4,5-g]quinoline derivatives were published as interesting BVDV inhibitors and the most active derivative of that series, 4-(4-chloro-3H-imidazo[4,5-g]quinolin-2-yl)benzonitrile (A) (EC50 0.3µM; CC50 >100 µM) [32Carta, A.; Briguglio, I.; Piras, S.; Corona, P.; Ibba, R.; Laurini, E.; Fermeglia, M.; Pricl, S.; Desideri, N.; Atzori, E.M.M.; La Colla, P.; Collu, G.; Delogu, I.; Loddo, R. A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems. Eur. J. Med. Chem., 2016, 117, 321-334.
[http://dx.doi.org/10.1016/j.ejmech.2016.03.080] [PMID: 27161176]
, 33Carta, A.; La Colla, P.; Loddo, R.; Paglietti, G. Compounds for the Treatment or Prevention of Infection by Flaviviridae., 2008.] was docked and scored for affinity towards the binding site of the BVDV RdRp, the resulting receptor/ligand complex was relaxed by energy minimisation, followed by MD simulation. The naphthoimidazole core showed significant H-bond interaction between nitrogen atoms and key amino acids (R295, Y674 and S411). In detail, arginine 295 interacts with N5 of the lead compound as H-bond donor, while N1 resulted as H-bond acceptor in the interaction with tyrosine 674 and the nitrogen of the benzonitrile moiety gives H-bond with serine 411. Has also been predicted the decrease of affinity of some molecules with the substrate when R295 and Y674 – critical residues for compound binding - are mutated into alanine and the prediction has been confirmed by in vitro assays on the mutant RdRp [32Carta, A.; Briguglio, I.; Piras, S.; Corona, P.; Ibba, R.; Laurini, E.; Fermeglia, M.; Pricl, S.; Desideri, N.; Atzori, E.M.M.; La Colla, P.; Collu, G.; Delogu, I.; Loddo, R. A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems. Eur. J. Med. Chem., 2016, 117, 321-334.
[http://dx.doi.org/10.1016/j.ejmech.2016.03.080] [PMID: 27161176]
]. In order to have a further negative control for these simulations, we now designed some chemical modification on the lead compounds and parent compounds similarly active in BVDV RdRp inhibition, and we synthesised the designed compounds. The replacement of the nitrogen atom in position 5 of imidazo [4,5-g] quinoline derivatives with a carbon atom, resulted in naphtho [2,3-d] imidazole derivatives (3a-j) designed to confirm the essential interaction between N5 and the arginine 295. The 2-phenyl moiety was modified in order to assess the relevance of interactions with serine 411.

2. MATERIALS AND METHODS

2.1. Synthetic Methods

Synthesis of derivatives 3a-j was carried on by using the general procedure for imidazole ring closure as described in literature [34Bahrami, K.; Khodaei, M. M.; Kavianinia, I. A Simple and Efficient One-Pot Synthesis of 2-Substituted Benzimidazoles. Synthesis (Stuttg)., 2007, 4, 547-550.
[http://dx.doi.org/10.1055/s-2007-965878]
] by mixing commercial naphthalene-2,3-diamine (1) and corresponding commercial aldehydes (2a-j) in a ratio 1:1 dissolved in acetonitrile (CH3CN), added with H2O2 30% (ratio 1:7) and HCl 37% (ratio 1:3,5) at room temperature overnight, solid was obtained (Scheme. I). The solid has been filtered off with vacuum, washed with acetonitrile first and with water then, till neutral pH of the filtrate. The crude solid has been stored and dried in the oven overnight and were obtained pure by crystallisation from ethanol.

2.2. Chemical Characterisation

Compounds melting points (m.p.) were taken in open capillaries in a Köfler hot stage or Digital Electrothermal melting point apparatus and are uncorrected. Retention factors (Rf) were measured by Thin Layer Chromatography (TLC) using Merck F-254 commercial plates and a proper mixture of petroleum spirit (PS) and ethyl acetate (EA) as eluent. Nuclear Magnetic Resonance (NMR) spectra were registered in solutions in deuterated DMSO and were recorded with a Bruker Avance III 400 NanoBay (400 MHz) instrument. 1H-NMR chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane (TMS) used as internal standard. Chemical shift values are reported in ppm (δ) and coupling constants (J) are reported in Hertz (Hz). Signal multiplicities are represented as s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), t (triplet), td (triplet of doublets), q (quadruplet) and m (multiplet). The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O. 13C-NMR chemical shifts are reported in downfield from tetramethylsilane (TMS) used as internal standard. Suitable method among APT (Attached Proton Test) and jmod (J-modulated spin-echo for X-nuclei coupled to H-1 to determine the number of attached protons) was selected for each compound. The solutions for ESI-MS measurements were prepared by dissolving the solid compounds in HPLC acetonitrile to obtain a concentration of 1.0-2.0 ppm. Mass spectra in the positive-ion mode were obtained on a Q Exactive Plus Hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific) mass spectrometer. The solutions were infused at a flow rate of 5.00 μl/min into the ESI chamber. The spectra were recorded in the m/z range 150–800 at a resolution of 140 000 and accumulated for at least 2 min in order to increase the signal-to-noise ratio. The instrumental conditions used for the measurements were as follows: spray voltage 2300 V, capillary temperature 250 °C, sheath gas 10 (arbitrary units), auxiliary gas 3 (arbitrary units), sweep gas 0 (arbitrary units), and probe heater temperature 50 °C. ESI-MS spectra were analysed by using Thermo Xcalibur 3.0.63 software (Thermo Fisher Scientific), and the average deconvoluted monoisotopic masses were obtained through the Xtract tool integrated with the software.

2.3. Cells and Viruses

Cell lines were purchased from American Type Culture Collection (ATCC). The absence of mycoplasma contamination was checked periodically by the Hoechst staining method. Cell line supporting the multiplication of BVDV was the following: Madin-Darby Bovine Kidney (MDBK) [ATCC CCL 22 (NBL-1) Bos Taurus]. The virus was purchased from American Type Culture Collection (ATCC), Bovine Viral Diarrhoea Virus (BVDV) [strain NADL (ATCC VR-534)].

2.4. Cytotoxicity Assays

Cytotoxicity assays were run in parallel with antiviral assays. MDBK cells were seeded at an initial density of 6x105 cells/cm3 in 96-well plates, in culture medium [Minimum Essential Medium with Earle’s salts (MEM-E) with L-glutamine, supplemented with 10% horse serum and 1 mM sodium pyruvate, and 0.025 mg/L kanamycin]. Cell cultures were then incubated at 37 °C in a humidified, 5% CO2 atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability was determined after 48–96 h at 37 °C by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl- tetrazolium bromide (MTT) method [35Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.; Desmyter, J.; De Clercq, E. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J. Virol. Methods, 1988, 20(4), 309-321.
[http://dx.doi.org/10.1016/0166-0934(88)90134-6] [PMID: 2460479]
].

2.5. Antiviral Assay

Compounds activity against BVDV was based on inhibition of virus-induced cytopathogenicity in MDBK cells acutely infected with an m.o.i. of 0.01. Briefly, MDBK cells were seeded in 96-well plates at a density of 3x104 cells/well and were allowed to form confluent monolayers by incubating overnight in growth medium at 37 °C in a humidified CO2 (5%) atmosphere. Cell monolayers were then infected with 50 µL of virus dilution in maintenance medium (MEM-E with L-glutamine, supplemented with 0.5% inactivated FBS, 1 mM sodium pyruvate and 0.025 g/L kanamycin) to give an m.o.i of 0.01. After 2 h, 50 µL of maintenance medium, without or with serial dilutions of test compounds, were added. After a 3 days incubation at 37 °C, cell viability was determined by the MTT method. Linear regression analysis: viral and cell growth at each drug concentration was expressed as a percentage of untreated controls and concentrations, resulting in 50% (EC50 and CC50) growth inhibition. NM 108 (2’-C-methylguanosine) and ribavirin are used as reference controls.

2.6. Experimental Section

2.6.1. 2-phenyl-1H-naphtho[2,3-d]imidazole (3a)

Compound 3a (C17H12N2, MW 244.291) was obtained in total yield 95%; m.p. 254.6-255 °C; TLC (PS/EA 6/4): Rf 0.55. 1H-NMR (DMSO-d6): δ 8.49 (2H, d, J= 6.8 Hz, H-2’,6’), 8.37 (2H, s, H-4,9), 8.19 (2H, dd, J= 3.2 Hz, H-5,8), 7.77 (3H, m, H-3’,4’,5’), 7.56 (2H, dd, J= 3.2 Hz, H-6,7). 13C-NMR (APT, DMSO-d6): δ 152.9 (C-2), 133.7 (CH-4), 132.4 (C-9a), 130.9 (3C, C-3a,4a,8a), 129.6 (3CH, C-3’,4’,5’), 128.6 (2CH, C-2’,6’), 128.1 (2CH, C-6,7), 125.4 (2CH, C-5,8), 123.8 (C-1’), 110.7 (CH-9). ESI-MS (m/z): calcd. for C17H12N2 245.1073, found 245.1072 [M+H]+.

2.6.2. 2-(p-tolyl)-1H-naphtho[2,3-d]imidazole (3b)

Compound 3b (C18H14N2, MW 258.317) was obtained in total yield 74%; m.p. 276.6-278 °C; TLC (PS/EA 6/4): Rf 0.62. 1H-NMR (DMSO-d6): δ 8.35 (2H, s, H-4,9), 8.26 (2H, d, J= 8 Hz, H-2’,6’), 8.19 (2H, dd, J= 3.2 Hz, H-5,8), 7.57 (4H, m, H-6,7,3’,5’), 2.47 (3H, s, CH3). 13C-NMR (APT, DMSO-d6): δ 152.9 (C-2), 144.6 (C-3a), 131.9 (C-4’), 130.9 (2C, C-4a,8a), 130.2 (2CH, C-3’,5’), 128.5 (2CH, C-2’,6’), 128.0 (2CH, C-6,7), 126.3 (C-9a), 125.5 (2CH, C-5,8), 120.6 (C-1’), 110.5 (2CH, C-4,9), 21.3 (CH3). ESI-MS (m/z): calcd. for C18H14N3 259.1230, found 259.1231 [M+H]+.

2.6.3. 2-(4-nitrophenyl)-1H-naphtho[2,3-d]imidazole (3c)

Compound 3c (C17H11N3O2) was obtained in total yield 72%; m.p. 236-238 °C; TLC (PS/EA 6/4): Rf 0.63. 1H-NMR (DMSO-d6): δ 8.57 (2H, d, J= 8.8 Hz, H-3’,5’), 8.51 (2H, d, J= 8.8 Hz, H-2’,6’), 8.28 (2H, s, H-4,9), 8.10 (2H, q, J= 3.6 Hz, H-5,8), 7.47 (2H, q, J= 3.2 Hz, H-6,7). 13C-NMR (APT, DMSO-d6): δ 152.5 (C-2), 148.8 (C-3a), 137.8 (C-4’), 133.9 (C-9a), 130.6 (2C, C-4a,8a), 128.4 (2CH, C-6,7), 127.9 (2CH, C-5,8), 127.3 (C-1’), 124.4 (2CH, C-2’,6’), 124.3 (2CH, C-3’,5’), 111.5 (2CH, C-4,9). ESI-MS (m/z): calcd. for C17H11N3O2 290.0924, found 290.0922 [M+H]+.

2.6.4. 4-(1H-naphtho[2,3-d]imidazol-2-yl)benzonitrile (3d)

Compound 3d (C18H11N3, MW 269.300) was obtained in total yield 85%; m.p. 237.7-239.1 °C; TLC (PS/EA 6/4): Rf 0.58. 1H-NMR (DMSO-d6): δ 8.53 (2H, d, J= 8.4 Hz, H-3’,5’), 8.29 (2H, s, H-4,9), 8.17 (2H, d, J= 8 Hz, H-2’,6’), 8.12 (2H, dd, J= 3.2 Hz, H-5,8), 7.49 (2H, dd, J= 3.2 Hz, H-6,7). 13C-NMR (APT, DMSO-d6): δ 152.5 (C-2), 136.6 (C-3a), 133.2 (2CH, C-3’,5’), 131.3 (C-9a), 130.7 (2C, C-4a,8a), 128.3 (2CH, C-2’,6’), 127.9 (2CH, C-6,7), 124.5 (2CH, C-5,8), 118.2 (CN), 113.8 (C-4’), 111.3 (2CH, C-4,9). ESI-MS (m/z): calcd. for C18H11N3 270.1026, found 270.1025 [M+H]+.

2.6.5. 2-(4-(trifluoromethyl)phenyl)-1H-naphtho[2,3-d]imida- zole (3e)

Compound 3e (C18H11F3N2, MW 312.289) was obtained in total yield 89%; m.p. 211.3-212 °C; TLC (PS/EA 6/4): Rf 0.78. 1H-NMR (DMSO-d6): δ 8.66 (2H, d, J= 8 Hz, H-2’,6’), 8.38 (2H, s, H-4,9), 8.17 (2H, dd, J= 3.2 Hz, H-5,9), 8.14 (2H, d, J= 8.4 Hz, H-3’,5’), 7.54 (2H, dd, J= 3.2 Hz, H-6,7). 13C-NMR (APT, DMSO-d6): δ 151.6 (C-2), 133.0 (C-3a), 132.4 (1C, m, CF3), 131.0 (2C-4a,8a), 129.3 (2CH, C-2’,6’), 128.0 (2CH, C-6,7), 127.1 (C-9a), 126.4 (CH-5), 125.4 (2CH, C-3’,5’), 125.0 (C-4’), 123.4 (CH-8), 122.3 (C-1’), 111.0 (2CH, C-4,9). ESI-MS (m/z): calcd. for C18H11F3N2 313.0947, found 313.0946 [M+H]+.

2.6.6. 2-(4-methoxyphenyl)-1H-naphtho[2,3-d]imidazole (3f)

Compound 3f (C18H14N2O, MW 274.317) was obtained in total yield 91%; m.p. 251-252.2 °C; TLC (PS/EA 6/4): Rf 0.40. 1H-NMR (DMSO-d6): δ 8.40 (2H, d, J= 8.8 Hz, H-2’,6’), 8.32 (2H, s, H-4,9), 8.19 (2H, dd, J= 3.2 Hz, H-5,8), 7.57 (2H, dd, J= 3.2 Hz, H-6,7), 7.35 (2H, d, J= 8.8 Hz, H-3’,5’), 3.94 (3H, s, CH3). 13C-NMR (APT, DMSO-d6): δ 163.7 (C-4’), 152.9 (C-2), 132.1 (2C, C-3a,9a), 130.9 (3C, C-4a,8a,1’), 130.6 (2CH, C-2’,6’), 128.0 (2CH, C-6,7), 125.4 (2CH, C-5,8), 115.3 (2CH, C-3’,5’), 110.2 (2CH, C-4,9), 55.9 (CH3). ESI-MS (m/z): calcd. for C18H14N2O 275.1179, found 275.1176 [M+H]+.

2.6.7. 2-(5-nitrofuran-2-yl)-1H-naphtho[2,3-d]imidazole (3g)

Compound 3g (C15H9N3O3, MW 279.250) was obtained in total yield 79%; m.p. 217-217.4 °C; TLC (PS/EA 6/4): Rf 0.46. 1H-NMR (DMSO-d6): δ 8.19 (2H, s, H-4,9), 8.05 (2H, dd, J= 3.2 Hz, H-5,8), 7.93 (1H, d, J= 3.6 Hz, H-4’), 7.68 (1H, d, J= 3.6, H-3’), 7.42 (2H, dd, J= 3.2 Hz, H-6,7). 13C-NMR (jmod, DMSO-d6): δ 152.1 (C-5’), 146.6 (C-2’), 144.7 (C-2), 138.9 (2C, C-3a,9a), 130.6 (2C, C-4a,8a), 127.8 (2CH, C-6,7), 124.0 (2CH, C-5,8), 114.8 (2CH, C-3’,4’), 111.80 (2CH, C-4,9). ESI-MS (m/z): calcd. for C15H9N3O3 280.0717, found 280.0717 [M+H]+.

2.6.8. 2-(5-nitrothiophen-2-yl)-1H-naphtho[2,3-d]imidazole (3h)

Compound 3h (C15H9N3O2S, MW 295.316) was obtained in total yield 71%; m.p. 253-254.1 °C; TLC (PS/EA 6/4): Rf 0.54. 1H-NMR (DMSO-d6): δ 8.28 (1H, d, J= 4.4 Hz, H-4’), 8.18 (2H, s, H-4,9), 8.12 (1H, d, J= 4.4 Hz, H-3’), 8.04 (2H, dd, J= 3.2 Hz, H-5,8), 7.42 (2H, dd, J= 3.2 Hz, H-6,7). 13C-NMR (APT, DMSO-d6+TFA-d): δ 153.1 (C-5’), 149.2 (C-2), 147.4 (C-3a), 139.0 (C-9a), 136.4 (C-2’), 130.8 (2C, C-4a,8a), 130.4 (CH-3’), 129.2 (CH-4’), 1270.8 (2CH, C-6,7), 124.6 (2CH, C-5,8), 111.3 (2CH, C-4,9). ESI-MS (m/z): calcd. for C15H9N3O2S 296.0488, found 296.0489 [M+H]+.

2.6.9. 2-(pyridin-4-yl)-1H-naphtho[2,3-d]imidazole (3i)

Compound 3i (C16H11N3, MW 245.279) was obtained in total yield 85%; m.p. 250.9-251.6 °C; TLC (PS/EA 6/4): Rf 0.11. 1H-NMR (DMSO-d6): δ 9.03 (2H, d, J= 6 Hz, H-3’,5’), 8.60 (2H, d, J= 5.6 Hz, H-2’,6’), 8.31 (2H, s, H-4,9), 8.09 (2H, dd, J= 3.2 Hz, H-5,8), 7.46 (2H, dd, J= 3.2 Hz, H-6,7). 13C-NMR (APT, DMSO-d6): δ 151.0 (C-2), 146.6 (2CH, C-2’6’), 139.7 (C-3a), 138.0 (C-9a), 130.8 (2C, C-4a,8a), 128.0 (2CH, C-6,7), 127.5 (C-4’), 124.5 (2CH, C-5,8), 122.6 (2CH, C-3’,5’), 112.0 (2CH, C-4,9). ESI-MS (m/z): calcd. for C16H11N3 246.1026, found 246.1025 [M+H]+.

2.6.10. 2-(pyridin-2-yl)-1H-naphtho[2,3-d]imidazole (3j)

Compound 3j (C16H11N3, MW 245.279) was obtained in total yield 65%; m.p. 238.3-239 °C; TLC (PS/EA 6/4): Rf 0.20. 1H-NMR (DMSO-d6): δ 8.79 (1H, d, J= 4.8 Hz, H-3’), 8.46 (1H, d, J= 8 Hz, H-6’), 8.16 (2H, s, H-4,8), 8.05 (1H, td, 1J= 7.6 Hz, 2J= 1.6 Hz, H-5’), 8.01 (2H, dd, J= 3.2 Hz, H-5,8), 7.59 (1H, ddd, 1J= 7.6 Hz, 2J= 4.8 Hz, 3J= 1.2 Hz, H-4’), 7.38 (2H, dd, J= 3.2 Hz). 13C-NMR (APT, DMSO-d6): δ 155.2 (C-2), 150.1 (CH-6’), 148.4 (C-2’), 138.2 (CH-4’), 137.1 (C-3a), 130.7 (2C, C-4a,8a), 129.1 (C-9a), 128.3 (2CH, C-6,7), 126.0 (CH-3’), 124.1 (2CH, C-5,8), 122.2 (2CH, C-4,9), 108.7 (CH-5’). ESI-MS (m/z): calcd. for C16H11N3 246.1026, found 246.1026 [M+H]+.

3. RESULTS AND DISCUSSION

As described above, the driven idea for this project is the isosteric substitution of a nitrogen atom with a carbon atom and evaluate the antiviral activity of the resulted molecules compared with the parental compounds.

Scheme I
Synthetic route for derivatives 3a-j.


Table 1
Antiviral activity and cytotoxicity of naphthoimidazole derivatives (3a-j), derivative A [28] was reported for comparison; NM 108 and ribavirin were used as positive controls.


Naphthoimidazole derivatives ( 3a - j ) were designed and synthesised in order to confirm the relevance of nitrogen atom in position 5 of the imidazoquinoline derivatives for the interaction with the target, these molecule’s antiviral results confirmed the predicted affinity data previously published since they showed a consistent loss of antiviral activity with respect to parental compounds [32Carta, A.; Briguglio, I.; Piras, S.; Corona, P.; Ibba, R.; Laurini, E.; Fermeglia, M.; Pricl, S.; Desideri, N.; Atzori, E.M.M.; La Colla, P.; Collu, G.; Delogu, I.; Loddo, R. A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems. Eur. J. Med. Chem., 2016, 117, 321-334.
[http://dx.doi.org/10.1016/j.ejmech.2016.03.080] [PMID: 27161176]
]. Derivative 3a still maintains a weak antiviral activity, EC50 at 16 µM, but related at cytotoxicity CC50 value of 37 µM. Cytotoxicity is consistent in the greater part of analysed compounds, contrary to what is detected for parental imidazoquinoline derivatives, 3b, 3e, 3f, 3h presented CC50 values ranging between 2.8 and 11.8 µM, (Table 1). Derivative 3d, which is as much like compound A, revealed a total loss of activity and, therefore, loss of affinity with the target. Clearly, the interactions between the aromatic moiety - here labelled as phenyl-R or R’ - and serine 411 of the target RdRp are not responsible for the anti-BVDV activity and are only supportive to it.

CONCLUSION

The naphthoimidazole derivatives were designed and synthesized as isostere of imidazoquinoline derivatives (previously reported) to evaluate the interactions between heteroatoms of the chemical scaffold of these derivatives and the key amino acids of the BVDV RdRp. The quinoline nitrogen atom was substituted with a carbon atom, which cannot make H-bond interactions with R295 amino acid, and this resulted in total loss of activity, as showed by the antiviral assay results. We can then confirm the predicted relevance of N5 - R295 interaction from the docking studies published. This isosteric substitution led to not active derivatives against BVDV. At the same time, the biochemical interaction between the aromatic moiety - here labelled as R - and serine 411 of the RdRp target is clearly not responsible but only bearing, for the anti-BVDV activity.

ETHICAL APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN RIGHTS AND ANIMAL RIGHTS

No humans or Animals were the basis of this research.

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

This study is supported by (P.O.R. Sardegna F.S.E. - Operational Programme of the Autonomous Region of Sardinia, European Social Fund 2014-2020 - Axis III Education and training, Thematic goal 10, Investment Priority 10ii), Specific goal 10.5.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

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(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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