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Abstract

Background

Herpes simplex virus 1 (HSV-1) affects a large part of the adult population. Anti-HSV-1 drugs, such as acyclovir, target thymidine kinase and viral DNA polymerase. However, the emerging of resistance of HSV-1 alerts for the urgency in developing new antivirals with other therapeutic targets. Thus, this study evaluated a series of 1,4-disubstituted-1,2,3-triazole derivatives against HSV-1 acute infection and provided deeper insights into the possible mechanisms of action.

Methods

Human fibroblast cells (HFL-1) were infected with HSV-1 17syn+ and treated with the triazole compounds at 50 μM for 24 h. The 50% effective drug concentration (EC₅₀) was determined for the active compounds. Their cytotoxicity was also evaluated in HFL-1 with the 50% cytotoxic concentration (CC₅₀) determined using CellTiter-Glo^® solution. The most promising compounds were evaluated by virucidal activity and influence on virus egress, DNA replication and transcription, and effect on an acyclovir-resistant HSV-1 strain.

Results

Compounds 3 ((E)-4-methyl-N′-(2-(4-(phenoxymethyl)-1H-1,2,3-triazol1yl)benzylidene)benzenesulfonohydrazide) and 4 (2,2′-(4,4′-((1,3-phenylenebis(oxy))bis(methylene))bis(1H-1,2,3-triazole-4,1 diyl)) dibenzaldehyde) were the most promising, with an EC₅₀ of 16 and 21 μM and CC₅₀ of 285 and 2,593 μM, respectively. Compound 3 was able to inhibit acyclovir-resistant strain replication and to interfere with virus egress. Both compounds did not affect viral DNA replication, but inhibited significantly the expression of ICP0, ICP4 and gC. Compound 4 also affected the transcription of UL30 and ICP34.5.

Conclusions

Our findings demonstrated that these compounds are promising antiviral candidates with different mechanisms of action from acyclovir and further studies are merited.

Introduction

Herpes simplex virus type-1 (HSV-1) infects epithelial cells with formation of water bubbles and wounds mainly in the oral and ocular mucosa [1]. It affects a large part of population, as shown in a recent epidemiological study in Latin America and the Caribbean that estimated about 90% of adults and 60% of children are infected by HSV-1 [2].

HSV-1 can also lead to severe complications such as blindness [3] and encephalitis [4]. In addition, several studies have associated HSV-1 to Alzheimer's disease [3–8]. In 1997, Itzhaki et al. [7] suggested the apolipoprotein E ε4 (APOE-ε4) allele, that is a risk factor for Alzheimer's disease, is also a risk of cold sores, and HSV-1 in brain of APOE-ε4 carriers confers higher risk of Alzheimer's disease. More recently, an epidemiological study carried out in Taiwan revealed that the risk of senile dementia is greater in those who are HSV-seropositive than HSV-seronegative, and that antiviral treatment causes a decrease in number of people who later develop senile dementia [8].

Currently, apart from acyclovir (ACV) and other nucleosides analogues, there are few commercially available drugs for the treatment of HSV-1 [9]. ACV is monophosphorylated by viral thymidine kinase, followed by phosphorylation by cellular kinases to become the active form to inhibit DNA polymerase, an enzyme that catalyses the elongation of viral DNA [10]. ACV and related drugs are undoubtedly effective in treating HSV-1 infections [11]. However, due to the widespread use of ACV, cases of ACV-resistant HSV-1 infections have been increasing, frequently in immunocompromised patients [12–14]. Approximately 95% of resistant cases are due to mutations in the UL23 gene encoding for thymidine kinase and 5% are due to mutations in the UL30 gene encoding for viral DNA polymerase [15]. A single mutation in DNA polymerase may confer resistance to many anti-HSV agents [16]. Since most of the anti-HSV drugs have these enzymes as targets, the treatment of resistant HSV-1 infection is problematic and restricted [17]. For this reason, it is necessary to discover new drugs with anti-HSV-1 activity and with a different mechanism of action [18]. Pires de Mello et al. [19] have shown several antiviral targets through the infection phases, from the entry of HSV-1 into epithelial cells, the lytic cycle, to latency and reactivation. Thus, essential viral proteins and some cellular targets could be new strategies in antiviral discovery.

Some series of 1,4 disubstituted-1,2,3-triazoles with antiviral activity have been described in literature. Ribavirin, for instance, is a known antiviral against HIV-1, HSV and HCV, and 1,2,3-triazole analogue increased pharmacological activity and reduced ribavirin cytotoxicity [20,21]. Regarding anti-HSV-1 activity, some authors have reported good in vitro activity [22–24]. Jordão et al. [22], for example, elaborated on a series of arysulfonylhydrazide-1H-1,2,3-triazoles and described 50% effective drug concentration (EC₅₀) values of 1.30 and 1.26 μM against this virus. The effectiveness of 1,2,3-triazole derivatives was also reported in the series of 5-(benzylthio)-4-carbamyl-1,2,3-triazoles where the authors obtained EC₅₀ values around 9.9-16.5 μM against HSV-1 [23]. Recently, Cunha et al. [24] showed that a series of 1,2,3-triazole linked nitroxyl radical derived from TEMPOL inhibited in vitro replication of HSV-1. Four hybrids showed important anti-HSV-1 activity with 50% inhibitory concentration (IC₅₀) values that ranged from 0.80 to 1.32 μM. The potential of the 1,2,3-triazole scaffold present in some compounds with antiviral activity prompted us to investigate the anti-HSV-1 activity of a series of 1,4 disubstituted-1,2,3-triazoles, their cytotoxicity and the effects on viral particle, DNA replication and transcription and viral egress.

Methods

Chemistry

Compounds 1-4 (Figure 1) were previously synthesized and reported with potency against highly resistant glioblastoma cells [25]. Compounds 5a-c were recently synthesized as described below.

Figure 1

Cytotoxicity, antiviral activity and selectivity index^a

Selectivity index (SI) = CC₅₀/EC₅₀. CC₅₀, concentration that reduces the viability of host cells by 50%; EC₅₀, 50% effective drug concentration.

For the structural elucidation of the synthesized compounds, ¹H NMR and ¹³C NMR spectra were recorded at ambient temperature on a Bruker Avance III spectrometer (operating at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR; Billerica, MA, USA). The chemical shifts (δ) were given in parts per million (ppm) from internal tetramethylsilane on the d scale, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet). All coupling constants (J values) were given in Hz. Melting points were determined with an electrothermal, analogue model. Infrared spectra were performed using a Varian-3100 spectrometer (Agilent Technologies, Santa Clara, CA, USA). High-resolution mass spectra (HRMS) were obtained by Bruker MicrOTOF II instrument. Reactions were monitored by thin-layer chromatography using Merck TLC Silica gel 60 F254 (Merck, Darmstadt, Germany). Silica gel column chromatography was performed over Merck Silica gel 60 Å (particle size: 0.040-0.063 mm, 230-400 mesh ASTM). All reagents used were commercially obtained.

Synthesis of alkynes

The alkyne (2-(prop-2-yn-1-yloxy)benzaldehyde; 6) were prepared following the procedure described by Silva et al. [25].

2-Hydroxybenzaldehyde (5.3 mmol) and anhydrous K₂CO₃ (10.6 mmol) were added in a 50 ml two-necked flask and dissolved in 15 ml acetonitrile. After 15 min, propargyl bromide (80% in toluene, 6.4 mmol) was slowly added and the reaction mixture was refluxed under N₂ atmosphere for 4.5 h. Then the mixture was diluted with water (30 ml) and extracted with dichloromethane (3x20 ml). The combined organic layers were dried over Na₂SO₄ and the solvent was concentrated under reduced pressure. The desired alkyne (5) was obtained without further purification.

2-(prop-2-yn-1-yloxy)benzaldehyde

Yield 90%; white solid; MP: 67-69°C. ¹H NMR (400 MHz, CDCl₃) δ 10.49 (s, 1H, CHO), 7.87 (dd, J=7.7, 1.8 Hz, 1H, Ar-H), 7.58 (ddd, J=8.5, 7.3, 1.8 Hz, 1H, Ar-H), 7.16-6.95 (m, 2H, Ar-H), 4.84 (d, J=2.4 Hz, 2H, CH), 2.57 (t, J=2.4 Hz, 1H, CH). IR (KBr, v_max): 3,270 (°CH), 2,118 (C°C), 1,684 (C=O) cm-¹.

Synthesis of aryl-azides

The aryl-azides (7a-d) were prepared following the procedure of Wilkening et al. [26]. The aniline derivative (7.5 mmol) was dissolved in 5 ml of water and concentrated sulfuric acid (98%, 1.5 ml) and additional water (1.5 ml) was added. The suspension was cooled to 0°C and a solution of NaNO₂ (7.6 mmol) of water (1.5 ml) was slowly added under constant stirring. After 15 min, NaN₃ (9.3 mmol) was added and the mixture was stirred for additional 0.5-1 h. The reaction mixture was extracted with ethyl acetate (3x20 ml) and the combined organic fractions were washed with water (50 ml). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The desired azides (7a-d) were obtained without further purification.

4-azidobenzoic acid (7a)

Yield 80%; light yellow solid. MP: 185°C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H, OH), 7.96 (d, J=8.7 Hz, 2H, Ar-H), 7.22 (d, J=8.7 Hz, 2H, Ar-H). IR (KBr, v_max): 2,105 (N°N), 1,284 (C-O) cm⁻¹.

1-azido-4-nitrobenzene (7b)

Yield 60%; dark yellow solid. MP: 65-67°C. ¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J=9.1 Hz, 2H, Ar-H), 7.14 (d, J=9.1 Hz, 2H, Ar-H). IR (KBr, v_max): 2,110 (N°N) cm⁻¹.

1-azido-4-bromobenzene (7c)

Yield 75%; brown liquid. ¹H NMR (400 MHz, CDCl₃) δ 7.48 (d, J=8.8 Hz, 2H, Ar-H), 6.93 (d, J=8.8 Hz, 2H, Ar-H). IR (KBr, v_max): 2,114 (N°N) cm⁻¹.

General procedure for the synthesis of triazoles

The triazoles (5a-c) were prepared following the procedure described by Rostovtsev et al. [27]. The alkyne (0.6 mmol) and aryl-azide (0.6 mmol) were added to a 1:1 mixture of water and tert-butyl alcohol (6 ml). Sodium ascorbate (0.06 mmol, in 200 m l of water) was added, followed by copper (II) sulphate pentahydrate (0.006 mmol, in 100 m l of water). The reaction mixture was stirred vigorously at room temperature and monitored by thin-layer chromatography until the reagents were completely consumed. At the end of the reaction the mixture was diluted with ice water (50 ml), the precipitate was collected by filtration, washed with cold water (2x25 ml) and dried under vacuum. The procedures for compounds 1-4 are described by Silva et al. [25].

4-(4-((4-formyl-2-methoxyphenoxy) methyl)-1H-1,2,3-triazol-1-yl)benzoic acid (1)

Yield 80%; white solid; reaction time: 8 h, MP: 238-239°C. ¹H NMR (400 MHz, DMSO) δ 13.27 (s, 1H), 9.87 (s, 1H), 9.11 (s, 1H, triazole-H), 8.18-8.05 (m, 4H), 7.59 (dd, J=8.2, 1.8 Hz, 1H), 7.46-7.41 (m, 2H), 5.38 (s, 2H), 3.82 (s, 3H). ¹³C NMR (101 MHz, DMSO-d6) δ 191.65, 152.88, 149.32, 143.67, 139.51, 129.93, 125.81, 123.43, 112.72, 109.72, 61.14, 55.14. HRMS(ESI) m/z calculated for C₁₈H₁5N₃O₅+Na [M+Na]⁺, 376.0903; found 376.0904.

(E)-4-(phenoxymethyl)-1-(2-((2-phenylhydrazono) methyl)phenyl)-1H-1,2,3-triazole (2)

Yield 70%; white solid; MP: 128-129°C. ¹H NMR (400 MHz, CDCl₃) δ 8.25 (dd, J=8.0, 1.1 Hz, 1H), 7.89 (s, 1H), 7.77 (s, 1H), 7.56 (t, J=7.1 Hz, 1H), 7.44 (td, J=7.6, 1.4 Hz, 1H), 7.39-7.33 (m, 4H), 7.32-7.29 (m, 1H), 7.11-7.01 (m, 5H), 6.92 (t, J=7.3 Hz, 1H), 5.37 (s, 2H).¹³C NMR (101 MHz, CDCl₃) d 158.11, 144.05, 134.23, 131.29, 130.81, 130.14, 129.66, 129.32, 128.51, 126.57, 126.07, 121.45, 120.61, 114.92, 112.87, 61.85. HRMS(ESI) m/z calculated for C₂₂H₁₉N₅O+Na [M+Na]⁺, 392.1481; found 392.1482.

(E)-4-methyl-N′-(2-(4-(phenoxymethyl)-1H-1,2,3-triazol1yl)benzylidene)benzenesulfonohydrazide (3)

Yield 65%; white solid; MP: 167-169°C. ¹H NMR (400 MHz, CDCl3) δ 8.57 (s, 1H), 8.09 (dd, J=7.3, 2.1 Hz, 1H), 7.82–7.79 (m, 3H), 7.54–7.48 (m, 3H), 7.35–7.27 (m, 4H), 7.05–6.95 (m, 3H), 5.26 (s, 2H), 2.39 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 157.97, 144.63, 144.14, 141.29, 135.47, 130.66, 130.21, 129.65, 129.12, 127.84, 127.32, 125.74, 125.29, 121.51, 114.82, 61.58, 21.58. HRMS(ESI) m/z calculated for C₂₃H₂₁N₅O₃S+Na [M+Na]⁺, 470.1265; found 470.1261.

2,2′-(4,4′-((1,3-phenylenebis(oxy))bis(methylene)) bis(1H-1,2,3-triazole-4,1 diyl)) dibenzaldehyde (4)

The crude product was purified by fash column chromatography using ethyl acetate/hexane (20:80) as eluent. The product was obtained as a light yellow solid in 50% yield. Reaction time: 16 h, MP: 106-107°C. ¹H NMR (400 MHz, CDCl₃) δ 9.91 (s, 2H), 8.11 (dd, J=7.8, 1.5 Hz, 2H), 8.05 (s, 2H, triazole-H), 7.77 (td, J=7.7, 1.6 Hz, 2H), 7.68 (t, J=7.5 Hz, 2H), 7.54 (dd, J=7.9, 0.9 Hz, 2H), 7.25 (t, J=8.2 Hz, 1H), 6.73 (t, J=2.3 Hz, 1H), 6.69 (dd, J=8.2, 2.4 Hz, 1H), 5.33 (s, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 189.17, 160.18, 145.69, 138.87, 135.30, 131.14, 131.08, 130.83, 130.34, 126.18, 125.51, 108.51, 102.95, 62.87. HRMS(ESI) m/z calculated for C₂₆H₂₀N₆O₄+Na [M+Na]⁺, 503.1438; found 503.1438.

4-(4-((2-formylphenoxy) methyl)-1H-1,2,3-triazol-1-yl) benzoic acid (5a)

Yield 85%; white solid; reaction time: 8 h; MP: 265-267°C. RMN ¹H (400 MHz, DMSO) d 10.44 (s, 1H), 9.13 (s, 1H, triazole-H) 8.12 (d, J=12.3 Hz, 4H), 7.82–7.62 (m, 2H), 7.48 (d, J=8.3 Hz, 1H), 7.13 (t, J=7.5 Hz, 1H), 5.47 (s, 2H). ¹³C NMR (101 MHz, DMSO) d 189.41, 160.36, 136.39, 127.61, 124.53, 122.82, 121.29, 114.15, 62.17. HRMS(ESI) m/z calculated for C₁₇H₁₃N₃O₄+Na [M+Na]⁺, 346.0798; found 346.0795.

2-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl) methoxy) benzaldehyde (5b)

Yield 60%; yellow solid; reaction time: 24 h; MP: 240-241°C. ¹H NMR (400 MHz, DMSO) d 10.45 (s, 1H), 9.23 (s, 1H, triazole-H), 8.47 (d, J=9.1 Hz, 2H), 8.26 (d, J=9.1 Hz, 2H), 7.76–7.67 (m, 2H), 7.48 (d, J=8.4 Hz, 1H), 7.13 (t, J=7.5 Hz, 1H), 5.48 (s, 2H). ¹³C NMR (101 MHz, DMSO) d 189.89, 160.79, 147.29, 144.84, 141.25, 136.87, 128.11, 126.06, 125.06, 123.60, 121.83, 121.22, 114.63, 62.65. HRMS(ESI) m/z calculated for C₁₆H₁₂N₄O₄+Na [M+Na]⁺, 347.0756; found 347.0750.

2-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl) methoxy)benzaldehyde (5c)

Yield 65%; light yellow solid; Reaction time: 8 h; ¹H NMR (400 MHz, DMSO) d 10.43 (s, 1H), 9.06 (s, 1H, triazole-H), 7.92 (d, J=8.9 Hz, 2H), 7.82 (d, J=8.9 Hz, 2H), 7.74-7.67 (m, 2H), 7.48 (d, J=8.3 Hz, 1H), 7.14 (dd, J=12.4, 4.9 Hz, 1H), 5.46 (s, 2H). ¹³C NMR (101 MHz, DMSO) d 189.37, 160.27, 143.84, 136.38, 132.78, 127.60, 124.51, 122.74, 122.06, 121.26, 114.13, 62.16. HRMS(ESI) m/z calculated for C₁₆H₁₂BrN₃O2+Na [M+Na]⁺, 380.0011; found 380.0004.

Cell lines and HSV-1 strains

The 1,2,3-triazole derivatives were diluted at the concentration of 50 mM in dimethylsulfoxide (DMSO) and stored at -20°C. We tested ACV as a reference antiviral drug to compare our data.

These compounds were evaluated in vitro on human fibroblast (HFL1 ATCC® CCL-153^™) cells which were cultivated at 37°C and 5% CO₂ with Dulbecco's Modification of Eagle's medium (DMEM), with 4.5 g/l glucose, l-glutamine and sodium pyruvate (Sigma-Aldrich, Saint Louis, MO, USA), supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), and 1% of l-glutamine-penicillin-streptomycin solution (Sigma-Aldrich).

To perform the plaque assay, we used rabbit skin (RS) cells cultivated at 37°C and 5% CO₂ with Minimum Essential Medium Eagle (MEM), with Earle's salts and l-glutamine (Sigma-Aldrich), supplemented with 5% bovine serum (Gibco) and 1% of l-glutamine-penicillin-streptomycin solution (Sigma-Aldrich).

The cells were cultivated until they achieved around 90% of confluence when they were transferred to plates of 24 or 96 wells depending on the assay.

HSV-1 strain 17syn+ was used for the experiments. We also tested the compounds on the ACV-resistant clinical strain HO-1 (kindly provided by the D Phelan, College of Medicine, University of Florida, personal communication). Virus stock cultures were prepared from supernatants of infected cells and stored at -80°C until use.

Antiviral activity evaluation

Antiviral activity of the compounds was first assayed by acute HSV-1 infection on HFL-1 cells. HFL cells were seeded in 24-multiwell plates (1x10⁵ cell/well) and incubated at 37°C and 5% CO₂ atmosphere until a complete confluence of the cells (24 h approximately). Then, we infected the cells with HSV-1 strain 17syn+, in a multiplicity of infection (MOI) of 1.0, for 1 h at 37°C and 5% CO₂ Three wells were not infected to be the control of cell growth. Then, the viral inoculum was removed, the cells were washed with phosphate-buffered saline (PBS), and treated with 50 μM of the compounds in DMEM. DMEM without any compound was used as a control of HSV-1 yield and DMSO was also tested to verify its influence in the HSV-1 yield. At 24 h postinfection (hpi), cells were lysed with 3 cycles of freezing and thawing to collect the extra and intracellular content.

Plaque assay

To determine the viral titre of the experimental groups, the plaque assay was performed according to Lucero et al. [28], with few modifications. RS cells were seeded on 24-multiwell plates in a density of 1x10⁵ cells/well and incubated at 37°C and 5% CO₂ until the confluence of the cells. Then, the samples were serial diluted (1:10) and RS cells were infected and incubated for 1 h at 37°C and 5% CO₂. The supernatant was aspirated, the cells were washed with PBS and the monolayer was covered with MEM. MEM and 0.3% of immunoglobulin G (IgG) from human serum (Sigma) for 48 h. Then, the monolayer was fixed and stained with 0.5% violet crystal and 20% methanol. The viral titre was determined according to the number of viral plaque-forming units per ml (PFU/ml).

Determination of EC₅₀

In order to determine the EC₅₀ of the active compounds, HFL1 cells were seeded in 24-multiwell plates (1x10⁵ cells/well) and after cell confluence they were infected with HSV-1 strain 17syn+ (MOI of 1.0) for 1 h at 37°C and 5% CO₂. Then, the viral inoculum was removed, the cells were washed and treated with different concentrations (6.25, 12.5, 25 and 50 μM) of the compounds for 24 h. We lysed the cells with 3 cycles of freezing and thawing to collect the samples for the plaque assay that was performed as described under Plaque assay. The EC₅₀ value was determined by linear regression compared with an infected and untreated control.

Cytotoxicity assay

Cytotoxicity values of the compounds were determined using CellTiter-Glo(R) assay (Promega, Madison, WI, USA). This assay determines the number of viable cells based on quantitation of ATP present, which indicates the presence of metabolically active cells, according to the manufacturer [29]. HFL1 cells were cultivated in 96-multiwell plates (5x10⁴ cells/well) and incubated at 37°C for 24 h and 5% CO₂. The cells were treated with the compounds in different concentrations (50, 250, 500 and 1,000 μM) diluted in DMEM, and the plates were incubated at 37°C for 24 h and 5% CO₂. Then, 100 m l of the CellTiter-Glo® solution was added to each well. After 12 min stirring at room temperature, the luminescence was recorded in the GloMax® Luminometer (Promega). The concentration that reduces the viability of host cells by 50% (CC₅₀) was calculated by linear regression analysis of the dose-response curves.

Antiviral activity on resistant HSV-1 strain

The most promising compounds 3 and 4 were tested on the ACV-resistant strain HO-1. HFL1 cells were infected with HO-1 (MOI 0.1) for 1 h. Then, the cells were washed and treated with the compound at 50 μM for 24 h at 37°C and 5% CO₂. After lysing of the cells, the content was harvested and titred as described in Antiviral activity evaluation.

Virucidal assay

The virucidal assay was carried out according to Schuhmacher et al. [30] with some modifications. HSV-1 strain 17syn+ diluted in DMEM (MOI 1.0) was pretreated with the compounds 1 and 4 or remained untreated for 4 h at 4°C. Then, HFL1 cells on the 24-wells plate were infected with the virus for 1 h at 37°C. Viral suspension was removed, the cells were washed with PBS and covered with DMEM. After 24 h at 37°C and 5% CO₂, the cells were lysed and the content was harvested to be titred as described in Antiviral activity evaluation. The number of PFU/ml of the treated groups was compared with the untreated group.

Egress assay

In order to evaluate the interference of the compounds on virus egress from the cells, we infected HFL1 cells (1x10⁵ cells/well in 24-well plates) for 1 h with a MOI of 1.0. The viral inoculum was removed, the cells were washed and treated with the compounds (50 μM) for 24 h, at 37°C and 5% CO₂. Then, the supernatant correspondent to the virus on the extracellular environment was collected. Cells were washed with PBS, covered with DMEM and lysed by 3 cycles of freezing and thawing to harvest the intracellular viral particles. These samples were diluted and submitted to the plaque assay, as described in Plaque assay.

Time-course assay

In order to determine in which phase of infection the compounds would be more effective, we performed time-course assays. HFL1 cells on 24-well plates were infected with HSV-1 strain 17syn+ (MOI of 1.0) for 1 h at 37°C and 5% CO₂. Then, the viral inoculum was removed, the cells were washed and treated with the compounds at 50 μM for 2, 4 or 8 hpi. Then, we harvested the content for the plaque assay.

Real-time quantitative PCR

HFL1 cells were infected with HSV-1 17syn+ (MOI of 1.0) for 1 h. Then, they were treated with compounds at 50 μM for 8 h. The supernatant was removed, the cells were washed with PBS and the total RNA and DNA were extracted using 500 ml of TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA).

Then, 100 μl of chloroform was added into each tube and after 3 min the samples were centrifuged at 12,000xg for 15 min at 4°C. The upper aqueous phase was collected in another tube for RNA extraction and the bottom organic phase was directed for DNA extraction (Zymo Research, Irvine, CA, USA).

RNA extraction was performed using Direct-zol® reagents according to the manufacturer instructions. The RNA samples were treated with DNase (Turbo DNA-free^™ Kit; Thermo Fisher Scientific, Carlsbad, CA, USA) and cDNA was obtained by reverse transcriptase reactions using Omniscript® Reverse Transcription Kit (Qiagen, Germantown, MD, USA) according to its protocol.

For DNA extraction the bottom phase was incubated for 3 min with 100% ethanol, centrifuged at 2,000xg for 5 min at 4°C. The supernatant was discarded and the pellet was resuspended in 0.1 M sodium citrate in 10% ethanol (pH 8.5). After 30 min, the samples were centrifuged at 2,000xg for 5 min at 4°C. This step was repeated. Then, the pellet was resuspended in 75% ethanol and after 20 min at room temperature, the samples were centrifuged at 2,000xg for 5 min at 4°C. The supernatant was discarded and the DNA pellet was dried and resuspended in 8 mM NaOH.

Quantitative PCR (qPCR) was performed using 20 ng input DNA, TaqMan® Fast Universal PCR 2X Master Mix (Applied Biosystems, Austin, TX, USA) with TaqMan® probes (Applied Biosystems) and target-specific primers (Applied Biosystems) and HSV-1 polymerase. Samples were running on a 7900HT Fast Real-Time PCR System (Applied Biosystems).

Results

Synthesis of 1,4-disubstituted-1,2,3-triazoles

As described by Silva et al. [25], the key step for the obtention of 1,4-disubstituted-1,2,3-triazoles (1-4) was the copper-catalysed azide-alkyne cycloaddition, click chemistry reaction. Compounds 5a-c were also obtained in good yields, by click reaction between 2-(prop-2-yn-1-yloxy(benzaldehyde) (6) and arylazides (7a-c), using one equivalent of corresponding azides and alkynes (Figure 2). These kinds of 1,4-disubstituted-1,2,3-triazoles, similar to compounds 5a-c, have been also described and characterized by Gupta et al. [31].

Figure 2

Chemical structure and synthesis of 1,4-disubstituted-1,2,3-triazoles compounds evaluated against HSV-1 [25]

Antiviral activity

All 1,4 disubstituted-1,2,3-triazoles were tested at 50 μM on HSV-1 17syn+ for 24 hpi and compounds 1, 2, 3, 4 and 5a showed antiviral activity, while 5b and 5c had no effect on viral yield. The active compounds were tested in different concentrations on HFL1 cells infected with HSV-1 17syn+ to determine the EC₅₀. The compounds with the higher antiviral activity were 3 and 4, with EC₅₀ of 16 and 21 μM, respectively (Figure 1).

Cytotoxicity assay

To determine the CC₅₀ of the compounds with antiviral activity, we evaluated them in different concentrations on HFL1 cells and the viability of the cells was determined indirectly by means of the quantification of ATP, which indicates the presence of metabolically active cells [29]. The CC₅₀ of the compounds were between 285 and 2,593 μM (Figure 1).

We also calculated the selectivity index (SI), obtained by the ratio between CC₅₀ and EC₅₀. The SI of the compounds were between 6.5 and 123.5.

Antiviral activity on resistant HSV-1 strains

Compounds 3 and 4 were tested on the ACV-resistant HSV-1 strain HO-1. Compound 4 was not able to control the yield of this strain. However, compound 3 showed remarkable antiviral activity against HO-1, with 75% reduction of PFU, whereas ACV reduced only 8.8%.

Effect on the viral particles

In order to evaluate whether the compounds could affect the viral particles, we pretreated HSV-1 17syn+ for 4 h with compounds 3 and 4 before infection and estimated the viral titre 24 hpi. Although the group treated with compound 3 showed fewer PFU/ml than the untreated group and compound 4 slightly higher, these differences were not significant. Therefore, the compounds did not affect the viral particles and the viral titre was similar in the untreated and treated groups.

Effects on Viral Egress from the Cells

We performed the egress assay to analyse whether the compounds could disturb the release of virus from the cells. We could verify that the compounds had a lower viral yield in intracellular content than the untreated group. However, compound 4 showed similar viral yield in the extracellular content, while 3 decreased the viral titre in the extracellular content compared with the untreated group (Figure 3). Additionally, the ratio of the intracellular/extracellular contents was 1.8 for the untreated group, 0.98 for 4 and 9.3 for 3, which suggests that compound 3 may interfere on the virus egress from the cell, or in some related process.

Figure 3

Viral titre on intra- and extracellular content to evaluate the effect of the compounds on the virus egress from cells

Time-course assay

To evaluate which infection phase the compounds could affect, we performed viral plaques at 2, 4 and 8 hpi. The compounds did not affect the virus titre at 2 hpi and they had some effect at 4 hpi, but it was not significant. However, at 8 hpi compound 3 reduced 96% ±0.6% of the virus titre and compound 4 reduced 95% ±2% (Figure 4).

Figure 4

Time-course for viral titre^a

Multiplicity of infection 1.0 on HFL cells.

Effects on HSV-1 DNA replication and transcription

In order to investigate whether these compounds may alter HSV-1 DNA replication and transcription, we performed qPCR during acute infection in HFL cells at 8 hpi, when the compounds showed significant antiviral activity. The number of DNA relative quantity was similar in the treated and untreated groups (Figure 5A), which means that the compounds did not affect DNA replication.

Figure 5

Quantitative PCR assays for HSV-1 cDNA copy number normalized with GAPDH

After production of cDNA from the RNA extraction, qPCR was performed for ICP0 and ICP4 (immediate early genes), UL30 and TK (early genes) and ICP34.5 and gC (late genes). Compounds 3 and 4 were able to suppress significantly the transcription of the immediate early genes ICP0 and ICP4, and the late gene gC.

Compound 4 also reduced significantly the transcription of UL30 and ICP34.5 (Figure 5B).

Discussion

This study evaluated a series of 1,2,3-triazole derivatives, which showed notable antiviral activity against HSV-1 17syn+, and the most promising compounds were 3 and 4. Compound 4 caused low cytotoxicity and showed the highest SI (SI=CC₅₀/EC₅₀), which is a pharmaceutical parameter to determine the security range for in vitro use. Compound 3 had moderate cytotoxicity, although its CC₅₀ was more than 17-fold the EC₅₀, offering a security range to manage dosing.

Similarly, other triazole derivatives were evaluated for anti-HSV-1 activity. Arylsulfonylhydrazide-1,2,3-triazoles derivatives presented EC₅₀ between 1.3 and 37 μM on viral infection in vitro [32]. Pandey et al. [33] found 2,3,4-triazole derivatives with EC₅₀ between 150 and 250 μg/ml. In another study, among 18 1,2,4-triazole derivatives, only one showed promising antiviral activity against HSV-1, with 88% reduction in plaque formation at 50 μg/ml [34]. However, none of these studies have addressed the possible mechanisms of action of these compounds.

Compounds 3 and 4 were also tested on a clinical ACV-resistant strain, HO-1, and compound 3 had satisfactory antiviral activity against this strain. This fact demonstrates the importance of searching for new compounds with antiviral activity that could serve as alternative treatments of resistant strains, which are prevalent especially in immunocompromised patients [12–14]. Additionally, we suggest that compound 3 may have a different mechanism of action from ACV and other nucleosides analogues.

In order to understand the mode of action of these compounds responsible for the antiviral effect, we performed experiments, such as virucidal assay, time-course, egress assay and qPCR. We verified that the compounds did not have virucidal activity because the pretreatment of viruses did not result in reduction of plaques, suggesting the compounds did not affect the virion envelope structure, and the free viruses were not sensitive to them. Also, we can infer that the molecules did not affect the processes of adsorption and entry into host cells.

The results from the time-course assay showed the both compounds had effective antiviral activity at 8 hpi. The cellular replication cycle of HSV takes 3–12 h [35], thus after 8 h most of the viral particles might be in the late phase of the lytic infection. In this phase, there is, predominately, the production of the proteins from tegument and envelope, and proteins for the assembly of the viral particle [36].

Our compounds did not affect DNA replication. This is the main mechanism of action of ACV and its analogues, which inhibit the DNA polymerase and interrupt viral DNA elongation [10]. Thus, this further supports that the 1,2,3-triazole derivatives presented in this study have a different mechanism of action from the current anti-HSV-1 drugs.

1,2,3-Triazole derivatives showed efficacy on gene transcription suppression. Compounds 3 and 4 had significant activity on the immediate early genes ICP0 and ICP4, which are upstream genes of the expression cascade of viral genes and their proteins regulate the transcription of genes from the following phases [37]. Both compounds also reduced significantly the expression of gC gene. gC is one of the glycoproteins of the viral envelope that is produced during the late phase and it is involved in the initial attachment phase through the binding to glycosaminoglycan of the plasma membrane [38].

Compound 4 also had significant activity on the expression of UL30 and ICP34.5. UL30 is a gene of the early phase of the lytic infection and with UL42 encode the viral DNA polymerase [39], an enzyme that catalyses the elongation of viral DNA and it is the main target for anti-HSV drugs [10]. The suppression of UL30 gene is relevant for the development of a new antiviral agent that will be able to have a possible synergic effect with ACV. Inhibiting the ICP34.5 gene is also interesting as an antiviral candidate because this is a major viral neurovirulence factor and is essential for efficient viral replication in neurons [40]. Furthermore, previous studies reported the importance of ICP34.5 in the viral spread and in the progress from lytic infection to latency and reactivation [41,42].

Additionally, in the egress assay, we noted that compound 3 induced the highest reduction of extracellular virus titre and the intra- and extracellular ratio was 9.3, whereas it was 1.8 in the untreated group, suggesting that this compound interfered with virus release from the cells or in some maturation process. It is plausible that this compound has extensive activity in the late phase during which structural proteins that compose the virion are produced [43]. Consequently, this compound may be interfering in the previous step of egress, the envelopment of capsids by Golgi vesicles, which is mediated by interactions between tegument proteins and cytoplasmatic portions of viral glycoproteins binding to the Golgi membrane [44]. Although the mechanism of action of drugs that disturb viral egress is unclear, brefeldin A, a drug that inhibits transport from the endoplasmic reticulum to the Golgi could block HSV-1 nuclear egress [45]. Combined, these results suggest that compounds 3 and 4 have promising profiles as antiviral agents with possible targets related to transcription and the viral egress for compound 3.

In conclusion, we presented a series of 1,2,3-triazole derivatives with antiviral activity and compounds 3 and 4 were the most promising. Our findings revealed that antiviral activity of these compounds is closely related to the inhibition of the expression of some important genes such as ICP0, ICP4 and gC. Compound 3 also affected the release of the viral particle from the host cell and showed activity against the ACV-resistant HSV-1 strain. These results suggest that the mechanism of action of 1,2,3-triazole derivatives is distinct from those of ACV and its analogues, which makes them even more interesting for antiviral development because they might be an alternative for HSV-1 treatment, particularly for resistant strains.

Footnotes

Acknowledgements

Daiane de Jesus Viegas had postgraduate scholarships from the Brazilian agency: Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES) by the Doctoral Program Sandwich Abroad (Programa de Doutorado Sanduíche no Exterior – PDSE; process: 88881.132603/2016-01).

The authors declare no competing interests.

Additional file 1: Further information on ¹H NMR and ¹³C NMR spectra of all the final compounds can be found at

References

Fatahzadeh

, Schwartz

R.A.

Human herpes simplex virus infections: Epidemiology, pathogenesis, symptomatology, diagnosis, and management. J Am Acad Dermatol 2007; 57: 737–763.

Sukik

, Alyafei

, Harfouche

, Abu-Raddad

L.J.

Herpes simplex virus type 1 epidemiology in Latin America and the Caribbean: systematic review and meta-analytics. PLoS One 2019; 14: e0215487.

Flores

, Nakayama

, Whisnant

A.W.

, Javanbakht

, Cullen

B.R.

, Bloom

D.C.

Mutational inactivation of herpes simplex virus 1 microRNAs identifies viral mRNA targets and reveals phenotypic effects in culture. J Virol 2013; 87: 6589–6603.

Piacentini

, De Chiara

, Li Puma

D.D.

HSV-1 and Alzheimer's disease: more than a hypothesis. Front Pharmacol 2014; 5: 97.

Agostini

, Mancuso

, Baglio

High avidity HSV-1 antibodies correlate with absence of amnestic mild cognitive impairment conversion to Alzheimer's disease. Brain Behav Immun 2016; 58: 254–260.

Itzhaki

R.F.

Corroboration of a major role for herpes simplex virus type 1 in Alzheimer's disease. Front Aging Neurosci 2018; 10: 324.

Itzhaki

R.F.

, Lin

W-R

, Shang

, Wilcock

G.K.

, Faragher

, Jamieson

G.A.

Herpes simplex virus type 1 in brain and risk of Alzheimer's disease. Lancet 1997; 349: 241–244.

Itzhaki

R.F.

, Lathe

Herpes viruses and senile dementia: first population evidence for a causal link. J Alzheimers Dis 2018; 64: 363–366.

Strasfeld

, Chou

Antiviral drug resistance: mechanisms and clinical implications. Infect Dis Clin North Am 2010; 24: 413–437.

10.

Elion

G.B.

Acyclovir: Discovery, mechanism of action, and selectivity. J Med Virol 1993; 41 Suppl 2: 2–6.

11.

De Clercq

A 40-year journey in search of selective antiviral chemotherapy. Annu Rev Pharmacol Toxicol 2011; 51: 1–24.

12.

Christophers

, Clayton

, Craske

Survey of resistance of herpes simplex virus to acyclovir in northwest England. Antimicrob Agents Chemother 1998; 42: 868–872.

13.

Stránská

, Schuurman

, Nienhuis

Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization. J Clin Virol 2005; 32: 7–18.

14.

Frobert

, Burrel

, Ducastelle-Lepretre

Resistance of herpes simplex viruses to acyclovir: an update from a ten-year survey in France. Antiviral Res 2014; 111: 36–41.

15.

Piret

, Boivin

Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management. Antimicrob Agents Chemother 2011; 55: 459–472.

16.

Bacon

T.H.

, Levin

M.J.

, Leary

J.J.

, Sarisky

R.T.

, Sutton

Herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy. Clin Microbiol Rev 2003; 16: 114–128.

17.

Jiang

Y-C

, Feng

, Lin

Y-C

, Guo

X-R

. New strategies against drug resistance to herpes simplex virus. Int J Oral Sci 2016; 8: 1–6.

18.

De Clercq

Dancing with chemical formulae of antivirals: a panoramic view (part 2). Biochem Pharmacol 2013; 86: 1397–1410.

19.

Pires de Mello

C.P.

, Bloom

D.C.

, Paixão

I.C.

Herpes simplex virus type-1: replication, latency, reactivation and its antiviral targets. Antivir Ther 2016; 21: 277–286.

20.

Ferreira M de

L.G.

, Pinheiro

L.C.S.

, Santos-Filho

O.A.

Design, synthesis, and antiviral activity of new 1H-1,2,3-triazole nucleoside ribavirin analogs. Med Chem Res 2014; 23: 1501–1511.

21.

Bonandi

, Christodoulou

M.S.

, Fumagalli

, Perdicchia

, Rastelli

, Passarella

The 1,2,3-triazole ring as a bioisostere in medicinal chemistry. Drug Discov Today 2017; 22: 1572–1581.

22.

Jordão

A.K.

, Ferreira

V.F.

, Souza

T.M.L.

Synthesis and anti-HSV-1 activity of new 1,2,3-triazole derivatives. Bioorg Med Chem 2011; 19: 1860–1865.

23.

Wen

, Zhang

, Liu

N-N

. Synthesis and antiviral activity of 5-(Benzylthio)-4-carbamyl-1,2,3-triazoles against human cytomegalovirus (Cmv) and varicella-zoster virus (Vzv). Med Chem 2017; 13: 453–464.

24.

Cunha

A.C.

, Ferreira

V.F.

, Vaz

M.G.F.

Chemistry and anti-herpes simplex virus type 1 evaluation of 4-substituted-1H-1,2,3-triazole-nitroxyl-linked hybrids. Mol Divers 2020; doi: 10.1007/s11030-020-10094-2.

25.

da Silva

V.D.

, de Faria

B.M.

, Colombo

Design, synthesis, structural characterization and in vitro evaluation of new 1,4-disubstituted-1,2,3-triazole derivatives against glioblastoma cells. Bioorg Chem 2019; 83: 87–97.

26.

Wilkening

, del Signore

, Hackenberger

C.P.R.

Synthesis of phosphonamidate peptides by Staudinger reactions of silylated phosphinic acids and esters. Chem Commun (Camb) 2011; 47: 349–351.

27.

Rostovtsev

V.V.

, Green

L.G.

, Fokin

V.V.

, Sharpless

K.B.

A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed Engl 2002; 41: 2596–2599.

28.

d'A Lucero

, Gomes

C.R.B.

, de

P.P.

, Frugulhetti

I.C.

Synthesis and anti-HSV-1 activity of quinolonic acyclovir analogues. Bioorg Med Chem Lett 2006; 16: 1010–1013.

29.

Promega. CellTiter-Glo® One solution Assay. Technical bulletin TB370. (Updated February 2016. Accessed 01 November 2017.) Available from www.promega.com/products/cell-health-assays/cell-viability-andcytotoxicityassays/celltiter_glo-one-solution-assay/?catNum=G8461.

30.

Schuhmacher

, Reichling

, Schnitzler

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro. Phytomedicine 2003; 10: 504–510.

31.

Gupta

, Jamatia

, Mahato

, Pal

A.K.

Metalloprotein-inspired ruthenium polymeric complex: a highly efficient catalyst in parts per million level for 1,3-dipolar huisgen's reaction in aqueous medium at room temperature. Ind Eng Chem Res 2017; 56: 2375–2382.

32.

Jordão

A.K.

, Ferreira

V.F.

, Souza

T.M.L.

Synthesis and anti-HSV-1 activity of new 1,2,3-triazole derivatives. Bioorg Med Chem 2011; 19: 1860–1865.

33.

Pandey

V.K.

, Tusi

, Joshi

Synthesis and biological evaluation of some novel 5-[(3-aralkyl amido/imidoalkyl) phenyl]-1,2,4-triazolo[3,4- b]-1,3,4-thiadiazines as antiviral agents. ISRN Org Chem 2012; 2012: 760517.

34.

Abdel-Hafez

A.A.

, Elsherief

H.A.H.

, Jo

Synthesis and evaluation of anti-HIV-1 and anti-HSV-1 activities of 4H-[1,2,4]-triazolo [1,5-a]pyrimidin-5-one derivatives. Arzneimittelforschung 2002; 52: 833–839.

35.

Wiedbrauk

D.L.

Herpes simplex virus. In Grody

W.W.

, Nakamura

R.M.

, Kiechle

F.L.

, Strom

C.M.

(Editors). Molecular Diagnostics. 1st ed. San Diego: Elsevier 2010: pp. 453–460.

36.

Boehmer

P.E.

, Lehman

I.R.

Herpes simplex virus DNA replication. Annu Rev Biochem 1997; 66: 347–384.

37.

Smibert

C.A.

, Smiley

J.R.

Differential regulation of endogenous and transduced beta-globin genes during infection of erythroid cells with a herpes simplex virus type 1 recombinant. J Virol 1990; 64: 3882–3894.

38.

Laquerre

, Argnani

, Anderson

D.B.

, Zucchini

, Manservigi

, Glorioso

J.C.

Heparan sulfate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J Virol 1998; 72: 6119–6130.

39.

Parry

M.E.

, Stow

N.D.

, Marsden

H.S.

Purification and properties of the herpes simplex virus type 1 UL8 protein. J Gen Virol 1993; 74: 607–612.

40.

Tang

, Guo

, Patel

, Krause

P.R.

Herpes simplex virus 2 expresses a novel form of icp34. 5, a major viral neurovirulence factor, through regulated alternative splicing. J Virol 2013; 87: 5820–5830.

41.

Whitley

R.J.

, Kern

E.R.

, Chatterjee

, Chou

, Roizman

Replication, establishment of latency, and induced reactivation of herpes simplex virus gamma 1 34.5 deletion mutants in rodent models. J Clin Invest 1993; 91: 2837–2843.

42.

Spivack

J.G.

, Fareed

M.U.

, Valyi-Nagy

Replication, establishment of latent infection, expression of the latency-associated transcripts and explant reactivation of herpes simplex virus type 1 34.5 mutants in a mouse eye model. J Gen Virol 1995; 76: 321–332.

43.

Whitley

R.J.

, Roizman

Herpes simplex virus infections. Lancet 2001; 357: 1513–1518.

44.

Melancon

J.M.

, Foster

T.P.

, Kousoulas

K.G.

Genetic analysis of the herpes simplex virus type 1 ul20 protein domains involved in cytoplasmic virion envelopment and virus-induced cell fusion. J Virol 2004; 78: 7329–7343.

45.

Sciaky

, Presley

, Smith

Golgi tubule traffic and the effects of brefeldin a visualized in living cells. J Cell Biol 1997; 139: 1137–1155.

46.

Livak

K.J.

, Schmittgen

T.D.

Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods 2001; 25: 402–408.

Antiviral Activity of 1,4-Disubstituted-1,2,3-Triazoles against HSV-1 in vitro

Abstract

Background

Methods

Results

Conclusions

Introduction

Methods

Chemistry

Synthesis of alkynes

2-(prop-2-yn-1-yloxy)benzaldehyde

Synthesis of aryl-azides

4-azidobenzoic acid (7a)

1-azido-4-nitrobenzene (7b)

1-azido-4-bromobenzene (7c)

General procedure for the synthesis of triazoles

4-(4-((4-formyl-2-methoxyphenoxy) methyl)-1H-1,2,3-triazol-1-yl)benzoic acid (1)

(E)-4-(phenoxymethyl)-1-(2-((2-phenylhydrazono) methyl)phenyl)-1H-1,2,3-triazole (2)

(E)-4-methyl-N′-(2-(4-(phenoxymethyl)-1H-1,2,3-triazol1yl)benzylidene)benzenesulfonohydrazide (3)

2,2′-(4,4′-((1,3-phenylenebis(oxy))bis(methylene)) bis(1H-1,2,3-triazole-4,1 diyl)) dibenzaldehyde (4)

4-(4-((2-formylphenoxy) methyl)-1H-1,2,3-triazol-1-yl) benzoic acid (5a)

2-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl) methoxy) benzaldehyde (5b)

2-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl) methoxy)benzaldehyde (5c)

Cell lines and HSV-1 strains

Antiviral activity evaluation

Plaque assay

Determination of EC50

Cytotoxicity assay

Antiviral activity on resistant HSV-1 strain

Virucidal assay

Egress assay

Time-course assay

Real-time quantitative PCR

Results

Synthesis of 1,4-disubstituted-1,2,3-triazoles

Antiviral activity

Cytotoxicity assay

Antiviral activity on resistant HSV-1 strains

Effect on the viral particles

Effects on Viral Egress from the Cells

Time-course assay

Effects on HSV-1 DNA replication and transcription

Discussion

Footnotes

Acknowledgements

References

Determination of EC₅₀