Benzocaine-N-acylindoline Conjugates: Synthesis and Antiviral Activity Against Coxsackievirus B3

doi:10.21203/rs.3.rs-3447939/v1

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Research Article

Benzocaine-N-acylindoline Conjugates: Synthesis and Antiviral Activity Against Coxsackievirus B3

https://doi.org/10.21203/rs.3.rs-3447939/v1

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published 13 Feb, 2024

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Indoline-5-sulfonamide derivatives of benzocaine have been synthesized using a sequence of three reactions: N-acylation, sulfochlorination, and sulfamidation, and their antienteroviral activity has been evaluated. Two compounds, namely, ethyl 4-((1-(cyclobutanecarbonyl)indoline)-5-sulfonamido)benzoate and ethyl 4-((1-benzoylindoline)-5-sulfonamido)benzoate exhibited a medium level of activity against coxsackievirus B3 (Nancy strain) in vitro. Their antiviral potential is exerted upon prophylactic application when added to cell culture before infection with the virus.

enteroviruses

coxsackievirus

antivirals

indoline

heterocycles

sulfonamides

Enteroviruses (EVs) represent a diverse cohort of small icosahedral non-enveloped viruses with single-stranded non-segmented positive RNA genome belonging to genus Enterovirus (Picornaviridae family). Enterovirus genus currently encompasses 15 species: Enteroviruses A–L and Rhinoviruses A–C [1]. Rhinoviruses are the leading cause of both upper and lower respiratory tract infections. Infection with enteroviruses can be mild and asymptomatic, but in some patients more severe diseases can develop: poliomyelitis, myocarditis, pancreatitis, encephalitis, meningitis [2–5]. Non-polio enteroviral infections (EVIs) are a serious challenge of public health world-wide [6]. The main consequences of EVI include hand-foot-mouth disease (HFMD), herpangina, acute hemorrhagic conjunctivitis [7]. EVI are more severe in neonates and young infants, who have a higher risk of systemic illness, including meningitis and meningoencephalitis [8–10]. The transmission of EVs is performed via fecal-oral and the respiratory routes. The indirect transmission may also occur via contaminated water, food, and fomites [2].

Enteroviruses cause outbreaks in small groups or communities (schools or hospital wards), or at a regional, national, or even international level. The climate is likely to drive the seasonal pattern of EVI cases. In tropical climate enteroviruses circulate year-round with peak in rainy season, in temperate climate EVI are primarily detected from April to October. The emergence of dominant serotypes which responsible for the seasonal illness rising is, generally, unpredictable [11]. Enterovirus A71 (EV-A71), as well as coxsackieviruses A6 (CVA6), A10 (CVA10), and A16 (CVA16) are the 4 major EVs which cause HFMD world-wide [4]. Moreover, in Asia, EV-A71 causes major outbreaks every 3–4 years since 1990s [12]. According to US Centers for Disease Control and Prevention (CDC) data, EV68 caused huge outbreaks of EVI in the form of SARS associated with acute flaccid myelitis were noted in the USA in 2014 and 2016 in children [13]. Treatment of EVI is mainly supportive; vaccine development is complicated due to high genetic and serological diversity [2]. Only vaccines against EV-A71 have been approved by the National Medical Products Administration of PR China [14].

The non-polio enteroviruses life cycle has been extensively studied and described in many reviews [15–17]. Progress in viral life cycle understanding has allowed scientists to move on to the search for enterovirus inhibitors targeting defined stages of EV reproduction. Among the previously studied molecules of EV inhibitors with known mechanism of action, several types can be mentioned: 1) capsid binders (for example, pleconaril, pokapavir, pirodavir, vapendavir, disoxaril), which prevent virus penetration into the cell; 2) inhibitors of viral proteases (namely, rupintrivir and AG7404); 3) inhibitors of viral polymerase (ribavirin, gemcitabine, amiloride); 4) viral ATPase inhibitors such as dibucaine and fluoxetine [18–21]. Despite their encouraging activity in vitro and further preclinical and clinical studies, none of them have been approved by FDA for EVI medication due to lack of efficacy, poor pharmacokinetic properties, and/or adverse effects [18, 20, 22].

Capsid binders are one of the most widely studied group of EV antivirals [18]. Their mechanism of action is well investigated. Particularly, they associate with a virus capsid made of VP1–VP4 proteins, causing the conformational reorganization of a capsid which interferes the binding of the virus to the cell surface. As a result, the virus penetrating into the cell is prevent. Although the use of capsid binders in clinical trials was marred by the emergence of virus resistance, they are still of high interest because they target crucial step in virus reproduction [18].

The rational design of capsid binders requires precise knowledge of virion surface morphology. To date two druggable pockets on the surface of EV particles were described: the hydrophobic pocket in VP1 at the bottom of the canyon formed around each of the 12 vertices of the icosahedral viral capsid and newly identified pocket located between the VP1–VP3 protomers [23–25]. VP1 capsid binders (such as pleconaril, Fig. 1) failed in clinical trials, whereas the progress has been made in the development of VP1–VP3 interprotomer pocket binders [25]. Particularly, 4-(4-(1,3-dioxoisoindolin-2-yl)phenylsulfonamido)benzoic acid (compound 1, Fig. 1) was identified as a highly perspective VP1–VP3 pocket inhibitor with promising in vitro activity [25]. This scaffold consists of two key structural elements: sulfonamide and phthalimide moieties. Since we were interested in the medicinal application of sulfonamides [26–29] and azaheterocycles [30–35], an attempt of continued exploration of this scaffold was made by our group and compound 2 with improved inhibitory action was discovered (Fig. 1) [36]. In present work, we propose a new chemotype using the scaffold-hopping approach [37] in order to increase the metabolic stability and reduce the potential toxicity of inhibitors for the future in vivo application. The peripheral phthalimide fragment was replaced with the N-acylindoline moiety and a number of new compounds (5a–d, 6a–d, and 7) based on this backbone were synthesized. Their antiviral properties were explored in vitro against Coxsackievirus B3 (Nancy) followed by mechanistic studies for the hits (5c and 5d). It was found they lack capsid binder properties and acted as prophylactic agents. The obtained results can be employed to develop novel antivirals for the EVI treatment.

Chemistry

The synthesis of target indoline derivatives 5a–d, 6a–d, and 7 presented in Scheme 1. Initially, N-acylindolines 3a–d were synthesized via N-acylation of indoline using the well-known protocol [43]. Further, compounds 3a–d were treated with chlorosulfonic acid according to the previously developed method [40] to give The reaction was preceded in the medium of chlorosulfonic acid. To increase the yield of the desired sulfonyl chlorides 4a–d, equivalent amount of thionyl chloride was added to the reaction mixture. This reagent provided a more complete conversion of the initially formed indoline-5-sulfonic acids into the corresponding sulfonyl chlorides 4. The reaction of sulfonyl chlorides 4 with benzocaine (ethyl ester of 4-aminobenzoic acid) was carried out in acetonitrile in the presence of pyridine as a base at RT. Resulted esters 5a–d were converted into acids 6a–d by the hydrolysis in an aqueous–ethanol solution of NaOH at RT. At the same time, the refluxing of esters 5 in the same NaOH solution led to acid 7.

The calculated toxicity values and quantitative estimate of druglikeness (QED) [44] according to the Syntelly platform data [45] for synthesized indoline derivatives 5a–d, 6a–d, and 7, as well as literature compounds 1 and 2 are presented in Table 1.

Table 1

The calculation values of toxicity and QED for compounds 1, ,2, **5a–d**, **6a–d**, and 7.¹.
Compound	Toxicity Mouse Oral LD₅₀, mg/kg	QED
1 2 5a 5b 5c 5d 6a 6b 6c 6d 7	4729 4764 2220 1952 2378 2339 2982 3323 3141 3190 2830	0.61 0.48 0.56 0.75 0.71 0.56 0.87 0.84 0.8 0.66 0.8

¹Data were obtained using synthelly.ru [45–47].

All evaluated compounds (both newly prepared and known) are characterized by high calculated OED values (especially 6a–c). According to calculated LD₅₀ data, they can be classified as low-toxic compounds (4–5 class). The obtained values indicated the prospects for their further consideration as potential drug candidates. So, esters 5a–d and acids 6a–d,7 were tested for antiviral activity against a representative of the genus of enteroviruses – Coxsackie virus B3, Nancy strain. The results of biological experiments are presented in section 3.2

Results of in vitro biological studies

At the beginning of the antiviral study, cytoprotection properties of compounds 5, 6, and 7 against Coxsackievirus B3 (Nancy strain) were evaluated using cytoprotection assay. In this cell-based assay cell survival depending on the concentration of tested compounds after virus infection is assessed using MTT assay. The literature inhibitor 1 was used as the reference compound. The results of the cytoprotection assay are presented below in Table 2.

Table 2

Results of cytoprotection assay of compounds **5a–d**, **6a–d**, and 7 against CVB3 Nancy (see description below).¹.
Compound	CC₅₀, µM²	EC₅₀, µM³			SI⁴
Compound	CC₅₀, µM²	MOI 0.1	MOI 0.01	MOI 0.001	MOI 0.1	MOI 0.01	MOI 0.001
1	> 710.2	78.9 ± 8.3	55.1 ± 6.5	34.3 ± 4.1	> 9	> 13	> 21
5a	257.5 ± 22.1	> 257.5	> 257.5	> 257.5	< 1	< 1	< 1
5b	313.1 ± 28.2	> 313	> 313	> 313	< 1	< 1	< 1
5c	210.3 ± 22.4	> 210.3	> 210.3	37.2 ± 4.1	> 1	> 1	6
5d	688.1 ± 55.2	> 688.1	> 688.1	27.5 ± 2.8	> 1	> 1	> 25
6a	> 832.4	> 832.4	> 832.4	> 832.4	> 1	> 1	> 1
6b	> 801.3	> 801.3	> 801.3	> 801.3	> 1	> 1	> 1
6c	> 749.2	> 759.1	> 759.1	> 759.1	> 1	> 1	> 1
6d	> 710.1	> 710.1	> 710.1	> 710.1	> 1	> 1	> 1
7	> 942.4	> 942.4	> 942.4	> 942.4	> 1	> 1	> 1
¹Compound 1 was used as a reference; ”>” indicates more than the highest concentration tested; the data presented were obtained from three independent experiments and the values for CC₅₀ and EC₅₀ are mean ± SD. ²CC₅₀ is the cytotoxic concentration resulting in the death of 50% of the cells; CC₅₀ was evaluated after 72 h of incubation of Vero cells with the compound only. ³EC₅₀ is the 50% cytoprotective concentration leading to 50% cytoprotection after infection of cells with CVB3 at corresponding MOI. ⁴SI is the selectivity index, the ratio of CC₅₀/EC₅₀ for each MOI.

As shown in Table 2, EC₅₀ values of all tested compounds (except the reference compound 1) were higher than the maximum concentrations used in the assay at MOI = 0.1 and MOI = 0.01. At the lowest MOI = 0.001 compounds 5c and 5d demonstrated cytoprotective activity upon infection with CVB3 (SI = 6 and SI > 25, respectively) in addition to compound 1 (SI > 21). For five compounds (6a–d and 7) CC₅₀ values exceeded the maximum concentration tested in the assay (the same as for the reference compound 1).

Next, in order to confirm virus-inhibition activity for 5c and 5d and verify the lack of activity of the others compounds, viral yield reduction test was performed. As described above in the materials and methods section, in this test the titer of viral progeny in the supernatant of the infected culture depending on the tested compound concentration is measured. Therefore, it is possible to assess the ability of the compounds to depress the virus reproduction The results of the viral yield reduction assay are summarized in the Table 3 below.

Table 3

Virus-inhibiting properties of compounds **5a–d**, **6a–d**, and 7 against Coxsackievirus B3 (Nancy) in viral yield reduction assay (see description below).¹.
Compound	CC₅₀, µM²	IC₅₀, µM³	SI⁴
1	> 1183.7	5.5 ± 0.9	> 215
5a	1288.1 ± 13.2	> 257.4	< 5
5b	786.2 ± 81.3	> 248.8	< 3
5c	253.4 ± 25.2	7.5 ± 2.1	34
5d	> 1109.9	20.3 ± 2.1	> 55
6a	> 1388.9	> 138.9	> 10
6b	1388.9 ± 15.3	> 267.1	< 5
6c	1273.6 ± 100.2	> 249.7	< 5
6d	> 1183.6	> 236.7	> 5
7	> 1570.4	> 313.1	> 5

¹MOI = 0.01; compound 1 was used as a reference; “>” indicates more than the highest concentration tested; the data presented were obtained from three independent experiments and the values for CC₅₀ and IC₅₀ are mean ± SD of the experiment.

²CC₅₀ is the cytotoxic concentration resulting in the death of 50% of the cells; CC₅₀ was evaluated after 24 h of incubation of Vero cells with the compound only.

³IC₅₀ is the 50% virus-inhibiting concentration leading to 50% inhibition of virus replication after infection of cells with CVB3 at MOI 0.01.

⁴SI is the selectivity index, the ratio of CC₅₀/IC₅₀.

The results of the viral yield assay were in accordance with the cytoprotection assay results. The two compounds (5c and 5d) demonstrated potent virus inhibiting properties, but their IC₅₀ were higher than that of the reference compound.

To elucidate the possible mechanism of action for 5c and 5d two cell-based assays were performed. At first, we checked whether they belonged to the capsid binders like the reference compound 1 in thermostability assay. For this aim CVB3 Nancy was pre-incubated with the tested compounds and thereafter the viral suspension was gradually heated and cooled down. Then the infectious titer of CVB3 at each temperature was determined and plotted at the diagram in Fig. 2 below.

As follows from the obtained results, the reference compound 1 increased thermal stability of the viral capsid in accordance with its capsid-binding nature. In contrast to the reference inhibitor 1, esters 5c and 5d demonstrated no thermostabilizing activity towards CVB3, which is consistent with their chemical structure, namely, the absence of a carboxyl group responsible for capsid binding.

Then we focused on the identification of the viral cycle stage which responsible for the antiviral action of our agents and performed a time-of-addition assay. The lifecycle of Coxsackievirus is relatively short, approximately 6 to 8 hours, and its timing has been extensively studied previously in cell culture. Viral genomic RNA is detected inside the cell until 1–2 hpi, the replication of the viral RNA starts 2–3 h after infection and the translation — at 3–4 hpi, viral progeny capsid proteins are visible after 4 hpi [48]. The evaluated compounds were added to the cell before CVB3 infection or at different time points (hours) after the infection. After one cycle of reproduction (8 hpi), the infectious titer of viral progeny in cell supernatants was determined in end-point dilution assay. The results of time-of-addition test are represented below at Fig. 3.

The most pronounced effect of the reference compound 1 was reached when it was present in the culture medium from − 2 to 1 hour post-infection (hpi) and most of the antiviral activity was lost when it was added 2 hpi (as described previously) [25]. This period corresponds to the virus’ attachment to the cell surface and penetration the cell which corresponds to its capsid-binder properties. In contrast to the reference compound 1, our esters 5c and 5d demonstrated efficacy only when added 1 hour before the addition of the virus to the cell (-2 time points), acting as prophylaxis agents, which is in accordance with the results of thermostability assay above.

Molecular docking studies

The cryo-electron microscopy analysis of VP1-VP3 interprotomer pocket in the capsid protein VP1 of Coxsackievirus B3 (strain Nancy) was reported before [25] with a derivative of sulfonylaminobenzoic acid in the binding site. It was reported that the carboxylic acid group is the crucial for the small molecule binding, however modifications of it’s moiety are to be explored in order to expand the chemical diversity of the binders. A molecular docking study of a compound 1 (PDB code: 6GZV) and 9 novel compounds was accomplished using Glide tool on Maestro (Release 2020-3) [51]. The results demonstrated that all the esters despite their larger size are binding in the interprotomer pocket stronger than their carboxylic acids analogs. The allowance of binding is caused by the flexible shape of the cavity formed between two VP1 units and one VP3 unit at an interprotomer interface. While the carboxylic acid 6d reflects the binding pose of the native ligand by an interaction of carboxylic group with Arg234 residue of the VP1 unit, the ethyl ester 5d is more bulky and finding it’s place in in a hydrophilic cavity between Cys73 and Gln99 (Fig. 4).

Enteroviral infections represent a significant threat to public health worldwide. The main affected population is newborn and children under 3 years of age. Despite many efforts by scientists, there are currently no approved antiviral drugs against EV infections. Vaccination option exists only for EV-A71 infection in eastern part of the world. Capsid binders are one of the most extensively studied antivirals targeting the very early steps in the life cycle of enteroviruses. Recently, a novel target for the design of capsid binder inhibitors was detected at VP1–VP3 interprotomer interface on the surface of most enteroviruses and derivatives of 4-substituted sulfonamidobenzoic acid were identified as novel class of capsid binders [25].

In the present study we prepared 9 novel antiviral compounds based on the scaffold of 4-substituted sulfonamidobenzoic acid. The peripheral phthalimide fragment was replaced by the molecular backbone of N-acylindoline. Antiviral properties, cytotoxicity and possible mechanism of action were described for two leading compounds in cell-based assays. It appeared, that the replacement of the peripheral phthalimide fragment by N-acylindoline led to abrogation of antiviral activity and thermo-stabilizing properties, even if the hydroxyl fragment of the carboxylic group of aminobenzoic acid primary responsible for target binding was retained [36]. At the same time, the combination of two structural changes: the carboxyl group to alkoxy carbonyl function and the phthalimide fragment to bulky N-cyclobutyloxyindoline (5c) and N-phenoxyindoline (5d) moieties led to the preservation of moderate antiviral activity. Interestingly, these compounds did not belong to the group of capsid-binding agents according to the results of thermostability assay. They exert their antiviral potential only upon prophylactic application when added to cell culture before the infection with Coxsackievirus. Based on these results, we propose that these compounds possibly target cell component crucial for virus attachment.

Targeting host cell component is potentially an attractive approach for antiviral design, since it suggests broad activity spectrum and high genetic barrier to drug resistance in contrast to direct acting antivirals [49]. Although cases of the resistant emergence to inhibitors of cellular enzymes have been described previously [50]. A potential problem of host-targeted antivirals is cytotoxicity and associated adverse effects. For a better understanding of the mechanisms of action of the hit compounds (5c and 5d), further research should be performed, including studies of the activity spectrum. Nevertheless, the results of this study can be applied in the design of new antiviral agents. The results obtained also expand our scope of knowledge on biological effects of benzocaine derivatives.

We have synthesized a series of indoline-5-sulfonamide derivatives of 4-aminobenzoic acid and these compounds were evaluated for the enteroviral replication using cytoprotection, virus inhibition, capsid-thermostability and time-of-addition assays. Two hits (5c, 5d) were shown to possess medium cytoprotective and virus inhibition properties against Coxsackievirus B3 with the absence of thermostabilizing effect on viral capsid. They exert antiviral activity only being added to the cells before addition of the virus in contrast to the reference capsid binder molecules.

Analysis of structure-activity relationship showed that esters 5a–d were more active against Coxsackievirus B3 in cytoprotection and virus inhibition tests than corresponding carboxylic acid (6a–d). The presence of a bulky and lipophilic cyclobutyl group or a phenyl ring in the acyl fragment at the nitrogen atom of indoline is more preferable for the manifestation of antiviral activity than the presence of a methyl or ethyl groups.

Thus, this finding offers promise on the right path towards design of drug-like small molecules for the prevention and treatment of enteroviral infections.

Reagents and instrumentation

2.1. General

Indoline, acylchlorides, chlorosulfonic acid, other reagents and solvents were obtained from commercial source (Acros, Merck) and used without additional purification.

All reactions were performed in an open flask. Reactions were monitored by analytical thin layer chromatography (TLC) Macherey-Nagel, TLC plates Polygram® Sil G/UV254. Visualization of the developed chromatograms was performed by fluorescence quenching at 254 nm. ¹H and ¹³C NMR spectra were measured on Bruker AVANCE DPX 400 (400 MHz for ¹H and 101 MHz for ¹³C, respectively). All chemical shifts (δ) are given in parts per million (ppm) with reference to solvent residues in DMSO-d₆ (2.50 for proton and 39.52 for carbon) and coupling constant (J) are reported in hertz (Hz). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Melting points were determined on Electrothermal IA 9300 series Digital Melting Point Apparatus. Mass spectra were recorded on microTOF spectrometers (ESI ionization).

2.2. Synthetic work

2.2.1. Synthesis of N–acylindolines 3a–d

To a solution of indoline (2 g, 16.8 mmol) in dry dichloromethane (20 mL) triethylamine (1.74 g, 17.2 mmol) was added. The reaction mixture was cooled to 0–5°C and corresponding acyl chloride (17.6 mmol, 1.05 equiv) was added. The reaction mixture was stirred at RT for 18 h (TLC monitoring). Then the reaction mixture was diluted with water (10 mL). The organic phase was separated and washed with 1% solution of NaHCO₃, brine and dried under anhydrous Na₂SO₄. The organic phase was filtered on the silica gel pad and solvent was removed in vacuo give the product 3 as a white or pale powder. It was further used without any additional purification.

1-(Indolin-1-yl)ethan-1-one(3a)[39]. Yield 95% (2.57 g), beige powder, mp 102–104 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.05 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 7.4 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 4.04 (t, J = 8.6 Hz, 2H), 3.11 (t, J = 8.6 Hz, 2H), 2.13 (s, 3H).

1-(Indolin-1-yl)propan-1-one(3b) [39]. Yield 91% (2.68 g), beige powder, mp 101–102 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.08 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 7.4 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 4.03 (t, J = 8.5 Hz, 2H), 3.11 (t, J = 8.5 Hz, 2H), 2.43 (q, J = 7.3 Hz, 2H), 1.05 (t, J = 7.3 Hz, 3H).

Cyclobutyl(indolin-1-yl)methanone(3c). Yield 97% (3.14 g), beige powder, mp 110–113 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.10 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 7.4 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 3.94 (t, J = 8.5 Hz, 2H), 3.40 (p, J = 8.3 Hz, 1H), 3.09 (t, J = 8.5 Hz, 2H), 2.35–2.07 (m, 4H), 2.04–1.85 (m, 1H), 1.86–1.70 (m, 1H).

Indolin-1-yl(phenyl)methanone(3d)[39]. Yield 93% (3.48 g), beige powder, mp 125–130 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.06 (s, 1H), 7.61–7.46 (m, 5H), 7.27 (d, J = 7.4 Hz, 1H), 7.16 (s, 1H), 7.03 (t, J = 7.5 Hz,1), 3.99 (t, J = 8.3 Hz, 2H), 3.07 (t, J = 8.3 Hz, 2H).

2.2.2. Synthesis of sulfonyl chlorides 4a–d

To a cooled to 0–5°C chlorosulfonic acid (1 mL, 15 mmol, 3 equiv) N-acylindoline 3 (5 mmol, 1 equiv) was added under vigorous stirring. The reaction mixture was stirred at RT for 30 min and then heated at 80°C for 2 h. Then thionyl chloride (0.47 mL, 6.5 mmol, 1.3 equiv) was added and the reaction mixture was stirred at 80°C for another 3 h. After completion of the reaction (TLC monitoring), the reaction mixture was poured onto ice and extracted with chloroform (2x 15 mL). The organic phase was washed with water (2 x 40 mL) and dried under Na₂SO₄. The resulting solution was purified by flash chromatography on silica gel using chloroform as an eluent.

1-Acetylindoline-5-sulfonyl chloride(4a) [40].Yield 79% (1.03 g), beige powder, mp 170–172 °С. ¹H NMR (400 MHz, CDCl₃) δ: 8.36 (d, J = 8.7 Hz, 1H), 7.86 (dd, J = 8.7, 2.2 Hz, 1H), 7.78 (s, 1H), 4.19 (t, J = 8.7 Hz, 2H), 3.30 (t, J = 8.8 Hz, 2H), 2.28 (s, 3H).

1-Propionylindoline-5-sulfonyl chloride(4b)[41]. Yield 80% (1.09 g), beige powder, mp 130–132 °С. ¹H NMR (400 MHz, CDCl₃) δ: 8.39 (d, J = 5.6 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.78 (s, 1H), 4.17 (t, J = 8.5 Hz, 2H), 3.29 (t, J = 8.5 Hz, 2H), 2.50 (q, J = 6.8 Hz, 2H), 1.23 (t, J = 7.3 Hz, 3H).

1-(Cyclobutanecarbonyl)indoline-5-sulfonyl chloride(4c).Yield 82% (1.22 g), beige powder, mp 130–134 °С. ¹H NMR (400 MHz, CDCl₃) δ: 8.39 (d, J = 7.3 Hz, 1H), 7.89–7.83 (m, 1H), 7.77 (s, 1H), 4.08 (t, J = 8.6 Hz, 2H), 3.34 (p, J = 9.1 Hz, 1H), 3.26 (t, J = 8.6 Hz, 2H), 2.42 (p, J = 9.2 Hz, 2H), 2.32–2.18 (m, 2H), 2.12–1.96 (m, 1H), 1.98–1.88 (m, 1H).

1-Benzoylindoline-5-sulfonyl chloride(4d).Yield 78% (1.25 g), beige powder, mp 139–143 °С. ¹H NMR (400 MHz, CDCl₃) δ: 8.11–7.84 (m, 1H), 7.83 (s, 2H), 7.63–7.38 (m, 5H), 4.18 (t, J = 8.5 Hz, 2H), 3.22 (t, J = 8.4 Hz, 2H).

2.2.3. Synthesis of sulfonamides 5a–d

To a cooled to 10°C solution of ethyl 4-aminobenzoate (165 mg, 1 mmol) and pyridine (0.11 mL, 1.3 mmol) in acetonitrile (10 mL) N-acylindoline-5-sulfonyl chloride 3 (1 mmol) was added. The reaction mixture was stirred at RT for 18 h (TLC monitoring). The reaction mixture was filtered; the collected solid was washed twice with water (2 x 5 mL) and dried at 50°C. The residue was purified by recrystallization from ethanol or ethanol/DMF mixture to afford the desired sulfonamide 5.

Ethyl 4-((1-acetylindoline)-5-sulfonamido)benzoate(5a).Yield 85% (331 mg), white powder, mp 257–261 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.69 (s, 1H), 8.09 (d, J = 8.3 Hz, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.69–7.59 (m, 2H), 7.21 (d, J = 7.7 Hz, 2H), 4.23 (q, J = 7.0, 6.4 Hz, 2H), 4.09 (t, J = 8.7 Hz, 2H), 3.14 (t, J = 8.7 Hz, 2H), 2.14 (s, 3H), 1.26 (t, J = 6.4 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.2, 165.8, 147.5, 143.1, 134.0, 133.5, 131.2 (2C), 127.9, 125.1, 124.0, 118.6 (2C), 115.9, 61.1, 49.3, 27.6, 24.7, 14.8. HRMS (ESI), calc. for C₁₉H₂₁N₂O₅S⁺ ([M + H]⁺) 389.1166; found m/z 389.1169.

Ethyl 4-((1-propionylindoline)-5-sulfonamido)benzoate(5b).Yield 82% (330 mg), white powder, mp 205–207 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.72 (s, 1H), 8.12 (d, J = 10.2 Hz, 1H), 7.80 (d, J = 7.9 Hz, 2H), 7.70–7.58 (m, J = 8.6 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H), 4.23 (q, J = 6.2, 4.9 Hz, 2H), 4.08 (t, J = 8.3 Hz, 2H), 3.14 (t, J = 8.8 Hz, 2H), 2.44 (q, J = 8.2, 7.7 Hz, 2H), 1.26 (t, J = 6.9 Hz, 3H), 1.02 (t, J = 6.9 Hz, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ: 173.3, 165.8, 147.7, 143.1, 133.9, 133.4, 131.2 (2 C), 127.9, 125.1, 124.0, 118.6 (2 C), 115.8, 61.1, 48.4, 28.9, 27.6, 14.8, 9.0. HRMS (ESI), calc. for C₂₀H₂₃N₂O₅S⁺ ([M + H]⁺): 403.1322; found m/z 403.1317.

Ethyl 4-((1-(cyclobutanecarbonyl)indoline)-5-sulfonamido)benzoate(5c).Yield 68% (292 mg), white powder, mp 225–227 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.71 (s, 1H), 8.14 (d, J = 9.7 Hz, 1H), 7.81 (d, J = 8.5 Hz, 2H), 7.72–7.56 (m, 2H), 7.21 (d, J = 8.5 Hz, 2H), 4.36–4.15 (m, 2H), 3.99 (t, J = 8.5 Hz, 2H), 3.49–3.36 (m, 1H), 3.13 (t, J = 8.8 Hz, 2H), 2.28–2.17 (m, 2H), 2.19–2.10 (m, 2H), 1.92 (dq, J = 20.0, 10.5, 9.6 Hz, 1H), 1.83–1.69 (m, 1H), 1.26 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 173.9, 165.8, 147.8, 143.1, 134.0, 133.4, 131.2 (2C), 127.9, 125.0, 123.9, 118.5 (2C), 115.9, 61.1, 47.9, 39.2, 27.7, 24.5 (2C), 17.9, 14.8. HRMS (ESI), calc. for C₂₂H₂₅N₂O₅S⁺ ([M + H]⁺): 429.1479; found m/z 429.1477.

Ethyl 4-((1-benzoylindoline)-5-sulfonamido)benzoate(5d).Yield 70% (316 mg), white powder, mp 215–218 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 10.78 (s, 1H), 7.95 (br s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.73–7.66 (m, 2H), 7.60–7.45 (m, 5H), 7.23 (d, J = 8.3 Hz, 2H), 4.24 (q, J = 7.1 Hz, 2H), 4.01 (t, J = 8.4 Hz, 2H), 3.10 (t, J = 8.4 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.5, 165.8, 147.4, 143.1, 137.0, 135.0, 134.2, 131.2 (2С), 131.2, 129.2 (2С), 127.6, 127.6 (2С), 125.1, 124.2, 118.6 (2С), 116.8, 61.8, 51.5, 28.0, 14.8. HRMS (ESI), calc. for C₂₄H₂₃N₂O₅S⁺ ([M + H]⁺): 451.1322; found m/z 451.1321.

2.2.4. Synthesis of acids 6a–dand 7

To the suspension of ester 5 (1 mmol) in 50% EtOH (5 mL) powdered NaOH (80 mg, 2 mmol) was added. The reaction mixture was stirred at RT for 18 h. The reaction mixture was filtered, cooled to 5–7 ᵒC, and acidified by conc. hydrochloric acid. The formed precipitate was collected by filtration and purified by recrystallization from ethanol or ethanol/DMF mixture to afford the desired product 6.

4-((1-Acetylindoline)-5-sulfonamido)benzoic acid(6a).Yield 65% (235 mg), white powder, mp 255–260 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 12.70 (s, 1H), 10.70 (s, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.71–7.55 (m, 2H), 7.18 (d, J = 8.7 Hz, 2H), 4.10 (t, J = 8.6 Hz, 2H), 3.15 (t, J = 9.1 Hz, 2H), 2.14 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 170.2, 167.4, 147.4, 142.8, 134.0, 133.6, 131.4 (2С), 127.8, 126.0, 124.0, 118.6 (2С), 115.9, 49.3, 27.6, 24.7. HRMS (ESI), calc. for C₁₇H₁₇N₂O₅S⁺ ([M + H]⁺): 361.0853; found m/z 361.0850.

4-((1-Propionylindoline)-5-sulfonamido)benzoic acid(6b).Yield 67% (251 mg), white powder, mp 254–257 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 12.72 (br s, 1H), 10.68 (s, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.80 (d, J = 8.7 Hz, 2H), 7.69–7.61 (m, 2H), 7.19 (d, J = 8.5 Hz, 2H), 4.08 (t, J = 8.6 Hz, 2H), 3.14 (t, J = 8.6 Hz, 2H), 2.44 (q, J = 7.4 Hz, 2H), 1.02 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 173.3, 167.4, 147.6, 142.8, 133.9, 133.4, 131.4 (2C), 127.9, 125.9, 124.0, 118.5 (2C), 115.8, 48.4, 28.9, 27.6, 9.1. HRMS (ESI), calc. for C₁₈H₁₉N₂O₅S⁺ ([M + H]⁺): 375.1009; found m/z 375.1007.

4-((1-(Cyclobutanecarbonyl)indoline)-5-sulfonamido)benzoic acid(6c).Yield 75% (301 mg), white powder, mp 259–262 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 12.73 (s, 1H), 10.70 (s, 1H), 8.14 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 9.2 Hz, 1H), 7.63 (s, 1H), 7.18 (d, J = 8.4 Hz, 2H), 4.00 (t, J = 8.5 Hz, 2H), 3.40 (p, J = 16.3, 7.7 Hz, 1H), 3.14 (t, J = 8.6 Hz, 2H), 2.28–2.17 (m, 2H), 2.19–2.08 (m, 2H), 2.00–1.84 (m, 1H), 1.85–1.68 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 173.8, 167.4, 147.8, 142.8, 134.0, 133.6, 131.4 (2С), 127.8, 126.0, 123.9, 118.5 (2С), 116.0, 47.9, 39.2, 27.7, 24.6 (2С), 17.9. HRMS (ESI), calc. for C₂₀H₂₁N₂O₅S⁺ ([M + H]⁺): 401.1166; found m/z 401.1178.

4-((1-Benzoylindoline)-5-sulfonamido)benzoic acid(6d).Yield 60% (254 mg), white powder, mp 250–255 °С. ¹H NMR (400 MHz, DMSO-d₆) δ: 12.75 (s, 1H), 10.73 (s, 1H), 7.96 (br s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.75–7.63 (m, 2H), 7.60–7.45 (m, 5H), 7.20 (d, J = 8.0 Hz, 2H), 4.01 (t, J = 8.6 Hz, 2H), 3.11 (t, J = 8.9 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 169.4, 167.8, 147.2, 143.0, 137.1, 134.9, 134.7, 131.3 (2C), 131.1, 129.2 (2C), 127.6 (2C), 126.5, 124.5, 124.2, 118.6 (2C), 116.7, 51.5, 28.0. HRMS (ESI), calc. for C₂₂H₁₉N₂O₅S⁺ ([M + H]⁺): 423.1009; found m/z 423.1006.

4-(Indoline-5-sulfonamido)benzoic acid(7).To the suspension of ester 6 (1 mmol) in 50% EtOH (5 mL) powdered NaOH (160 mg, 4 mmol) was added. The reaction mixture was refluxed for 18 h. The reaction mixture as filtered, cooled to 5–7 ᵒC, and acidified by conc. hydrochloric acid. The formed precipitate was collected by filtration and purified by recrystallization from ethanol or ethanol/DMF mixture to afford the desired product 7. Yield 55% (38 mg), beige powder, mp 227–229°С. ¹H NMR (400 MHz, DMSO-d₆) δ: 12.75 (s, 1H), 10.43 (s, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.40–7.37 (m, 2H), 7.15 (d, J = 8.4 Hz, 2H), 6.42 (d, J = 8.2 Hz, 1H), 3.49(t, J = 8.7 Hz, 2H), 2.93 (t, J = 8.8 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ: 167.5, 157.3, 143.4, 131.3 (2C), 129.8, 129.0, 125.6, 125.4, 123.5, 118.0 (2C), 106.6, 46.9, 28.7. HRMS (ESI), calc. for C₁₅H₁₅N₂O₄S⁺ ([M + H]⁺): 319.0747; found m/z 319.0744.

2.3. Viruses and Cells

Coxsackievirus B3 (CVB3, strain Nancy) was obtained from the collection of viruses of St. Petersburg Pasteur Institute (St. Petersburg, Russia). The virus was propagated in Vero cell line (ATCC #CCL-81). Infectious titers (in 50%-tissue culture infection dose, TCID50) were determined in Vero cells by endpoint dilution assay using Spearmen-Carber method essentially as described before [36].

2.4. Cytotoxicity Assay

The microtetrazolium test (MTT) was used to study the cytotoxicity of the compounds [42]. The permissive cell line Vero seeded in 96-well plates in Eagles minimal essential medium (MEM) supplemented with 10% FBS was used for cytotoxicity testing. After 24 h, the media was removed, and the wells were washed with saline. Compounds were dissolved in DMSO, and a series of two-fold dilutions of each compound (starting from 500 ug/ml) in MEM without FBS were prepared and added to the cells in triplicates. Cells were incubated for 24 h at 37°C in 5% CO₂. Thereafter the MTT was performed as described in [30]. The 50% cytotoxic dose (CC₅₀) of each compound (i.e., the compound concentration that causes the death of 50% of cells in a culture, or decreases the optical density twice as compared to the control wells upon 24 h of exposition) was calculated using four-parameter logistic nonlinear regression model (GraphPad Prism 6.01). CC50 values in µg/mL were then converted into micromoles.

2.5. Cytoprotection assay

Vero cells were seeded in 96-well plates in 24 hours before the assay. On the assay day, the cells were washed with saline and tested compounds in appropriate concentrations (300–3 µg/mL) in MEM without FBS were added to cells. No compounds were added to virus control wells. The plates were incubated for 1 hour at 37°C at 5% CO₂. Then the cells were infected with CVB3 Nancy (multiplicity of infection (MOI) range of 0.1–0.001) and incubated for 72 hours at 37°C at 5% CO₂. No virus was added to cytotoxicity control wells. After that cell viability and the cytoprotective activity of compounds was assessed by MTT test as described before [30].

Based on the results obtained, the values of EC₅₀, i.e., concentration of compounds that result in 50% cells protection were calculated for MOI 0.1–0.001 using four-parameter logistic nonlinear regression model (GraphPad Prism 6.01 software). EC₅₀ values in µg/mL were then converted into micromoles. From the ratio of CC₅₀ to EC₅₀ values selectivity index (SI) for cytoprotection was calculated for each compound tested.

2.6. Antiviral Activity Determination

Viral yield reduction assay was used to evaluate the antiviral activity of the compounds in Vero cell line seeded in MEM supplemented with 5% FBS in 24-well plates as described before [30]. The following concentration range of compounds (100–1 µg/mL) and CVB3 at MOI 0.01 was used. After 24 h of incubation at 37°C in 5% CO₂, the infectious titer of viral progeny (in TCID50) for each compound concentration, cell control, and virus control wells were determined in Vero cells by endpoint dilution assay as described above (in 2.3). Then the titer of virus progeny was plotted against log concentration of the compounds tested to generate the dose–response curve. The 50% inhibition concentration (IC₅₀) of each compound tested (i.e., the compound concentration that decreases the infectious viral progeny titer twice as compared to the control wells) was calculated using four-parameter logistic nonlinear regression model (GraphPad Prism 6.01). IC₅₀ values in µg/mL were then converted into micromoles.

Selectivity index (SI) was calculated for each compound tested as a ratio of CC₅₀ (at 24 h time point) to IC₅₀ values.

2.7. Thermostability assay

Thermostability assay for CVB3 Nancy and compounds tested was performed as described before [30]. The thermal gradient of 37–55°C for 2 min with cooling to 4°C was performed in BioRad CFX PCR-machine. The infectious titer of CVB3 at each temperature was calculated using end-point titration assay.

2.8. Time-of-addition assay

Time-of-addition assay was performed in Vero cells seeded in 24-well plates in 24 h before the beginning of the assay as described before in [43]. Given the 8-hours long life cycle of coxsackieviruses in vitro, the following time point were used for addition of the tested compounds to each well of the cell plate: (–2), (–1), 0, 2, 4, 6, where (–2) means 1 h before addition of the virus, (–1) — addition of the virus, 0 — completion of viral sorption on the cell surface, 2, 4, 6 — 2, 4 or 6 h after completion of virus sorption and washout of non-adsorbed virus. At (–1) suspension of 10⁴ TCID50 of CVB3 was added to all the wells except cell control and the plate was incubated at + 4°C for 1 h in order to synchronize the infection in all conditions, the unbound virus was washed thereafter and the plate was returned to + 37°C. 8 h after 0 time point the procedure was stopped and the infectious titer of viral progeny in each well was evaluated in Vero cells using end-point titration.

2.9. Statistics

All in vitro experiments were repeated three times and primary results were analyzed using GraphPad Prism 6.01 software (Mann–Whitney U-test was used to compare differences between groups).

3.0. Molecular docking

Molecular docking was carried out using Glide docking (Schrödinger’s Maestro,release 2020-3). The three-dimensional (3D) structure of the Coxsackievirus B3 (strain Nancy) capsid protein with a ligand 4-[[4-[1,3-bis(oxidanylidene)isoindol-2-yl]phenyl]sulfonylamino]benzoic acid was obtained from the Protein Data Bank (PDB code: 6GZV). Protein structure was refined and optimized by protein preparation wizard tool of Maestro with the correction of missing residues at pH = 7.4, and then minimized using OPLS-3e force field. The glide grid was generated using receptor grid generation panelusing a native ligand in the active side of the protein as a center which Glide uses as a region for the calculation.A library of 9 novel antiviral compounds was prepared using LigPrep tool generating the low-energy 3D structures of the molecules and their ionized statesat a given pH (7.4 ± 2.0). All molecules as well as the native ligand were further introduced into the Glide docking tool to evaluate the affinity of binding of these compounds to the interprotomer pocket. Glide Extra Precision (EP) was used with no bonding constraints given during the calculation, and binding affinity was evaluated by the Glide docking score. Post-docking minimization was performed restricting the output to two poses per ligand.

Supplementary information

The online version of supplementary materials available at https://doi.org/10.1007/xxxxxx-xxx-xxxxx-x.

Acknowledgements

The authors wish to thank Dr. S. Kalinin for kind help with manuscript preparation.

Funding

This study was supported by the Russian Science Foundation (project number 22-23-20158).

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Scheme 1 is available in the Supplementary Files section.

Scheme1.png
Scheme 1. Four-stepsynthesis of indoline derivatives 5a–d, 6a–d, and 7. Detailed experimental data presented in section 2.2.
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Benzocaine-N-acylindoline Conjugates: Synthesis and Antiviral Activity Against Coxsackievirus B3

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Abstract

Figures

Introduction

Results and discussion

Chemistry

Results of in vitro biological studies

Molecular docking studies

Discussion

Conclusion

Experimental section

Reagents and instrumentation

2.1. General

2.2. Synthetic work

2.2.2. Synthesis of sulfonyl chlorides 4a–d

2.2.3. Synthesis of sulfonamides 5a–d

2.3. Viruses and Cells

2.4. Cytotoxicity Assay

2.5. Cytoprotection assay

2.6. Antiviral Activity Determination

2.7. Thermostability assay

2.8. Time-of-addition assay

2.9. Statistics

3.0. Molecular docking

Declarations

References

Scheme

Supplementary Files

Status:

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