Ramified derivatives of 5-(perylen-3-ylethynyl)uracil-1-acetic acid and their antiviral properties

The propargylamide of N3-Pom-protected 5-(perylen-3-ylethynyl)uracil acetic acid, a universal precursor, was used in a CuAAC click reaction for the synthesis of several derivatives, including three ramified molecules with high activities against tick-borne encephalitis virus (TBEV). Pentaerythritol-based polyazides were used for the assembly of molecules containing 2⋯4 antiviral 5-(perylen-3-ylethynyl)uracil scaffolds, the first examples of polyvalent perylene antivirals. Cluster compounds showed enhanced absorbance, however, their fluorescence was reduced due to self-quenching. Due to the solubility issues, Pom group removal succeeded only for compounds with one peryleneethynyluracil unit. Four compounds, including one ramified cluster 9f, showed remarkable 1⋯3 nM EC50 values against TBEV in cell culture.

Introduction 5-(Perylen-3-ylethynyl)-deoxy-uridine dUY11 (Fig. 1), prepared initially as a uorescent nucleoside, 1 had unexpectedly shown remarkably broad spectrum antiviral properties. 2 Nucleoside derivative dUY11 and its arabino analog aUY11 effectively suppressed reproduction of a number of enveloped viruses in cell cultures, thus constituting a new class of antivirals with a nonnucleoside mechanism of action. 2, 3 The presumable target for these compounds is the lipid membrane bilayer of the virion envelope. Compounds dUY11 and aUY11 are rigid amphipathic fusion inhibitors (RAFIs) 2c that target the viral envelope either by a shape-determined biophysical mechanism 2c,3,4 or by photosensitization. 5 Both shape-determined and photophysical properties of the conjugated aryl part of the molecule are probably crucial for the antiviral properties, and the carbohydrate part seems to be less important. Very recently, 6 we prepared 5-(perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11, Fig. 1) with a carboxymethyl group replacing the pentose moiety. The acid showed a pronounced antiviral activity against tick-borne encephalitis virus (TBEV), 6 herpes simplex virus type 1 (HSV-1), 7 and African swine fewer virus (ASFV). 8 Moreover, its 3-Pom-modied precursor cm1pUY11 (Fig. 1) and amides were also active against TBEV 6 and HSV-1. 7 Thus, the 5-(perylen-3-ylethynyl)uracil scaffold seems to be responsible for the antiviral activity of perylene RAFIs. Lipophilic perylene residue is expected to reside in the lipid bilayer upon binding between a RAFI molecule and an enveloped virion. Therefore, one can expect cooperative enhancement of lipid membrane anchoring for compounds containing 2/4 perylene residues, or other effects of clustering perylenes.
The concept of clusterized/multivalent (or dendrimeric) antivirals has been developed for decades, 9 both for specic and non-specic compounds and functional groups. Recent promising examples include polyanions, 10 polycations, 11 as well as dendrimers carrying amino acids, 12 peptides, 13 phenols, 14 terpenes, 15 mono-, 16 oligosaccharides, 17 and neuraminidase inhibitors. 18 The approach, however, has been never applied to RAFIs. To study the possible effects of combining several perylene cores in a single molecule on the antiviral activity, we synthesized small clusters of 5-(perylen-3-ylethynyl)-1-(carboxymethyl) uracil, quantied their uorescence properties, and measured the efficiency of TBEV reproduction inhibition by these clusters in PEK cell culture.

Synthesis of compounds
Recently, we used the azide-alkyne click reaction for the synthesis of dUY11 derivatives with enhanced activity. 19 Here we report the application of Huisgen-Meldal-Sharpless reaction (Cu(I)-catalyzed alkyne-azide cycloaddition, CuAAC) 20 for the synthesis of 5-(perylen-3-ylethynyl)-1-(carboxymethyl)uracil derivatives. First, we prepared a set of branched azides 2-5 (Scheme 1). The starting tetraol 1 was mesylated with a controlled excess of mesyl chloride (MsCl) in DCM in the presence of triethylamine as a base. Tuning the mesyl chloride/hydroxyl ratio leads to the desired distribution of products in the reaction. Earlier, we prepared azides 4 and 5 using 1 : 3 molar ratio of 1 to mesyl chloride 21 and used them for the assembly of complex oligonucleotide conjugates. 21,22 Here we report the synthesis of azides 2 and 3 as main products. Mesylation with 1 : 1.2 molar ratio (1 : MsCl), and subsequent nucleophilic substitution with sodium azide affords compounds 2 and 3 in 32% and 27% yield, respectively, together with some amount of triazide 4.
The alkaline removal of pivaloyloxymethyl (Pom) protection from compounds 7 and 9a-c yielded the corresponding amides 8, 10a-c (Scheme 2). However, the usual Pom group deprotection protocol with sodium methoxide or ammonia in methanol 23 was inappropriate because both the starting compounds and the desired products were insoluble in alcohol. Therefore, we modied the deprotection protocol by choosing NaOH as the base, and DMSO/MeOH/H 2 O mixture as the reaction medium. As a result, full conversion of the starting material occurred in 30 min (TLC control).
While the conversions of compounds 9a-c into 10a-c by TLC were excellent, their isolated yields in deprotection steps (Scheme 2) turned out to be poor because of the losses during the purication using column chromatography. The products showed low solubility and chromatographic isolations were carried out in the overloading mode. Thus, a number of fractions containing the products were overlapping with some minor impurities, and they were excluded to obtain high quality samples for antiviral activity studies.
We failed to prepare and isolate Pom-deprotected compounds from 9d-f because of the incomplete deprotection, and low solubility of the desired products.

Spectral studies
Compounds 9d-f contain 2/4 chromophoric units linked with exible spacers. To obtain some insight into the structure of the compounds in a solution and to reveal a possible inuence of spectral properties on antiviral activity, 2d we compared their spectra to each other, and to those of monomeric compounds 9c and 10c. UV-Vis spectra (Fig. 2) showed no signicant difference between Pom-protected compound 9c and the deprotected derivative 10c; the shape of the spectrum, and the positions of maxima were always identical.
The increase of the number of chromophoric units led to the corresponding increase of the molar attenuation coefficient in true solution (20% DMSO in 96% ethanol as a solvent). Upon the reduction of DMSO concentration to 2% and further to 0.2%, the bands in absorbance spectra of tetramer 9f, and then of trimer 9e became broader and less structured (Fig. 2). The effect, conrming the aggregation of chromophoric residues, was observed earlier for other perylene compounds. 19,24 Fluorescence spectra of compounds 9c-f and 10c were recorded at 0.1 mM concentrations in 96% EtOH (Fig. 3). Due to Scheme 1 Synthesis of azides. Conditions: (a) methanesulfonyl chloride, NEt 3 , DCM; (b) NaN 3 , DMSO, rt, 12 h, 32% (2), 27% (3), 8% (4).
self-quenching, uorescence intensity decreases with the increase in the number of uorophores comprising the 5-(perylen-3-ylethynyl)uracil groups. There were no drastic changes in uorescence spectra for compounds 9c-f and 10c, except for relative enhancement of 550 nm band, and proximal long wavelength emission for polychromophore molecules 9df, suggesting weak excimer uorescence from perylene residues located close to each other.

Antiviral properties
All prepared compounds, containing one (7-9b, 10a-c) or several (9d-f) perylene residues, were tested for cytotoxicity and for ability to inhibit TBEV (strain Absettarov) reproduction in PEK cell culture as measured by plaque reduction assay (Table  1). All experimental conditions were the same as in our previous studies. 2d ,6,19,25 All the compounds showed little to no cytotoxicity on PEK cells and selectivity indices (SI ¼ CC 50 /EC 50 ) up to 50 000. The alkyne precursor 7 appeared to be the most potent compound in the series, showing nanomolar EC 50 . Its unprotected analogue 8 and acid precursor 6 showed slightly lower activity, thus revealing low inuence of small substituents in the uracil moiety, similarly to one of the previous studies, 25a but contrary to another. 6 Although cluster compounds 9d-f inhibited TBEV reproduction in one or two digit nanomolar concentrations, their single-perylene analogues were more potent. Remarkably, Pom deprotection of branched compound 9c, affording 10c, led to a considerable activity decrease.

Molecular modeling
While the abbreviation RAFI stands for Rigid Amphipathic Fusion Inhibitor, the compounds synthesized here contain a large number of rotatable bonds. Thus, their antiviral activity and membrane interactions may be largely dened by their conformational space. To obtain some insight into this conformational space, we performed a simple conformational analysis for molecular models of the compounds, seeking the global minima of the compound energies using the Confort method 26 implemented in SYBYL-X 2.1. 27 The optimized structures are shown in Fig. 4, along with the molecular surfaces colored by the hydrophobic potential.
As the most important part of the RAFI molecules is the perylene moiety, 2d,25a its exposure and ability to interact with the 0.10 AE 0.05 >50 37 AE 13 viral membrane should play a crucial role in the potency of the compounds. It can be seen that compounds 6-8, as well as aUY11, do not show substantial exibility, and the perylene moiety of these compounds is completely exposed. Molecules 9 and 10, bearing longer and more exible moieties, show less uniform antiviral activity, which is more strongly affected by possible conformational behavior. For example, the inuence of Pom is opposite in the pairs 9a-10a and 9b-10b; in the former, the benzyl moiety may interact with perylene, whereas in the latter the hydroxyethyl group should prefer a more hydrophilic environment.
Using coloring by the hydrophobic potential, one can see the amphipathic nature of perylenylethynyluracil compounds. Perylenylethynyluracil units are probably responsible for lipid bilayer anchoring and for the general antiviral effect. The polar part of the molecules seems to have a modulating (within two orders of magnitude) 6 effect on the inhibition of a viral replication. Remarkably, in the most active compounds, the polar part of the molecule has a small hydrophobic unit, Pom or benzyl, but not both together. Although neither the target, nor the mechanism of antiviral action of RAFIs was directly proven to date, this simple observation can be used in a future design of antivirals.

General methods
Reagents and solvents were from commercial suppliers and used as received, except DMF, DMSO, CH 2 Cl 2 , CHCl 3 , EtOH and methanol were used freshly distilled from CaH 2 . Azides 4, 5, 21 tetrakis(5-hydroxy-2-oxapentyl)methane 21 and 3-Pom-5-(perylen-3-ylethynyl)uracil-1-acetic acid 6 6 were prepared as described. 500 MHz 1 H and 125.7 MHz 13 C NMR spectra were recorded on Bruker AMX-400 or Bruker Avance 500 spectrometers and referenced to DMSO-d 6 (2.50 ppm for 1 H and 39.5 ppm for 13 C) or CDCl 3 (7.26 ppm for 1 H and 77.16 ppm for 13 C). 1 H NMR coupling constants are reported in hertz (Hz) and refer to apparent multiplicities. Electrospray ionization high resolution mass spectra (ESI HRMS) of low molecular weight compounds were recorded using a Thermo Scientic Orbitrap Exactive mass spectrometer (positive or negative ion mode). UV spectra were recorded on a Varian Cary 100 spectrophotometer. Fluorescence spectra were recorded using a PerkinElmer LS 55 uorescence spectrometer. Analytical thin layer chromatography was performed on Kieselgel 60 F 254 precoated aluminum plates (Merck). Silica gel column chromatography was performed using Merck Kieselgel 60 0.040-0.063 mm.

Cell toxicity assay
A cytotoxicity test in porcine embryo kidney (PEK) cells was performed as described previously. 2a,6,19,25 In brief, PEK cells were seeded and incubated for 72 h at 37 C. Stock solutions of the compounds with the concentration of 5 mM were prepared in 100% DMSO (Sigma). Two-fold dilutions of studied compounds were prepared in medium 199 on Earle solution to obtain nal concentrations starting from 50 mM. Equal volumes of compound dilutions were added in four replicates to the cells. Cell control was treated with the same sequential concentrations of DMSO as in compounds dilutions in four replicates. Aer incubation at 37 C on days 1 or 7 CC 50 values were calculated according to the Kerber method. 28

Antiviral activity assays
Plaque reduction test was performed as previously described 2a,6,19,25 on tick-borne encephalitis virus strain Absettarov. Stock solutions of the compounds with concentration of 5 mM were prepared in 100% DMSO (Sigma). Compounds were added to PEK cells simultaneously with virus and incubated at 37 C for 1 h with gentle shaking every 15-20 min. Then, each well was overlaid with 1 mL of 1.26% methylcellulose (Sigma) containing 2% FBS (Gibco). Aer incubation at 37 C in CO 2 incubator for 6 days cells were xed with 96% ethanol. Plaques were stained with 0.4% gentian violet. EC 50 values were calculated according to  Statistical analysis Data are expressed as means AE standard deviations. The statistical signicance was analyzed using Student's t test for at least three independent experiments.

Molecular modelling
The molecules were drawn in InstantJChem 30 and transferred to SYBYL-X 2.1 (ref. 27) in MOL format. MMFF94 charges 31 were assigned to the atoms and the Powell 32 optimization (10 000 steps, gradient termination, threshold 0.05 kcal (mol À1 ÂÅ)) was performed in MMFF94s force eld. 31b Then Confort 26 global minimization was performed, sampling 2000 conformations (program maximum) and optimizing them with termination by negative change. Precision parameter was set to 0.001. Electrostatics was considered. The number of acyclic rotors concurrently sampled was set to 200, rotors in cycles were not sampled due to the aromaticity of all the studied cycles. Compound 9f contains more atoms than can be treated by Confort and thus was not optimized using this method. Fast Connolly surfaces 33 for the optimized structures were calculated by MOLCAD in SYBYL-X 2.1 and colored according to hydrophobic potential.

Conclusions
In summary, we used pentaerythritol-based azides for the preparation of clusters containing up to four residues of 5-(perylen-3-ylethynyl)uracil, an antiviral scaffold. UV-Vis, uorescence, and anti-TBEV properties of compounds were studied. For the rst time, the antiviral activity of RAFI clusters was demonstrated. Four compounds, including tetramer 9f, showed one-digit nanomolar activity against TBEV, being among the most potent anti-TBEV molecules to date. Some structural features of the most active compounds can be used in further design of antivirals.

Conflicts of interest
There are no conicts of interest to declare.