Alkyl Derivatives of Perylene Photosensitizing Antivirals: Towards Understanding the Influence of Lipophilicity

Abstract

Amphipathic perylene derivatives are broad-spectrum antivirals against enveloped viruses that act as fusion inhibitors in a light-dependent manner. The compounds target the lipid bilayer of the viral envelope using the lipophilic perylene moiety and photogenerating singlet oxygen, thereby causing damage to unsaturated lipids. Previous studies show that variation of the polar part of the molecule is important for antiviral activity. Here, we report modification of the lipophilic part of the molecule, perylene, by the introduction of 4-, 8-, and 12-carbon alkyls into position 9(10) of the perylene residue. Using Friedel-Crafts acylation and Wolff-Kishner reduction, three 3-acetyl-9(10)-alkylperylenes were synthesized from perylene and used to prepare 9 nucleoside and 12 non-nucleoside amphipathic derivatives. These compounds were characterized as fluorophores and singlet oxygen generators, as well as tested as antivirals against herpes virus-1 (HSV-1) and vesicular stomatitis virus (VSV), both known for causing superficial skin/mucosa lesions and thus serving as suitable candidates for photodynamic therapy. The results suggest that derivatives with a short alkyl chain (butyl) have strong antiviral activity, whereas the introduction of longer alkyl substituents (n = 8 and 12) to the perylenyethynyl scaffold results in a dramatic reduction of antiviral activity. This phenomenon is likely attributable to the increased lipophilicity of the compounds and their ability to form insoluble aggregates. Moreover, molecular dynamic studies revealed that alkylated perylene derivatives are predominately located closer to the middle of the bilayer compared to non-alkylated derivatives. The predicted probability of superficial positioning correlated with antiviral activity, suggesting that singlet oxygen generation is achieved in the subsurface layer of the membrane, where the perylene group is more accessible to dissolved oxygen.

Keywords: antivirals; lipophilicity; perylene; photosensitizers; singlet oxygen.

Figure 1

Photosensitizer- and intercalator-mediated fusion inhibition vs. normal viral fusion (viral fusion proteins are not shown).

Scheme 1

Synthesis of 3-ethynyl-9(10)-alkylperylenes 5a–c (perylene position numbers shown in red).

Scheme 2

Synthesis of 5-(alkylperylenylethynyl)uracil nucleosides; target compounds framed in green.

Scheme 3

Synthesis of alkylperylenylethynylphenols (framed in green).

Scheme 4

Synthesis of alkylperylenylthiophenecarboxylic acids (framed in green).

Figure 2

Normalized absorption spectra of types of compounds: nucleoside (9–11a–c) (blue), aggregated compounds (9c, 11c) (red), phenolic (12–14a–c) (green), and thiophene carboxylic (15a–c) (purple) in MeOH.

Figure 3

Normalized excitation (solid line) and emission (dashed line) spectra of compounds types: nucleoside (9–11a–c) (blue), aggregated compounds (9c, 11c) (red), phenolic (12–14a–c) (green), and thiophene carboxylic (15a–c) (purple) in MeOH.

Figure 4

Structures of previously reported parent perylene compounds (without alkyl chain) used as controls for newly prepared C₄, C₈, and C₁₂ alkyl derivatives.

Figure 5

Preferential orientations of perylene derivatives in the lipid bilayer in the course of MD. (A) The most populated membrane-bound modes: state 1 (purple) and state 2 (grey) of para-phenolic derivative 12 (C₀) (the same is true for other compounds). Molecule 12 is shown in ball representation, lipid molecules are shown as sticks with the orange spheres corresponding to phosphorus atoms that denote the water–membrane interface. (B) Fractions of MD states where state 2 occurs (tilt angle ≥ 60°). The data are averaged over 3 independent MD runs. (C) A 2D-histogram of the distribution of perylene derivatives in membrane-bound state: X-axis denotes the tilt angle, Y-axis shows Z-distance from the bilayer center (Z = 0 nm) to their center of mass (COM). Population of state 2 (high angle and Z values) notably decreases with increasing alkyl tail length in both sets of compounds. The color on the right-side scale ranges from black (no occurrence) to yellow (25 occurrences).

Figure 6

Cytotoxicity and anti-HSV-1 activity of alkylated perylene derivatives. (A) Cytotoxicity of the tested compounds (10 µM) to Vero cells after a 48 h incubation. (B) Anti-HSV-1 activity of the compounds (10 µM) in Vero cells after a 48 h incubation. Viral titers were determined from medium supernatant. (C) Selected alkylated perylene derivatives (10 µM) did not suppress virus replication inside HSV-1 infected Vero cell culture after a 48 h incubation. Data are expressed as the mean ± SD. The experiments were performed in triplicates or hexaplicates. The horizontal dashed line indicates the minimum detectable threshold of 1.44 log₁₀ PFU/mL.

Figure 7

Light-induced HSV-1-inactivating activity and photocytotoxicity of alkylated perylene derivatives. (A–C) HSV-1-inactivating activity of selected alkylated perylene derivatives in daylight. (A) Schematic representation of the experiment. (B) Dose–response curves showing HSV-1-inactivating activity of selected compounds in daylight. (C) Inhibition curves used to determine EC₅₀ values in daylight. Compounds 12a, 13a, and 15b are not plotted because they caused only partial or no viral inactivation (see Figure 7B), and, therefore, their EC₅₀ values cannot be properly determined. (D–F) HSV-1-inactivating activity of selected alkylated perylene derivatives after blue light irradiation (465–480 nm, power density of 30 mW/cm²). (D) Schematic representation of the experiment. (E) Dose–response curves showing HSV-1-inactivating activity of selected compounds after blue light irradiation. (F) Inhibition curves used to determine EC₅₀ values after blue light irradiation. Compounds 10, 12, and 15 are non-alkylated (control) derivatives. (G,I) HSV-1-inactivating activity of selected alkylated perylene derivatives after blue light irradiation; those compounds that showed no/negligible anti-HSV-1 activity in a general anti-HSV-1 screening (see Figure 6B). (H,J) HSV-1-inactivating activity of selected alkylated perylene derivatives in the absence of excitation light; the entire experiment was performed under red light (624 ± 20 nm). (K,L) Light-induced cytotoxicity of the selected compounds to Vero cells. Viability of compound-treated cells irradiated with blue light (full lines) compared with control cells (incubated in daylight, dashed lines). Data are expressed as mean ± SD. Experiments were performed in triplicate or hexaplicate. The horizontal dashed line indicates the minimum detectable threshold of 1.44 log₁₀ PFU/mL.

Figure 8

Mechanistic studies with alkylated perylene derivatives. (A,C,E) Demonstration of the interaction of alkylated perylene derivatives with the HSV-1 envelope using the intercalation assay. (A) Schematic representation of the experiment. (C,E) Quantification of virus infectivity after incorporation of the selected compounds (10 µM) into the HSV-1 envelope using Vero cells. (B,D,F) Inhibition of the virus–cell fusion process with alkylated perylene derivatives. (B) Schematic representation of the experiment. (D,F) Quantification of virus infectivity using the fusion assay (based on Vero cells) at compound concentration of 10 µM. Data are expressed as the mean ± SD. The experiments were performed in triplicate. n.d., not detected.

Figure 9

Fluorescence spectra of compounds 9 (A), 9a (B), 9b (C), and 9c (D) in PBS (blue dashed line) and after interaction with liposomes (red line). The excitation wavelength for the measurement of the fluorescence spectra is indicated.