Cationic Perylene Antivirals with Aqueous Solubility for Studies In Vivo

Perylene-based compounds are attracting significant attention due to their high broad-spectrum antiviral activity against enveloped viruses. Despite unambiguous results of in vitro studies and high selectivity index, the poor water solubility of these compounds prevented in vivo evaluation of their antiviral properties. In this work, we synthesized a series of compounds with a perylene pharmacophore bearing positively charged substituents to improve the aqueous solubility of this unique type of antivirals. Three types of charged groups were introduced: (1) quaternary morpholinium salts (3a–b); (2) a 2′-O-l-valinyl-uridine hydrochloride residue (8), and (3) a 3-methylbenzothiazolium cation (10). The synthesized compounds were evaluated based both on antiviral properties in vitro (CHIKV, SARS-CoV-2, and IAV) and on solubility in aqueous media. Compound 10 has the greatest aqueous solubility, making it preferable for pre-evaluation by intragastrical administration in a mouse model of lethal influenza pneumonia. The results indicate that the introduction of a positively charged group is a viable strategy for the design of drug candidates with a perylene scaffold for in vivo studies.


Introduction
Epidemics of diseases associated with various viruses, such as Influenza A (H1N1) and Ebola viruses, as well as middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and 2) occurred in 2009,2014,2012,2003 and 2019, respectively. Re-emerging EBOVs and new H1N1, MERS, and SARS viruses pose a great threat to humanity and easily cross country borders. The traditional "one-bug -one drug" approach is insufficient for addressing the issue of re-emerging and new viral pathogens, and few drugs are currently available to control epidemic viral diseases [1][2][3][4][5]. Thus, it is critical to develop a class of broad-spectrum antiviral agents.
Antivirals with broad-spectrum activity can be divided into two categories: directacting drugs and host-targeting antivirals that engage host-cell machinery important for maintaining various stages of the viral life cycle. Representatives of the former was then subjected to deblocking of 3′-and 5′-hydroxyls groups by treatment with TBAF·3H2O. The Boc protecting group from 7 was removed by hydrolysis in acidic conditions, yielding positively charged nucleoside derivative 8. Finally, 2,3-dimethylbenzo[d]thiazol-3-ium chloride 9 and perylene-3-carbaldehyde, as the carbanion-forming and the carbonyl-containing electrophilic components, respectively, were subjected to a Knoevenagel condensation reaction [35] by refluxing in acetic anhydride to give final compound 10. NMR spectroscopy and HRMS spectrometry were used to confirm the structure and elemental composition of intermediates and target compounds (see Experimental and Figures S1-S9). The purity of target positively charged derivatives 3a-b, 8, and 10 was >90%, as determined by HPLC (Figures S11-S14).

In Vitro Activity, Aqueous Solubility, and Serum Stability Assessment
Since determining the scope of applicability of perylene-based compounds as antivirals was the main purpose of this study, we first evaluated in vitro antiviral activity (Table 1)  First, N-(ω-azidoalkyl)morpholine 1 was condensed with 3-ethynylperylene under Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conditions [28]. Then, the N atom of the morpholine ring was alkylated with methyl iodide, affording target positively charged derivatives 3a and 3b, with an ethyl (n = 2) or propyl (n = 3) bridge, respectively. Nucleoside derivative 8 was prepared in four steps. 3 ,5 -O-silyl protected 5-iodouridine 4 [34] was subjected to Sonogashira coupling with 3-ethynylperylene, yielding 5. Condensation of 5 with N-Boc-L-valine using DCC as the condensing agent afforded 6, which was then subjected to deblocking of 3 -and 5 -hydroxyls groups by treatment with TBAF·3H 2 O. The Boc protecting group from 7 was removed by hydrolysis in acidic conditions, yielding positively charged nucleoside derivative 8. Finally, 2,3-dimethylbenzo[d]thiazol-3-ium chloride 9 and perylene-3-carbaldehyde, as the carbanion-forming and the carbonyl-containing electrophilic components, respectively, were subjected to a Knoevenagel condensation reaction [35] by refluxing in acetic anhydride to give final compound 10.
NMR spectroscopy and HRMS spectrometry were used to confirm the structure and elemental composition of intermediates and target compounds (see Experimental and Figures S1-S9). The purity of target positively charged derivatives 3a-b, 8, and 10 was >90%, as determined by HPLC (Figures S11-S14).

In Vitro Activity, Aqueous Solubility, and Serum Stability Assessment
Since determining the scope of applicability of perylene-based compounds as antivirals was the main purpose of this study, we first evaluated in vitro antiviral activity (Table 1) of target compounds 3a-b, 8, 10 and synthetic intermediates 2a-b, and 5-7 against a number of enveloped RNA viruses pathogenic for humans: Chikungunya virus (CHIKV), severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), and influenza A virus (IAV). Charged compounds retained antiviral activity in the low micromolar-submicromolar concentration range against CHIKV and SARS-CoV-2 with almost no evidence of cytotoxicity. At the same time, some uncharged intermediates did not demonstrate any activity against CHIKV (2b, 6) or SARS-CoV-2 (2b, 5, 6) in concentrations studied. In particular, replacement of the ethyl linker between perylenyltriazolyl and morpholine residues with a propyl one led to complete loss of activity against CHIKV and SARS-CoV-2 in the morpholine series, but the corresponding N-methylated derivative 3b had activity against these two viruses. Surprisingly, 5 ,3 -O-protected 5-(perylen-3-ylethynyl)uridine derivative 5 did not show any signs of efficacy against SARS-CoV-2, and the introduction of an additional lipophilic substituent at the 2 -OH function of the ribose residue also caused a complete loss of activity of 6 against CHIKV. Meanwhile, removal of the siloxane 5 ,3 -O-protective group restored the activity of 7, suggesting the importance of maintaining the lipophilic perylene residue and polar, in our case hydroxy, groups, for maximum activity. Activity against IAV was substantially lower compared to CHIKV and SARS-CoV-2, surprisingly decreasing with the introduction of the positive charge in the morpholine series (3a-b vs. 2a-b). In contrast, positively charged nucleoside derivative 8 has slightly better activity compared to its non-charged intermediates 6 and 7. Finally, all charged derivatives showed comparable-to-higher activity against CHIKV and SARS-CoV-2 vs. positive control Nhydroxycytidine (NHC) [36]. All compounds had lower activity against IAV vs. Tamiflu as the control antiviral.
Then, an assessment of aqueous solubility of the positively charged compounds was performed to select a candidate for studies in a model of lethal influenza pneumonia in mice ( Table 2). Positively charged derivatives 3a-b and 10 were found to have substantial solubility in water, whereas positively charged and uncharged nucleoside analogs 8 and aUY11 had poor and no solubility, respectively. Among the candidates synthesized, 8 had the highest antiviral activity against influenza A virus; however, its low aqueous solubility limits evaluation in vivo. Since, among the remaining three derivatives, compound 10 had the highest potency and solubility, it was chosen for subsequent evaluation in a mouse model of influenza A virus infection.
Next, human intestinal absorption (cHIA) [38] and blood-brain barrier permeability (clogBB) [39] were estimated using predictive QSAR models [40]. Most of the compounds were able to penetrate the blood-brain barrier effectively, and derivative 10 demonstrated the highest level of intestinal absorption, and even distribution between the brain and blood, which may be useful in the treatment of various brain lesion complications associated with influenza (Table S1) [41,42].
Since in vivo evaluation of the leader compound in mouse model involves intragastric administration, stability in stomach acid-mimicking 0.16 M aqueous HCl was assessed. The compound was stable, with slight signs of decomposition as determined by thin-layer chromatography (TLC) for at least 4 h, which exceeds the average residence time of drugs in the stomach [43]. Then, stability in fetal bovine serum (FBS) was evaluated. In vitro FBS half-life (t 1/2 ) was found to be 167 min ( Figure S10), which is comparable to approved drugs and other drug candidates [44][45][46].

In Vivo Studies
Protective efficacy for compound 10 was assessed by the ability to prevent the development of lethal influenza pneumonia in mice. BALB/c mice were treated intragastrically with the compound in two different dosing regimens, placebo and comparative drug Tamiflu [47,48] for the period between one day prior and three days after intranasal infection with IAV. Clinical symptom development and body weight were observed daily. Survival rates and curves are presented in Table 3 and Figure 1, respectively, and body weight changes in Figure 2.    Infection with the IAV strain A/California/7/09/MA caused mouse mortality starting from day 5 post infection (p.i.). By the end of the experiment, mortality was 90% in the Placebo group and 40% (p = 0.015) in the comparison group (Tamiflu), which corre-  Infection with the IAV strain A/California/7/09/MA caused mouse mortality starting from day 5 post infection (p.i.). By the end of the experiment, mortality was 90% in the Placebo group and 40% (p = 0.015) in the comparison group (Tamiflu), which corresponded to a protection index (PI) of 56% and was consistent with available information for this strain [49]. The use of compound 10 in a dose of 0.5 or 4.5 mg/kg did not cause a significant reduction in the mortality rate compared to the Placebo group (p > 0.05).
Influenza virus infection induced a reduction in body weight of all animals in all groups, reaching a maximum of 26.2% on day 9 p.i. and returning to the initial values on day 14 p.i. in the Placebo group. In the comparison group (Tamiflu), the reduction was less pronounced and reached 24.0% on day 10 p.i. The experimental group treated with 4.5 mg/kg of 10 showed less weight loss (up to 21.3% on day 9 p.i.) and faster weight recovery compared to the Placebo group.
To conclude, mice treated with 10 (4.5 mg/kg) showed a lower mortality rate and faster weight recovery, although the differences were not significant.

Solubility
The samples were dissolved in DMSO, diluted in series, and photometrically analyzed. The average molar absorption coefficient was calculated from the obtained optical densities of calibration solutions at the absorption maxima. Solubility values were determined as previously described [52] by adding weighed amounts of compounds to distilled water followed by sonication (30 min), centrifugation (12,000 rpm, 10 min), and photometry.

Cells and Viruses
The green monkey kidney (Vero) cell line originated from WHO Biologicals, Switzerland , and the Madin-Darby canine kidney (MDCK) cell line was obtained from the American Type Culture Collection (ATCC CCL-34).

Methods Cell viability Assay in Vero Cells
Two-fold dilutions of the compounds (starting from 100 µM) were prepared in DMEM (Chumakov FSC R&D IBP RAS, Russia) and added to confluent cell monolayers. After 5 days of incubation (37 • C, 5% CO 2 ), the cultural supernatant was substituted with a resazurin solution (25 µg/mL), followed by 4 h incubation (37 • C, 5% CO 2 ). Then, 20 µL of 10% SDS were added to stop the reaction. Fluorescence intensity was measured with Promega GloMax-Multi Detection System (Ex525/Em580-640). Cells treated with dilutions of the compounds and DMSO, but not resazurin, were used to subtract the background fluorescence. All experimental procedures were performed in two replicates. Fluorescence curves were analyzed with Microsoft Excel 2013. 50% Cytotoxic concentrations (CC 50 ) were calculated.

Cell Viability Assay in MDCK Cells
Compound solutions were prepared in DMSO (20 mg/mL), and then 2-fold dilutions in cultural medium were prepared starting from 1000 µg/mL. One-day MDCK cell monolayers (over 95% confluency) in 96-well plates (6×105 cells/well) were washed twice with serum-free alpha-MEM (Thermo-Fisher Scientific, USA) and treated with 100 µL of compound dilutions in 2 replicates. The plates were then incubated for 24 h at 37 • C in a CO 2 incubator. Cell viability was assessed using the tetrazolium test (MTT, ApplyChem) [53]. Optical density was measured on a Wallac 1420 Victor2 microplate reader (Perkin Elmer, Ramsey, MN, USA) at 535 nm, then CC 50 values were calculated.

IAV Virus Yield Reduction Assay
Compound solutions were prepared in DMSO (20 mg/mL). The compounds were weighed in 2 mg amounts and dissolved in 100 µL of DMSO. Then, a series of 3-fold dilutions was prepared, starting from 300 µg/mL, in a cultural medium. The MDCK cell monolayer was treated with compound dilutions and incubated for 1 h in a CO 2 incubator at 37 • C. Untreated cells were used as negative control. Afterward, the virus was added (MOI 1), and cells were incubated for 24 h at 37 • C in a CO 2 incubator. After incubation, the culture medium was collected, and viral yields were determined by titration. In brief, a series of 10-fold dilutions (10 −7 -10 −1 ) of viral supernatants were prepared in a cultural medium and applied to daily MDCK cell monolayers. Infected cells were incubated for 72 h at 37 • C in a CO 2 incubator. The presence of the virus in the culture supernatants was determined using the hemagglutination assay with a 1% suspension of chicken erythrocytes [54]. Viral titer was calculated according to the Reed-and-Muench method and was expressed as 50% tissue infectious doses (TID 50 ) per 100 µL of volume. Inhibition (%) was calculated by comparison with non-treated cells, and EC 50 was calculated from inhibitory curves.

SARS-CoV-2 and CHIKV Cytopathic Effect (CPE) Inhibition Test
The procedure was described previously [55]. In brief, two-fold dilutions of the compounds were prepared in Dulbecco MEM (Chumakov FSC R&D IBP RAS, Russia) and mixed 1:1 with a virus suspension containing 50-200 TCID 50 and incubated for 1 h at 37 • C in a CO 2 incubator. The final concentration series started from 100 µM. Then, compound-virus mixtures were added to confluent Vero cell monolayers in 2 replicates. After incubation at 37 • C in a CO 2 -incubator for 5 days, CPE signs (cell death) were assessed via microscope, and 50% effective concentration (EC 50 ) (compound concentration required to decrease CPE by 50%) was calculated using the Karber method [56].

Plasma Stability Test
In vitro stability of 10 was studied in Gibco FBS (Thermo-Fisher Scientific, USA) by following the reported procedure with minor modifications [57]. A 10 mM solution of the compound in DMSO (20 µL) was added to 80% FBS in 0.05 M PBS (pH 7.4) at 37 • C to yield the final concentration of 200 µM. The assays were carried out in triplicate in a water bath at 37 • C. Samples (100 µL) were taken at 0, 15, 30, 45, 60, 90, 120, 180, 240, and 300 min and added to 400 µL acetonitrile in order to deproteinize the plasma. The samples were then shaken for 1 min and centrifuged at 37 • C at 15,000 rpm for 5 min. The supernatants were analyzed by HPLC (detection at 530 nm). AUC values were determined and averaged from three independent experiments. In vitro serum half-life (t 1/2 ) was calculated using the equation t 1/2 = 0.693/b, where b is the slope found by linear fitting of the negative natural logarithm of the fraction remaining (AUC 0-300 /AUC 0 ) vs. incubation time.

Animals
Female BALB/c mice (5-7 weeks old) were obtained from the "Stolbovaya" branch of the Scientific center for biomedical technology of the Federal medical-biological agency (Moscow region, Russia). All animal procedures were performed in accordance with the Helsinki declaration and Russian animal protection regulations.

Virus
Influenza A virus strain A/California/7/09/MA adapted to mice by 8 consecutive passages and accumulated on chicken embryos was used.

Design of the Experiment to Determine the Antiviral Activity of Compounds
The test compound (0.5 and 4.5 mg/kg), placebo (PBS as a negative control), and reference drug Tamiflu (20 mg/kg, oseltamivir phosphate as a positive control) were administered once per day to the animals using a gastric tube in a 0.

Conclusions
While previously reported data on perylene RAFIs have demonstrated remarkable subnanomolar antiviral activity in vitro, low water solubility and a rather controversial mode of action have discouraged in vivo studies. The introduction of positively charged moieties led to a dramatic improvement in the solubility of these compounds without a significant reduction in efficacy in vitro. We demonstrated 22% protective activity and accelerated weight recovery upon intragastrical administration of the most promising compound 10 in a mouse model of lethal influenza pneumonia, but the differences were not significant. This is the first example of in vivo efficacy assessment of RAFIs, paving the way for the development of water-soluble perylene-based antiviral agents and their preclinical studies.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15101178/s1, Figures S1-S14: 1 H and 13 C NMR spectra of the compounds, Plasma stability evaluation, HPLC trace for compounds; Table S1: Predicted ADMET profiles of the synthesized compounds.