key: cord-0823322-hhmde0cn
authors: Kozlovskaya, Liubov I.; Volok, Viktor P.; Shtro, Anna A.; Nikolaeva, Yulia V.; Chistov, Alexey A.; Matyugina, Elena S.; Belyaev, Evgeny S.; Jegorov, Artjom V.; Snoeck, Robert; Korshun, Vladimir A.; Andrei, Graciela; Osolodkin, Dmitry I.; Ishmukhametov, Aydar A.; Aralov, Andrey V.
title: Phenoxazine nucleoside derivatives with a multiple activity against RNA and DNA viruses
date: 2021-04-15
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2021.113467
sha: 5686fef1c2345d2f4fbbcdbbefea8db3ce53f7a0
doc_id: 823322
cord_uid: hhmde0cn

Emerging and re-emerging viruses periodically cause outbreaks and epidemics all over the world, eventually leading to global events such as the current pandemic of the novel SARS-CoV-2 coronavirus infection COVID-19. Therefore, an urgent need for novel antivirals, is crystal clear. Here we present the synthesis and evaluation of an antiviral activity of phenoxazine-based nucleoside analogs divided into three groups: (1) 8-alkoxy-substituted, (2) acyclic, and (3) carbocyclic. The antiviral activity was assessed against a structurally and phylogenetically diverse panel of RNA and DNA viruses from 25 species. Four compounds (11a-c, 12c) inhibited 4 DNA/RNA viruses with EC(50) ≤ 20 μM. Toxicity of the compounds for the cell lines used for virus cultivation was negligible in most cases. In addition, previously reported and newly synthesized phenoxazine derivatives were evaluated against SARS-CoV-2, and some of them showed promising inhibition of reproduction with EC(50) values in low micromolar range, although, accompanied by commensurate cytotoxicity.

Historically, naturally occurring nucleoside and nucleotide analogs have undergone different modifications during the search for drug candidates and tools for molecular biology, bioorganic chemistry, and medicine. Several effective drugs for the treatment of viral diseases and cancers have been found among these compounds [1] [2] [3] , and some possess multiple activities [4] . Therefore, nucleoside and nucleotide analogs were among the first molecules to test against novel emerging viruses, such as SARS-CoV-2 [5] .

Nucleosides, consisting of a heterocyclic base and a pentafuranose residue, usually get modifications in one of these structural fragments or both of them at the same time. Exemplary modifications of the carbohydrate moiety, which led to the compounds used in the medical practice, are present in the acyclic nucleosides acyclovir 1a and ganciclovir 1b (Fig. 1) , specific and potent inhibitors of infections caused by herpes simplex viruses HSV-1 and HSV-2 as well as by varicella-zoster virus (VZV), all belong to Herpesviridae family [6, 7] . Another example of sugar fragment modification leading to a class of carbocyclic nucleoside analogs is the replacement of the furanose oxygen atom by a methylene group [8] . Among these molecules, several drugs were found, including the ones against the diseases caused by the viruses utilizing reverse transcription: abacavir 2 [9] against the RNA-containing human immunodeficiency virus (HIV) and entecavir 3 [10] against the DNA-containing hepatitis B virus (HBV). Antiviral activity of a nucleoside analog can be achieved by changing the size and shape of not only a sugar residue, but also the aromatic heterocyclic base. For example, acyclic nucleoside analogs with expanded heterocyclic bases, ethenonucleosides 4a-b, are active against various herpesviruses, namely HSV-1 and HSV-2 (MIC reached 1.52 and 0.07 μM for 4a and 4b, respectively), VZV and cytomegalovirus (CMV) (4b MIC 14.3 μM for both viruses) [11, 12] . Bicyclic pyrimidine nucleoside analogs (BCNAs) demonstrated potent and specific inhibition of VZV reproduction. Compounds 5 and 6 from this series inhibited VZV replication with EC 50 of 0.027 and 0.0002 µM, respectively (clinically used drugs brivudine and acyclovir showed EC 50 values of 0.009 µM and 3.4 µM, respectively, in that assay) [13, 14] . In addition, the analogs of 5 with longer C8-C10 chains were even more active then the parent derivative with the C8 being the most favorite length for maximal activity [13] . Similar ribonucleoside derivatives inhibited reproduction of enterovirus A71 and coxsackievirus A16 on a single-digit micromolar level [15] .

J o u r n a l P r e -p r o o f We have recently reported a group of ribo-and deoxyribonucleoside analogs bearing an expanded heterocyclic system -1,3-diaza-2-oxophenoxazine -and showing inhibitory activity against VZV and CMV reproduction [16] . EC 50 for the most potent compound 7a (Fig. 1) against the wild type and thymidine kinase (TK) deficient VZV strains was 0.06 and 10 µM, respectively. Thus, to expand the repertoire of phenoxazine-based antiviral candidates, we mainly used experience in anti-VZV drugs design. Indeed, acyclic analogs and BCNA are substrates of HSV-1 and VZV TKs, which phosphorylate them thereby providing the first step of the activation process [17] . Dimetoxytritylated analogues based on 7b structure showed an efficient inhibition of RNA-containing tick-borne encephalitis virus (TBEV) in the range of 0.4 to 3.4 µM [16] .

Accordingly, we present a synthesis of a new series of 1,3-diaza-2-oxophenoxazine nucleosides with fatty C 8-10 alkoxy substituents in the aromatic moiety, as well as derivatives with acyclic and carbocyclic moieties instead of the ribose residue. Antiviral activity of the synthesized compounds was evaluated against not only DNA but additionally a broad panel of RNA viruses, including TBEV, Powassan virus (POWV), and Omsk hemorrhagic fever virus (OHFV) (flaviviruses), chikungunya virus (CHIKV, alphavirus), respiratory syncytial virus (RSV, pneumovirus), influenza viruses H1N1 and H3N2. While this work was in preparation, COVID-19 pandemic emerged, and Russian isolate of SARS coronavirus

The compounds belonging to three classes were synthesized in the current work: (1) seven 8alkoxy-substituted derivatives (11a-c, 12a-d); (2) five acyclic derivatives (16a-b, 17, 19, and 20) along with their synthetic precursors (13, 14, and 18), and (3) one phenoxazine-containing representative (23) of carbocyclic nucleoside analogs.

The series of phenoxazine nucleoside analogs with fatty alkoxy substituents in position 8 of phenoxazine system was synthesized in the following way. 4-(Alkoxy)-2-nitrophenols 9 were prepared from previously reported 4-alkoxyphenols 8 [19] by nitration in benzene (Scheme 1). Then, the reduction of the nitro group afforded anilines 10, which were used without purification in the reaction with 3',5'-O-acetyl-4-N-(1,2,4-triazol-1-yl)-5-bromo-2'-deoxycytidine in the presence of DIPEA. Subsequent cyclization by refluxing in the mixture of TEA/C 2 H 5 OH and aqueous ammonolysis gave desired derivatives 11. Since 1,3-diaza-2-oxophenoxazine is an analog of cytosine and maintains its H-bonding pattern, we also prepared N 10 -methyl substituted derivatives 12 in order to study the effect of the pattern disturbance. Additionally, we also carried out N 10 -methylation of 7, the most potent against VZV [16] , to obtain its derivative 12d. Acyclic uracil and phenoxazine nucleoside analogs and a carbocyclic phenoxazine nucleoside analog were synthesized according to Scheme 2. 1-(2-Hydroxyethoxymethyl)-5-bromouracil 13 [20] was acetylated to yield 14. Then, C4 of 14 was activated by the Appel reaction followed by the substitu- Reproduction of RNA tick-borne flaviviruses (TBEV, POWV, OHFV) was inhibited in plaquereduction assay by several compounds, but for mosquito-borne flaviviruses (YFV, ZIKV) such an effect was not observed in cytopathic effect inhibition assay ( Table 2) . The most potent ones were 11a, 11b, 12b, and 12c with EC 50 values less than 10 µM. Similarly, the same compounds showed promising activity against CHIKV in plaque-reduction assay, but not against Sindbis virus in cytopathic effect inhibition assay, both from Alphavirus genus. Several compounds (11c, 16a, 17) inhibited reproduction of H1N1 in a yield reduction assay with EC 50 less than 10 µM. Moreover, carbocyclic compound 23 was active against RSV. Toxicity of the compounds in the cell lines used for virus cultivation was negligible in most cases. [18] . Virus reproduction caused a pronounced cytopathic signs in Vero cells that allowed us to establish a cytopathic effect inhibition assay. The assay was calibrated using convalescent sera and N-hydroxycytidine (NHC), well-known inhibitor of SARS-CoV-2 reproduction [22] . Serum antibodies and NHC showed a dose-depended inhibition of the strain PIK35 cytopathic effect with EC 50 < 10 µM. To expand the activity spectrum study for the new compounds, the series was enriched with the previously reported phenoxazine nucleosides ( Table 3 ) [16] . The results are summarized in Table 3 .

Only compounds reported earlier showed promising inhibition of SARS-CoV-2 reproduction with EC 50 values in the low micromolar range. The activity was accompanied by rather pronounced cytotoxicity, which was in line with the previous findings. 

Broad-spectrum phenotypic antiviral activity screening is a powerful technique of boosted discovery of both new antiviral scaffolds and their target viruses. In this work we applied this approach to 16 new phenoxazine nucleoside analogs, extending our previous study [16] to employ modifications of the scaffold used in the successful antiviral drugs [1] , or addition of aliphatic chains, reported to enhance antiviral activity of certain nucleosides [13] [14] [15] . Virus panel consisted of 24 viruses pathogenic for humans, belonging to different realms and Baltimore classes. Additionally, 25 th virus, now known as SARS-CoV-2, emerged while this work was in preparation, and the panel was extended to include it.

Thus, more than 400 new antiviral activity values were determined here.

Phenoxazine deoxyribonucleoside series was extended to include 8-alkylated (11, 12) trast to BCNAs [13, 14] these derivatives were less (about 20-60 fold) but still active against TK -VZV strain suggesting TK-independent mechanism of action. 10-Methylation of these compounds decreased their potency, suggesting mechanistical role of mimicking hydrogen bond pattern of parent nucleosides.

On the other hand, 10-methylation did not have a pronounced effect on the activity against RNA viruses, suggesting a non-nucleoside-like mechanism of action, consistent with other observations for alkylated nucleosides [15] . 10-Methylation of non-alkylated phenoxazine nucleoside 7, which showed EC 50 of 0.06 against TK + VZV [16] , gave 12d, which did not inhibit reproduction of the studied viruses in the promising concentration range.

Acyclic nucleoside analogs were represented in our series by brominated uracil (13, 14, and 18) and phenoxazine (16, 17, 19, and 20 VZV strains and TBEV with EC 50 in the range of 0.9 to 2.7 μM [16] , and further highlights the need to reduce the toxicity of this compound. It should be also noted that dimethoxytritylation of the distal hydroxyl does not have a consistent effect on anti-SARS-CoV-2 activity, contrary to a generally positive effect observed for RNA viruses both in this study and earlier experiments [16] .

In 

All reagents were commercially available unless otherwise mentioned and used without further purification. All solvents were purchased from commercial sources. Thin layer chromatography (TLC) was performed on plates (Merck) precoated with silica gel (60 μm, F254) and visualized using UV light 

To a solution of 11 or 7 (0.22 mmol) in CH 2 Cl 2 (10 mL) DBU (2 equiv, 0.44 mmol) was added at rt. After 15 min iodomethane (2 equiv, 0.44 mmol) was added in one portion and the resulting mixture was stirred at rt for 1 hour and then poured into 5% aqueous citric acid solution (10 ml). The organics

were extracted with 10% 1-butanol in CH 2 Cl 2 (3 × 10 mL), dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (0-3% MeOH in CH 2 Cl 2 ) yielding 12 as light brown solid.

(77 mg, 0.17 mmol, yield 76%). 1 

To a solution of 13 (3.10 g, 11.7 mmol) in dry pyridine (60 mL) acetic anhydride (2.2 mL, 23.4 mmol) was added. After 3 hours at room temperature the reaction mixture was poured into saturated aqueous NaHCO 3 (50 mL) and extracted with EtOAc (2 × 50 mL). The oily residue was co-evaporated with toluene (2 × 10 mL) and the residue was purified by column chromatography on silica gel (75% J o u r n a l P r e -p r o o f 

To a solution of 14 (2.76 g, 9.0 mmol) and PPh 3 (4.72 g, 18 mmol) in CH 2 Cl 2 (50 mL) carbon tetrachloride (10 mL) was added and the resulting solution was refluxed for 5 hours. Then, the reaction mixture was evaporated to a foam in vacuo, dissolved in CH 2 Cl 2 (50 mL) and 2-aminophenol (1.47 g, 13.5 mmol) was added, followed by TEA (1.9 mL, 13.5 mmol). After 3 hours at room temperature the reaction mixture was concentrated to brown foam. The foam was dissolved in a mixture of absolute C 2 H 5 OH (60 mL) and TEA (12 mL) and refluxed under N 2 for 48 hours. After cooling to room temperature aqueous NH 3 (10 mL) was added and the mixture was heated at 40°C overnight followed by concentration and then co-evaporation with acetonitrile (2 × 10 mL) in vacuo. The residue was purified by column chromatography on silica gel (0-4% MeOH in CH 2 Cl 2 ) yielding 16a (0.57 g, 2.07 mmol, 23%) as light brownish solid. 1 

0.85 (t, J = 6.4 Hz, 3H, CH 3 ). 13 C NMR (151 MHz

3-(4-Hydroxy-2-oxabutyl)-10-methyl-1,3-diaza-2-oxophenoxazine (17)

The organics were extracted with 10% 1-butanol in CH 2 Cl 2 (3 × 10 mL), dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (0-3% MeOH in CH 2 Cl 2 ) yielding 17 as light brownish solid (0.10 g, 0.34 mmol, 68%). 1 H NMR (600 MHz

HRMS (ESI) m/z: calcd for

3 mmol) was co-evaporated with anhydrous pyridine (5 mL), then dissolved in anhydrous pyridine (5 mL) and 4,4'-dimethoxytrityl chloride (120 mg, 0.36 mmol) was added in one portion at rt. After 4 hours at rt the reaction was stopped by the addition of 5% aqueous NaHCO 3 solution (10 mL) and the organics were extracted with DCM (2x15 mL). The combined organic layers were dried over Na 2 SO 4 , filtered

Purification was performed by silica gel column chromatography on silica gel (0-0.5% MeOH in CH 2 Cl 2 with 0.1% TEA) yielding 17 (129 mg, 0.23 mmol, 76%) as yellowish foam

16 (s, 2H, N-CH 2 -O), 3.73 (s, 6H, 2 × OCH 3 ), 3.71-3.67 (m, 2H, -CH 2 -), 3.06-3.02 (m, 2H, -CH 2 -). 13 C NMR (151 MHz, DMSO-4.1.11. 3-[4-(4,4'-Dimethoxytrityloxy)-2-oxabutyl]-1,3-diaza-2-oxophenoxazine (19) This derivative (yield 71%, yellowish foam) was prepared starting from 16a as described for the preparation of 18. 1 H NMR (600 MHz

(m, 8H, 2 × OCH 3 /-CH 2 -), 3.32 (s, 3H

1-(4'-Acetoxy-2'-cyclopenten-1'-yl)-5-bromouracil (22)

57 (s, 1H, H6), 6.34 (dd

After 3 days of incubation at 37 °C, the cell number was determined with a Beckman Coulter counter. The cytostatic concentrations or CC 50 (compound concentration required reducing cell proliferation by 50%) were estimated from graphic plots of the number of cells

Russia) appropriate for a chosen cell line. Cell suspensions were added to the wells with compound dilutions and DMSO control (approx. 105 cells per well) in the appropriate cultural medium supplemented with 5% FBS (Invitrogen, South America). The final concentration series of eight dilutions started from 100 μM. After incubation at 36.5 °C in a CO 2 -incubator for 5 days cultural medium was substituted with resazurin solution (25 μg/ml). Cells were incubated at 36.5°C in a CO 2 -incubator for 4 h

Fluorescence was measured with Promega GloMax-Multi Detection System 525 nm Ex

As additional controls we used the same series of cells treated with compounds and DMSO dilutions but without resazurin solution to subtract the background fluorescence; and a medium with resazurin solution to set up a minimal value of non-reduced resazurin. All experimental procedures were performed in two replicates. Statistical analysis and fluorescence curves were prepared with MS Excel 2013. The 50% cytotoxic concentration (CC 50 ) was calculated

Following a 2 h adsorption period, viral inoculum was removed and the cell cultures were incubated in the presence of varying concentrations of the test compounds starting at 100 µM. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC 50 (compound concentration required reducing virus-induced cytopathicity or viral plaque formation by 50%)

Five-fold dilutions of studied compounds and DMSO as a control were prepared in cell culture medium (FSBSI "Chumakov FSC R&D IBP RAS

PFU/well) at final concentration series starting from 4 or 50 μM and incubated at 37 °C for 1 h. Then compound+virus mixtures were added to the cell monolayers (Vero for CHIKV, PEK for tick-borne flaviviruses) and incubated at 37 °C for 1 h for infectious virus adsorption. Then, each well was overlaid with 1 mL of 1.26% methylcellulose (Sigma) containing 2% FBS (Invitrogen, South America)

Plaques were stained with 0.4% crystal violet and counted. EC 50 were calculated according to the Reed-and-Muench method

Enteroviruses cytopathic effect inhibition test (FSBSI "Chumakov FSC R&D IBP RAS")

After 1 h incubation at 36.5 °C the RD cell suspension (approx.10 5 cells per well) in 2×EMEM containing 5% FBS (Invitrogen, South America) was added to experimental mixtures. A final concentration series started from approx. 100 μM. Each experiment contained virus dose titration in the inoculate to assure the acceptable dose-range. After a 5-day incubation at 37 °C, cytopathic effect (CPE) was visually accessed via microscope

SARS-CoV-2 cytopathic effect inhibition test (FSBSI "Chumakov FSC R&D IBP RAS")

Eight 2-fold dilutions of 5mM stock solutions of the compounds were prepared in DMEM (FSBSI "Chumakov FSC R&D IBP RAS", Russia). containing 100 CCID 50 per well. A final concentration series started from approx

Each experiment contained a positive control compound NHC and virus dose titration to assure the acceptable dose-range. 4.2.2.7. Virus yield reduction assay (Smorodintsev Research Institute of Influenza). The assay was performed as described before [27]. In brief, a series of 3-fold dilutions were prepared from DMSO solution of the compound (2000 µg/mL), added to MDCK cell monolayer and incubated for 1 h in CO 2 -incubator at 37°C. Then, the virus was added (MOI 1) and then incubated for 24 h in a CO 2 -incubator at 37°C. At the end of the incubation period, a series of consecutive 10-fold dilutions in a supporting medium was prepared from culture supernatant and added to the cell monolayers incubated for 72 h in CO 2 -incubator at 37°C. The virus titers were determined by hemagglutination reaction in U-bottom immunological 96-well plates with the equal volume of 1% suspension of chicken red blood cells in physiological solution. The virus titer was calculated using the Reed-and-Muench method (Reed and Muench, 1938) and expressed in 50% tissue infection doses

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Financial support from Ministry of Science and Higher Education of the Russian Federation 

Supplementary data (Tables with the viruses studied and data for the inactive compounds, HPLC data of the key compounds and NMR spectra) can be found at J o u r n a l P r e -p r o o f

x The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:J o u r n a l P r e -p r o o f