Synthesis of Antiviral Perfluoroalkyl Derivatives of Teicoplanin and Vancomycin

Abstract The limited scope of antiviral drugs and increasing problem of antiviral drug resistance represent a global health threat. Glycopeptide antibiotics and their lipophilic derivatives have emerged as relevant inhibitors of diverse viruses. Herein, we describe a new strategy for the synthesis of dual hydrophobic and lipophobic derivatives of glycopeptides to produce selective antiviral agents without membrane‐disrupting activity. Perfluorobutyl and perfluorooctyl moieties were attached through linkers of different length to azido derivatives of vancomycin aglycone and teicoplanin pseudoaglycone, and the new derivatives were evaluated against a diverse panel of viruses. The teicoplanin derivatives displayed strong anti‐influenza virus activity at nontoxic concentrations. Some of the perfluoroalkylated glycopeptides were also active against a few other viruses such as herpes simplex virus or coronavirus. These data encourage further exploration of glycopeptide analogues for broad antiviral application.


Introduction
Many biologically active small molecules contain one or more fluorine substituents, owing to the beneficial effect of fluorination on pharmacokinetic and pharmacodynamic properties. [1][2][3][4][5][6][7] The use of fluorine substituents in medicinal chemistry has continuously increased, [8][9][10] and currently more than 50 % of blockbuster drugs are fluorinated. In these compounds, the presence of a few fluorine atoms strongly modifies the biological activity and chemical reactivity, while having relatively low impact on the physical properties. [11,12] On the other hand, the physical characteristics of perfluori-nated compounds are very different from those of the hydrogen-containing analogues. Most importantly, perfluorination simultaneously enhances hydrophobicity and lipophobicity, [13,14] a unique characteristic that creates exceptional biomedical possibilities. Although the number of pharmacologically applicable molecules containing perfluoroalkyl substituents is currently still limited, polyfluorinated compounds have shown promise in several areas of medicinal chemistry. [15][16][17] In recent years, we conducted a systematic study on the synthesis of lipophilic derivatives of the glycopeptide antibiotics vancomycin, teicoplanin and ristocetin, and this yielded several new antibiotics with promising antibacterial and antiviral activity. [18][19][20][21][22] We demonstrated that the high lipophilicity of the side chains (C 8 À C 10 alkyl groups) in these molecules is essential for antiviral activity against, among others, influenza virus. However, antiviral activity proved accompanied by high cytotoxicity, probably due to a membrane-disrupting effect of the highly lipophilic side chains. [22] In addition, we found that cytotoxicity was reduced by decreasing the overall lipophilicity of the compounds following incorporation of a tetra(ethylene glycol) linker between the peptide core and the lipophilic group. [22] Hence, we assumed that attaching perfluoroalkyl groups that are highly hydro-and lipophobic might confer antiviral activity without creating a membrane-disrupting effect. Namely, it has been shown that triphilic polymers bearing bulky and lipophobic perfluorinated blocks do not mix well with the hydrocarbon chains of lipids, explaining their low membrane partition coefficients. [23,24] This is the reason why fluorinated surfactants are non-cytolytic, that is, they are unable to solubilize membranes, as opposed to their hydrogenated counterparts. [25] Herein, we report on the synthesis and antiviral plus antibacterial evaluation of perfluoroalkyl derivatives of vancomycin aglycone and teicoplanin pseudoaglycone.
Although synthetic modification of glycopeptide antibiotics is increasingly recognized in the context of antiviral [26] and antibacterial [27] drug design, our study is the first to explore conjugation of glycopeptides with perfluoroalkyl groups.

Synthesis
Our recent results on semisynthetic lipoglycopeptides revealed that the type and length of the linkers significantly modifies the antiviral activity and cytotoxicity. [22] Herein, we have chosen the 1 a and 1 b allyl ethers of ethylene and tetra (ethylene glycol)s as linker chains between perfluoroalkyl substituents and the antibiotic molecules (Scheme 1). A lightpromoted atom-transfer radical addition reaction [28] of the commercially available perfluorobutyl iodide and perfluorooctyl iodide onto the double bond of 1 a and 1 b resulted in 2 a and 2 b, as well as 5 a and 5 b in good yields. Attempted reductive removal of iodo substituent with LiAlH 4 proceeded with low efficacy. Fortunately, catalytic hydrogenation of the iodo derivatives gave 3 a and 3 b, as well as 6 a and 6 b with moderate to high yields. Finally, these derivatives were reacted with propargyl bromide, respectively, to produce propargylated compounds 4 a and 4 b and 7 a and 7 b that are suitable for the azide alkyne cycloaddition click reaction.

Structure elucidation and testing oligomerization
In addition to NMR structure validation (see the Supporting Information, including 13 C HSQC, HMBC, 13 C and 19 F spectra and spectral assignments) we tested compound 17 for possible oligomerization. Strong head to tail dimers are commonly found of glycopeptide antibiotics in aqueous solutions, that is co-operatively enhanced by ligand binding. [31][32][33] As 17 is not soluble in water, it was dissolved in MeOD for NMR diffusion experiments. [34] We used the internal mass standard method as described earlier. [35] TMS (tetramethylsilane, MW = 88.2) and β-cyclodextrin (β-CD, MW = 1135) were used alternatively. About 20 mg of sample 17 was dissolved in 500 μL MeOD and measured at 300 K temperature. Spectra were recorded with 32 or 64 linear gradient steps and evaluated with topspin 2.1 software using inverse Laplace transformation to obtain diffusion domain.
These experiments yielded MW between 7-8 kDa when referenced to internal TMS. ( Figure S1). However, in repeated measurements with~3 mg of 17 dissolved in MeOD and referenced to internal β-CD, DOSY yielded only 2.4 kDa mass for 17, that is closer to monomeric state ( Figure S2). In control DOSY experiments of teicoplanin and vancomycin dissolved in [D 6 ]DMSO with β-CD mass reference, DOSY yielded the monomeric molecular masses as expected. Hence, we do not suppose the presence of oligomers in case of dilute MeOD solutions of 17.

Biological evaluation
Vancomycin aglycone derivatives 12 and 13 were inactive against various influenza strains and both were highly cytotoxic in MDCK cells (Table 1). Perfluorobutyl and perfluorooctyl derivatives 15, 16 and 17 of teicoplanin pseudoaglycone displayed robust activity against influenza A and B viruses with a favorable selectivity (i. e., ratio between cytotoxicity and activity). In contrast, the similar perfluorooctyl derivative 18 with a tetra(ethylene glycol) linker displayed high cytotoxicity without antiviral activity. Although this marked difference between vancomycin and teicoplanin derivatives may seem surprising, we previously demonstrated that even a slight difference in the peptide core can lead to very different antiviral properties. [36] Besides, activity was noted against herpes simplex virus and vaccinia virus (compounds 15, 16, 17 and 18), adenovirus (15 and 17) and coronavirus (17 and 18; Table 2). Two other sensitive viruses were respiratory syncytial virus (EC 50 values: 9.9 μM for 15; 11 μM for 16 and 4.6 μM for 17; MTS-based CPE assay in HeLa cells) and Zika virus (EC50 for 15: 10 μM; MTS-based CPE assay in Vero cells; Tables S1 and S2).
The broad activity of teicoplanin pseudoaglycone derivatives 15, 16 and 17 against different viruses is consistent with our hypothesis that these derivatives may act by disrupting the viral endocytosis process, similarly to what we reported for a glycopeptide active against influenza virus. [37] Recently, it has been described that glycopeptide antibiotics like teicoplanin, dalbavancin, oritavancin and telavancin are able to prevent the host cell entry processes of Ebola virus, Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), which results also fit with our surmise. [38,39] Finally, the antibacterial activity was evaluated on a panel of Gram-positive bacteria (Table 3). Vancomycin derivatives 12 and 13 displayed moderate activity. Perfluorooctyl derivatives 17 and 18 of teicoplanin pseudoaglycone were inactive, but perfluorobutyl compounds 15 and 16 had excellent antibacterial activity against sensitive and resistant staph-  ylococci and good activity against resistant enterococci having vanA and vanB genes.

Conclusion
We have designed and synthesized the first members of fluoroglycopeptides, a novel type of glycopeptide antibiotic derivatives bearing perfluoroalkyl side chains. Such substituents are not only hydrophobic, but at the same time lipophobic endowing the antibiotic molecules with unique physicochemical properties that are worth exploring and exploiting from an antimicrobial point of view. As the lipophobic perfluoroalkyl blocks are known to have no membrane activity, low cytotoxicity of the perfluoroalkylated glycopeptides were expected. However, the vancomycin derivatives and one of the teicoplanin molecules showed cytotoxicity on canine kidney cells whereas they were inactive against influenza viruses. At the same time, three of the new teicoplanin derivatives displayed high activity against the influenza strains studied, two were active against human corona and adenoviruses, and all teicoplanins proved to be active against vaccinia and herpes viruses without showing cytotoxicity. Moreover, perfluorobutyl derivatives of teicoplanin pseudoaglycone displayed excellent antibacterial activity against a panel of Gram-positive bacteria. We hope that these results can open a new way in finding more effective antivirals based on glycopeptide antibiotics.

General information
Compounds 1 b, [40] 8, [29] 9 [30] and 14 [18] were synthesized according to the literature. Compound 1 a was purchased from Sigma-Aldrich, nonafluoro-1-iodobutane from TCI and heptadecafluoro-1-iodooctane from Alfa Aesar. TLC was performed on Kieselgel 60 F 254 (Merck) with detection either by immersing into ammonium molybdate-sulfuric acid solution followed by heating or by using Pauly's reagent for detection. Flash column chromatography was performed using Silica gel 60 (Merck 0.040-0.063 mm). The photoinitiated reactions were carried out in a borosilicate vessel by irradiation with a Hg lamp giving maximum emission at 365 nm. The 1 H NMR (500, 400 and 360 MHz) 13 C NMR (125, 100 and 90 MHz) and 2D NMR spectra were recorded with a Bruker DRX-360, Bruker DRX-400 and Bruker Avance II 500 spectrometer at 298 or 300 K. For DOSY (Diffusion Ordered Spectroscopy) experiments, Bruker AVANCE-II, 500 MHz spectrometer was applied, with manufacturer's "ledbpgp2 s" pulse sequence. Chemical shifts are referenced to Me 4 Si (0.00 ppm for 1 H) and to the solvent residual signals. MALDI-TOF MS analyses of the compounds were carried out in the positive reflectron mode (20 kV) using a BIFLEX III mass spectrometer (Bruker) equipped with delayed-ion extraction. A nitrogen laser (337 nm, 3 ns pulse width, 106-107 W/cm 2 ) operating at 4 Hz was applied to produce laser desorption. 2,5-Dihydroxybenzoic acid (DHB) was used as matrix and F 3 CCOONa as cationizing agent in DMF. ESI-QTOF MS measurements were carried out on a maXis II UHR ESI-QTOF MS instrument (Bruker), in positive ionization mode. The following parameters were applied for the electrospray ion source: capillary voltage: 3.5 kV; end plate offset: 500 V; nebulizer pressure: 0.8 bar; dry gas temperature: 200°C and dry gas flow rate: 4.5 L/min. Constant background correction was applied for each spectrum, the background was recorded before each sample by  2- ((4,4,5,5,6,6,7,7,7-Nonafluoro-2-iodoheptyl)

11-Heptadecafluoroundecyl)oxy)ethan-1-ol (6 a)
To a solution of 5 a (1.296 g, 2 mmol) in methanol (15 mL) 10 % palladium on activated charcoal (270 mg) and NaHCO 3 (420 mg, 5 mmol) were added. The reaction mixture was stirred overnight under H 2 atmosphere, then filtered through Celite, and the solvent was evaporated. The residue was dissolved in dichloromethane (50 mL) and the solution was washed with distilled water (10 mL) two times, dried with anhydrous Na 2 SO 4 , filtered and the solvent was evaporated in vacuum.   (4 a, 4 b, 7 a and 7

b)
NaH (60 % dispersion in mineral oil; 2 mmol) was washed with hexane, abs. THF (10 mL) was added and 3 a, 3 b, 6 a or 6 b (1 mmol) was dissolved in it. After 30 min of stirring, propargyl bromide (80 % solution in toluene; 1.2 mmol) was added, and the reaction mixture was stirred for 3 h. Then ethyl acetate (5 mL) and methanol (1 mL) were added to the mixture, and it was stirred for 15 min. The solvent was evaporated, the residue was dissolved in dichloromethane and the solution was washed with distilled water (3x15 mL). The organic phase was dried on anhydrous Na 2 SO 4 , then it was filtered and evaporated in vacuum. The product was purified by flash column chromatography (hexane/acetone 8 : 2) to yield 4 a, 4 b, 7 a or 7 b as yellowish liquids.