Fluorination of Naturally Occurring N6

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Fluorination of Naturally Occurring N 6-Benzyladenosine Remarkably Increased Its Antiviral Activity and Selectivity Vladimir E. Oslovsky 1,† , Mikhail S. Drenichev 1,† , Liang Sun 2 , Nikolay N. Kurochkin 1 , Vladislav E. Kunetsky 1 , Carmen Mirabelli 2 , Johan Neyts 2 , Pieter Leyssen 2 and Sergey N. Mikhailov 1, * ID 1

2

* †

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia; vladimirosl[email protected] (V.E.O.); [email protected] (M.S.D.); [email protected] (N.N.K.); [email protected] (V.E.K.) Laboratory for Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven-University of Leuven, Minderbroedersstraat 10, Leuven 3000, Belgium; [email protected] (L.S.); [email protected] (C.M.); [email protected] (J.N.); [email protected] (P.L.) Correspondence: [email protected]; Tel.: +7-499-135-9733 The authors contributed equally.

Received: 27 June 2017; Accepted: 17 July 2017; Published: 20 July 2017

Abstract: Recently, we demonstrated that the natural cytokinin nucleosides N6 -isopentenyladenosine (iPR) and N6 -benzyladenosine (BAPR) exert a potent and selective antiviral effect on the replication of human enterovirus 71. In order to further characterize the antiviral profile of this class of compounds, we generated a series of fluorinated derivatives of BAPR and evaluated their activity on the replication of human enterovirus 71 in a cytopathic effect (CPE) reduction assay. The monofluorination of the BAPR-phenyl group changed the selectivity index (SI) slightly because of the concomitant high cell toxicity. Interestingly, the incorporation of a second fluorine atom resulted in a dramatic improvement of selectivity. Moreover, N6 -trifluoromethylbenzyladenosines derivatives (9–11) exhibited also a very interesting profile, with low cytotoxicity observed. In particular, the analogue N6 -(3-trifluoromethylbenzyl)-adenosine (10) with a four-fold gain in potency as compared to BAPR and the best SI in the class represents a promising candidate for further development. Keywords: fluorinated N6 -benzyladenosines; synthesis and antiviral activity; SAR; enterovirus 71

1. Introduction For many years, natural products (NPs) have been a leading source for the majority of the approved drugs, and their structures are a valuable source of inspiration for medicinal chemists [1]. Interestingly, only 36% of the new chemical entities discovered between 1981 and 2010 were developed without inspiration from a natural product [2]. Among natural products, the development of nucleosides is by far the most fruitful field of investigation. About one hundred drugs derive from nucleoside structures: the vast majority of them were developed as antiviral drugs, and a consistent proportion as antitumor drugs. Natural nucleosides are isolated from DNA, RNA, nucleotides, and coenzymes of various natural sources. Nowadays, the nucleoside library consists of about 550 compounds, and is a promising pool for the development of new biologically active compounds [3–5]. N6 -Modified purine nucleosides (cytokinin nucleosides) are an important group of biologically active natural compounds with a unique spectrum of biological activities [3]. Cytokinin nucleosides contain a hydrophilic ribofuranose moiety and a purine heterocyclic scaffold modified with a Molecules 2017, 22, 1219; doi:10.3390/molecules22071219

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contain a hydrophilic ribofuranose moiety and a purine heterocyclic scaffold modified with a hydrophobic residue residue at at the the N N66 position. position. tRNA tRNA contains contains N N66-isopentenyladenosine -isopentenyladenosine and some related related hydrophobic and some 6 6 nucleosides [6,7]. [6,7]. N nucleosides N -Substituted -Substituted adenosines adenosines are are naturally naturally present present in in plants plants [8–10] [8–10] and and bacteria bacteria [11]. [11]. 6-benzyladenosine (BAPR) exhibited a pronounced 6 In 2008, Arita and co-workers found that N In 2008, Arita and co-workers found that N -benzyladenosine (BAPR) exhibited a pronounced antiviral activity activityagainst against replication of human enterovirus 71 (EV71) [12]. is a nonantiviral thethe replication of human enterovirus 71 (EV71) [12]. EV71 is aEV71 non-enveloped, enveloped, single-stranded, positive-sense RNA virus to the Enterovirus within the single-stranded, positive-sense RNA virus belonging to belonging the Enterovirus genus within genus the Picornaviridae Picornaviridae family. EV71 commonly hand-, foot-,disease and mouth disease (HFMD), a mild and family. EV71 commonly causes hand-,causes foot-, and mouth (HFMD), a mild and self-limiting self-limiting illness mostly affecting children under the In agesome of five. In some patients, EV71associated has been illness mostly affecting children under the age of five. patients, EV71 has been associated with severe neurological complications including encephalitis, aseptic meningitis, and with severe neurological complications including encephalitis, aseptic meningitis, and acute flaccid acute flaccid paralysis [13–15]. EV71 is prevalent worldwide, but most of the large outbreaks of paralysis [13–15]. EV71 is prevalent worldwide, but most of the large outbreaks of neurotropic EV71 neurotropic EV71 have occurred area in the Pacific-Asia areain[15–17]. However, recent years, such have occurred in the Pacific-Asia [15–17]. However, recent years, such in epidemic tracts have epidemic tracts have been reported also in America and in Europe [15,17]. The World Health been reported also in America and in Europe [15,17]. The World Health Organization has placed EV71 Organization hasbiggest placedworldwide EV71 as one of to thepublic next health, biggestespecially worldwide threatschildren, to public as one of the next threats to young duehealth, to the especially to young children, due to [18,19]. the lack of effective antiviral treatments [18,19]. lack of effective antiviral treatments Recently, we we showed showed that, that, similarly similarly to to BAPR, BAPR, two two other other naturally naturally occurring occurring plant plant cytokinin cytokinin Recently, 6 6 nucleosides, namely namely N N -isopentenyladenosine and N N -furfuryladenosine nucleosides, -isopentenyladenosine and -furfuryladenosine (kinetin), (kinetin), possessed possessed aa potent potent and selective antiviral effect on EV71 [20,21]. Unfortunately, these compounds were rather cytotoxic, and selective antiviral effect on EV71 [20,21]. Unfortunately, these compounds were rather cytotoxic, with CC CC50 50 values were able able to to improve improve the the selectivity selectivity of of with values in in the the low low micromolar micromolar range range (4–8 (4–8 μM). µM). We We were this group group of of compounds by modifying this compounds by modifying the the size size and and the the nature nature of of the the linker. linker. In In particular, particular, aa modified modified BAPR with a two-to-three atom-long linker had a very pronounced antiviral activity, and 50-fold BAPR with a two-to-three atom-long linker had a very pronounced antiviral activity, and aa 50-fold improvement of of the the selectivity selectivity index index (SI) (SI) as as result result of of aa lower lower cytotoxicity cytotoxicity [21]. [21]. improvement The introduction of fluorine in order to improve the pharmacological The introduction of fluorine in order to improve the pharmacologicalproperties propertiesofofa adrug drugis isa modern trend in medicinal chemistry. Currently, there are about 200 fluorinated drugs on the a modern trend in medicinal chemistry. Currently, there are about 200 fluorinated drugs on the market market (~20% of (~20% of all all pharmaceuticals), pharmaceuticals), with with even even higher higher figures figures for for agrochemicals agrochemicals (up (up to to 30%) 30%) [22,23]. [22,23]. Therefore, Therefore, in the the present present study, study, we we report report on on the the modification modification of of natural natural BAPR BAPR by by the the substitution substitution in in the the phenyl phenyl in ring with fluoro-, difluoro-, and trifluorometyl groups to evaluate the eventual improvement the ring with fluoro-, difluoro-, and trifluorometyl groups to evaluate the eventual improvement in in the antiviral profile antiviral profile of of these these fluorinated fluorinatedcompounds compoundsin inthe thecontext contextofofEV71 EV71replication replication(Figure (Figure1).1).

Figure 1. Strategy of modification of natural cytokinin nucleoside BAPR.

2. Results 2. Results and and Discussion Discussion 2.1. Chemistry 6versatile approach for for the the preparation of Nof Recently, we we have have developed developedaanew newuseful usefuland and versatile approach preparation 6 6 0 0 0 6 6 N -alkylation -acetyl-2′,3′,5′-tri-O-alkylation of NN -acetyl-2 ,3 ,5 -tri-Oacetyladenosine with withalcohols alcoholsunder underMitsunobu Mitsunobureaction reactionconditions conditions with alkyl halides promoted acetyladenosine oror with alkyl halides promoted by a base [20,21,24,25]. main advantage of our method is possibility the possibility to both use both halides abybase [20,21,24,25]. TheThe main advantage of our method is the to use alkylalkyl halides and 6 -modification. and alcohols N6-modification. is important, especially in the when an amine is not stable alcohols for Nfor ThisThis is important, especially in the casecase when an amine is not stable or

6 -modified modified adenosine derivatives N adenosine derivativesbybythe the regioselective regioselective

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3 of 12 have been Using this methodology, several hundred N6 -substituted adenosines 3 of 12 synthesized in one of our laboratories. Molecules 2017, 22, 1219 of 12 6-substituted adenosines have3been or is hardly available. Using this methodology, several hundred N 6-substituted Theavailable. traditional approach for the preparation of NN6 -alkylated or N6 -arylated is the or is hardly Using this methodology, several hundred adenosines haveadenosines been synthesized in one of our laboratories. 6-substituted adenosines have been synthesized in one our laboratories. or is hardly available. Using this methodology, several hundred N substitution ofofthe chlorine atom in commercially available 6-chloropurine riboside with alkylThe traditional approach for the preparation of N66-alkylated or N66-arylated adenosines is the The traditional approach for the preparation of N -alkylated or N -arylated adenosines isdeacetylation the synthesized in one of our laboratories. or arylamines [26,27]. 6-hloropurine riboside can be readily prepared by the of substitution of the chlorine atom in commercially available 6-chloropurine riboside with alkyl- or 6-alkylated 6-arylated 0 0 0 substitution of the chlorine atom in commercially available 6-chloropurine riboside with alkylor The traditional approach for the preparation of N or N adenosines is the 2 ,3 ,5 -tri-O-acetyl-6-chloropurine [28]. prepared by the deacetylation of 2′,3′,5′-triarylamines [26,27]. 6-hloropurine ribosideriboside can be readily arylamines [26,27]. can be readily by the deacetylation of 2′,3′,5′-trisubstitution of the 6-hloropurine chlorine atom riboside in commercially available riboside with alkyl- or 0 ,50 -tri-O-acetyl-6-chloropurine To simplify the separation procedure, we usedprepared 20 ,36-chloropurine riboside directly O-acetyl-6-chloropurine riboside [28]. O-acetyl-6-chloropurine riboside [28]. arylamines [26,27]. 6-hloropurine riboside can be readily prepared by the deacetylation of 2′,3′,5′-trithe separation usedare 2′,3′,5′-tri-O-acetyl-6-chloropurine riboside with aniline, inTo thesimplify substitution reactions. procedure, The acetyl we groups completely preserved in the reaction To simplify the separation procedure, we used 2′,3′,5′-tri-O-acetyl-6-chloropurine riboside O-acetyl-6-chloropurine riboside [28]. directly in the substitution reactions. Thecan acetyl groups areby completely preserved in the reaction with and the protected intermediate be isolated silica gel chromatography and characterized. directly the substitution reactions. The acetylwe groups completely preserved in the reaction with To in simplify the separation procedure, usedare 2′,3′,5′-tri-O-acetyl-6-chloropurine riboside aniline, and removal the protected intermediate can be isolated byN6silica gel chromatography and in overall After the of the acetyl groups by ammonolysis, -phenyladenosine was obtained aniline, in and the protected intermediate cangroups be isolated by silica gel 6 chromatography and directly the substitution reactions. The acetyl are completely preserved in the reaction with characterized. After On the removal of the acetyl groups by ammonolysis, N -phenyladenosine was 0 ,30 ,50 -tri-O-acetyl-6-chloropurine high yield [21]. other the of 2ammonolysis, riboside with characterized. After the the removal ofhand, the acetyl groups by N6-phenyladenosine aniline, and the protected intermediate canreaction be isolated by silica gel chromatography was and obtained in overall high yield [21]. On the other hand, the reaction of 2′,3′,5′-tri-O-acetyl-66due benzylamines was accompanied byacetyl the by-products to the partialwas removal of the obtained in overall high yield [21]. the formation other hand, the reaction Nof 2′,3′,5′-tri-O-acetyl-6characterized. After the removal of theOn groups by of ammonolysis, -phenyladenosine chloropurine riboside with benzylamines was accompanied by the formation of by-products due to chloropurine riboside with benzylamines thereaction formation of2′,3′,5′-tri-O-acetyl-6by-products duerequired to obtained in overall yield [21]. Onwas the other hand,bythe of the protective groups,high which complicated theaccompanied chromatographic control of reaction, and a large the partial removal of the protective groups, which complicated the chromatographic control of the theexcess partial of removal of the protective groups, which complicated the chromatographic control of the chloropurine riboside with benzylamines was accompanied by the formation of by-products due to the full conversion the compound. we decided to study reaction, andamines requiredfor a large excess of aminesoffor thestarting full conversion of theTherefore, starting compound. reaction, and required a large excessgroups, of amines forcomplicated thethe fullone conversion offor theour starting compound. the partial removal of the protective which the chromatographic control of theresults of the the stability of different O-acyl groups to select optimal purposes. The Therefore, we decided to study the stability of different O-acyl groups to select the one optimal for Therefore,and we required decided to studyexcess the stability of different O-acyl groups to select the one optimal for reaction, a large of amines for the full conversion of the starting compound. 0 0 ,50 -tri-O-acylinosine are summarized in Table 1. experiments ourO-deacylation purposes. The results of of the2 ,3 O-deacylation experiments of 2′,3′,5′-tri-O-acylinosine are Molecules 2017, 22, 1219 is hardly available. Molecules 2017, 22, 1219

our purposes. The results of the thestability O-deacylation experiments of 2′,3′,5′-tri-O-acylinosine Therefore, we decided to study of different O-acyl groups to select the one optimal are for summarized in Table 1. summarized in Table 1. our purposes. The results of the O-deacylation experiments of 2′,3′,5′-tri-O-acylinosine are Table 1. Stability of O-acyl protecting groups under different deblocking conditions. summarizedTable in Table 1. 1. Stability of O-acyl protecting groups under different deblocking conditions.

Table 1. Stability of O-acyl protecting groups under different deblocking conditions. t1. h Substrate O-Deacylation Conditions, 20 a◦ C ta½. h Complete Complete O-Deacylation, h Substrate Conditions, 20 °C O-Deacylation, h 2 Table 1. Stability of O-Deacylation O-acyl protecting groups under different deblocking conditions. Substrate O-Deacylation Conditions, 20 °C a t½. h Complete O-Deacylation, h 3NH 2/C22H 5OH 0.25 2 CHCH /C OH (4M) 0.25 2 3 NH 2 H5(4M) 2H5OH (4M) 2 CH3NH2/C Substrate O-Deacylation Conditions, 20 °C a t0.25 ½. h Complete O-Deacylation, h CH3NH2/C2H5OH (4M) NHNH 3/MeOH (4M)(4M) 3 /MeOH NH3/MeOH (4M)

1 1

NH3/MeOH (4M)

1

CH3NH2/C2H5OH (4M) 3NH 2/C22H 5OH CHCH /C OH (4M) 3 NH 2 H5(4M) CH3NH2/C2H5OH (4M) NH3/MeOH (4M) NHNH 3/MeOH (4M) 3 /MeOH (4M) NH3/MeOH (4M)

a a

0.25

3 3

2 1

3

15 15

15

75 75 75

CH3NH2/C2H5OH (4M) CH3NH2/C2H5OH (4M)

6 6

26 26

3NH 2/C22H 5OH CHCH /C OH (4M) 3 NH 2 H5(4M)

6 19 19

NHNH 3/MeOH (4M)(4M) 3 /MeOH

19

15

15

15

NH3/MeOH (4M) NH3/MeOH (4M)

5

5

3 15 15

5 5

6

26

75

26

96 96 19

96

96

The reagent was used in at least 400-fold excess. The reagent was used in at least 400-fold excess.

The reagent was used in at least 400-fold excess. According to the data in Table 1, the acetyl group is rather labile under basic conditions, and the According to the data in Table 1,a The the reagent acetyl group is rather labile under basic conditions, and the was used in at least 400-fold excess. benzoyl group is the most stable. The properties of the iso-butyroyl group exhibited the best behavior benzoyl group istothe The of the iso-butyroyl group exhibited the best behavior According themost datastable. in Table 1, properties the acetyl group is rather labile under basic conditions, and the for our aims, since it is fairly resistant to the action of benzylamines, and its synthesis is more for our According aims, since is fairly resistant to the action of benzylamines, and its synthesis is more conditions, benzoyl group is the it most stable. The properties of the iso-butyroyl group exhibited the best behavior theofdata Table derivatives. 1, the acetyl is rather labile under basic straightforward thantothat the in benzoyl Thegroup compound 2′,3′,5′-tri-O-isobutyroyl-6straightforward thanit that of the benzoyl compound and 2′,3′,5′-tri-O-isobutyroyl-6for our aims, since is fairly resistant to derivatives. the action ofThe benzylamines, its synthesis is more and the benzoyl group is been the used mostasstable. The properties the iso-butyroyl group exhibited chloropurine riboside (4) has then the starting substrate in the of reactions with a small excess chloropurine riboside hasof then used asderivatives. the starting The substrate in the reactions with a small excess straightforward than (4) that thebeen benzoyl compound 2′,3′,5′-tri-O-isobutyroyl-6of the benzylamines with fluoroandaims, trifluoromethyl groups 1). The protective were best behavior for our since it is fairly(Scheme resistant to the action groups of benzylamines, and of benzylamines with(4) fluoroand trifluoromethyl groupssubstrate (Schemein1). protective were chloropurine riboside has then been used as the starting theThe reactions with agroups small excess removed in the presence of MeNH 2 /EtOH at room temperature with the subsequent chromatographic its synthesis is more straightforward than that ofwith thethe benzoyl derivatives. The compound removed in the presence of MeNH at room temperature chromatographic of benzylamines with fluoroand2/EtOH trifluoromethyl groups (Scheme 1). subsequent The protective groups were 0 ,30 ,50 -tri-O-isobutyroyl-6-chloropurine purification of the resulting products. Compounds 5–11 were obtained in been overallused goodas yield (50–98%). 2 riboside (4) has then the starting purification of the resulting products. Compounds 5–11 were obtained insubsequent overall good yield (50–98%). substrate in removed in the presence of MeNH 2/EtOH at room temperature with the chromatographic It should be mentioned that some of these compounds were previously prepared starting from 6the reactions a small excess of benzylamines with fluoro-inand trifluoromethyl groups It should beofmentioned that some ofCompounds these compounds were previously prepared from 6- (Scheme 1). purification thewith resulting products. 5–11 were obtained overall goodstarting yield (50–98%). chloropurine riboside [26]. The protective groups were removed the presence MeNH2 /EtOH at room temperature with the chloropurine riboside [26]. It should be mentioned that some of these in compounds wereofpreviously prepared starting from 6chloropurine riboside [26]. subsequent chromatographic purification of the resulting products. Compounds 5–11 were obtained a

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in overall good yield (50–98%). It should be mentioned that some of these compounds were previously Molecules 2017, 22, 1219from 6-chloropurine riboside [26]. 4 of 12 prepared starting

6 0 ,30 ,50 -tri-OScheme 1. Synthesis by the the substitution Scheme Synthesis of of N N6-alkyladenosines -alkyladenosines by substitution of the the chlorine chlorine atom atom in in22′,3′,5′-tri-Oisobutyroyl-6-chloropurineriboside.Reagents Reagentsand andconditions: conditions: RNH MeCN, 70 ◦10–24 C, 10–24 h; isobutyroyl-6-chloropurineriboside. (i)(i) RNH 2, DIPEA, MeCN, 70 °C, h; (ii) 2 , DIPEA, (ii) MeNH /EtOH, room temperature., 24 h, 50–98% (overall yields); (The structure of R is given in 2 room temperature., 24 h, 50–98% (overall yields); (The structure of R is given in Table 1). MeNH2/EtOH, Table 1).

The structure of the obtained compounds was confirmed by NMR and mass spectroscopy. The presence of fluorineofatoms in the phenyl residuewas wasconfirmed confirmedby byNMR spin-spin coupling constants The structure the obtained compounds and mass spectroscopy. 19 1 1 19 13 13 between F and H in H-NMR (JH-F) residue and between F and C C-NMRcoupling spectra (J C-F). The The presence of fluorine atoms inspectra the phenyl was confirmed byinspin-spin constants 1H-NMR 19 1 Hthe 1 H-NMR spectra 19 F and 13 Cininthe 13 C-NMR N6-benzyladenosine analogues (5–8) low fieldspectra region (J were between spectra F and of in fluorinated (JH-F ) and between C-F ). 19F-1H couplings: 3JH-F–8.0–9.0 Hz, 4JH-F–6.7–5.5 Hz, and 5JH-F ˂ 2.0 Hz. 1 6 complicated by the presence of The H-NMR spectra of the fluorinated N -benzyladenosine analogues (5–8) in the low field region 13C-NMR spectra, three types of coupling 19 F-1 H couplings: 4J In thecomplicated constants J3C-F were present, are characteristic were by the presence JH-F –8.0–9.0 Hz,which Hz, and H-F –6.7–5.5 1JC-F–240–248 Hz, 2JC-F–12–24 Hz, and 3JC-F–7.5 Hz. The 5 J fluorinated 13 of aromatic compounds [29]: H-F < 2.0 Hz. In the C-NMR spectra, three types of coupling constants JC-F were present, which presence of trifluoromethyl residue in nucleosidic derivatives was confirmed low-intensive are characteristic of fluorinated aromatic compounds [29]: 1 J(9–11) Hz, 2 JC-Fby –12–24 Hz, and C-F –240–248 13 3 quartet coupling constant of ~30 Hz in residue C-NMR Thisderivatives constant was consistent with the JC-F –7.5with Hz. aThe presence of trifluoromethyl inspectra. nucleosidic (9–11) was confirmed 13 literature data for trifluoromethylated aromatic compounds [29]. Despite the majority of was the by low-intensive quartet with a coupling constant of ~30 Hz in C-NMR spectra. This constant synthesized compounds having been characterized by aromatic NMR earlier, their detailed analysis and the consistent with the literature data for trifluoromethylated compounds [29]. Despite the majority assignment of all chemical shifts and coupling constants has not been presented. Therefore, we of the synthesized compounds having been characterized by NMR earlier, their detailed analysis and the provided in the Supplementary section a detailed NMR analysis for each analogue produced. assignment of all chemical shifts and coupling constants has not been presented. Therefore, we provided in the Supplementary section a detailed NMR analysis for each analogue produced. 2.2. Biological Activity on EV71 and Structure-Activity Relationship (SAR) 2.2. Biological Activity on EV71 and Structure-Activity Relationship (SAR) We have shown earlier that three natural cytokinin nucleosides (compound 1–3) exerted a potent We have that three natural 1–3)also exerted a potent antiviral effectshown on theearlier replication of EV71 withcytokinin an EC50 ofnucleosides 0.3–1.4 μM,(compound but exhibited a rather high antiviral effect on the replication of EV71 with an EC50 of 0.3–1.4 µM, but exhibited also a rather high (linker) of cytotoxicity [20,21] (Table 2). As previously mentioned, modifications of the N6-substituent 6 cytotoxicity [20,21]led (Table As previously mentioned, modifications theproduced N -substituent of the BAPR scaffold to a 2). remarkable increase of selectivity [21]. Here,ofwe a series(linker) of BAPR the BAPR scaffold led to remarkable of selectivity Here, we produced a series of BAPR analogues to evaluate thea effect of the increase fluorination of BAPR[21]. on the replication of EV71. A cytopathic analogues evaluate assay the effect the fluorination ofnewly BAPRsynthetized on the replication of EV71. A cytopathic effect (CPE)toreduction wasof performed with the analogues (compounds 5–11) 6 6 effect (CPE) reduction assay the newly synthetized analogues (compounds 5–11) in rhabdomyosarcoma (RD) was cells.performed BAPR, N with -isopenthenyladenosine, and N -furfuryladenosine were 6 -isopenthenyladenosine, and N 6 -furfuryladenosine were in rhabdomyosarcoma (RD) cells. included in the screening, and theBAPR, toxicityNof all of the aforementioned compounds was evaluated in includedonintreated-uninfected the screening, andcells. the toxicity of all of the aforementioned compounds was evaluated in parallel parallel on treated-uninfected cells.

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6-substituted adenosines on the replication of the EV71 strain BrCr 6 -substituted Table 2.2. Antiviral Antiviral effect effectofofNN Table adenosines on the replication of the EV71 strain BrCr in in RD cells. RD cells.

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Table 2. Antiviral effect of N66-substituted adenosines on the replication of the EV71 strain BrCr in RD cells.

No. 1 2

Compound Name Compound Name Compound Name

No. No. 1

1

2

2

3

Compound Name

6 N6-benzyladenosine -benzyladenosine NN66-benzyladenosine (BAPR) (BAPR) (BAPR)

N666-Isopenthenyladenosine

-Isopenthenyladenosine NN -Isopenthenyladenosine N6-Isopenthenyladenosine (iPR) (iPR) (iPR) 6 -furfuryladenosine NN66-furfuryladenosine (KINR) (KINR) N6-furfuryladenosine

3

3

Substituent Substituent (R) Substituent (R)

a,b с a,b±с SD a,b (R) CC 50a,b± SD EC SI c SI CC5050±± SD SD 50 a,b a,b a,b 50 ±a,b CC SD EC50EC ± SD SI с

CC50 ± SD

4.3 1.61.6 4.3 4.3±±

NN666-(2-fluorobenzyl) -(2-fluorobenzyl)adenosine adenosine

5

6

6

-(3-fluorobenzyl)adenosine adenosine NN666-(3-fluorobenzyl)

6

7

7

adenosine N6-(3-fluorobenzyl) -(4-fluorobenzyl) adenosine NN666-(4-fluorobenzyl) adenosine

7

8

8

-(2,6-difluorobenzyl) adenosine NN666-(2,6-difluorobenzyl) adenosine N6-(4-fluorobenzyl) adenosine

9

9

5

N6-(2-fluorobenzyl) adenosine

8

10 10

9

6.0

7.8 3.43.4 7.8±±

1.4 ±1.4 0.3± 0.3 5.6

5.6

7.8 ± 3.4

a

aa

13.3±± 13.3 3.73.7

13.3 ± 3.7

>254 >254 2.7

>235 >235

>235 >235

5.6 44

0.30 ± 0.05

0.24 0.24 ± 0.09 ± 0.09 26

6.2 2.7±± 2.7 0.90.9

6.0

1.4 ± 0.3

± 0.05 44 0.30 0.30 ± 0.05

6.2 1.81.8 6.2±±

6 -(3-trifluoromethylbenzyl)adenosine adenosine NN66-(3-trifluoromethylbenzyl)

-(4-trifluoromethylbenzyl)adenosine adenosine NN66-(4-trifluoromethylbenzyl)

1.0 ± 0.2

15

15

1.0 ±1.0 0.2± 0.2 6.0

>235 >235

adenosine N6-(2-trifluoromethylbenzyl) 6

11 11

± 0.05 ± 0.28 1.6 0.28 0.2815± 0.05 ± 0.05

6.0 ± 0.6

6 -(2-trifluoromethylbenzyl)adenosine adenosine NN66-(2-trifluoromethylbenzyl)

N6-(2,6-difluorobenzyl) adenosine

SI

6.0 0.60.6 6.0±±

(KINR) 5

EC50 ± SD

44 26

± 0.14 1.8 0.14 0.2419± 0.09 ± 0.05 ± 0.05

26

19

± 0.01 >1210 ± 0.01 ± 0.21 0.9 0.21 0.14 ± 0.05>1210 19

1.0 ±1.0 0.1± 0.1 >235

>235

0.0680.068 ± 0.001 >3456 ± 0.001

>3456

>254

0.21 ± 0.01

>235

1.0 ± 0.1

1.0 ±1.0 0.1± 0.1 >235

All values are in μM and are based on at least three independent dose-response curves;

b b

>1210

>235 >235

On

All values are in µM and are based on at least three independent dose-response curves; b On rhabdomyosarcoma c

c 50/EC50 50; SD, standard deviation. 6-(3-trifluoromethylbenzyl) rhabdomyosarcoma (RD) cells; adenosine (SI); SI = CC50 c Selectivity Index 10 (RD)Ncells; >235 0.068 ± 0.001 (SI); SI =Selectivity CC50 /ECIndex 50 ; SD, standard deviation.

>3456

Overall, the incorporation of fluoro- and trifluoromethyl groups significantly improved the selectivity of BAPR (Table 2). In particular, the monofluorination of significantly the phenyl group Overall, theindex incorporation ofadenosine fluoroand trifluoromethyl groups improved the 6-(4-trifluoromethylbenzyl) 11 N >235 1.0 ± 0.1 >235 (compounds 5–7) slightly changed the SI because of the concomitant cytotoxicity of such compounds. selectivity index of BAPR (Table 2). In particular, the monofluorination of the phenyl group Surprisingly, the incorporation of a second fluorine atom resulted in a substantial improvement over (compounds 5–7)are slightly SI 8because ofanthe concomitant such compounds. a All b On thevalues selectivity. In presented EC50 50 comparable tocytotoxicity BAPR with a of dramatic inparticular, μMchanged and compound are the based on at least three independent dose-response curves; c 50 50 Surprisingly, the incorporation of a second fluorine atom resulted in a substantial improvement reduction of cell toxcity: from a CC 50 of 13.3 ± 3.7 μM for the monofluorinated analogue to a CC 50 rhabdomyosarcoma (RD) cells; Selectivity Index (SI); SI = CC50/EC50; SD, standard deviation. over the selectivity. In particular, compound 8 presented an EC50 comparable to BAPR with a Overall, the incorporation of from fluoroand trifluoromethyl improved the dramatic reduction of cell toxcity: a CC 13.3 ± 3.7 µMgroups for the significantly monofluorinated analogue 50 of selectivity index of BAPR particular, counterpart. the monofluorination of the group to a CC50 higher than 250 (Table µM for2). theIndifluorinate We wanted alsophenyl to assess the (compounds 5–7) slightly changed the the SI because the of concomitant of such Compounds compounds. effect of a trifluoromethyl group on phenylofring BAPR on cytotoxicity EV71 replication. Surprisingly, the incorporation a second fluorine resulted in a substantial over 9, 10 and 11 did not show anyofcytotoxicity at the atom highest concentration tested, improvement and the analogue the selectivity. In particular, compound 8 presented an EC50 comparable to BAPR with a dramatic reduction of cell toxcity: from a CC50 of 13.3 ± 3.7 μM for the monofluorinated analogue to a CC50

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N6 -(3-trifluoromethylbenzyl)-adenosine (compound 10) exhibited also a four-fold improvement in potency as compared to BAPR. Previous reports showed that the halogenation (and, in particular, the addition of I or Cl atoms) on a BAPR scaffold increased its selectivity by reducing the cell toxicity in cancer cell lines [26,30]. In line with these findings, we observed that the gain in selectivity in our model was mostly due to a decreased cell toxicity. In particular, only the analogues containing two fluorine or a trifluoromethyl group dramatically improved the cytotoxicity. In spite of our interest in understanding compound-driven cell toxicity, addressing this question was beyond our scientific scope. Future works on the optimization of this class of analogues may shed light on the mechanism of action and their metabolization within an infected cell. Altogether, our data revealed that the introduction of at least two fluorine atoms or a trifluoromethyl group on the phenyl ring of BAPR dramatically improved its selectivity by reducing the cytotoxicity, and in case of compound 10, also by increasing the potency. 3. Materials and Methods 3.1. General The solvents and materials were reagent grade and were used without additional purification. Column chromatography was performed on silica gel (Kieselgel 60 Merck, Germany, 0.063–0.200 mm). TLC was performed on an Alugram SIL G/UV254 (Macherey-Nagel, Düren, Germany) with UV visualization. The melting points were determined with Electrothermal Melting Point Apparatus IA6301 and are uncorrected. The 1 H and 13 C (with complete proton decoupling) NMR spectra were recorded on a Bruker (Karlsruhe, Germany) AMX 400 NMR instrument at 303 K. The 1 H-NMR-spectra were recorded at 400 MHz and the 13 C-NMR-spectra at 100 MHz. The chemical shifts in ppm were measured relative to the residual solvent signals as internal standards (CDCl3 , 1H: 7.26 ppm, 13C: 77.1 ppm; DMSO-d6 , 1H: 2.50 ppm, 13C: 39.5 ppm). Spin-spin coupling constants (J) are given in Hz. The high resolution mass spectra (HRMS) were registered on a Bruker Daltonics (Madison, WI, USA) micrOTOF-Q II instrument using electrospray ionization (ESI). The measurements were done in positive ion mode. Interface capillary voltage: 4500 V; mass range from m/z 50 to 3000; external calibration (Electrospray Calibrant Solution, Fluka); nebulizer pressure: 0.4 Bar; flow rate: 3 µL/min; dry gas: nitrogen (4 L/min); interface temperature: 200 ◦ C. Samples were injected into the mass spectrometer chamber from the Agilent 1260 HPLC system equipped with an Agilent (Palo Alto, CA, USA) Poroshell 120 EC-C18 (3.0 × 50 mm; 2.7 µm) column: the flow rate was 200 µL/min; and the samples were injected from the acetonitrile–water (1:1) solution and eluted in a linear gradient of acetonitrile concentrations (50→100%). 3.2. 9-(2,3,5-Tri-O-isobutyroyl-β-D-ribofuranosyl)-6-chloropurine (4) Isobutyric anhydride (5.6 mL, 33.8 mmol) was added in portions to a suspension of 3 g (11.2 mmol) of inosine in 18 mL of dry pyridine. The reaction mixture was stirred for 24 h at room temperature, and then evaporated in vacuum. The residue was diluted with the mixture ethanol:water (50 mL), and the suspension was filtered. The resulting powder was washed with the mixture ethanol:water (50 mL) and dried in a vacuum dessicator over phosphorous pentoxide for 2 days. The yield was 5 g (93%) of 20 ,30 ,50 -tri-O-isobutyroylinosine as a white powder. Rf 0.39 (CH2 Cl2 :EtOH—98:2). 1 H-NMR (400 MHz, CDCl3 ): δ = 1.23–1.12 (m, 18H, Me-i-Bu), 2.66–2.50 (m, 3H, CH-i-Bu), 4.40 (d, 2H, J50 40 = 3.7 Hz, H50 ), 4.45 (dt, 1H, J40 50 = 3.7 Hz, J40 30 = 4.6 Hz, H40 ), 5.60 (dd, 1H, J30 40 = 4.6 Hz, J30 20 = 5.4 Hz, H30 ), 5.84 (dd, 1H, J20 30 = 5.4 Hz, J20 10 = 5.3 Hz, H20 ), 6.18 (d, 1H, J10 20 = 5.3 Hz, H10 ), 8.21 (br s, 2H, H2, H8 Hyp), 12.99 (br s, 1H, NH Hyp). The compound 20 ,30 ,50 -tri-O-isobutyroylinosine (2.9 g, 6.05 mmol) was then dissolved in 30 mL of a DMF:dichloroethane (1:15) mixture, and thionyl chloride (1.14 mL, 15.7 mmol) was added dropwise to the mixture under intensive stirring. After stirring at 65 ◦ C for 15 min, the reaction mixture was diluted

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with dichloromethane (60 mL) and washed successively with 10% sodium bicarbonate (4 × 50 mL) and water (2 × 50 mL). The organic layer was separated, dried over anhydrous Na2 SO4 , filtered, and evaporated in a vacuum. The residue was purified by column chromatography on silica gel (200 mL). The column was washed with dichloromethane (200 mL). The product was eluted with the system CH2 Cl2 :EtOH—96:4. The yield was 2.8 g (93%) as a slightly yellow syrup. Rf 0.52 (CH2 Cl2 :EtOH—98:2). 1 H-NMR (400 MHz, CDCl ): δ = 1.09–1.23 (m, 18H, Me-i-Bu), 2.49–2.69 (m, 3H, CH-i-Bu), 4.40 (d, 3 2H, J50 ,40 = 3.7 Hz, H50 ), 4.27 (dd, 1H, J40 ,50 = 3.7 Hz, J40 ,30 = 4.5 Hz, H40 ), 5.63 (dd, 1H, J30 ,20 = 5.3 Hz, J40 ,30 = 4.5 Hz, H30 ), 5.89 (t, 1H, J20 ,30 = 5.3 Hz, J20 ,10 = 5.3 Hz, H20 ), 6.22 (d, 1H, J10 ,20 = 5.3 Hz, H10 ), 8.30 (s, 1H, H2), 8.76 (s, 1H, H8). 13 C-NMR (100 MHz, CDCl3 ): δ = 18.77, 18.85, 18.93, 18.99, 19.01, 19.11 (CH3 -i-Bu), 33.80, 33.92, 34.05 (CH-i-Bu), 63.05 (C50 ), 70.55 (C30 ), 73.46 (C20 ), 81.12 (C40 ), 87.10 (C10 ), 132.40 (C6), 143.57 (C5), 151.42 (C8), 151.73 (C4), 152.44 (C2), 175.51, 175.71, 176.57 (C=O). HRMS: m/z [M + H]+ calculated C22 H30 ClN4 O7 + 497.1798, found 497.1798. 3.3. N6 -(2-Fluorobenzyl)adenosine (5) A mixture of 4 (200 mg, 0.4 mmol) and 2-fluorobenzylamine (0.091 mL, 0.8 mmol) was dissolved in MeCN (3 mL), and then DIPEA (0.14 mL, 0.8 mmol) was added in one portion. The solution was stirred at 70 ◦ C. The reaction was monitored by TLC (CH2 Cl2 :EtOH—99.5:0.5). After 22 h, the reaction mixture was evaporated in a vacuum and the residue was diluted with methylene chloride (30 mL) and washed with water (2 × 15 mL). The organic layer was separated, dried over anhydrous Na2 SO4 , and evaporated in a vacuum. The residue was purified by column chromatography on silica gel. The product was eluted with CH2 Cl2 :EtOH—99:1. The yield was 208 mg (89%) of N6 -(2-fluorobenzyl)-20 ,30 ,50 -tri-O-isobutyroyladenosine as a syrup. Rf 0.6 (CH2 Cl2 :EtOH—99.5:0.5). 1 H-NMR (400 MHz, DMSO-d ): δ = 1.0–1.2 (m, 18H, Me-i-Bu), 2.51–2.66 (m, 3H, CH-i-Bu), 4.35–4.41 (m, 6 2H, H50 ), 4.27 (ddd, 1H, J40 ,50 = 6.1 Hz, H40 ), 4.76 (br s, 2H, CH2 ), 5.75 (dd, 1H, J30 ,20 = 5.4 Hz, H30 ), 6.03 (dd, 1H, J20 ,30 = 5.4 Hz, J20 ,10 = 4.9 Hz, H20 ), 6.21 (d, 1H, J10 ,20 = 4.9 Hz, H10 ), 7.11 (td, 1H, 3 J5-6 = 7.5 Hz, 3J 4 3 3 4 5-4 = 7.5 Hz, J 5-3 = 1.0 Hz, 5H-2-F-Ph), 7.16 (ddd, 1H, J H-F = 8.2 Hz, J 3-4 = 8.8 Hz, J 3-5 = 1.0 Hz, 4 3 3 4 3H-2-F-Ph), 7.28 (dddd, 1H, JH-F = 5.5 Hz, J4-3 = 8.8 Hz, J4-5 = 7.5 Hz, J4-6 = 1.6 Hz, 4H-2-F-Ph), 7.32 (ddd, 1H, 4 JH-F = 7.5 Hz, 3 J6-5 = 7.5 Hz, 4 J6-4 = 1.6 Hz, 6H-2-F-Ph), 7.23 (s, 1H, H2 Ade), 8.36 (s, 1H, H8 Ade), 8.42 (1H, NH). The resulting N6 -(2-fluorobenzyl)-20 ,30 ,50 -tri-O-isobutyroyladenosine (206 mg, 0.352 mmol) was treated with 8 M MeNH2 in EtOH solution (4.5 mL). After 2 days, the mixture was evaporated in a vacuum and the residue was purified by column chromatography on silica gel. The column was washed with CH2 Cl2 :EtOH—95:5, and then eluted with CH2 Cl2 :EtOH—90:10 to give 5 as a white powder. The yield was 118 mg (79% for two steps). Rf 0.15 (CH2 Cl2 :EtOH—95:5). m.p. 194–195 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ = 3.55 (ddd, 1H, J50 b,50 a = −12.0 Hz, J50 b,40 = 3.4 Hz, J50 b,OH = 6.9 Hz, H50 b), 3.68 (ddd, 1H, J50 a,50 b = −12.0 Hz, J50 a,40 = 3.4 Hz, J50 a,OH = 4.7 Hz, H50 a), 3.96 (ddd, 1H, J40 ,50 b = 3.4, J40 ,50 a = 3.4 Hz, J40 ,30 = 3.4 Hz, H40 ), 4.15 (ddd, 1H, J30 ,40 = 3.4 Hz, J30 ,20 = 4.9 Hz, J30 ,OH = 4.7 Hz, H30 ), 4.61 (ddd, 1H, J20 ,30 = 4.9 Hz, J20 ,10 = 6.1 Hz, J20 ,OH = 6.2 Hz, H20 ), 4.77 (br s, 2H, CH2 ), 5.15 (d, 1H, JOH-30 = 4.7 Hz, 30 OH), 5.31 (dd, 1H, JOH-50 b = 6.9 Hz, JOH-50 a = 4.7 Hz, 50 OH), 5.41 (d, 1H, JOH-20 = 6.2 Hz, 20 OH), 5.90 (d, 1H, J10 ,20 = 6.1 Hz, H10 ), 7.11 (td, 1H, 3 J5-6 = 7.5 Hz, 3 J5-4 = 7.5 Hz, 4 J5-3 = 1.0 Hz, 5J 3 3 4 H-F < 1.0 Hz, 5H-2-F-Ph), 7.16 (ddd, 1H, J H-F = 9.4 Hz, J 3-4 = 8.2 Hz, J 3-5 = 1.0 Hz, 3H-2-F-Ph), 4 3 3 4 7.26 (dddd, 1H, JH-F = 5.5 Hz, J4-3 = 8.2 Hz, J4-5 = 7.5 Hz, J4-6 = 1.7 Hz, 4H-2-F-Ph), 7.32 (ddd, 1H, 4 JH-F = 6.7 Hz, 3 J6-5 = 7.5 Hz, 4 J6-4 = 1.7 Hz, 6H-2-F-Ph), 8.21 (s, 2H, H2, NH), 8.39 (s, 1H, H8). 13 C-NMR (100 MHz, DMSO-d ): δ = 36.78 (CH ), 61.62 (C50 ), 70.60 (C30 ), 73.49 (C20 ), 85.86 (C40 ), 87.92 6 2 (C10 ), 114.95 (d, 2 JC-F = 21.1 Hz, C3-Ph), 119.77 (C5), 124.18 (C5-Ph), 126.52 (d, 2 JC-F = 12.6 Hz, C1-Ph), 128.52 (d, 3 JC-F = 7.4 Hz, C4-Ph), 128.85 (br s, C6-Ph), 140.00 (C8), 148.59 (C4), 152.29 (C2), 154.49 (C6), 160.00 (d, 1 JC-F = 244.0 Hz, C2-Ph). HRMS: m/z [M + H]+ calculated C17 H19 FN5 O4 + 376.1416, found 376.1417; m/z [M + Na]+ calculated C17 H18 FN5 O4 Na+ 398.1235, found 398.1238.

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3.4. N6 -(3-Fluorobenzyl)adenosine (6) Following the procedure for the preparation of 5, the condensation of 4 (200 mg, 0.4 mmol) with 3-fluorobenzylamine (0.091 mL, 0.8 mmol) in the presence of DIPEA (0.14 mL, 0.8 mmol) in MeCN (3 mL) for 22 h at 70 ◦ C with a subsequent deblocking in 8 M MeNH2 in EtOH solution (4.5 mL) at room temperature gave 6 as a white powder. The overall yield was 111 mg (74%). Rf 0.15 (CH2 Cl2 :EtOH—95:5). m.p. 159–160 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ = 3.56 (ddd, 1H, J50 b,50 a = −12.0 Hz, J50 b,40 = 3.4 Hz, J50 b,OH = 6.9 Hz, H50 b), 3.67 (ddd, 1H, J50 a,50 b = −12.0 Hz, J50 a,40 = 3.4 Hz, J50 a,OH = 4.7 Hz, H50 a), 3.96 (ddd, 1H, J40 ,50 b = 3.4 Hz, J40 ,50 a = 3.4 Hz, J40 ,30 = 3.4 Hz, H40 ), 4.15 (ddd, 1H, J30 ,40 = 3.4 Hz, J30 ,20 = 4.7 Hz, J30 ,OH = 4.7 Hz, H30 ), 4.61 (ddd, 1H, J20 ,30 = 4.7 Hz, J20 ,10 = 6.1 Hz, J20 ,OH = 6.2 Hz, H20 ), 4.77 (br s, 2H, CH2 ), 5.15 (d, 1H, JOH-30 = 4.7 Hz, 30 OH), 5.32 (dd, 1H, JOH-50 b = 6.9 Hz, JOH-50 a = 4.7 Hz, 50 OH), 5.41 (d, 1H, JOH-20 = 6.2 Hz, 20 OH), 5.91 (d, 1H, J10 ,20 = 6.1 Hz, H10 ), 7.03 (dd, 1H, 3 JH-F = 8.9 Hz, 3 J4-5 = 8.2 Hz, 4H-3-F-Ph), 7.13 (d, 1H, 3 JH-F = 8.9 Hz, 2H-3-F-Ph), 7.18 (dd, 1H, 3 J6-5 = 8.2 Hz, 5 JH-F = 2.2 Hz, 6H-3-F-Ph), 7.33 (td, 1H, 4 JH-F = 6.3 Hz, 3 J5-4 = 8.2 Hz, 3J 13 5-6 = 8.2 Hz, 5H-3-F-Ph), 8.21 (s, 1H, H2 Ade), 8.39 (s, 1H, H8 Ade), 8.46 (br s, 1H, NH). C-NMR (100 MHz, DMSO-d6 ): δ = 42.51 (CH2 ), 61.61 (C50 ), 70.60 (C30 ), 73.49 (C20 ), 85.86 (C40 ), 87.93 (C10 ), 113.29 (d, 2 JC-F = 21.0 Hz, C2-Ph), 113.71 (d, 2 JC-F = 21.5 Hz, C4-Ph), 119.76 (C5), 123.06 (C6-Ph), 130.10 (d, 3 JC-F = 7.5 Hz, C5-Ph), 139.99 (C8), 143.09 (br s, C1-Ph), 148.54 (C4), 152.30 (C2), 154.44 (C6), 162.16 (d, 1 JC-F = 243.1 Hz, C3-Ph). HRMS: m/z [M + H]+ calculated C17 H19 FN5 O4 + 376.1416, found 376.1418; m/z [M + Na]+ calculated C17 H18 FN5 O4 Na+ 398.1235, found 398.1239. 3.5. N6 -(4-Fluorobenzyl)adenosine (7) Following the procedure for the preparation of 5, the condensation of 4 (200 mg, 0.4 mmol) with 4-fluorobenzylamine (0.091 mL, 0.8 mmol) in the presence of DIPEA (0.14 mL, 0.8 mmol) in MeCN (3 mL) for 22 h at 70 ◦ C with a subsequent deblocking in 8 M MeNH2 in EtOH solution (4.5 mL) at room temperature gave 7 as a white powder. The overall yield was 81 mg (54%). Rf 0.15 (CH2 Cl2 :EtOH—95:5). mp 181–182 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ = 3.55 (ddd, 1H, J50 b,50 a = −12.0 Hz, J50 b,40 = 3.4 Hz, J50 b,OH = 6.9 Hz, H50 b), 3.68 (ddd, 1H, J50 a,50 b = −12.0 Hz, J50 a,40 = 3.4 Hz, J50 a,OH = 4.6 Hz, H50 a), 3.96 (ddd, 1H, J40 ,50 b = 3.4 Hz, J40 ,50 a = 3.4 Hz, J40 ,30 = 3.4 Hz, H40 ), 4.15 (ddd, 1H, J30 ,40 = 3.4 Hz, J30 ,20 = 4.7 Hz, J30 ,OH = 4.7 Hz, H30 ), 4.62 (ddd, 1H, J20 ,30 = 4.7 Hz, J20 ,10 = 6.1 Hz, J20 ,OH = 6.2 Hz, H20 ), 4.69 (br s, 2H, CH2 ), 5.15 (d, 1H, JOH-30 = 4.7 Hz, 30 OH), 5.32 (dd, 1H, JOH-50 b = 6.9 Hz, JOH-50 a = 4.6 Hz, 50 OH), 5.41 (d, 1H, JOH-20 = 6.2 Hz, 20 OH), 5.89 (d, 1H, J10 ,20 = 6.1 Hz, H10 ), 7.11 (t, 2H, 3 JH-F = 8.9 Hz, 3 J3-2 = 8.9 Hz, 3H-4-F-Ph, 5H-4-F-Ph), 7.37 (dd, 2H, 3 J2-3 = 8.9, 4 JH-F = 6.0 Hz, 2H-4-F-Ph, 6H-4-F-Ph), 8.21 (s, 1H, H2 Ade), 8.37 (s, 1H, H8 Ade), 8.43 (br s, 1H, NH). 13 C-NMR (100 MHz, DMSO-d6 ): δ = 42.21 (CH2 ), 61.62 (C50 ), 70.61 (C30 ), 73.48 (C20 ), 85.87 (C40 ), 87.93 (C10 ), 114.87 (d, 2 JC-F = 21.2 Hz, C3-Ph, C5-Ph), 119.74 (C5), 129.05 (d, 3 JC-F = 7.5 Hz, C2-Ph, C6-Ph), 136.15 (C1-Ph), 139.91 (C8), 148.52 (C4), 152.30 (C2), 154.43 (C6), 161.07 (d, 1 JC-F = 241.9 Hz, C4-Ph). HRMS: m/z [M + H]+ calculated C17 H19 FN5 O4 + 376.1416, found 376.1407; m/z [M + Na]+ calculated C17 H18 FN5 O4 Na+ 398.1235, found 398.1227. 3.6. N6 -(2,6-Difluorobenzyl)adenosine (8) Following the procedure for the preparation of 5, the condensation of 4 (191 mg, 0.384 mmol) with 2,6-difluorobenzylamine (0.092 mL, 0.77 mmol) in the presence of DIPEA (0.134 mL, 0.77 mmol) in MeCN (2.5 mL) for 11 h at 70 ◦ C with a subsequent deblocking in 8 M MeNH2 in EtOH solution (2 mL) at room temperature gave 8 as a white powder. The overall yield was 129 mg (85%). Rf 0.15 (CH2 Cl2 :EtOH—95:5). m.p. 192–194 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ = 3.56 (ddd, 1H, J50 b,50 a = −12.0 Hz, J50 b,40 = 3.5 Hz, J50 b,OH = 6.8 Hz, H50 b), 3.68 (ddd, 1H, J50 a,50 b = −12.0 Hz, J50 a,40 = 3.5 Hz, J50 a,OH = 4.7 Hz, H50 a), 3.97 (ddd, 1H, J40 ,50 b = 3.5 Hz, J40 ,50 a = 3.5 Hz, J40 ,30 = 3.3 Hz, H40 ), 4.15 (ddd, 1H, J30 ,40 = 3.3 Hz, J30 ,20 = 5.2 Hz, J30 ,OH = 4.6 Hz, H30 ), 4.61 (ddd, 1H, J20 ,30 = 5.2 Hz, J20 ,10 = 6.1 Hz, J20 ,OH = 6.1 Hz, H20 ), 4.80 (br s, 2H, CH2 ), 5.15 (d, 1H, JOH-30 = 4.6 Hz, 30 OH), 5.31 (dd, 1H, JOH-50 b = 7.0 Hz, JOH-50 a = 4.7 Hz, 50 OH), 5.41 (d, 1H, JOH-20 = 6.1 Hz, 20 OH), 5.89 (d, 1H, J10 ,20 = 6.1 Hz,

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H10 ), 7.05 (dd, 2H, 3 JH-F =7.6 Hz, Jm-H-p-H = 8.9 Hz, m-H-2,6-di-F-Ph), 7.37 (tt, 1H, Jp-H-m-H =8.9 Hz, 4J H-F = 6.0 Hz, p-H-2,6-di-F-Ph), 8.19 (br s, 1H, NH), 8.24 (s, 1H, H8 Ade), 8.36 (s, 1H, H2 Ade). 13 C-NMR (100 MHz, DMSO-d ): δ = 32.17 (CH ), 61.61 (C50 ), 70.59 (C30 ), 73.50 (C20 ), 85.83 (C40 ), 87.93 6 2 (C10 ), 111.38 (d, 2 JC-F = 23.6 Hz, C3-Ph, C5-Ph), 114.44 (t, 2 JC-F = 17.6 Hz, C1-Ph), 119.70 (C5), 129.60 (C4-Ph), 139.85 (C8), 148.64 (C4), 152.17 (C2), 154.16 (C6), 161.23 (d, 1 JC-F = 248.0 Hz, C2-Ph, C6-Ph). HRMS: m/z [M + H]+ calculated C17 H18 F2 N5 O4 + 394.1321, found 394.1325; m/z [M + Na]+ calculated C17 H17 F2 N5 O4 Na+ 416.1141, found 416.1143. 3.7. N6 -(2-Trifluoromethylbenzyl)adenosine (9) Following the procedure for the preparation of 5, the condensation of 4 (213 mg, 0.43 mmol) with 2-trifluoromethylbenzylamine (0.12 mL, 0.86 mmol) in the presence of DIPEA (0.15 mL, 0.86 mmol) in MeCN (3 mL) for 10 h at 70 ◦ C with a subsequent deblocking in 8 M MeNH2 in EtOH solution (3 mL) at room temperature gave 9 as a white powder. The overall yield was 168 mg (92%). Rf 0.13 (CH2 Cl2 :EtOH—95:5). m.p. 204–205 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ = 3.56 (ddd, 1H, J50 b,50 a = −12.0 Hz, J50 b,40 = 3.5 Hz, J50 b,OH = 6.9 Hz, H50 b), 3.68 (ddd, 1H, J50 a,50 b = −12.0 Hz, J50 a,40 = 3.5 Hz, J50 a,OH = 4.7 Hz, H50 a), 3.97 (ddd, 1H, J40 ,50 b = 3.5 Hz, J40 ,50 a = 3.5 Hz, J40 ,30 = 3.5 Hz, H40 ), 4.16 (ddd, 1H, J30 ,40 = 3.5 Hz, J30 ,20 = 5.2 Hz, J30 ,OH = 4.7 Hz, H30 ), 4.63 (ddd, 1H, J20 ,30 = 5.2 Hz, J20 ,10 = 6.1 Hz, J20 ,OH = 6.2 Hz, H20 ), 4.93 (br s, 2H, CH2 ), 5.15 (d, 1H, JOH-30 = 4.7 Hz, 30 OH), 5.29 (dd, 1H, JOH-50 b = 6.9 Hz, JOH-50 a = 4.7 Hz, 50 OH), 5.41 (d, 1H, JOH-20 = 6.2 Hz, 20 OH), 5.91 (d, 1H, J10 ,20 = 6.1 Hz, H10 ), 7.40–7.75 (m, 4H, Ph), 8.19 (s, 1H, H8), 8.42 (s, 1H, H2), 8.45 (br s, 1H, NH). 13 C-NMR (100 MHz, DMSO-d6 ): δ = 39.52 (CH2, overlapping with the solvent peak), 61.62 (C50 ), 70.62 (C30 ), 73.51 (C20 ), 85.89 (C40 ), 87.92 (C10 ), 119.83 (C5), 123.24 (C2 Ph), 125.77 (C6 Ph), 125.95 (q, JC-F = 30.7 Hz, CF3 ), 127.03 (C3 Ph), 127.54 (C4 Ph), 132.55 (C5 Ph), 138.10 (C1 Ph), 140.17 (C8), 146.68 (C4), 152.38 (C2), 154.52 (C6). HRMS: m/z [M + H]+ calculated C18 H19 F3 N5 O4 + 426.1384, found 426.1387; m/z [M + Na]+ calculated C18 H18 F3 N5 O4 Na+ 448.1203, found 448.1210. 3.8. N6 -(3-Trifluoromethylbenzyl)adenosine (10) Following the procedure for the preparation of 5, the condensation of 4 (137 mg, 0.276 mmol) with 3-trifluoromethylbenzylamine (0.08 mL, 0.55 mmol) in the presence of DIPEA (0.096 mL, 0.55 mmol) in MeCN (2 mL) for 20 h at 70 ◦ C with a subsequent deblocking in 8 M MeNH2 in EtOH solution (2 mL) at room temperature gave 10 as a white powder. The overall yield was 59 mg (50%). Rf 0.11 (CH2 Cl2 :EtOH—95:5). m.p. 139–140 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ = 3.55 (ddd, 1H, J50 b,50 a = −12.0 Hz, J50 b,40 = 3.6 Hz, J50 b,OH = 7.0 Hz, H50 b), 3.68 (ddd, 1H, J50 a,50 b = −12.0 Hz, J50 a,40 = 3.6 Hz, J50 a,OH = 4.7 Hz, H50 a), 3.97 (ddd, 1H, J40 ,50 b = 3.6 Hz, J40 ,50 a = 3.6 Hz, J40 ,30 = 3.6 Hz, H40 ), 4.16 (ddd, 1H, J30 ,40 = 3.6 Hz, J30 ,20 = 5.2 Hz, J30 ,OH = 4.7 Hz, H30 ), 4.62 (ddd, 1H, J20 ,30 = 5.2 Hz, J20 ,10 = 6.1 Hz, J20 ,OH = 6.2 Hz, H20 ), 4.80 (br s, 2H, CH2 ), 5.14 (d, 1H, JOH-30 = 4.7 Hz, 30 OH), 5.30 (dd, 1H, JOH-50 b = 7.0 Hz, JOH-50 a = 4.7 Hz, 50 OH), 5.40 (d, 1H, JOH-20 = 6.2 Hz, 20 OH), 5.90 (d, 1H, J10 ,20 = 6.1 Hz, H10 ), 7.5–7.75 (m, 4H, Ph), 8.21 (s, 1H, H8), 8.39 (s, 1H, H2), 8.53 (br s, 1H, NH). 13 C-NMR (100 MHz, DMSO-d6 ): δ = 42.60 (CH2 ), 61.63 (C50 ), 70.63 (C30 ), 73.54 (C20 ), 85.89 (C40 ), 87.97 (C10 ), 119.79 (C5), 123.41 (C4 Ph), 123.69 (C2 Ph), 125.64 (C6 Ph), 128.96 (q, JC-F = 31.4 Hz, CF3 ), 129.30 (C5 Ph), 131.32 (C3 Ph), 140.08 (C8), 141.54 (C1 Ph), 148.58 (C4), 152.33 (C2), 154.43 (C6). HRMS: m/z [M + H]+ calculated C18 H19 F3 N5 O4 + 426.1384, found 426.1384; m/z [M + Na]+ calculated C18 H18 F3 N5 O4 Na+ 448.1203, found 448.1204 3.9. N6 -(4-Trifluoromethylbenzyl)adenosine (11) Following the procedure for the preparation of 5, the condensation of 4 (190 mg, 0.382 mmol) with 4-trifluoromethylbenzylamine (0.11 mL, 0.76 mmol) in the presence of DIPEA (0.132 mL, 0.76 mmol) in MeCN (3 mL) for 10 h at 70 ◦ C with a subsequent deblocking in 8 M MeNH2 in EtOH solution (3 mL) at room temperature gave 11 as a white powder. The overall yield was 160 mg (98%). Rf 0.09 (CH2 Cl2 :EtOH—95:5). m.p. 152–153 ◦ C. 1 H-NMR (400 MHz, DMSO-d6 ): δ = 3.55 (ddd,

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1H, J50 b,50 a = −12.0 Hz, J50 b,40 = 3.6 Hz, J50 b,OH = 6.9 Hz, H50 b), 3.68 (ddd, 1H, J50 a,50 b = −12.0 Hz, J50 a,40 = 3.6 Hz, J50 a,OH = 4.7 Hz, H50 a), 3.97 (ddd, 1H, J40 ,50 b = 3.6, J40 ,50 a = 3.6 Hz, J40 ,30 = 3.6 Hz, H40 ), 4.16 (ddd, 1H, J30 ,40 = 3.6 Hz, J30 ,20 = 5.2 Hz, J30 ,OH = 4.7 Hz, H30 ), 4.62 (ddd, 1H, J20 ,30 = 5.2 Hz, J20 ,10 = 6.1 Hz, J20 ,OH = 6.2 Hz, H20 ), 4.80 (br s, 2H, CH2 ), 5.14 (d, 1H, JOH-30 = 4.7 Hz, 30 OH), 5.30 (dd, 1H, JOH-50 b = 6.9 Hz, JOH-50 a = 4.7 Hz, 50 OH), 5.40 (d, 1H, JOH-20 = 6.2 Hz, 20 OH), 5.90 (d, 1H, J10 ,20 = 6.1 Hz, H10 ), 7.5–7.75 (m, 4H, Ph), 8.20 (s, 1H, H8), 8.39 (s, 1H, H2), 8.51 (br s, 1H, NH). 13 C-NMR (100 MHz, DMSO-d6 ): δ = 42.65 (CH2 ), 61.60 (C50 ), 70.59 (C30 ), 73.49 (C20 ), 85.86 (C40 ), 87.91 (C10 ), 119.79 (C5), 122.98 (C4 Ph), 125.07 (C3 Ph, C5 Ph), 127.48 (q, JC-F = 30.7 Hz, CF3 ), 127.67 (C2 Ph, C6 Ph), 140.02 (C8), 144.92 (C1 Ph), 148.59 (C4), 152.29 (C2), 154.42 (C6). HRMS: m/z [M + H]+ calculated C18 H19 F3 N5 O4 + 426.1384, found 426.1383; m/z [M + Na]+ calculated C18 H18 F3 N5 O4 Na+ 448.1203, found 448.1203. 3.10. Antiviral Assay Against EV71 in RD Cells An EV71 BrCr laboratory-adapted strain was used at a low multiplicity of infection (MOI) in a standardized antiviral assay [31]. Briefly, freshly harvested rhadbdosarcoma (RD) cells were seeded in a 96-well plate (2 × 104 cells/well) and incubated at 37 ◦ C in 5% CO2 . The next day, a serial dilution of the compounds was prepared in assay medium and added to the RD cells. Then, the cells were supplemented with the viral suspension. The assay plates were incubated until full virus–induced cell death was observed in the untreated, infected controls (3–4 days post-infection). Subsequently, the antiviral effect was quantified using a colorimetric readout with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate (MTS/PMS method), and the concentration of compound at which a 50% inhibition of virus-induced cell death would be observed (EC50 ) was calculated from the antiviral dose-response curves. A similar assay setup was used to determine the adverse effect of the compound on uninfected, treated cells for the calculation of the CC50 (concentration of compound that reduces overall cell health with 50% as determined by the MTS/PMS method). The selectivity index (SI) was calculated as a ratio of EC50 /CC50 . 4. Conclusions We reported here the antiviral profile of a class of analogues of the cytokinin nucleoside BAPR, previously described by our groups as a potent and selective inhibitor of EV71 replication. Interestingly, we showed that the replacement of hydrogen with fluorine atoms or a trifluoromethyl group in the aromatic moiety of BAPR overall increased its SI. In particular, the least successful analogue (compound 7) with the addition of one fluorine atom at position 4 of the BAPR phenyl showed a 1.25-times increase of SI, whereas the best analogue of the class (compound 10) with a trifluoromethyl at position 3 exhibited an SI that was 230 times higher than BAPR. Fluorinated analogues of natural substances are of interest, since often the biological activity of the parent compounds is improved. However, the tremendous gain in selectivity reported here represents one of the most spectacular examples of structure optimization of a lead natural compound by introducing fluoro- or trifluoromethyl groups into an aromatic moiety. Supplementary Materials: Supplementary Materials are available online. Acknowledgments: This work was supported by the Russian Foundation for Basic Research (16-04-01594 and 17-04-01939) and Russian Academy of Sciences (Program ‘Molecular and Cell Biology’) and by Russian Federation President Program for young scientists (MK-8496.2016.4). Liang Sun is supported by a CSC: Project of Europe grant from the China Scholarship Council (CSC No. 201403250056). Kim Donckers and Caroline Collard are acknowledged for their excellent assistance in the acquisition of the antiviral data. Author Contributions: Liang Sun, Carmen Mirabelli and Sergey N. Mikhailov conceived and designed the experiments; in particular, Liang Sun, Mikhail S. Drenichev, Vladimir E. Oslovsky, Nikolay N. Kurochkin and Vladislav E. Kunetsky performed the experiments; Johan Neyts contributed reagents/materials/analysis tools; Carmen Mirabelli, Pieter Leyssen and Sergey N. Mikhailov wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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