Chemical modification of the plant isoprenoid cytokinin N 6-isopentenyladenosine yields a selective inhibitor of human enterovirus 71 replication

Article abstract of DOI:10.1016/j.ejmech.2014.11.048

In this study, we demonstrate that N⁶-isopentenyladenosine, which essentially is a plant cytokinin-like compound, exerts a potent and selective antiviral effect on the replication of human enterovirus 71 with an EC₅₀ of 1.0 ± 0.2 ?1/4M and a selectivity index (SI) of 5.7. The synthesis of analogs with modification of the N⁶-position did not result in a lower EC₅₀ value. However, in particular with the synthesis of N⁶-(5-hexene-2-yne-1-yl)adenosine (EC₅₀ Combining double low line 4.3 ± 1.5 ?1/4M), the selectivity index was significantly increased: because of a reduction in the adverse effect of this compound on the host cells, an SI > 101 could be calculated. With this study, we for the first time provide proof that a compound class that is based on the plant cytokinin skeleton offers an interesting starting point for the development of novel antivirals against mammalian viruses, in the present context in particular against enterovirus 71.

Full text of DOI:10.1016/j.ejmech.2014.11.048

Accepted Manuscript
6
Chemical modification of the plant isoprenoid cytokinin N -isopentenyladenosine
yields a selective inhibitor of human enterovirus 71 replication
Vitali I. Tararov , Aloys Tijsma , Svetlana V. Kolyachkina , Vladimir E. Oslovsky ,
Johan Neyts , Mikhail S. Drenichev , Pieter Leyssen , Sergey N. Mikhailov
PII:
S0223-5234(14)01080-0
10.1016/j.ejmech.2014.11.048
EJMECH 7541
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 October 2014
Revised Date: 20 November 2014
Accepted Date: 24 November 2014
Please cite this article as: V.I. Tararov, A. Tijsma, S.V. Kolyachkina, V.E. Oslovsky, J. Neyts, M.S.
6
Drenichev, P. Leyssen, S.N. Mikhailov, Chemical modification of the plant isoprenoid cytokinin N -
isopentenyladenosine yields a selective inhibitor of human enterovirus 71 replication, European Journal
of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.11.048.
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ACCEPTED MANUSCRIPT
Graphical abstract
Chemical modification of the plant isoprenoid cytokinin N6-isopentenyladenosine yields a
selective inhibitor of human enterovirus 71 replication
Vitali I. Tararov1, Aloys Tijsma2, Svetlana V. Kolyachkina1, Vladimir E. Oslovsky1,
Johan Neyts2, Mikhail S. Drenichev1, Pieter Leyssen2, Sergey N. Mikhailov1,*

ACCEPTED MANUSCRIPT
Chemical modification of the plant isoprenoid cytokinin N6-
isopentenyladenosine yields a selective inhibitor of human enterovirus 71
replication
Vitali I. Tararov1, Aloys Tijsma2, Svetlana V. Kolyachkina1, Vladimir E. Oslovsky1, Johan Neyts2,
Mikhail S. Drenichev1, Pieter Leyssen2, Sergey N. Mikhailov1,*
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences. Address: Vavilov str. 32, Moscow 119991, Russian
Federation
2KU Leuven – University of Leuven, Department of Microbiology and Immunology, Laboratory for Virology and
Chemotherapy, Rega Institute for Medical Research, Minderbroedersstraat 10, 3000 Leuven, Belgium
Corresponding author: Sergey N. Mikhailov
Dedicated to Prof. Dr. Piet Herdewijn on the occasion of his 60th anniversary
Abstract
In this study, we demonstrate that N6-isopentenyladenosine, which essentially is a plant cytokinin-like
compound, exerts a potent and selective antiviral effect on the replication of human enterovirus 71 with
an EC50 of 1.0 ± 0.2 µM and a selectivity index (SI) of 5.7. The synthesis of analogs with modification of
the N6-position did not result in a lower EC50 value. However, in particular with the synthesis of N6-(5-
hexene-2-yne-1-yl)adenosine (EC50 = 4.3 ± 1.5µM), the selectivity index was significantly increased:
because of a reduction in the adverse effect of this compound on the host cells, an SI >101 could be
calculated. With this study, we for the first time provide proof that a compound class that is based on the
plant cytokinin skeleton offers an interesting starting point for the development of novel antivirals against
mammalian viruses, in the present context in particular against enterovirus 71.
Key words: adenosine, N6-derivatives, synthesis, antiviral activity, enterovirus 71
1. Introduction
Common natural isoprenoid cytokinins are N6-isopentenyladenine, trans- and cis-zeatin, and
dihydrozeatin. The first step in isoprenoid cytokinin biosynthesis is carried out by the adenosine
phosphate isopentenyltransferase (EC 2.5.1.27), which catalyzes the N6-prenylation of adenosine 5′-
(mono-, di-, or tri-) phosphates with 3,3-dimethylallyldiphosphate [1]. The resulting nucleotides are
readily dephosphorylated to their corresponding nucleoside derivatives. These in turn serve as substrate
for two other enzymes, namely adenosine nucleosidase (EC 3.2.2.7) and purine nucleoside phosphorylase
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(EC 2.4.2.1), which convert the cytokinin nucleosides into their cytokinin bases. Accordingly, to these
transformations N6-substituted adenines and adenosines might have similar cytokinin activities [1].
Several N6-substituted adenosines, such as N6-isopentenyladenosine, N6-methyladenosine, and some
others have been detected in, amongst others, tRNA [2]. Cytokinin ribosides were found to inhibit the
growth of mammalian neoplastic cells in vitro [3,4], and, in limited clinical trials in the 1970’s, N6-
isopentenyladenosine showed some antitumor chemotherapeutic effect [5]. These findings initiated an
interest to synthesize novel N6-adenosine analogs in an attempt to identify compounds with enhance
antitumor activity. However, biological evaluation of a wide variety of such derivatives did not yield
compounds with activity higher than that of N6-isopentenyladenosine itself [6].
Nonetheless, encouraged by the fact that N6-substituted adenosines have been shown to elicit biological
activity in mammalian assay systems for malaria [7], African sleeping sickness [8], toxoplasmosis [9],
and some others, we decided to explore the potential of N6-isopentenyladenosine and derivatives as
inhibitors of virus replication.
2. Results and discussion
2.1. Chemistry
Recently, we developed a new and versatile method for the preparation of N6-adenosine derivatives by
regioselective N6-alkylation of N6-acetyl-2′,3′,5′-tri-O-acetyladenosine (1) [10,11]. One of the most
common approaches for the preparation of N6-alkylated adenosines is the reaction of alkylamines with 6-
chloropurine riboside. [12,13] The advantage of our method is the possibility of usage both alkyl halides
and alcohols for N6-modification. This is important especially in the case when an amine is hardly
available. Compounds 2a-c were synthesized by alkylation of tetraacetate 1 with appropriate alkyl halides
(Scheme 1, method A) and they were deprotected with ammonia in methanol according to well-
established procedures reported in the literature.
In other cases, tetraacetate 1 was transformed into nucleosides 2d-k using the Mitsunobu protocol
(Scheme 1, method B). Although the Mitsunobu coupling promoted with Ph3P and DEAD (diethyl
azodicarboxylate) is a very popular mild chemical transformation that occurs under essentially neutral
conditions, it produces at least equivalent amounts of Ph3PO and (NHCOOEt)2. It is almost impossible to
isolate pure substances 2 without repeated column chromatography on silica gel which resulted in low
yields of 2. The partially purified acetates 2 were subjected to deacetylation with the following column
chromatography to give pure nucleosides 3 with reasonable overall yields.
Deacylation with 7M NH3 in methanol at room temperature proceeded completely during 48 h. However,
the rate of deprotection of N-acetyl and O-acetyl groups in nucleosides 2 are quite different: the first one
is much more stable and, as a result, an additional amount of acetamide was formed under deblocking
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conditions. Acetamide caused some difficulties in the purification of the final products using column
chromatography on silica gel, because it was often eluted together with nucleosides 3.
We have also tried to use 0.1M MeONa in MeOH at room temperature for removal of acetyl groups.
After 30 minutes, we were able to isolate N6-acetyl derivatives of 3 in good yields and their structure was
confirmed by NMR and MS methods. Prolonged treatment results in substantial decomposition of the
desired products.
Finally, we found that the use of 4M PrNH2 in MeOH [14] at room temperature for one day is the method
of choice for the preparation of nucleosides 3. The formed N-propylacetamide have higher mobility on
silica gel than the desired nucleosides 3.
N6-Substituted adenine derivatives 4b-c were obtained by acid hydrolysis of the nucleosides 3b-c. The
treatment of corresponding nucleosides with 0.5M НСl at 100°С leads to cleavage of the glycoside bond
and removal of N-acetyl group. Unfortunately, this method was not applicable to isoprenoid derivatives
due to the side reactions [15,16]. Isopentenyladenine (4a) was obtained via the enzymatic phosphorolysis
of 3a in the presence of recombinant purine nucleoside phosphorylase in KH2PO4 buffer (pH 7.5) [10].
The structure of all synthesized compounds was confirmed by NMR and MS methods.
Among the synthesized derivatives, N6-acetyl-N6-propargyl-2′,3′,5′-tri-O-acetyladenosine (2c) may be
used for introduction of new structural motifs in the adenine moiety according to Scheme 2.
No literature examples of allylation of acetylenic nucleic base derivatives were found, though successful
examples carried out on simple alkynes are available [17,18,19]. We found that reaction of allyl bromide
and 3,3-dimethylallyl bromide with compound 2c can be carried out in the presence of CuCl and a base at
ambient temperature affording 5 and 6 in 70 – 80 % yield. It is noteworthy that no reaction occurred with
methyl iodide or benzyl bromide. This can be a piece of evidence that the mechanism of acetylenic
allylation, catalyzed with Cu (I), is more complicated than it was suggested [17], since it is limited only to
allylic halides. Obviously, the participation of η3-allylic complex should also be taken into consideration.
The participation of binuclear copper species is also highly probable in a way similar to Cu (I) catalyzed
[3+2] cycloaddition of azides to acetylenes [20].
The reaction of 2c with allylbromide and 3,3-dimethylallylbromide is to be carried out at ambient
conditions but in anaerobic (N2) atmosphere to observe a sufficient increase in yields. We were unable to
monitor both reactions by TLC on silica gel plates because in the wide range of the used solvents, the
starting compound 2c and the products 5 or 6 have quite the same mobility. To ensure the complete
consumption of 2c and for simplification of chromatographic isolation of 5 and 6, the reaction mixtures,
after stirring for 16 h in N2 atmosphere, were additionally stirred for 6-8 h on air. This causes the
oxidation of the unreacted initial compound 2c with the formation of more polar products, thus
simplifying the isolation procedure. The oxidation of 2c in solution in the presence of Cu(I) was
confirmed by independent experiment in which 2c and CuCl were stirred in MeCN in the open flask and
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complete conversion of 2c to polar compounds was observed within several hours. After the removal of
acetyl groups by ammonolysis of peracetylated compounds 5 and 6, free nucleosides 7 and 8 can be
isolated in good yields.
The structure of the side chain of the compounds was unambiguously assigned by 1H NMR spectral data
(the numeration of side chain carbon atoms is depicted in Figure 1. Long-range spin-spin coupling
constants were observed. In compounds 5 and 6, the 1-CH2 groups appears as triplets with 5J = 2.2 Hz. In
the free nucleosides 7 and 8, significant broadening of the 1-CH2 signals is observed. Such effect is
general for free N6-substituted nucleosides. A possible explanation can be found in the literature and is
ascribed to ‘a chemical exchange process connected to the restricted conformational change’ [21,22]. As
a result, spin-spin coupling between 1-CH2 and 4-CH2 is not detectable. Nevertheless, the sets of
constants for other hydrogen atoms remain the same as for acetylated nucleosides 5 and 6 (Supporting
information). Thus, no principle structural changes of N6-residues occurred during deprotection. The
chemical purity of 7 and 8 was established to be higher than 99 % on the basis of LC-MS analysis.
2.2. Biological activity
In a first round, the compounds were evaluated for their potential as inhibitors of virus replication in cell-
based assays for hepatitis C virus (HCV, replicon-based assay), chikungunya virus (CHIKV, virus-cell-
based assay) and enterovirus 71 (EV71, virus-cell-based assay). No antiviral activity was observed in the
assays for HCV and CHIKV. In contrast, a clear cell protective effect was apparent in the EV71 assay for
compounds 3a, 3b, 3e, 3j, 3k, and 7, for which an EC50 value could be derived from the dose-response
curves (Table 1). This value represents the concentration of compound that induces a cell protective effect
of 50% and is indicative of the antiviral potency of the compound. In parallel, also uninfected cells were
treated with the compounds to quantify the adverse effect of treatment on host cell metabolism. From
these dose-response curves, CC50 values equaling the concentration of compound that reduces overall
host cell metabolism by 50%, are calculated. From both values, the selectivity index (SI= CC50/EC50) can
be derived, a value that is indicative for the therapeutic window of a particular compound in this assay
(the larger the SI, the larger the therapeutic window). However, a cell protective effect that is obtained in
a virus-cell-based assay does not necessarily prove that a compound selectively inhibits virus replication.
Therefore, all assay wells with a cytoprotective effect were inspected by microscope for minor signs of
virus-induced cell death, or for treatment-induced host cell or monolayer abnormalities. Only compounds
for which the cells at least at one concentration of compound resemble the untreated, uninfected control
cells are considered as selective inhibitors of EV71 virus replication. Compound 3a proved to be the most
potent compound of this series (EC50 of 1.0 ± 0.2 µM) while compound 7 was the most selective
compound (SI>101). Both matched the selection criteria for a selective inhibitor of EV71 replication as
outlined above, and were selected for further evaluation. The N6-substituted adenines 4a-c do not elicit an
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antiviral effect in this assay, which indicates that the ribofuranose residue is most likely essential for
antiviral activity.
In a second round, the antiviral effect of compound 3a and 7 was evaluated in virus-cell-based assays for
a panel of other enteroviruses. Interestingly, except for a very faint cell protective effect in the assay for
echovirus 11 (ECHO11, EC50=230 ± 45 µM), no antiviral activity was observed against any of the other
viruses. This indicates that the antiviral activity of this compound class is most likely very selective for
enterovirus 71 (Table 2).
To confirm that the compounds are selective inhibitors for EV71, their antiviral effect was evaluated in
virus-cell-based assays for a panel of clinical isolates of EV71 that belong to different EV71 genogroups
(Table 3).
Compound 3a proved to be equipotent against all viruses of this panel, while for 7, the activity ranged
from 0.9 ± 1.8µM for EV71 strain H08300 461 #812 (genogroup C2) to 17 ± 4 µM for EV71 strain
TW/70902/08 (genogroup B5). Also the other virus isolate that belongs to genogroup B5 appeared to be
less sensitive to the antiviral effect of this compound. Because all EV71 assays have been conducted
using RD (rhabdomyosarcoma) cells, these results may indicate that the compound most likely has a
mechanism of action that also involves a specific viral protein rather than only a cellular factor.
3. Conclusions
In the present study, we reported on the synthesis of N6-isopentenyladenosine analogues and
demonstrated that these cytokinin-like compounds have potential as inhibitors of the replication of EV71
in a mammalian bio-assay. To obtain a deeper insight into the structure-activity relationship of these
compounds against EV71, additional sets should be prepared, which hopefully will allow the
identification of a compound with a large window of selectivity. The availability of such a compound will
be essential to initiate studies into the precise molecular mechanism of action by which enterovirus 71
replication is inhibited.
4. Experimental section
4.1. General
The solvents and materials were reagent grade and were used without additional purification. Column
chromatography was performed on silica gel (Kieselgel 60 Merck, 0.063-0.200 mm). TLC was performed
on Alugram SIL G/UV254 (Macherey-Nagel) with UV visualization. Melting points were determined on
1
13
a Electrothermal apparatus and are uncorrected. H and C (with complete proton decoupling) NMR
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spectra were recorded on Bruker AMX 400 NMR instrument at 300K. 1H-NMR-spectra were recorded at
13
400 MHz and C-NMR-spectra at 100 MHz. Chemical shifts in ppm were measured relative to the
1
13
1
residual solvent signals as internal standards (CDCl3, H: 7.26 ppm, C: 77.1 ppm; DMSO-d6, H: 2.50
13
ppm, C: 39.5 ppm) [23]. Spin-spin coupling constants (J) are given in Hz. Double resonance technique
was applied for assigning the resonances. High resolution mass spectra (HRMS) were measured on a
Bruker micrOTOF II instrument using electrospray ionization (ESI) [24]. The measurements were done in
a positive ion mode (interface capillary voltage – 4500 V) or in a negative ion mode (3200 V); mass range
from m/z 50 to m/z 3000 Da; external or internal calibration was done with Electrospray Calibrant
Solution (Fluka). A syringe injection was used for solutions in acetonitrile, methanol, or water (flow rate
3 µL/min). Nitrogen was applied as a dry gas; interface temperature was set at 180°C. LC-MS analysis
was performed on a Surveyor MSQ instrument (Thermo Finnigan, USA), operating in APCI (atmospheric
pressure chemical ionization) mode with detection of positive and negative ions, and equipped with Onyx
Monollithic C18 25×4.6 mm Part No CHO-7645 column. The eluent was 0.1% HCOOH in water with a
gradient of solution in MeCN. Chromatographic peaks were detected simultaneously with ELSD
(evaporative light scattering detector), PAD (photodiode array detector), and TIC (total ion current)
detector. In all cases, only one peak was revealed and the chromatographic purity of compounds was
more than 99%.
4.2. Typical procedure for alkylation of tetraacetate 1 in DMF in the presence of K2CO3 (Method A)
To a stirred mixture of tetraacetate 1 (500 mg, 1.15 mmol), K2CO3 (0.32 g, 2.3 mmol) in 5 ml of DMF in
one portion alkyl bromide (1.4 mmol) was added at room temperature. The reaction was monitored by
TLC. After stirring for 20 h at ambient temperature, the conversion was completed. The reaction mixture
was diluted with AcOEt and the solution was washed with brine (3×20 ml). The extract was dried over
Na2SO4 and evaporated. The residue was applied to column chromatography on silica gel.
4.2.1. N6-Acetyl-N6-isopentenyl-2′,3′,5′-tri-O-acetyladenosine (2a)
A commercial isopentenyl bromide was used for the reaction. The yield of 2a was 410 mg (67%) as a
foam; Rf 0.41 (CH2Cl2-EtOH, 25:1). 1H NMR (400 MHz, CDCl3): δ = 1.61 (s, 6Н, Ме), 2.10 (s, 3H, AcO),
2.13 (s, 3H, AcO), 2.16 (s, 3H, AcO), 2.28 (s, 3H, AcN), 4.39 (dd, 1H, J5′b,5′a = 12.9, J5′b,4′ = 5.2, H-5′b),
4.45-4.49 (m, 2H, H-5′a + H-4′), 4.84 (d, 2Н, J = 6.7, NCH2), 5.22-5.26 (m, 1Н, СН2СН=С), 5.69 (dd,
1H, J3′,4′ = 4.8, J3′,2′ = 5.6, H-3′), 5.96 (dd, 1H, J2′,3′ = 5.6, J2′,1′ = 5.0, H-2′), 6.23 (d, 1H, J1′,2′ = 5.0, H-1′),
8.19 (s, 1H, H-2), 8.79 (s, 1H, H-8).
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4.2.2. N6-Acetyl-N6-allyl-2′,3′,5′-tri-O-acetyladenosine (2b)
A commercial allyl bromide was used for the reaction. The yield of 2b (420 mg, 77%) as a foam; Rf 0.58
1
(CH2Cl2-EtOH, 25:1). H NMR (400 MHz, CDCl3): δ = 2.10 (s, 3H, AcO), 2.12 (s, 3H, AcO), 2.15 (s,
3H, AcO), 2.32 (s, 3H, AcN), 4.39 (dd, 1H, J5′b,5′a = 12.8, J5′b,4′ = 5.2, H-5′b), 4.46 (dd, 1H, J5′a,5′b = 12.8,
J5′a,4′ = 3.0, H-5′a), 4.47 (ddd, 1H, J4′,5′b = 5.2, J5′a,4′ = 3.0, J4′,3′ = 4.9, H-4′), 4.88 (d, 1Н, 3J = 5.1, NCH2),
5.04 (dd, 1Н, 2J = 1.0, 3J = 10.3, С=СН-cis), 5.17 (dd, 2J = 1.0, 3J = 17.2, С=СН-trans), 5.69 (dd, 1H, J3′,4′
3
= 4.9, J3′,2′ = 5.4, H-3′), 5.89 (ddd, J = 5.1, 10.3 and 17.2, СН2СН=С), 5.96 (dd, 1H, J2′,3′ = 5.4, J2′,1′
5.0, H-2′), 6.23 (d, 1H, J1′,2′ = 5.0, H-1′), 8.19 (s, 1H, H-2), 8.78 (s, 1H, H-8).
=
4.2.3. N6-Acetyl-N6-propargyl-2′,3′,5′-tri-O-acetyladenosine (2c)
A commercial solution of propargyl bromide in toluene (80% wt) was used for the reaction. The yield of
2c was 463 mg (85%) as a foam; Rf 0.42 (CH2Cl2-EtOH, 25:1). 1H NMR (400 MHz, CDCl3): δ = 2.10 (s,
3H, AcO), 2.12 (t, 1H, J = 2.5, HC≡C), 2.13 (s, 3H, AcO), 2.16 (s, 3H, AcO), 2.39 (s, 3H, AcN), 4.40
(dd, 1H, J5′b,5′a = 12.9, J5′b,4′ = 5.2, H-5′b), 4.47 (dd, 1H, J5′a,5′b = 12.9, J5′a,4′ = 3.1, H-5′a), 4.48 (ddd, 1H,
J4′,5′b = 5.2, J4′,5′a = 3.1, J4′,3′ = 4.8, H-4′), 5.10 (d, 2H, J = 2.5, CH2N,) 5.68 (dd, 1H, J3′,4′ = 4.8, J3′,2′ = 5.6,
H-3′), 5.97 (dd, 1H, J2′,3′ = 5.6, J2′,1′ = 5.0, H-2′), 6.25 (d, 1H, J1′,2′ = 5.0, H-1′), 8.22 (s, 1H, H-2), 8.82 (s,
1H, H-8).
4.3. N6-Isopentenyladenosine (3a)
The tetraacetate 2a (500 mg, 0.99 mmol) was dissolved in 7M NH3 in MeOH (3 ml, 21 mmol) and the
solution was left for 48 h at ambient temperature. The final reaction mixture was evaporated in vacuum
and the residue was crystallized from water. The product was dried in vacuum over P2O5.
The yield of 3a was 124 mg, (33 %) as a white powder; Rf 0.45 (CH2Cl2- EtOH, 4:1). mp 130-132oC
(H2O); Lit.: Mp 134-136oC [16]. 1H NMR (400 MHz, DMSO-d6): δ = 1.66 (s, 3H, Me), 1.69 (s, 3H, Me),
3.55 (ddd, 1H, J5′b,5′a = 12.1, J5′b,4′ = 3.0, J5′b,OH = 7.9, H-5′b), 3.67 (ddd, 1H, J5′a,5′b = 12.1, J5′a,4′ = 3.6,
J5′a,OH = 4.6, H-5′a), 3.96 (ddd, 1H, J4′,5′b = 3.0, J4′,5′a = 3.6, J4′,3′ = 2.8, H-4′), 4.14 (ddd, 1H, J3′,4′ = 2.8,
J3′,2′ = 5.3, J3′,OH = 4.8, H-3′), 4.08 (br s, 2H, NCH2C), 4.60 (ddd, 1H, J2′,3′ = 5.3, J2′,1′ = 6.0, J2′,OH = 6.2,
H-2′), 5.16 (d, 1H, JOH,3′ = 4.8, 3′-OH, exchangeable with D2O), 5.30 (dd, 1H, JOH,5′b = 7.9, JOH,5′a = 4.6,
5′-OH, exchangeable with D2O), 5.37-5.47 (m, 2H, JOH,2′ = 6.2, CCH=C, 2′-OH, exchangeable with D2O),
5.87 (d, 1H, J1′,2′ = 6.0, H-1′), 7.82 (br s, 1H, NH, exchangeable with D2O), 8.19 (br s, 1H, H-2), 8.32 (s,
13
1H, H-8). C NMR (100 MHz, DMSO-d6): δ = 18.48 (Me), 26.05 (Me), 38.57 (CH2N), 62.24 (C-5'),
71.22 (C-3'), 74.24 (C-2'), 86.59 (C-4'), 88.82 (C-1'), 120.13 (C-5), 121.76 (CH=), 135.44 (=CMe2),
140.52 (C-8), 148.77 (C-4), 153.17 (C-2), 154.97 (C-6). MS (APCI): m/z [M+H+] calculated
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[C15H22N5O4] 336.17, found 336.23; m/z [M-H‒+HCOOH] calculated [C16H22N5O6] 380.16, found
380.20.
4.4. N6-Allyladenosine (3b)
Tetraacetate 2b (400 mg, 0.841 mmol) was dissolved in 7M NH3 in MeOH (2.4 ml, 16.8 mmol) and the
solution was left for 48 h at ambient temperature. The precipitate formed was filtered off and washed with
ether (10 ml). The product was dried in vacuum over P2O5. The yield of 3b was 182 mg (70%) as a white
o
1
powder; Rf 0.25 (CH2Cl2 – EtOH, 9:1). mp 165-167 C (MeOH); Lit.: Mp 167oC [25]. H NMR (400
MHz, DMSO-d6): 3.56 (m, 1H, H-5′b), 3.66 (m 1H, H-5′a), 3.97 (m 1H, = 2.8, H-4′), 4.15 br s CH2N, H-
3′), 4.62 (m, 1H, OH), 5.05 (d, J = 10.3, С=СН-cis), 5.13 (m, 1H, OH), 5.15 (d, J = 17.0, С=СН-trans),
5.34 (m, 1H, H-2′), 5.40 (m, 1H, OH), 5.94-6.13 (m, 2H, overlapping signals of СН2СН=С and H-1′),
13
7.98 (br s, 1Н, NH), 8.20 (s, 1H, H-2), 8.34 (s, 1H, H-8). C NMR (100 MHz, DMSO-d6): δ = 33.56
(NCH2), 61.66 (C-5'), 70.64 (C-3'), 73.49 (C-2'), 85.89 (C-4'), 87.95 (C-1'), 115.02 (CH=), 118.75 (C-5),
135.60 (=CH2), 139.78 (C8), 152.30 (C-2), 154.49 (C-6). HRMS: m/z [M+H]+ calculated [C13H18N5O4]
308.1353, found 308.1357; m/z [M+Na]+ calculated [C13H17N5O4Na] 330.1173, found 330.1176.
4.5. N6-Propargyladenosine (3c)
The tetraacetate 2c (500 mg, 1.06 mmol) was dissolved in 7M NH3 in MeOH (3 ml, 21 mmol) and the
solution was left for 48 h at ambient temperature. The final reaction mixture was evaporated in vacuum
and the residue was crystallized from EtOH – H2O. The product was dried in vacuum over P2O5.
The yield of 3c was 145 mg (41%) as a white powder; Rf 0.33 (CH2Cl2-EtOH, 9:1). mp 171-174oC (EtOH
- H2O); Lit.: Mp 169oC [25]. 1H NMR (400 MHz, DMSO-d6): δ = 3.01 (t, 1H, J = 2.3, HC≡C), 3.56 (ddd,
1H, J5′b,5′a = 12.0, J5′b,4′ = 3.7, J5′b,OH = 7.2, H-5′b), 3.68 (ddd, 1H, J5′a,5′b = 12.0, J5′a,4′ = 3.9, J5′a,OH = 4.3,
H-5′a), 3.97 (ddd, 1H, J4′,5′b = 3.7, J4′,5′a = 3.9, J4′,3′ = 2.8, H-4′), 4.16 (ddd, 1H, J3′,4′ = 2.8, J3′,2′ = 5.0, J3′,OH
= 4.7, H-3′), 4.29 (br s, 2H, NCH2Ph), 4.60 (ddd, 1H, J2′,3′ = 5.0, J2′,1′ = 5.9, J2′,OH = 6.2, H-2′), 5.14 (d,
1H, JOH,3′ = 4.7, 3′-OH, exchangeable with D2O), 5.28(dd, 1H, JOH,5′b = 7.2, JOH,5′a = 4.3, 5′-OH,
exchangeable with D2O), 5.41 (d, 1H, JOH,2′ = 6.2, 2′-OH, exchangeable with D2O), 5.90 (d, 1H, J1′,2′
=
5.9, H-1′), 8.18 (br s, 1H, NH, exchangeable with D2O), 8.28 (br s, 1H, H-2), 8.39 (s, 1H, H-8). 13C NMR
(100 MHz, DMSO-d6): δ = 29.38 (CH2N), 61.69 (C-5'), 70.67 (C-3'), 72.47 (HC≡), 73.66 (C-2'), 81.88 (-
C≡), 85.94 (C-4'), 88.03 (C-1'), 119.93 (C-5), 140.25 (C-8), 148.88 (C-4), 152.32 (C-2), 154.07 (C-6). MS
(APCI): m/z [M+H+] calculated [C13H16N5O4] 306.12, found 306.20; m/z [M-H-+HCOOH] calculated
[C14H16N5O6] 350.11, found 350.14.
8

ACCEPTED MANUSCRIPT
4.6. Typical procedure for preparation of nucleosides 3d-k by Mitsunobu reaction of tetraacetate 1 with
alcohols (Method B)
A mixture of 1 (435 mg, 1 mmol), Ph3P (393 mg, 1.5 mmol) and corresponding alcohol (1.5 mmol) in 5
ml of THF was stirred at room temperature until a homogeneous solution was formed. After DEAD (0.24
ml, 1.5 mmol) was added in one portion, the stirring was continued at ambient temperature. The reaction
was monitored by TLC (silica gel, CH2Cl2-EtOH = 97:3). If the conversion of 1 was not completed after
20h, the additional portions of reagents (Ph3P, alcohol and DEAD) in above indicated quantities were
added to achieve full conversion of 1. After 4-5 h, the reaction mixture was evaporated and the residue
was applied to column chromatography (silica gel, CH2Cl2-EtOH = 97:3). Partially purified compound
was dissolved in 4M PrNH2 in MeOH solution (50 mmol) and was left for 24 h, after which the mixture
was evaporated and the residue was applied to column chromatography. The product was eluted with
CH2Cl2-EtOH (9:1 v/v) mixture.
4.6.1. N6-(4-Hydroxy-2-butynyl)adenosine (3d)
The yield of 3d was 203 mg (61%, white powder); Rf 0.13 (CH2Cl2- EtOH, 9:1). mp 160-162oC (EtOH -
H2O). 1H NMR (400 MHz, DMSO-d6): δ = 3.56 (ddd, 1H, J5′b,5′a = 11.9, J5′b,4′ = 4.1, J5′b,OH = 7.0, H-5′b),
3.68 (ddd, 1H, J5′a,5′b = 12.0, J5′a,4′ = 3.6, J5′a,OH = 4.6, H-5′a), 3.97 (ddd, 1H, J4′,5′b = 4.1, J4′,5′a = 3.6, J4′,3′
= 2.8, H-4′), 4.02 (dt, 2H, 5J = 1.7, 3JCH-OH = 5.8, OCH2C≡), 4.15 (ddd, 1H, J3′,4′ = 2.8, J3′,2′ = 5.1, J3′,OH
=
4.8, H-3′), 4.32 (br s, 2H, NCH2C≡, H-3′), 4.60 (ddd, 1H, J2′,3′ = 5.1, J2′,1′ = 6.0, J2′,OH = 6.2, H-2′), 5.10
3
(t, JHO-CH = 5.8, ω-OH, exchangeable with D2O), 5.17 (d, 1H, JOH,3′ = 4.8, 3′-OH, exchangeable with
D2O), 5.34 (dd, 1H, JOH,5′b = 7.0, JOH,5′a = 4.6, 5′-OH, exchangeable with D2O), 5.43 (d, 1H, JOH,2′ = 6.2,
2′-OH, exchangeable with D2O), 5.90 (d, 1H, J1′,2′ = 6.0, H-1′), 8.14 (br s, 1H, NH, exchangeable with
13
D2O), 8.27 (s, 1H, H-2), 8.38 (s, 1H, H-8). C NMR (100 MHz, DMSO-d6): δ = 29.48 (CH2N), 49.12
(CH2OH), 61.70 (C-5'), 70.67 (C-3'), 73.66 (C-2'), 81.41 (C≡), 81.73 (C≡), 85.96 (C-4'), 88.04 (C-1'),
119.94 (C-5), 140.21 (C-8), 148.83 (C-4), 152.35 (C-2), 154.06 (C-6). MS (APCI): m/z [M+H+]
calculated [C14H18N5O5] 336.13, found 336.18; m/z [M-H-+HCOOH] calculated [C15H18N5O7] 380.12,
found 380.14.
4.6.2. N6-(Z)-(4-Hydroxy-2-butenyl)adenosine (3e)
The yield of 3e was 141 mg (42%, white powder); Rf 0.43 (CH2Cl2-EtOH, 9:1). mp 159-160oC (EtOH -
H2O). 1H NMR (400 MHz, DMSO-d6): δ = 3.55 (ddd, 1H, J5′b,5′a = 12.0, J5′b,4′ = 3.6, J5′b,OH = 7.2, H-5′b),
3.67 (ddd, 1H, J5′a,5′b = 12.1, J5′a,4′ = 3.8, J5′a,OH = 4.6, H-5′a), 3.96 (ddd, 1H, J4′,5′b = 3.6, J4′,5′a = 3.8, J4′,3′
=
2.8, H-4′), 4.07-4.23 (m, 5H, overlapping OCH2C=, NCH2C=, H-3′), 4.60 (ddd, 1H, J2′,3′ = 4.9, J2′,1′ = 6.0,
9

ACCEPTED MANUSCRIPT
J2′,OH = 6.2, H-2′), 4.71 (t, J = 5.3, ω-OH, exchangeable with D2O), 5.15 (d, 1H, JOH,3′ = 4.6, 3′-OH,
exchangeable with D2O), 5.37 (dd, 1H, JOH,5′b = 7.2, JOH,5′a = 4.6, 5′-OH, exchangeable with D2O), 5.41
(d, 1H, JOH,2′ = 6.2, 2′-OH, exchangeable with D2O), 5.88 (d, 1H, J1′,2′ = 6.0, H-1′), 7.91 (br s, 1H, NH,
exchangeable with D2O), 8.20 (s, 1H, H-2), 8.34 (s, 1H, H-8). 13C NMR (100 MHz, DMSO-d6): δ = 37.86
(CH2N), 57.70 (CH2OH), 62.28 (C-5'), 71.26 (C-3'), 74.29 (C-2'), 86.64 (C-4'), 88.85 (C-1'), 119.83 (C-
5), 127.99 (CH=), 132.56 (CH=), 140.75 (C-8), 148.68 (C-4), 153.19 (C-2), 155.07 (C-6). MS (APCI):
m/z [M+H+] calculated [C14H20N5O5] 338.13, found 338.20; m/z [M-H-+HCOOH] calculated
[C15H20N5O7] 382.12, found 382.15.
4.6.3. N6-Geranyladenosine (3f)
The yield of 3f was 155 mg (39%) as a white powder; Rf 0.13 (CH2Cl2-EtOH, 97:3). mp 137-140oC (with
1
decomposition). H NMR (400 MHz, DMSO-d6): δ = 1.54 (s, 3H, CH3), 1.59 (s, 3H, CH3), 1.70 (s, 3H,
CH3), 1.92-1.99 (m, 2H, CH2CH2CH=), 2.00-2.08 (m, 2H, CH2CH2CH), 3.55 (ddd, 1H, J5′b,4′ = 3.6, J5′b,5′a
= – 12.0, JOH,5′b = 7.3, H5'b), 3.68 (ddd, 1H, J5′a,5′b = 12.0, J5′a,4′ = 3.6, JOH,5′a = 4.6, H5'a), 3.96 (q, 1H, J4′,3′
= 3.6, J4′,5′a = 3.6, J4′,5′b = 3.6 Hz, H4'), 4.09 (br s, 2H, NHCH2), 4.15 (ddd, 1H, J3′,2′ = 5.2, J3′,4′ = 3.6, JOH,3′
= 4.6, H3'), 4.60 (ddd, 1H, J2′,1′ = 6.1, J2′,3′ = 5.2, JOH,2′ = 6.2, H2'), 5.06 (tt, 1H, JCH-CH2 = 7.0, JCH-Me = 1.4
(trans) CHCMe2), 5.13 (d, 1H, JOH,3′ = 4.6, 3'OH, exchangeable with D2O), 5.31 (br. t, JCH-CH2 = 6.5 Hz,
NHCH2CH), 5.36 (dd, 1H, J5′a,5′b = 12.0, JOH,5′b = 7.2 Hz, JOH,5′a = 4.6 Hz, 5'OH, exchangeable with D2O),
5.39 (d, 1H, JOH,2′ = 6.2 Hz, 2'OH, exchangeable with D2O), 5.88 (d, 1H, J1′,2′ = 6.1 Hz, H1'), 7.84 (br s,
13
1H, NH), 8.19 (s, 1H, H8), 8.32 (s, 1H, H2). C NMR (100 MHz, DMSO-d6): δ = 16.12, 17.48, 25.38,
25.94, (CH2, CH2, CH3, CH3, CH3), 37.68 (NHCH2), 61.63 (C5'), 70.65 (C3'), 73.48 (C2'), 85.84 (C4'),
87.94 (C1'), 119.75 (C5), 121.73 (CH=), 123.91 (CH=), 130.75 (=C(Me)CH2), 136.64 (=CMe2), 139.59
(C8), 148.30 (C4), 152.27 (C2), 154.35 (C6). HRMS: m/z [M+H]+ calculated [C20H30N5O4] 404.2292,
found 404.2284; m/z [M+Na]+ calculated [C20H29N5O4Na] 426.2112, found 426.2102.
4.6.4. N6-Neryladenosine (3g)
The yield of 3g was 192 mg (48%) as a white powder; Rf 0.15 (CH2Cl2-EtOH, 97:3). mp 129-130oC (with
1
decomposition). H NMR (400 MHz, DMSO-d6): δ = 1.58 (s, 3H, CH3), 1.64 (s, 3H, CH3), 1.67 (s, 3H,
CH3), 2.03-2.16 (m, 4H, =C(Me)CH2CH2, CH2CH2CH=), 3.55 (ddd, 1H, J5′b,5′a = 12.2, J5′b,4′ = 3.5, J5′b,OH
= 6.5, H5'b), 3.67 (ddd, 1H, J5′a,5′b = 12.2, J5′a,4′ = 3.5, J5′a,OH = 3.8, H5'a), 3.96 (q, 1H, J4′,3′ = 3.5, J4′,5′a
=
3.5, J4′,5′b = 3.5, H4'), 4.09 (br s, 2H, NHCH2), 4.15 (ddd, 1H, JOH,3′ = 4.6, J3′,2′ = 4.5, J3′,4′ = 3.5, H3'), 4.60
(ddd, 1H, J2′,1′ = 6.1, J2′,3′ = 4.5, JOH,2′ = 6.3, H2'), 5.10-5.16 (m, 2H, overlapping 3'OH, CHCMe2), 5.31
(br. t, JCH-CH2 = 6.6, NHCH2CH), 5.35 (dd, 1H, JOH,5′b = 6.5, JOH,5′a = 3.8, 5'OH, exchangeable with D2O),
5.38 (d, 1H, JOH,2′ = 6.3 Hz, 2'OH, exchangeable with D2O), 5.88 (d, 1H, J1′,2′ = 6.1, H1'), 7.80 (br s, 1H,
NH), 8.18 (s, 1H, H8), 8.32 (s, 1H, H2). 13C NMR (100 MHz, DMSO-d6): δ = 17.52, 23.09, 25.48, 26.08,
10

ACCEPTED MANUSCRIPT
31.67, (CH2, CH2, CH3, CH3, CH3), 37.36 (NHCH2), 61.67 (C5'), 70.64 (C3'), 73.50 (C2'), 85.88 (C4'),
87.95 (C1'), 119.80 (C5), 122.68 (CH=), 124.05 (CH=), 131.07 (=C(Me)CH2), 136.85 (=CMe2), 139.64
(C8), 148.30 (C4), 152.29 (C2), 154.39 (C6). HRMS: m/z [M+H]+ calculated [C20H30N5O4] 404.2292,
found 404.2286; m/z [M+Na]+ calculated [C20H29N5O4Na] 426.2112, found 426.2105.
4.6.5. N6-[(S)-(-)-Perillyl]-adenosine (3h)
The yield of 3h was 241 mg (60%) as a white powder; Rf 0.11 (CH2Cl2-EtOH, 97:3). mp 158-160oC (with
1
decomposition). H NMR (400 MHz, DMSO-d6): δ = 1.32–1.45 (m, 1H, H-perillyl), 1.68 (s, 3H, CH3-
perillyl), 1.71–1.91 (m, 2H, CH2-perillyl), 1.98–2.13 (m, 4H, 2×CH2-perillyl), 3.55 (dd, 1H, J5′b,5′a = 12.1,
J5′b,4′ = 3.2, H5'b), 3.68 (dd, 1H, J5′a,5′b = 12.1 J5′a,4′ = 3.4, H5'a), 3.96 (ddd, 1H, J4′,3′ = 3.3, J4′,5′a = 3.4, J4′,5′b
= 3.2, H4'), 4.03 (br.s, 2H, NHCH2), 4.12–4.18 (m, 1H, H3'), 4.62 (dd, 1H, J2′,1′ = 6.1, J2′,3′ = 5.2, H2'),
4.68 (s, 2H, C(CH3)=CH2 - perillyl), 5.13 (br s, 1H, 3'OH, exchangeable with D2O), 5.43–5.30 (m, 2H,
overlapping 5'OH, =CHCH2 - perillyl), 5.53 (br s, 1H, 2'OH, exchangeable with D2O), 5.88 (d, 1H, J1′,2′
=
13
6.1, H1'), 7.89 (br s, 1H, NH), 8.19 (s, 1H, H8), 8.34 (s, 1H, H2). C NMR (100 MHz, DMSO-d6): δ =
20.54, 26.57, 27.06, 29.81, (CH2, CH2, CH2, CH3), 40.52 (CHC(Me)=), 44.53 (NHCH2), 61.65 (C5'),
70.63 (C3'), 73.42 (C2'), 85.88 (C4'), 87.94 (C1'), 108.77 (CH(Me)C=CH2), 119.68 (C5), 120.23 (=CH),
134.77 (C(Me)=), 139.67 (C8), 148.32 (C4), 149.25 (NHCH2C=), 152.28 (C2), 154.68 (C6). HRMS: m/z
[M+H]+ calculated [C20H28N5O4] 402.2136, found 402.2135; m/z [M+Na]+ calculated [C20H27N5O4Na]
424.1955, found 424.1948.
4.6.6. N6-[(1R)-(-)-Myrtenyl]-adenosine (3i)
The yield of 3i was 130 mg (33%) as white foam; Rf 0.11 (CH2Cl2-EtOH, 97:3). mp 168-169oC. 1H NMR
(400 MHz, DMSO-d6): δ = 0.76 (s, 3H, CH3-myr), 1.10 (d, 1H, J = 8.4, H-myrtenyl), 1.22 (s, 3H, CH3-
myrtenyl), 2.26–2.00 (m, 4H, myrtenyl), 2.34 (dt, 1H, JCHaHb = 8.4, JCHa-CH1 = JCHa-CH2 = 5.7, CHaHb-
myrtenyl), 3.55 (ddd, 1H, J5′b,5′a = 12.1, J5′b,4′ = 3.7 Hz, JOH,5′b = 7.3, H5'b), 3.67 (ddd, 1H, J5′a,5′b = 12.1,
JOH,5′a = 4.3, J5′a,4′ = 3.7, H5'a), 3.96 (td, 1H, J4′,3′ = 3.4, J4′,5′a = 3.7, J4′,5′b = 3.7, H4'), 4.07 (br.s, 2H,
NHCH2), 4.14 (ddd, 1H, J3′,2′ = 4.7, J3′,4′ = 3.7, JOH,3′ = 4.6, H3'), 4.62 (ddd, 1H, J2′,1′ = 6.3 Hz, J2′,3′ = 4.7
Hz, JOH,2′ = 6.2 Hz, H2'), 5.13 (d, 1H, JOH,3′ = 4.6 Hz, 3'OH, exchangeable with D2O), 5.32-5.38 (m, 2H,
overlaping =CH-myrtenyl, 5'OH), 5.39 (d, 1H, JOH,2′ = 6.2 Hz, 2'OH), 5.88 (d, 1H, J1′,2′ = 6.3 Hz, H1'),
13
7.83 (br s, 1H, NH), 8.17 (s, 1H, H8), 8.32 (s, 1H, H2). C NMR (100 MHz, DMSO-d6): δ = 20.89,
26.04, 30.61, 31.02, 37.61, 43.29, (myrtenyl), 43.79 (NHCH2), 61.65 (C5'), 70.63 (C3'), 73.40 (C2'),
85.87 (C4'), 87.88 (C1'), 116.41 (=CHCH2–), 119.55 (C5), 139.61 (C8), 145.30 (NHCH2C=), 148.50
(C4), 152.20 (C2), 154.65 (C6). HRMS: m/z [M+H]+ calculated [C20H28N5O4] 402.2136, found 402.2133;
m/z [M+Na]+ calculated [C20H27N5O4Na] 424.1955, found 424.1946.
11

ACCEPTED MANUSCRIPT
4.6.7. N6-(Cyclopropylmethyl)adenosine (3j)
The yield of 3j was 110 mg (36%) as a white powder; Rf = 0.08 (CH2Cl2-EtOH, 97:3). mp 176-177oC. 1H
NMR (400 MHz, DMSO-d6): δ = 0.27 (td, 2H, JCH2CH2 = 5.8 Hz, JCH2CH = 4.2 Hz, CH2-cyclopropyl), 0.41
(td, 2H, JCH2CH2 = 6.0 Hz, JCH2CH = 4.2 Hz, CH2-cyclopropyl), 1.21–1.08 (m, 1H, NHCH2CH), 3.17 (d,
1H, JOH,2′ = 5.5 Hz, 2'OH), 3.37 (br s, 1H, 3'OH, exchangeable with D2O), 3.55 (ddd, 1H, J5′b,4′ = 3.5 Hz,
J5′b,5′a = 12.0 Hz, JOH,5′b = 6.8 Hz, H5'b), 3.68 (ddd, 1H, J5′a,4′ = 4.0 Hz, J5′a,5′b = 12.0 Hz, JOH,5′a = 4.9 Hz,
H5'a), 3.95 (ddd, 1H, J4′,3′ = 7.2 Hz, J4′,5′a = 4.0 Hz, J4′,5′b = 3.5 Hz, H4'), 4.15 (dd, J3′,2′ = 4.7 Hz, J3′,4′ = 7.4
Hz, H3'), 4.51 (dd, 1H, JOH,5′a = 4.9 Hz, JOH,5′b = 6.8 Hz, 5'OH), 4.61 (ddd, 1H, J2′,1′ = 6.0 Hz, J2′,3′ = 4.7
Hz, JOH,2′ = 5.5 Hz, H2'), 5.48–5.34 (m, 2H, NHCH2), 5.90 (d, 1H, J1′,2′ = 6.0 Hz, H1'), 7.88 (br s, 1H,
13
NH), 8.18 (s, 1H, H8), 8.33 (s, 1H, H2). C NMR (100 MHz, DMSO-d6): δ =3.21 (CH2, CH2), 11.13
(CH2CH), 43.97 (NHCH2), 61.66 (C5'), 70.63 (C3'), 73.46 (C2'), 85.88 (C4'), 87.96 (C1'), 119.68 (C5),
139.63 (C8), 148.39 (C4), 152.29 (C2), 154.64 (C6). HRMS: m/z [M+H]+ calculated [C14H20N5O4]
322.1509, found 322.1510; m/z [M+Na]+ calculated [C14H19N5O4Na] 344.1329, found 344.1326.
4.6.8. N6-(Tetrahydropyran-2-methyl)-adenosine (3k)
The yield of 3e was 187 mg (51%) as a white powder; Rf = 0.02 (CH2Cl2- EtOH, 97:3). mp 145-147oC.
1H NMR (400 MHz, DMSO-d6): δ = 1.85–1.13 (m, 6H, tetrahydropyran), 3.37–3.25 (m, 1H,
tetrahydropyran, overlaping with D2O), 3.50–3.40 (m, 1H, tetrahydropyran), 3.51 (br s, 2H, NHCH2),
3.57 (ddd, 1H, J5′b,4′ = 3.6 Hz, J5′b,5′a = – 12.1 Hz, JOH,5′b = 6.9 Hz, H5'b), 3.68 (ddd, 1H, J5′a,4′ = 3.8 Hz,
J5′a,5′b = – 12.1 Hz, JOH,5′a = 4.4 Hz, H5'a), 3.90–3.83 (m, 1H, tetrahydropyran), 3.96 (ddd, 1H, J4′,3′ = 3.4
Hz, J4′,5′a = 3.4 Hz, J4′,5′b = 3.4 Hz, H4'), 4.15 (ddd, 1H, J3′,2′ = 4.7 Hz, J3′,4′ = 3.2 Hz, JOH,3′ = 4.6 Hz, H3'),
4.61 (dd, 1H, J2′,1′ = 6.0 Hz, J2′,3′ = 4.7 Hz, JOH,2′ = 6.2 Hz, H2'), 5.13 (d, 1H, JOH,3′ = 4.6 Hz, 3'OH), 5.34
(dd, 1H, JOH,5′b = 6.9 Hz, JOH,5′a = 4.4 Hz, 5'OH), 5.39 (d, 1H, JOH,2′ = 6.2 Hz, 2'OH), 5.88 (d, 1H, J1′,2′
=
6.1 Hz, H1'), 7.59 (br s, 1H, NH), 8.20 (s, 1H, H8), 8.34 (s, 1H, H2). 13C NMR (100 MHz, DMSO-d6): δ
= 29.07, 25.65, 22.59 (tetrahydropyran), 44.67 (NHCH2), 61.64 (C5'), 67.26 (tetrahydropyran), 70.61
(C3'), 73.47 (C2'), 75.48 (NHCH2CHO tetrahydropyran), 85.87 (C4'), 87.94 (C1'), 119.67 (C5), 139.76
(C8), 148.32 (C4), 152.25 (C2), 154.64 (C6). HRMS: m/z [M+H]+ calculated [C16H24N5O5] 366.1772,
found 366.1768; m/z [M+Na]+ calculated [C16H23N5O5Na] 388.1591, found 388.1586.
4.7. N6-Isopentenyladenine (4a)
Compound 4a was obtained by the enzymatic phosphorolysis of 3a in the presence of E. Coli
Recombinant Purine Nucleoside Phosphorylase (PNP) in KH2PO4 buffer (pH 7.5) [10].
1
The yield of 4a was 15 mg (50%) as a white powder; Rf 0.43 (CH2Cl2-EtOH, 9:1). mp 218-220oC. H
NMR (400 MHz, DMSO-d6): δ = 1.67 (s, 3H, Me), 1.70 (s, 3H, Me), 4.08 (br s, 1H, NCH2), 5.31 (s, 1H,
HC=C), 7.55 (br s, 1H, NH), 8.04 (s, 1H, H-2), 8.16 (s. 1H, H-8), 12.84 (br s, 1H, NH).
12

ACCEPTED MANUSCRIPT
4.8. Procedure for preparation of nucleobases 4b-c by acid hydrolysis of nucleosides 3b-c
The solution of corresponding nucleoside 3b-c (0.2 mmol) in 0.5M НСl (2.5 ml, 1.26 mmol) was stirred
at 100°С for 8 h. The reaction was monitored by TLC. (acetonitrile - 25% ammonia, 9:1). When the traces
of initial nucleoside 3 had disappeared, the reaction was cooled down and neutralized to pH 7 with
concentrated NH3. The resulting precipitate was filtered, washed with cold water (2×5 ml), acetone (2×5
ml) and dried in vacuum over P2O5.
4.8.1. N6-Allyladenine (4b)
The yield of 4b was 26 mg (70%) as a white powder. Rf 0.45 (acetonitrile-ammonia, 9:1). mp 223-224 oC.
1H NMR (400 MHz, DMSO-d6): δ = 4.16 (br s, 2Н, NСH2), 5.06 (d, 1H, 3J = 9.5 Hz, С=СН-cis), 5.17 (d,
1H, 3J = 17 Hz, С=СН-trans), 5.93-6.00 (m, 1H, -CH=СH2), 7.71 (br s, 1H, H-6), 8.07 (s, 1H, H-2), 8.17
(s, 1H, H-8), 12.87 (br s, 1H, 9-NH). 13C NMR (100 MHz, DMSO-d6): δ = 41.94 (CH2), 114.96 (=CH),
135.91 (=CH2), 138.73 (C-8), 149.83 (C-4), 152.30 (C-2), 154.26 (C-6).
4.8.2. N6-Propargyladenine (4c)
The yield of 4c was 35 mg (94%) as a white powder; Rf 0.67 (acetonitrile – 25% ammonia, 9:1). mp 255
oC (with decomposition). 1H NMR (400 MHz, DMSO-d6): δ = 3.01 (s, 1H, ≡CH), 4.30 (s, 2H, CH2), 7.92
(br s, 1H, H-9), 8.12 (s, 1H, H-2), 8.24 (s, 1H, H-8), 12.96 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-
d6): δ = 29.26 (CH2N), 72.38 (HC≡), 82.01 (-C≡), 117.95 (C-5), 139.51 (C-8), 147.58 (C-4), 152.16 (C-
2), 153.23 (C-6).
4.9. N6-Acetyl-N6-(hex-5-en-2-ynyl)-2′,3′,5′-tri-O-acetyladenosine (5)
The reaction was carried out in N2 atmosphere using standard Schlenk technique. A mixture of 2c (500
mg, 1.06 mmol), CuCl (105 mg, 1.06 mmol), K2CO3 (292 mg, 2.12 mmol), Bu4NBr (34 mg, 0.106
mmol), CH2=CHCH2Br (0.1 ml, 1.16 mmol) and DMF (5 ml) was stirred at room temperature in N2
atmosphere for 16 h. Subsequently, the flask was opened and stirring was continued for an additional 8 h.
The mixture was diluted with AcOEt (150 ml) and washed successively with water (30 ml) and 0.1 M
water solution of disodium EDTA (3x30 ml). The organic layer was separated, dried over Na2SO4 and
evaporated. The residue was applied to column chromatography on silica gel.
The yield of 5 was 477 mg (88 %) as a foam; Rf 0.47 (CH2Cl2-EtOH, 25:1). 1H NMR (400 MHz, CDCl3):
δ = 2.10 (s, 3H, AcO), 2.13 (s, 3H, AcO), 2.16 (s, 3H, AcO), 2.37 (s, 3H, AcN), 2.80-2.86 (m, 3J = 5.1, 4J
= 2 and 1.6, 5J = 2.2, 2Н, ≡ССН2С=), 4.40 (dd, 1H, J5′b,5′a = 12.9, J5′b,4′ = 5.3, H-5′b), 4.48 (dd, 1H, J5′a,5′b
13

ACCEPTED MANUSCRIPT
= 12.9, J5′a,4′ = 3.1, H-5′a), 4.48 (ddd, 1H, J4′,5′b = 5.3, J5′a,4′ = 3.1, J4′,3′ = 4.7, H-4′), 4.97 (ddt, 1Н, 2J = 1.7,
3J = 10, 4J = 1.6, С=СН-trans), 5.06 (ddt, 1Н, 2J = 1.7, 3J = 17.1, 4J = 2, С=СН-cis), 5.10 (t, 2Н, 5J = 2.2,
NCH2), 5.65 (ddt, 1Н, 3J = 5.1, 10 and 17.1, СН2СН=С), 5.69 (dd, 1H, J3′,4′ = 4.7, J3′,2′ = 5.6, H-3′), 5.97
(dd, 1H, J2′,3′ = 5.6, J2′,1′ = 5.0, H-2′), 6.25 (d, 1H, J1′,2′ = 5.0, H-1′), 8.20 (s, 1H, H-2), 8.81 (s, 1H, H-8).
13C NMR (100 MHz, CDCl3): δ = 20.50 (AcO), 20.62 (AcO), 20.85 (AcO), 23.08 (NAc), 23.08 (CH2),
37.06 (NCH2), 63.11 (C-5'), 70.65 (C-3'), 70.77 (≡C-), 73.28 (C-2'), 77.79 (-C≡), 80.54 (C-4'), 86.90 (C-
1'), 116.02 (-C=), 132.32 (=CH2), 138.50 (C-4), 142.19 (C-8), 152.34 (C-2), 152.72 (C-6), 169.47 (CO),
169.67 (CO), 170.39 (CO), 170.45 (CO). MS (APCI): m/z [M+H]+ calculated [C24H28N5O] 514.19, found
514.75.
4.10. N6-Acetyl-N6-(6-Methylhept-5-en-2-ynyl)-2′,3′,5′-tri-O-acetyladenosine (6)
The reaction was carried out in N2 atmosphere using standard Schlenk technique. To a stirred mixture of
2c (500 mg, 1.06 mmol), CuCl (105 mg, 1.06 mmol), K2CO3 (292 mg, 2.12 mmol) and Bu4NBr (34 mg
0.106 mmol) in DMF (5 ml), 80% solution of isopenthenyl bromide in toluene (0.274 ml, 2.33 mmol) was
added in one portion. The reaction mixture was stirred at room temperature for 48 h, after which it was
diluted with AcOEt (150 ml) and washed successively with water (30 ml) and 0.1 M water solution of
disodium EDTA (3x30 ml). The extract was dried over Na2SO4 and evaporated. The residue was applied
to column chromatography on silica gel.
The yield of 6 was 417 mg (72 %) as a foam; Rf 0.5 (CH2Cl2-EtOH, 25:1). 1H NMR (400 MHz, CDCl3): δ
4
= 1.49 (s, 3Н, Ме), 1.62 (d, 3Н, J = 1, Ме), 2.10 (s, 3H, AcO), 2.13 (s, 3H, AcO), 2.16 (s, 3H, AcO),
2.36 (s, 3H, AcN), 2.74 (m, 2Н, 3J = 6.9, 5J = 2.2, ≡ССН2С=), 4.39 (dd, 1H, J5′b,5′a = 12.9, J5′b,4′ = 5.4, H-
5′b), 4.47 (dd, 1H, J5′a,5′b = 12.9, J5′a,4′ = 3, H-5′a), 4.48 (ddd, 1H, J4′,5′b = 5.4, J5′a,4′ = 3, J4′,3′ = 4.7, H-4′),
5.00 (m, 1Н, 3J = 10, 4J = 1, СН2СН=С), 5.05 (t, 2Н, 5J = 2.2, NCH2), 5.69 (dd, 1H, J3′,4′ = 4.7, J3′,2′ = 5.6,
H-3′), 5.97 (dd, 1H, J2′,3′ = 5.6, J2′,1′ = 5.1, H-2′), 6.25 (d, 1H, J1′,2′ = 5.1, H-1′), 8.20 (s, 1H, H-2), 8.81 (s,
1H, H-8). 13C NMR (100 MHz, CDCl3): δ = 17.86 (Me), 20.33 (AcO), 20.44 (AcO), 20.66 (AcO), 24.21
(NAc), 25.54 (Me), 45.56 (NCH2), 45.56 (CH2), 62.95 (C5'), 70.63 (≡C-), 70.67 (-C≡), 70.46 (C-3'),
73.12 (C-2'), 80.30 (C-4'), 86.81 (C-1'), 120.10 (-CH=), 127.55 (C-5), 135.75 (CMe2), 142.15 (C-8),
152.19 (C-2), 152.48 (C-4), 153.83 (C-6), 169.34 (CO), 169.52 (CO), 170.21 (CO), 171.15 (CO). MS
(APCI): m/z [M+H]+ calculated [C26H31N5O8] 542.22, found 542.22.
4.11. N6-(Hex-5-en-2-ynyl)adenosine (7)
Compound 5 (1.2 g, 2.34 mmol) was dissolved in 7M NH3 in MeOH (6.7 ml, 46.8 mmol) and the
solution was left for 48 h at room temperature. The reaction mixture was evaporated and the residue was
treated with 5ml of cold MeOH. The solid was filtered, washed with MeOH (5 ml) and dried in vacuum
over Р2О5.
14

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1
The yield of 7 was 609 mg (75%) as a white powder; Rf 0.28 (CH2Cl2-EtOH, 9:1). mp 136-138oC. H
NMR (400 MHz, DMSO-d6 + D2O): δ = 2.95 (m, 2H, 3J = 5, 4J = 1.4 and 1.7, -CH2CH=), ), 3.56 (dd, 1H,
J5′a,5′b = 12.5, J5′a,4′ = 3.3, H-5′a), 3.67 (dd, 1H, J5′b,5′a = 12.5, J5′b,4′ = 3.4, H-5′b, 3.97 (ddd, 1H, J4′,5′b = 3.4,
J4′,5′a = 3.3, J4′,3′ = 2.9, H-4′), 4.15 (dd, 1H, J3′,4′ = 2.9, J3′,2′ = 5.3, H-3′), 4.31 (broad s, 2H, NCH2), 4.61
2
3
4
(dd, 1H, J2′,3′ = 5.3, J2′,1′ = 6.0, H-2′), 5.05 (ddt, 1H, J = 1.7, J = 10, J = 1.7, -С=CH-trans), 5.26 (ddt,
2
3
4
1H, J = 1.7, J = 17.3, J = 1.4, -С=CH-cis), 5.15 (d, 1H, 3′OH), 5.32 (dd, 1H, 5′OH), 5.42 (d, 1H,
2′OH), 5.77 (ddt, 1H, 3J = 5, 10 and 17.3, СН2CH=С), 5.90 (d, 1H, J1′,2′ = 6.0, H-1′), 8.18 (br s, 1H, NH),
13
8.27(br s, 1H, H-2), 8.39 (s, 1H, H-8). C NMR (100 MHz, CDCl3): δ = 22.26 (CH2), 29.44 (NCH2),
61.58 (C-5'), 70.55 (C-3'), 73.50 (C-2'), 78.12 (≡C-), 80.34 (-C≡), 85.81 (C-4'), 87.88 (C-1'), 115.79 (-
CH=), 119.80 (C-5), 132.95 (=CH2), 140.02 (C-8), 148.72 (C-4), 152.19 (C-2), 153.97 (C-6). MS (APCI):
m/z [M+H]+ calculated [C16H20N5O4] 346.14, found 346.25.
4.12. N6-(6-Methylhept-5-en-2-ynyl)adenosine (8)
Compound 6 (1 g, 1.9 mmol) was dissolved in 7M NH3 in MeOH (5.6 ml, 40.7 mmol) and the solution
was left for 48 h at room temperature. The reaction mixture was evaporated and the residue was treated
with 5 ml of cold MeOH. The solid was filtered, washed with MeOH (5 ml) and dried in vacuum over
Р2О5.
1
The yield of 8 was 510 mg (76%) as a white powder; Rf 0.67 (CH2Cl2-EtOH, 4:1). mp 128-130oC. H
NMR (400 MHz, DMSO-d6 + D2O): δ = 1.55 (s, 3H, Ме), 1.64 (d, 3H, 4J = 1, Ме), 2.85 (d, 2H, 3J = 6.8,
С-CH2-CH=), 3.55 (dd, 1H, J5′a,5′b = 12.2, J5′a,4′ = 3.5, H-5′a), 3.65 (dd, 1H, J5′b,5′a = 12.2, J5′b,4′ = 3.4, H-
5′b), 3.98 (ddd, 1H, J4′,5′b = 3.4, J4′,5′a = 3.5, J4′,3′ = 2.8, H-4′), 4.14 (dd, 1H, J3′,4′ = 2.8, J3′,2′ = 4.9, H-3′),
3
4
4.25 (br s, 2H, NCH2), 4.56 (dd, 1H, J2′,3′ = 4.9, J2′,1′ = 6.2, H-2′), 5.09 (m, 1H, J = 6.8, J = 1,
13
СН2СН=С) 5.86 (d, 1H, J1′,2′ = 6.2, H-1′), 8.23 (br s, 1H, H-2), 8.31 (с, 1H, H-8). C NMR (100 MHz,
DMSO-d6): δ = 17.16 (Me), 25.09 (Me), 29.07 (CH2), 29.38 (NCH2), 61.64 (C5'), 70.54 (C-3'), 70.61
(≡C-), 73.42 (-C≡), 73.51 (C-2'), 85.84 (C-4'), 87.89 (C-1'), 105.06 (-CH=), 119.09 (C-5), 139.85 (CMe2),
143.20 (C-8), 149.05 (C-4), 152.33 (C-2), 156.13 (C-6). MS (APCI): m/z [M+H]+ calculated
[C18H24N5O4] 374.41, found 374.30.
5. Antiviral assays
RD (rhabdomyosarcoma) cells were obtained from the European Collection of Cell Cultures and were
maintained in minimal essential medium (Gibco) supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 1% sodium bicarbonate (Gibco) and 1% L-glutamine (Gibco). Cells were grown at 37°C
and 5% CO2. EV71 strains that originated from Taiwan were a generous gift from Dr. Shih-Cheng Chang
(Chang Gung University, Taoyuan, Taiwan). Strain H08300 461 #812 was acquired from National
15

ACCEPTED MANUSCRIPT
Collection of Pathogenic Viruses (NCPV). Strain 11316 was a kind gift from the National Institute for
Public Health and the Environment (RIVM, The Netherlands). EV71 strain BrCr was kindly provided by
F. van Kuppeveld (UMCU, The Netherlands). Poliovirus 3 was a gift from J. Martin
(NIBSC,Hertfordshire, UK). All other viruses belonged to the virus collection of the Rega Institute for
Medical Research
The antiviral assay for HCV was essentially performed as described by Manfroni et al. [26] while that for
chikungunya virus was performed as described by Gigante et al. [27]. The antiviral assay for EV7 was
performed as described by Tijsma et al [28] Briefly, RD cells, grown to confluence in 96-well microtiter
plates were infected with ~100 CCID50 of EV71 and were treated with a dilution series of the different
compounds. Cultures were incubated for three days at 37°C (5% CO2) after which residual cell viability
was quantified using an MTS readout method according to the manufacturers’ instructions (Promega). A
similar assay setup was used for the other viruses, although with different cell lines (BGM cells for the
polioviruses as well as echovirus 11, Vero cells for Coxsackievirus B3, and HeLa cells for the human
rhinoviruses).
Acknowledgements
This work was supported by the Russian Foundation for Basic Research (14-04-00835_а) and Russian
Academy of Sciences (Program ‘Molecular and Cell Biology’), and the European Union 7th Framework
Program EUVIRNA Marie Curie Initial Training Network (264286). We would like to thank Piet
Herdewijn and Steven De Jonghe for initiating the contacts between both research groups. Kim Donckers
and Caroline Collared are acknowledged for their excellent assistance in the acquisition of the antiviral
data.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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LIST OF CAPTIONS
Scheme 1. Synthesis of adenosine derivatives with unsaturated N6-substituents. Reagents and conditions:
(i) Method A: RX, K2CO3, DMF, room temperature, 16 h; (ii) Method B: ROH, Ph3P, DEAD, THF,
room temperature, 20 h; (iii) 7M NH3 in MeOH, room temperature, 48 h; (iv) 4M PrNH2 in MeOH, room
temperature, 24 h; (v) 0.5M НСl, 100°С, 8 h; (vi) enzymatic phosphorolysis (E. coli PNP, KH2PO4, pH
7.5).
Scheme 2. Allylation of acetylenic adenosine derivative 2c. Reagents and conditions: (i)
(R2)2C=CHCH2Br, K2CO3, CuCl, BuN4Br, DMF, room temperature, 16 h; (ii) 7M NH3 in MeOH, room
temperature, 48 h.
Fig. 1. Numeration of carbon atoms in N6-substituents of compounds 5 and 6.
Table 1. Antiviral effect on the replication of EV71 strain BrCr in RD cells.
Table 2. Antiviral effect of 3a and 7 against a selection of enteroviruses.
Table 3. Antiviral effect of 3a and 7 against a selection of EV71 clinical isolates.
20

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Ten new N6-substituted adenosines have been synthesized.
N6-isopenthenyladenosine exerts antiviral effect on the replication of EV71.
The structure-activity relationship for N6-substituted adenosines has been studied.
The allylation of N6-propargyladenosine in the presence of Cu(I) was developed.
N6-(5-Hexene-2-yne-1-yl)adenosine exhibits the highest selectivity index.

ACCEPTED MANUSCRIPT
TABLES
Table 1
Antiviral effect on the replication of EV71 strain BrCr in RD cells.
Compound name
CC50 ± SDa b EC50 ± SDa b
SI
N6-isopenthenyladenosine
N6-isopenthenyladenine
N6-allyladenosine
N6-allyladenine
N6-propargyladenosine
N6-propargyladenine
5.7 ± 0.3
ND
1.0 ± 0.2
>492
5.7
ND
11
3a
4a
3b
4b
3c
4c
78 ± 20
ND
7.1 ± 1.6
>571
ND
ND
ND
166 ± 15
ND
>166
>577
N6-(4-hydroxy-2-butynyl)
adenosine
ND
>298
ND
26
3d
3e
N6-(Z)-(4-hydroxy-2-butenyl)
adenosine
122 ± 25
4.7 ± 0.7
N6-geranyladenosine
N6-neryladenosine
N6-(S)-(-)-perillyladenosine
N6-(1R)-(-)-myrtenyladenosine
N6-cyclopropylmethyladenosine
174 ± 10
>372
>124
>124
NA
NA
NA
NA
5
3f
3g
3h
3i
307 ± 7
>374
>125
>125
22 ± 3
4.6 ± 0.8
3j
N6-(tetrahydropyran-2-methyl)
adenosine
>274
>434
>402
33 ± 5
4.3 ± 1.5
>335
>8
>101
NA
3k
7
N6-(5-hexene-2-yne-1-
yl)adenosine
N6-(2-methylhept-2-en-4-yne-1-
yl) adenosine
8
a All values are in ꢀM and are based on at least three independent dose-response curves.
b On rhabdomyosarcoma (RD) cells
c Selectivity Index (SI); SI=CC50/EC50
ND = Not Determined; NA = Not Active within tested range

ACCEPTED MANUSCRIPT
Table 2
Antiviral effect of 3a and 7 against a selection of enteroviruses.
EC50 ± SDa
Virus
EV
Type
Strain
BrCr
3a
1.0 ± 0.2
>298
>298
>298
>298
>224
>298
>298
7
71
1
4.3 ± 1.5
>362
PV
2
Sabin
>290
3
>290
CV
B3
11
2
Nancy
>290
ECHO
Gregory
230 ± 45
>290
hRV
/
14
>290
a All values are in ꢀM and are based on at least three independent dose-response curves. EV = enterovirus, PV = poliovirus,
CV = Coxsackievirus, ECHO = echovirus, hRV = human rhinovirus

ACCEPTED MANUSCRIPT
Table 3
Antiviral effect of 3a and 7 against a selection of EV71 clinical isolates.
EC50 ± SDb
Genogroup
Strain
Genbank
3a
7
B2
B5
C2
C4
11316
AB575927
GQ231942
GQ231936
―
2.0 ± 0.1
2.3 ± 0.6
2.5 ± 0.5
1.2 ± 0.2
1.5 ± 0.2
1.4 ± 0.5
8.0 ± 2.0
12 ± 3
TW/96016/08
TW/70902/08
H08300 461/812
TW/1956/05
TW/2429/04
17 ± 4
0.9 ± 1.8
3.9 ± 1.1
4.5 ± 1.3
GQ231926
GQ231927
a All values are in ꢀM and are based on at least three independent dose-response curves.

ACCEPTED MANUSCRIPT
SCHEMES AND FIGURES
Scheme 1. Synthesis of adenosine derivatives with unsaturated N6-substituents. Reagents and conditions:
(i) Method A: RX, K2CO3, DMF, room temperature, 16 h; (ii) Method B: ROH, Ph3P, DEAD, THF,
room temperature, 20 h; (iii) 7M NH3 in MeOH, room temperature, 48 h; (iv) 4M PrNH2 in MeOH, room
temperature, 24 h; (v) 0.5M НСl, 100°С, 8 h; (vi) enzymatic phosphorolysis (E. coli PNP, KH2PO4, pH
7.5).

ACCEPTED MANUSCRIPT
Scheme 2. Allylation of acetylenic adenosine derivative 2c. Reagents and conditions: (i)
(R2)2C=CHCH2Br, K2CO3, CuCl, BuN4Br, DMF, room temperature, 16 h; (ii) 7M NH3 in MeOH, room
temperature, 48 h.

ACCEPTED MANUSCRIPT
Fig. 1. Numeration of carbon atoms in N6-substituents of compounds 5 and 6.

ACCEPTED MANUSCRIPT
Chemical modification of the plant isoprenoid cytokinin N6-isopentenyladenosine yields a selective
inhibitor of human enterovirus 71 replication
Vitali I. Tararov1, Aloys Tijsma2, Svetlana V. Kolyachkina1, Vladimir E. Oslovsky1, Johan Neyts2, Mikhail S. Drenichev1, Pieter Leyssen2, Sergey N.
Mikhailov1,*
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences. Address: Vavilov str. 32, Moscow 119991, Russian Federation
2KU Leuven – University of Leuven, Department of Microbiology and Immunology, Laboratory for Virology and Chemotherapy, Rega Institute for Medical Research,
Minderbroedersstraat 10, 3000 Leuven, Belgium
Corresponding author: Sergey N. Mikhailov
(Supporting information)
13
1H and C (with complete proton decoupling) NMR spectra were recorded on Bruker AMX 400 NMR instrument at 300 K relative to the residual
solvent signals as internal standards (CDCl3, 1H: δ = 7.26, 13C: δ = 77.16; DMSO-d6, 1H: δ = 2.50, 13C: δ = 39.52; CD3OD, 1H: δ = 3.31, 13C: δ =
49.00). 1H-NMR-spectra were recorded at 400 MHz and 13C-NMR-spectra at 100 MHz.