Synthesis of acyclic nucleoside analogues based on 1,2,4-triazolo[1,5-a] pyrimidin-7-ones by one-step Vorbrüggen glycosylation

Article abstract of DOI:10.1016/j.tet.2013.12.051

New acyclovir analogues were obtained by reaction of 1,2,4-triazolo[1,5-a] pyrimidin-7-ones 4a-i with (2-acetoxyethoxy)methyl acetate 5 in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) as catalyst (Vorbrüggen procedure). Coupling betwe

Full text of DOI:10.1016/j.tet.2013.12.051

Tetrahedron xxx (2014) 1e8
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Tetrahedron
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Synthesis of acyclic nucleoside analogues based on 1,2,4-triazolo
€
[1,5-a]pyrimidin-7-ones by one-step Vorbruggen glycosylation
,
Igor A. Khalymbadzha a, Tatyana S. Shestakova a, Julia O. Subbotina a b, Oleg S. Eltsov a,
Alexandra A. Musikhina a, Vladimir L. Rusinov a, Oleg N. Chupakhin a, Inna L. Karpenko c,
,
Maxim V. Jasko c, Marina K. Kukhanova c, Sergey L. Deeva
*
a Ural Federal University, 19 Mira Street, Ekaterinburg 620002, Russia
b I.Ya. Postovsky Institute of Organic Synthesis, Ural Branch of the Russian Academy of Sciences, 22 S. Kovalevskoy Street, Ekaterinburg 620219, Russia
c Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
New acyclovir analogues were obtained by reaction of 1,2,4-triazolo[1,5-a]pyrimidin-7-ones 4aei with
(2-acetoxyethoxy)methyl acetate 5 in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf)
Received 14 August 2013
Received in revised form 5 December 2013
Accepted 20 December 2013
Available online xxx
€
as catalyst (Vorbruggen procedure). Coupling between compounds 4aef and 5 led to a mixture of N3-
and N4-isomers 6 and 7, respectively. On the contrary, the reaction of compounds 4gei with 5 proceeded
selectively with formation of N3-isomers only. It was found that the ratio of 6aef and 7aef depends on
the presence or the absence of N,O-bis(trimethylsilyl)acetamide (BSA). Glycosylated products 6aef and
7aef underwent reversible isomerization under TMSOTf treatment. The ratio of glycosylated products of
the coupling reaction between 4 and 5 was thermodynamically controlled. A similar reaction occurred if
ZnCl2 was chosen as a catalyst, although lower yields of the acyclic analogues of nucleosides were ob-
served. The glycosylation of other purines (adenine and guanine) can be achieved via the non-BSA
Keywords:
Acyclovir analogues
1,2,4-Triazolo[1,5-a]pyrimidine-7-one
TMSOTf
€
Vorbruggen glycosylation
€
modification of the Vorbruggen procedure.
Herpes simplex virus
Regioselectivity
Ó 2013 Published by Elsevier Ltd.
Transglycosylation
1. Introduction
them became our current interest. Continuing our efforts to find
new potent inhibitors of HSV replication we have synthesized
Derivatives of 1,2,4-triazolo[1,5-a]pyrimidines represent the
core for many biologically active compounds.1 Inclusion of an N-
alkyl or ribofuranosyl fragment in the structure of heterocycles of
this class can be used to design antiviral and anticancer com-
pounds. For example, N3-benzyl-1,2,4-triazolo[1,5-a]pyrimidine
derivate 1 was described as potent growth inhibitor in a PTEN
deficient cancer cell line (Fig. 1).2 Compound 2, bearing a butyl
moiety, with a terminal hydroxyl group linked to the azine cycle
showed an antiherpetic activity. This compound suppressed effi-
ciently the replication of herpes simplex virus (HSV) in cell cul-
a series of new acyclic analogues of nucleosides based on 1,2,4-
triazolo[1,5-a]pyrimidin-7-ones 4aei.
2. Results and discussion
Nucleoside analogues based on 1,2,4-triazolo[1,5-a]pyrimidines
are typically prepared via coupling of NH-heterocycles with ribose
derivatives. Synthetic method, a so called ‘one-pot’ protocol of
tures.3
Ribosylated
1,2,4-triazolo[1,5-a]pyrimidin-7-ones
3
exhibited a moderate activity against the rhinovirus comparable
with ribavirin.4
Considering the therapeutic potential of nucleoside analogues
based on 1,2,4-triazolo[1,5-a]pyrimidin-7-ones, the development
of new efficient and easy to control synthetic routines to obtain
* Corresponding author. Tel.: þ7 343 375 4501; fax: þ7 343 374 0458; e-mail
Fig. 1. N-Substituted 1,2,4-triazolo[1,5-a]pyrimidines with antiviral and anticancer
activity.
address: deevsl@yandex.ru (S.L. Deev).
0040-4020/$ e see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.tet.2013.12.051
Please cite this article in press as: Khalymbadzha, I. A.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2013.12.051

2
I.A. Khalymbadzha et al. / Tetrahedron xxx (2014) 1e8
exhibit a cross peak between 10 proton and C2 and C3a atoms in
their HMBC spectra (see Supplementary data). This observation
confirms that the alkyl substituent is linked to the N3 atom of the
heterocycle. In the case of triazolopyrimidines 7aef, the N4 posi-
tion of acyclic fragment was supported by cross peak between the
C5 and H10 atoms. Additionally, the structure of compounds 6e and
7e was determined by X-ray crystallography (Figs. 2 and 3).
€
Vorbruggen glycosylation, is one of the most convenient pro-
cedures to obtain nucleosides and their analogues.5
Previously, we have described an example of successful ribo-
sylation of heterocycles 4def via the Vorbruggen method, although
€
the mechanistic details of the reaction and its dependency on the
reaction conditions were not investigated.6 Herein, we present
€
a study on the mechanism of Vorbruggen glycosylation of 1,2,4-
triazolo[1,5-a]pyrimidin-7-ones 4aei with (2-acetoxyethoxy)
methyl acetate 5 under varied reaction conditions (Table 1). The
introduction of acetate 5 into a coupling reaction with NH-
heterocycles enables the acquisition of the analogues of acyclovir,
which is still considered the gold standard for HSV therapy.7 Alto-
gether, two methods (A and B) were employed. In method A, the
reaction mixture in MeCN was treated with BSA, the silylation re-
agent, and TMSOTf, the catalyst for glycosylation. The coupling
between compounds 4aei and 5 was also observed if a mixture of
reagents in MeCN was treated with TMSOTf only (method B).
Table 1
Coupling reaction of 4aei and 5a
Fig. 2. ORTEP diagram of the X-ray structure of compound 6e.
Entry
1
Heterocycle
Methoda
Ratio 6/7b
Yieldc
%
4a
A
B
A
B
A
B
A
B
C
A
B
C
A
B
C
A
B
A
B
A
B
62:38
100:0
51:49
75:25
52:48
94:6
5:95
75:25
4:96
56
60
59
49
63
80
86
48
40
70
43
27
59
46
28
39
41
75
24
35
34
2
3
4
5
4b
4c
4d
4e
Fig. 3. ORTEP diagram of the X-ray structure of compound 7e.
40:60
70:30
8:92
6
4f
4:96
€
The possibility to obtain various regioisomers under Vorbrug-
60:40
0:100
100:0
100:0
100:0
100:0
100:0
100:0
gen reaction conditions is well documented.8 For example,
€
depending on the temperature regimen, Vorbruggen reaction with
7
8
9
4g
4h
4i
alloxazine resulted in two different products of glycosylation.9 This
phenomenon was explained by isomerization of the N-glycosyla-
tion products. Isomerization was registered for different nucleoside
analogues based on purines, imidazo[1,2-a][1,2,4]triazin-7-ones
and other non-natural NH-heterocycles.10 It is usually initiated by
addition of TMSOTf in reaction.
Indeed, dissolution of compounds 6aef in MeCN, containing
2 equiv of trimethylsilyl trifluoromethanesulfonate yielded a mix-
ture of N3- and N4-isomers, 6aef and 7aef. The reverse reaction
was also observed: the treatment of a solution of N4-isomers 7aef
with 2 equiv of TMSOTf led to a mixture of N4- and N3-isomers as
well. This observation allowed us to modify the ‘one-pot’
a
Legends for reaction conditions A: MeCN (7 mL), BSA (2 mmol), 4aei (1.8 mmol),
5 (2 mmol), TMSOTf (2 mmol), 0.5 h; B: MeCN (7 mL), 4aei (1.8 mmol), 5 (2 mmol),
TMSOTf (2 mmol), 0.2 h; C: 4aei (1.8 mmol), 5 (4 mmol), ZnCl2 (0.1 mmol),
140e160 ꢀC, 0.5 h.
b
Ratio 6/7 was determined by 1H NMR spectroscopy.
c
Isolated overall yields.
Glycosylation of 4aef in the presence of BSA and TMSOTf led to
a mixture of N3- and N4-isomers, 6aef and 7aef, respectively, in
different ratios. While the prevalence of N3-isomers 6aec was
observed in case of glycosylation of 4aec (see Table 1), the coupling
of 4def with 5 led to mixtures largely containing the products of
N4-glycosylation, 7def. It should be also highlighted that the re-
action between 4gei and 5 was selective and yielded only N3-
isomers 6gei. This selectivity was attributed to the bulky phenyl
group protecting the N4 position from coupling.
€
Vorbruggen procedure of coupling 4aef with 5 in order to achieve
a better ratio of N3/N4-isomers. Glycosylation of 4aef was carried
out in the presence of TMSOTf solely (method B) and yielded
a mixture of compounds 6aef containing the acyclic fragment in
the azole ring as major products (Table 1).
Based on the observation described above we have assumed
that compounds 7aef were the products of isomerization of 6aef
and the final ratio of isomers in the coupling reaction between 4aef
and 5 directly depends on the contribution of a reversible N3%N4
isomerization process in different conditions.
The structure of products 6aei and 7aef was studied by 1He13C
HMBC two-dimensional NMR experiments. Compounds 6aei
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I.A. Khalymbadzha et al. / Tetrahedron xxx (2014) 1e8
3
Thus, the general Scheme 1 describing the formation of glyco-
sylation products for triazolopyrimidines 4aef should include the
rearrangement step of compounds 6aef/7aef into 7aef/6aef. This
process occurs through the cleavage of the C10eN3/C10eN4 bond in
6aef/7aef and may lead to the cation 9 and to O-silylated de-
rivatives 8aef (reactions a and b in Scheme 1). Compounds 8aef
again react with cation 9 producing isomeric alkyl derivatives 7aef/
6aef. There is also an alternative pathway to form diglycosides
10aef as a result of secondary alkylation of 6aef/7aef by cation 9.
Next, diglycosides 10aef can be converted back to mono-N-alkyl
derivatives 7aed/6aef (reactions c and d in Scheme 1). Similar
transglycosylation was previously described for purine
nucleosides.8
Fig. 4. Fragment of 1D 1H NMR spectra for 4*d (A), 4#d (B), 6#d (C), 7#d (D). Unlabeled
components of signals H2 are marked by black diamond (A).
Scheme 1. Possible pathways of formation of 6aef and 7aef.
To prove the mechanism of transglycosylation in N4- and N3-
isomers, 6aef and 7aef, we have performed experiments with
15N-labeled compounds. The formation of cation 9 was confirmed
by experiments with [1-15N]-1,2,4-triazolo[1,5-a]pyrimidin-7-one
4*d (86%, 15N), which was obtained from 15N-amino-1,2,4-triazine
11* (Scheme 2). Reaction of 11* with formyl derivative 12 led to
azoloazine 13*. Refluxing of 13* with HCl gave 4*d. Isotope enrich-
ment of heterocycle 4*d was verified by analysis of the integrated
intensity of the H2 signal (Fig. 4A). Incorporation of 15N-labeled
1,2,4-triazolo[1,5-a]pyrimidin-7-one 4*d in isomerization of 6d/7d
led to the mixture of compounds 4#d, 6#d, and 7#d in 100:45:55
ratio (Scheme 3). The 15N enrichment for 4#d, 6#d, and 7#d was
w43%. Isotope enrichment was determined by integrated intensity
in the 1H NMR spectra (Fig. 4BeD). Inclusion of 15N in the products
of isomerization undoubtedly confirms the formation of cation
intermediate 9 during the rearrangement 6d%7d. It should be
noted that isotopic enrichment in 4#d, 6#d, and 7#d showed that
the equilibrium 6d%7d was reached in the isomerization process.
The state of equilibrium was achieved in 30 days. This fact can be
taken as proof that compounds 6d and 7d have equal thermody-
namic stability.
Scheme 3. Isomerization of 6d/7d in presence of [1-15N]-1,2,4-triazolo[1,5-a]pyr-
imidin-7-one 1*d. Observed 1He15N coupling constants in 1D 1H NMR spectra are
shown by blue (w86%, 15N) and red (w43%, 15N) arrows.
approximation) using Gaussian 09 package.11 All compounds were
initially optimized and the character of local minima was confirmed
by Hessian calculations. Obtained data and calculated heats of
formation for 6def and 7def confirmed that the N4-isomers as
glycosylation products of the coupling reaction of heterocycles
4def are slightly more stable when compared to the N3-isomers
(Fig. 5). The energy difference between the values of relative en-
thalpy of formation for N3- and N4-isomers was 0.3e2.4 kcal/mol.
Thus, the glycosylation is thermodynamically controlled. Obtained
values of Hf were in good agreement with N3/N4 isomeric ratio
observed in the reaction of glycosylation 4def (method A, Table 1).
Furthermore, the calculated relative stability of other possible
products of glycosylation 14def suggested that they were not
formed due to their low stability comparing to products 6def,
7aed.
An alternative route to acyclovir analogues includes the cou-
pling between 5 and NH-heterocycles catalyzed via Lewis acids.12
We found that heterocycles 4def react with 5 in the presence of
ZnCl2 at high temperatures. The glycosylation of 4d,e yielded 6d,e
and 7d,e in the mixture, and N4-isomer was a major product (Table
1). Regarding compound 4f, only N4-isomer 7f was isolated. It
should be mentioned that the total yields of glycosylated products
obtained through this procedure were lower than those observed
To evaluate the relative thermodynamic stability of isomers
6def and 7def we have performed DFT calculations (B3LYP/6-31G
*
€
for the ‘one-pot’ Vorbruggen glycosylation. Additionally, the ZnCl2
catalyst did not enable reaction direction control. As a result, this
catalyst it can be suitable for the synthesis of isomers with a high
Scheme 2. Synthesis of [1-15N]-1,2,4-triazolo[1,5-a]pyrimidin-7-one 4*d.
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4
I.A. Khalymbadzha et al. / Tetrahedron xxx (2014) 1e8
Scheme 6. Deacetylation of compounds 6aei.
Fig. 5. Values of relative enthalpy of formation for 6def, 7aed and possible products
of N1-glycosylation 14def.
Scheme 7. Deacetylation of compounds 7aef.
thermodynamic stability. Additionally, we found that the heating of
alkyl derivative 6d at 160 ꢀC in an excess of (2-acetoxyethoxy)
methyl acetate 5 with ZnCl2 gives 7d. This result confirms that
ZnCl2-catalyzed coupling between 4def and 5 includes the isom-
erization 6/7. This rearrangement is irreversible.
cells according to the previously reported procedure.3 Among these
acyclic analogues, compound 20f has exhibited weak anti-HSV
activity compared to acyclovir (Table 2). Its toxicity was compara-
ble with the toxicity of acyclovir and the selectivity index was
significantly lower than that of acyclovir.16
€
The scope of the ‘one-pot’ Vorbruggen procedure presented in
this paper was not limited by the type of NH-heterocycles 4aei. The
BSA free ‘one-pot’ coupling of adenine 15 and guanine 16 with
compound 5 (Schemes 4 and 5) was also successful. In the former
case, the product 17 with 30% yield was separated, while the latter
led to the products 18 and 19 in a 96:4 ratio. Compounds 17 and 18
were isolated and characterized by 1H NMR spectra. The structures
of 17 and 18 were confirmed via the analysis of the chemical shifts
of purine protons.13,14 Although compound 19 was not separated,
its structure was resolved via 1H NMR spectra.15 Despite the low
yields for the products of interest, this example reflects the po-
Table 2
Cytotoxicity and anti-HSV activity of new acyclovir analogues
Compound
CD50 (mM)
Multiplicity of infection
0.1 PFU/cell
0.01 PFU/cell
a
a
ID50 (mM)
SI
ID50 (mM)
SI
20a
20b
20c
20d
20e
20f
20g
20h
20i
21a
21b
21c
4.62
4.20
3.70
NA
NA
NA
NA
NA
>3.70
1.74
0.83
0.37
1.74
0.83
0.75
NA
>1
>2
>4
>8
>2
>4
>4
€
tential for the BSA free ‘one-pot’ Vorbruggen procedure to be
>3.49
>3.33
>3.01
>3.49
>3.33
>3.01
>4.62
>4.20
>3.70
>3.49
>3.33
>3.01
2.2
3.49
2.50
0.75
3.49
2.50
3.01
NA
>1
>1.33
4
>1
>1.33
>1
transferred to other types of NH-heterocycles. However, a careful
examination and the optimization of the reaction conditions
(temperature, solvents, etc.) are further required.
NA
NA
3.70
3.49
3.33
3.01
0.00282
>1
>1
>1
>1
780
1.35
1.74
1.66
1.50
>2
>2
>2
>2
21d
21e
21f
Acyclovir
Scheme 4. Coupling reaction of adenine 15 with 5.
CD50dcytotoxic dose, i.e., the agent concentration, at which 50% of uninfected cells
die; ID50dinhibitory dose, i.e., the agent concentration, at which the cytopathic
effect is decreased by 50%; SIdselectivity index, CD50/ID50; PFUdplaque forming
unit.
a
Mean values of three independent experiments are given.
3. Conclusions
We have studied the reaction of 1,2,4-triazolo[1,5-a]pyrimidin-
7-ones 4aei with (2-acetoxyethoxy)methyl acetate 5. This reaction
was susceptible to the nature of the substituents in 4aei and to the
reaction conditions. The appearance of a bulky group at position 5
of the pyrimidine ring of compounds 4gei leads to an exclusive
selectivity of glycosylation. In other cases a mixture of N3- and N4-
Scheme 5. Coupling reaction of guanine 16 with 5.
Deprotection of 6aei and 7aef yielded new acyclovir analogues
20aei and 21aef (Schemes 6 and 7). The cytotoxicity and antiviral
activity of 20aei and 21aef were examined against HSV-1 in Vero
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I.A. Khalymbadzha et al. / Tetrahedron xxx (2014) 1e8
5
isomers, 6aef and 7aef, was observed. The formation of products
7aef occurs through the isomerization of 6aef by a reaction similar
to the process of transglycosylation, previously described in purine
nucleosides. The final ratio of products 6/7 depends on their rela-
tive thermodynamic stability. Studied coupling reaction between
1,2,4-triazolo[1,5-a]pyrimidin-7-ones 4aei and (2-acetoxyethoxy)
methyl acetate 5 was found to be useful to obtain new acyclic an-
alogues of nucleosides.
(475 mg; 2.69 mmol) and TMSOTf (600 mg; 2.70 mmol) in MeCN
(10 mL) including BSA (549 mg; 2.70 mmol) was stirred at ambient
temperature for 3 h and then neutralized with NaHCO3, extracted
with CH2Cl2 (2ꢂ20 mL), dried (Na2SO4), and concentrated in vacuo.
The products were separated by column chromatography (hexane/
EtOAc 10:1/0:100).
Method B.
A
solution of 1,2,4-triazolo[1,5-a]pyrimidine
4
(1.80 mmol), (2-acetoxyethoxy)methyl acetate
5
(475 mg;
2.69 mmol), and TMSOTf (600 mg; 2.70 mmol) in MeCN (10 mL)
was stirred at ambient temperature for 0.2 h and then neutralized
with NaHCO3, extracted with CH2Cl2 (2ꢂ20 mL), dried (Na2SO4),
and concentrated in vacuo. The products were isolated by column
chromatography (hexane/EtOAc 10:1/0:100).
4. Experimental section
4.1. General experimental methods
Commercially available reagents and catalysts were used as
obtained, unless otherwise stated, from freshly opened containers
without further purification. MeCN was distilled over P2O5. NMR
spectra were recorded on a Bruker Avance II 400 MHz spectrom-
eter using DMSO-d6 as solvent. 1H NMR spectra were recorded at
400 MHz; multiplicities are designated as s (singlet), d (doublet), t
(triplet), br (broad), and m (multiplet). 1H chemical shifts were
referenced relative to the residual CD2H signal of DMSO-d6 at
2.50 ppm. 13C and 15N NMR spectra were registered at 100.6 and
40.5 MHz, respectively. 13C and 15N chemical shifts were refer-
enced relative to TMS and liquid NH3, respectively. The line posi-
tions of signals in 1H, 13C, and 15N NMR spectra are given in ppm
Method C. A mixture of 1,2,4-triazolo[1,5-a]pyrimidines 4def
(1.80 mmol), (2-acetoxyethoxy)methyl acetate
5 (700 mg;
3.97 mmol), and ZnCl2 (14 mg; 0.10 mmol) was heated at
140e160 ꢀC for 0.5 h in an oil bath. The reaction mixture was then
cooled, and product 6d,e or 7def was isolated via column chro-
matography on silica gel (hexane/EtOAc 10:1/0:100).
4.2.1.1. 3-[(2-Acetoxyethoxy)methyl]-5-methyl-1,2,4-triazolo[1,5-
a]pyrimidin-7-one (6a). White solid; yield 34% (162 mg, procedure
A)/60% (287 mg, procedure B); mp 140 ꢀC; 1H NMR
d: 1.94 (3H, s,
COeCH3), 2.29 (3H, s, Me), 3.80e3.82 (2H, m, OC30H2), 4.09e4.11
(2H, m, OC40H2), 5.49 (2H, s, NC10H2), 6.00 (1H, s, H6), 9.01 (1H, s,
(
d
). 1H and 13C resonance assignment was done using gradient
H2); 13C NMR : 20.23 (COeCH3), 23.7 (Me), 62.7 (C40), 67.5 (C30),
d
enhanced versions of 2D 1He13C HSQC and 1He13C HMBC spectra.
Column chromatography was carried out on silica gel 60
(230e400 mesh). Analytical TLC was performed using silica gel
aluminum sheets. HMRS for 15N-labeled compounds were ac-
quired on a 7-Tesla Finnigan LTQ FT spectrometer equipped with
an Ion Max electrospray ion source. Mass spectra (MS) for un-
73.0 (C10), 102.0 (C6), 142.6 (C2), 148.3 (C3a), 155.4(C7), 163.9 (C5),
170.1 (COeCH3); MS (ESI) m/z: 267.11 [MþH]þ; IR: 1535, 1585, 1682,
1733, 2943, 3099. Found: C, 49.58; H, 5.27; N, 20.96. C11H14N4O4
requires C, 49.62; H, 5.30; N, 21.04.
4.2.1.2. 3-[(2-Acetoxyethoxy)methyl]-6-phenyl-1,2,4-triazolo[1,5-
a]pyrimidin-7-one (6d). White solid; yield 4% (23 mg, procedure A)/
36% (213 mg, procedure B)/2% (9 mg, procedure C); mp 152 ꢀC; 1H
labeled compounds were recorded on
a Bruker Daltonics
micrOTOF-Q II mass spectrometer equipped with an orthogonal
electrospray ionization (ESI) source, a six-port divert valve, and
NMR d
: 1.96 (3H, s, COeCH3), 3.83e3.85 (2H, m, OC30H2), 4.10e4.13
syringe pump kd Scientific with a flow rate of 180
m
L/h. Melting
(2H, m, OC40H2), 5.56 (2H, s, NC10H2), 7.30e7.34 (1H, m, Hp(Ph)),
7.40e7.44 (2H, m, Hm(Ph)), 7.68e7.70 (2H, m, Ho(Ph)), 8.28 (1H, s,
points are uncorrected. IR spectra were recorded using a Bruker
Alpha (NPVO, ZnSe) IR-Fur spectrometer; absorbance frequencies
are given at maximum intensity in cmꢁ1. Elemental analyses were
carried out using a CHNS/O analyzer PerkineElmer 2400 Series II
instrument. Obtained compounds 6e and 7e were recrystallized
from i-propanol and gave single crystals suitable for X-ray crys-
H5), 9.13 (1H, s, H2); 13C NMR : 20.5 (COeCH3), 62.7 (C40), 67.6
d
(C30), 73.2 (C10), 116.2 (C6), 127.0 (Cp), 128.2 (Cm), 128.3 (Co), 134.3
(Ci), 143.4 (C2), 148.3 (C3a), 151.8 (C5), 154.8 (C7), 170.2 (COeCH3);
MS (ESI) m/z: 329.12 [MþH]þ; IR: 1549, 1605, 1674, 1688, 1737,
2954, 3134. Found: C, 58.28; H, 4.77; N, 17.12. C16H16N4O4 requires
C, 58.53; H, 4.91; N, 17.06.
tallographic analysis. The diffraction data were collected on X-ray
ꢀ
diffractometer Xcalibur S CCD with Mo K
graphite monochromator, /2
a
radiation (
l
¼0.71073 A,
u
q
-scanning technique). All struc-
4.2.1.3. 3-[(2-Acetoxyethoxy)methyl]-5-phenyl-1,2,4-triazolo[1,5-
tures were solved by direct methods implemented in the SHELXS-
97 program.17 The refinement was carried out by full matrix an-
isotropic least-squares methods on F2 for all reflections for non-H
atoms by using the SHELXL-97 program.18 The X-ray CIF files have
been deposited at the Cambridge Crystallographic Data Centre and
allocated with the deposition numbers CCDC 928809 (compound
6e) and CCDC 930137 (compound 7e). Copies of the data can be
obtained, free of charge, from CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (e-mail: deposit@ccdc.cam.ac.uk; Inter-
net: www.ccdc.cam.ac.uk). Experimental data for compounds
6b,c,e,f,h,i, 7b,c,e,f, 20b,c,e,f,h,i, and 21b,c,e,f are available in
Supplementary data.
a]pyrimidin-7-one (6g). White solid; 39% (218 mg, procedure A)/
41% (242 mg, procedure B); mp 145 ꢀC; 1H NMR
d: 1.94 (3H, s,
COeCH3), 3.88e3.90 (2H, m, OC30H2), 4.12e4.14 (2H, m, OC40H2),
5.61 (2H, s, NC10H2), 6.74 (1H, s, H6), 7.50e7.51 (3H, m, Hp(Ph)þ
Hm(Ph)), 8.11e8.14 (2H, m, Ho(Ph)), 9.11 (1H, s, H2); 13C NMR
d:
20.5 (COCH3), 62.8 (C40), 67.7 (C30), 73.1 (C10), 99.1 (C6), 127.1 (Co),
128.7 (Cm), 130.5 (Cp), 136.5 (Ci), 143.2 (C2), 148.9 (C3a), 156.1 (C7),
160.2 (C5), 170.2 (COeCH3); MS (ESI) m/z: 329.13 [MþH]þ; IR: 1544,
1674, 1733, 3046, 3109. Found: C, 58.45; H, 4.97; N, 16.93.
C16H16N4O4 requires C, 58.53; H, 4.91; N, 17.06.
4.2.1.4. 4-[(2-Acetoxyethoxy)methyl]-5-methyl-1,2,4-triazolo[1,5-
a]pyrimidin-7-one (7a). White solid; yield 22% (105 mg, procedure
A); mp 116ꢀC; 1H NMR
: 1.95 (3H, s, COeCH3), 2.50 (3H, s, Me),
3.76e3.79 (2H, m, C30H2), 4.09e4.11 (2H, m, C40H2), 5.72 (2H, s,
NC10H2), 6.05 (1H, s, H6), 8.23 (1H, s, H2); 13C NMR
: 17.6
4.2. Synthesis
d
Compounds 4aei6,19e24 and 525 were prepared as previously
d
described.
(COeCH3), 20.5 (Me), 62.7 (C30), 67.1 (C40), 76.5 (C10), 100.9 (C6),
151.6 (C2), 152.4 (C3a), 152.5 (C5), 154.9 (C7), 170.1 (COeCH3); MS
(ESI) m/z: 265.12 [MþH]þ; IR: 1314, 1563, 1689, 1732, 2949, 3099.
Found: C, 49.71; H, 5.38; N, 21.07. C11H14N4O4 requires C, 49.62; H,
5.30; N, 21.04.
4.2.1. General procedures for coupling reactions of NH-heterocycles
4aei with compound 5. Method A. A solution of 1,2,4-triazolo[1,5-
a]pyrimidine 4 (1.80 mmol), (2-acetoxyethoxy)methyl acetate 5
Please cite this article in press as: Khalymbadzha, I. A.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2013.12.051

6
I.A. Khalymbadzha et al. / Tetrahedron xxx (2014) 1e8
4.2.1.5. 4-[(2-Acetoxyethoxy)methyl]-6-phenyl-1,2,4-triazolo[1,5-
a]pyrimidin-7-one (7d). Colorless crystals; yield 82% (484 mg,
(2H, m, Ho(Ph)), 8.28 (1H, s, H5), 9.13 (1H, d, 2JNH 14.3 Hz, H2); 13
NMR
: 20.5 (COeCH3), 62.8 (C40), 67.6 (C30), 73.2 (C10), 116.3 (C6),
127.0 (Cp), 128.2 (Cm), 128.3 (Co), 134.3 (Ci), 143.4 (C2, 1JCN 1.1 Hz),
C
d
procedure A)/12% (71 mg, procedure B)/38% (227 mg, procedure C);
mp 161 ꢀC; 1H NMR
d
: 1.92 (3H, s, COeCH3), 3.85e3.87 (2H, m,
148.3 (C3a, JCN 1.7 Hz), 151.9 (C5), 154.8 (C7, JCN 3.0 Hz), 170.2
2
2
OC30H2), 4.10e4.12 (2H, m, OC40H2), 5.66 (2H, s, NC10H2), 7.37e7.40
(1H, m, Hp(Ph)), 7.45e7.48 (2H, m, Hm(Ph)), 7.65e7.67 (2H, m,
(COeCH3); 15N NMR
d
: 262.8; HRMS (ESI) m/z: [MþH]þ found
330.1220. C16H17N135NO4 requires 330.1216.
Ho(Ph)), 8.33 (1H, s, H2), 8.50 (1H, s, H5); 13C NMR
d: 20.5
(COeCH3), 62.8 (C40), 67.5 (C30), 80.1 (C10), 112.8 (C6), 127.8 (Cp),
128.3 (Cm), 128.7 (Co), 132.7 (Ci), 140.8 (C5), 150.5 (C3a), 152.3 (C2),
155.1 (C7), 170.2 (COeCH3); MS (ESI) m/z: 329.13 [MþH]þ; IR: 1574,
1668, 1727, 3062. Found: C, 58.31; H, 4.73; N, 17.04. C16H16N4O4
requires C, 58.53; H, 4.91; N, 17.06.
4.2.3.2. [1-15N]-4-[(2-Acetoxyethoxy)methyl]-6-phenyl-1,2,4-
triazolo[1,5-a]pyrimidin-7-one (7#d) (15N, 43%). Yield 119 mg (35%)/
20 mg (32%). 1H NMR
d: 1.92 (3H, s, COeCH3), 3.85e3.87 (2H, m,
OC30H2), 4.10e4.12 (2H, m, OC40H2), 5.66 (2H, s, NC10H2), 7.37e7.40
(1H, m, Hp(Ph)), 7.45e7.48 (2H, m, Hm(Ph)), 7.65e7.67 (2H, m,
Ho(Ph)), 8.33 (1H, d, 2JNH 16.0 Hz, H2), 8.50 (1H, s, H5); 13C NMR
d:
4.2.2. Synthesis of 15N-labeled compounds and isomerization of 6d/
20.6 (COeCH3), 62.9 (C40), 67.5 (C30), 80.2 (C10), 112.8 (C6), 127.8
(Cp), 128.4 (Cm), 128.7 (Co), 132.7 (Ci), 140.8 (C5), 150.6 (C3a), 152.3
7d in presence of 4*d. Compounds 11* 15N, w86%) and 12 were
(
obtained by described procedures.26,27
(C2), 155.2 (C7), 170.3 (COeCH3); 15N NMR
d: 274.5; HRMS (ESI) m/
z: [MþH]þ found 330.1220. C16H17N135NO4 requires 330.1216.
4.2.2.1. [1-15N]-3-Amino-1,2,4-triazole (11*)
(
15N, w86%). IR:
1045, 1352, 1615, 2776, 2814, 3151, 3324.
4.2.3.3. [1-15N]-6-Phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7-one
(4#d) (15N, 43%). Yield 50 mg (24%)/12 mg (30%). 1H NMR
d:
4.2.2.2. [1-15N]-7-Amino-6-phenyl-1,2,4-triazolo[1,5-a]pyrimi-
dine (13*) (15N, w86%). A solution of 15N-3-amino-1,2,4-triazole 11*
(900 mg; 10.59 mmol) and phenyl(formyl)acetonitrile 12 (1536 mg;
10.59 mmol) in acetic acid (6 mL) was refluxed for 3 h. The pre-
cipitate formed was filtered off and dried to give compound 13*,
which was used without further purification. Pale yellow crystals;
7.32e7.26 (1H, m, Hp(Ph)), 7.40e7.42 (2H, m, Hm(Ph)), 7.42e7.44
(2H, m, Ho(Ph)), 8.22 (1H, s, H5), 8.29 (1H, d, 2JNH 16.0 Hz, H2); 13
NMR : 111.9 (C6), 127.4 (Cp), 128.2 (Cm), 128.7 (Co), 133.4 (Ci), 138.8
(C5), 150.1 (C3a), 152.3 (C2, br s), 155.8 (C7).
C
d
4.2.4. Synthesis of compounds 17 and 18 by use of BSA free ‘one-pot’
yield 45% (1021 mg). 1H NMR
7.49e7.51 (4H, m, HoþHm(Ph)), 7.89 (2H, br s, NH2) 8.28 (1H, s, H5),
d: 7.41e7.44 (1H, m, Hp(Ph)),
Vorbruggen procedure. A solution of adenine 15 or guanine 16
€
(1.48 mmol), (2-acetoxyethoxy)methyl acetate
5 (405 mg;
2
8.49 (1H, d, JNH 16.0 Hz, H2). 13C NMR
d
: 104.9 (C6), 127.7 (Cp),
2.30 mmol), and TMSOTf (511 mg; 2.30 mmol) in anhydrous MeCN
(7 mL) was stirred at ambient temperature for 2 h and then diluted
with 10 mL acetonitrile with a few drops of water and neutralized
with NaHCO3. The resulting suspension was filtered. The filtrate
was concentrated in vacuo. The product was isolated by column
chromatography (EtOAc/EtOH 20:1 for 17 or CHCl3/EtOH 10:1
for 18).
129.0 (Cm), 129.3 (Co), 133.1 (Ci), 146.5 (C7, 2JCN 3.2 Hz), 153.8 (C5),
154.6 (C2, 1JCN 3.3 Hz), 154.7 (C3a); 15N NMR
d: 262.5; HRMS (ESI)
m/z: [MþH]þ found 213.0902. C11H10N145N requires 213.0906; IR:
705, 1201, 1240, 1461, 1562, 1589, 1632, 3118, 3413.
4.2.2.3. [1-15N]-6-Phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7-one
(4*d) (15N, w86%). A mixture of 15N-1,2,4-triazolo[1,5-a]pyrimidine
13* (700 mg; 3.30 mmol) and 11 M HCl (8 mL) was refluxed for 7 h.
After cooling, a precipitate formed was filtered off and recrystal-
4.2.4.1. 9-[(2-Acetoxyethoxy)methyl]adenine (17). White solid;
yield 39% (145 mg). 1H NMR
d: 1.95 (s, 3H, COeCH3), 3.71e3.73 (m,
lized from DMF. Colorless crystals; yield 44% (312 mg). 1H NMR
d:
2H, OCH2), 4.07e4.09 (m, 2H, OCH2), 5.57 (s, 2H, NCH2), 7.05 (s, 2H,
7.32e7.36 (1H, m, Hp(Ph)), 7.40e7.42 (2H, m, Hm(Ph)), 7.64e7.66
NH2), 8.11 (s, 1H, CH), 8.16 (s, 1H, CH).
(2H, m, Ho(Ph)) 8.22 (1H, s, H5), 8.29 (1H, d, 2JNH 16.0 Hz, H2); 13
C
3
NMR
d
: 111.9 (C6, JCN 1.0 Hz), 127.5 (Cp), 128.3 (Cm), 128.7 (Co),
4.2.4.2. 9-[(2-Acetoxyethoxy)methyl]guanine (18). White solid;
133.4 (Ci), 138.8 (C5), 150.1 (C3a), 152.3 (C2, 1JCN 2.8 Hz), 155.8 (C7,
yield 29% (115 mg). 1H NMR
d: 1.96 (s, 3H, COeCH3), 3.65e3.67 (m,
2JCN 3.5 Hz); 15N NMR
d
: 271.7; HRMS (ESI) found 214.0743.
2H, OCH2), 4.06e4.08 (m, 2H, OCH2), 5.35 (s, 2H, NCH2), 6.52 (s, 2H,
NH2), 7.82 (s, 1H, C8H), 10.65 (s, 1H, NH).
C11H9ON145N [MþH]þ requires 214.0746; IR: 684, 765, 1276, 1179,
1633, 2869, 3094.
4.2.5. Deprotection of N-(acetoxyethoxy)methyl-1,2,4-triazolo[1,5-a]
pyrimidin-7-ones 6aei and 7aef. A solution of 6 or 7 (1 mmol) in
NH3/MeOH (20 mL; saturated at ꢁ10 ꢀC) was stirred at ambient
temperature for 24 h in a sealed flask. After evaporation of solvent
under vacuum, the residue was purified by column chromatogra-
phy using ethyl acetate (100%) as an eluant to give 20 or 21.
4.2.3. Isomerization of compounds 6d/7d in the presence of 4*d. A
solution of 4*d (198 mg; 0.93 mmol)/(40 mg; 0.19 mmol), TMSOTf
(413 mg; 1.86 mmol)/(84 mg; 0.38 mmol) in anhydrous MeCN
(10 mL)/(3 mL) was stirred at ambient temperature for 0.5 h, and
compound 6d (305 mg; 0.93 mmol)/7d (61 mg, 0.19 mmol) was
added. The resulting solution was allowed to stand at room tem-
perature for 30 days and then 10% aqueous solution of Na2CO3
(10 mL/5 mL) was added. The resulting suspension was extracted
with CH2Cl2 (3ꢂ10 mL) and organic layers were separated and
concentrated in vacuo. Compounds 6#d and 7#d were isolated by
column chromatography (hexane/EtOAc 10:1/1:10). The aqueous
layer was acidified to pH 1e2 with 11 M HCl. The precipitate formed
was filtered off and dried to give compound 4#d.
4.2.5.1. 3-[(2-Hydroxyethoxy)methyl]-5-methyl-1,2,4-triazolo
[1,5-a]pyrimidin-7-one (20a). Colorless crystals; yield 80%
(179 mg); mp 186 ꢀC; 1H NMR
d
: 2.29 (3H, s, Me), 3.48e3.51 (2H, m,
C40H2), 3.61e3.63 (2H, m, C30H2), 4.64 (1H, t, J 5.4 Hz, OH), 5.49 (2H,
s, NC10H2), 6.00 (1H, s, H6), 8.99 (1H, s, H2); 13C NMR
: 23.7 (Me),
d
59.8 (C40), 71.4 (C30), 73.3 (C10), 101.9 (C6), 142.6 (C2), 148.2 (C3a),
155.3 (C7), 163.9 (C5); MS (ESI) m/z: 225.10 [MþH]þ; IR: 1220, 1547,
1592, 1697, 3124, 3344. Found: C, 48.21; H, 5.20; N, 25.15.
C9H12N4O3 requires C, 48.21; H, 5.39; N, 24.99.
4.2.3.1. [1-15N]-3-[(2-Acetoxyethoxy)methyl]-6-phenyl-1,2,4-
triazolo[1,5-a]pyrimidin-7-one (6#d)
(
15N, w43%). Yield 99 mg
(32%)/14 mg (22%). 1H NMR
d: 1.96 (3H, s, COeCH3), 3.83e3.85 (2H,
4.2.5.2. 3-[(2-Hydroxyethoxy)methyl]-6-phenyl-1,2,4-triazolo
m, OC30H2), 4.10e4.13 (2H, m, OC40H2), 5.56 (2H, s, NC10H2),
7.30e7.34 (1H, m, Hp(Ph)), 7.40e7.44 (2H, m, Hm(Ph)), 7.68e7.71
[1,5-a]pyrimidin-7-one (20d). Colorless crystals; yield 84%
(241 mg); mp 164 ꢀC; 1H NMR : 3.48e3.52 (2H, m, OC40H2),
d
Please cite this article in press as: Khalymbadzha, I. A.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2013.12.051

I.A. Khalymbadzha et al. / Tetrahedron xxx (2014) 1e8
7
3.64e3.67 (2H, m, OC30H2), 4.68 (1H, t, J 4.8 Hz, OH), 5.55 (2H, s,
C10H2), 7.32e7.34 (1H, m, Hp(Ph)), 7.40e7.44 (2H, m, Hm(Ph)),
7.69e7.71 (2H, m, Ho(Ph)), 8.28 (1H, s, H5), 9.12 (1H, s, H2); 13C
virus with varied multiplicity of infection (0.01 or 0.1 PFU/cell) and
incubated in the Eagle’s medium supplied with the 199 medium
(1:1) and 2% fetal calf serum. The tested compounds at concen-
NMR
d
: 59.9 (C40), 71.6 (C30), 73.5 (C10), 116.1 (C6), 127.0 (Cp), 128.2
trations up to 1000 mM were added directly before infecting. After
(Cm), 128.3 (Co), 134.3 (Ci), 143.4 (C2), 148.2 (C3a), 151.8 (C5), 154.8
(C7); MS (ESI) m/z: 287.12 [MþH]þ; IR: 1545,1596,1667, 2908, 3021,
3117, 3354. Found: C, 58.90; H, 4.81; N, 19.46. C14H14N4O3 requires
C, 58.73; H, 4.93; N, 19.57.
72 h incubation the number of living cells was measured using the
trypan blue exclusion method. Cytotoxicity (CD50) was estimated in
the presence of tested compounds at the concentrations of
0e1000 mM after 72 h incubation with uninfected cells and calcu-
lated as compound concentration, at which 50% cells died.24
4.2.5.3. 3-[(2-Hydroxyethoxy)methyl]-5-phenyl-1,2,4-triazolo
[1,5-a]pyrimidin-7-one (20g). Colorless crystals; yield 84%
Acknowledgements
(240 mg); mp 187 ꢀC; 1H NMR : 3.52 (2H, br s, C40H2), 3.68e3.71
d
(2H, m, C30H2), 4.71 (1H, br s, OH), 5.61 (2H, s, C10H2), 6.74 (1H, s,
This work was supported by the Federal Target Program ‘Sci-
entific and Science-Educational Personnel of Innovative Russia’
(State contract 14.A18.21.0806) and Russian Foundation for Basic
Research (projects 13-04-02059, 12-03-33144, 12-03-31476, and
12-04-00581). J.O.S thanks Compute CanadaeCalcul Canada and
West Grid for computing resources and Prof. Arvi Rauk (University
of Calgary, Canada) for his personal assistance.
H6), 7.50e7.51 (3H, m, Hm(Ph)þHp(Ph)), 8.12e8.13 (2H, m, Ho(Ph)),
9.10 (1H, s, H2); 13C NMR : 59.9 (C40), 71.7 (C30), 73.5 (C10), 99.0
d
(C6), 127.1 (Co), 128.7 (Cm), 130.5 (Cp), 136.5 (Ci), 143.2 (C2), 148.8
(C3a), 156.1 (C7), 160.2 (C5); MS (ESI) m/z: 287.11 [MþH]þ; IR: 1545,
1599, 1673, 2915, 3039, 3108, 3316. Found: C, 58.61; H, 5.08; N,
19.57. C14H14N4O3 requires C, 58.73; H, 4.93; N, 19.57.
4.2.5.4. 4-[(2-Hydroxyethoxy)methyl]-5-methyl-1,2,4-triazolo
Supplementary data
[1,5-a]pyrimidin-7-one (21a). Colorless crystals; yield 62%
(139 mg); mp 153 ꢀC; 1H NMR
d
: 2.52 (3H, s, Me), 3.46e3.50 (2H, m,
C40H2), 3.58e3.60 (2H, m, C30H2), 4.59 (1H, t, J 5.4 Hz, OH), 5.71 (2H,
s, NC10H2), 6.03 (1H, s, H6), 8.22 (1H, s, H2); 13C NMR
: 17.6 (Me),
Copies of 1H NMR, and 13C NMR spectra for all new compounds,
experimental data for compounds 6b,c,e,f,h,i, 7b,c,e,f, 20b,c,e,f,h,i,
and 21b,c,e,f, X-ray crystallography data for compounds 6e and 7e,
and computational details are available. Supplementary data as-
sociated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.2013.12.051
d
59.9 (C40), 71.0 (C30), 76.7 (C10), 100.7 (C6), 151.5 (C2), 152.3 (C3a),
152.6 (C5), 154.8 (C7); MS (ESI) m/z: 225.10 [MþH]þ; IR: 1113, 1472,
1564, 1702, 3096, 3404. Found: C, 48.39; H, 5.24; N, 25.21.
C9H12N4O3 requires C, 48.21; H, 5.39; N, 24.99.
References and notes
4.2.5.5. 4-[(2-Hydroxyethoxy)methyl]-6-phenyl-1,2,4-triazolo
[1,5-a]pyrimidin-7-one (21d). Colorless crystals; yield 84%
1. (a) Ai, Y.; Chen, Y.; Tang, C.; Yang, G.-Z.; Liang, Y.-J.; Fu, L.-W.; Liu, J.-C.; He, H.-W.
Eur. J. Med. Chem. 2012, 47, 206e213; (b) Boechat, N.; Pinheiro, L. C. S.; Silva, T.
S.; Carvalho, A. S.; Bastos, M. M.; Costa, C. C. P.; Mendonca, J. S.; Dutra, K. D. B.;
Santos-Filho, O. A.; Pinto, A. C.; Aguiar, A. C. C.; Ceravolo, I. P.; Krettli, A. U.;
Pinheiro, S.; Valverde, A. L. Molecules 2012, 17, 8285e8302; (c) Rusinov, V. L.;
Petrov, A. Yu.; Pilicheva, T. L.; Chupakhin, O. N.; Kovalev, G. V.; Komina, E. R.
Pharm. Chem. J. 1986, 20, 113e117; (d) El-Gendy, M. M. A.; Shaaban, M.; Shaaban,
K. A.; El-Bondkly, A. M.; Laatsch, H. J. Antibiot. 2008, 61, 149e157; (e) Yu, W.;
Goddard, C.; Clearfield, E.; Mills, C.; Xiao, T.; Guo, H.; Morrey, J. D.; Motter, N. E.;
Zhao, K.; Block, T. M.; Cuconati, A.; Xu, X. J. Med. Chem. 2011, 54, 5660e5670; (f)
Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Afragola, J.; Hernandez, R.; Lucas, J.;
Gibbons, J.; Beyer, C. J. Med. Chem. 2007, 50, 310e327; (g) Gujjar, R.; El Mazouni,
F.; White, K. L.; White, J.; Creason, S.; Shackleford, D. M.; Deng, X.; Charman, W.
N.; Bathurst, I.; Burrows, J.; Floyd, D. M.; Matthews, D.; Buckner, F. S.; Charman,
S. A.; Phillips, M. A.; Rathod, P. K. J. Med. Chem. 2011, 54, 3935e3949; (h)
Marwaha, A.; White, J.; El Mazouni, F.; Creason, S. A.; Kokkonda, S.; Buckner, F.
S.; Charman, S. A.; Phillips, M. A.; Rathod, P. K. J. Med. Chem. 2012, 55,
7425e7436; (i) Li, H.; Linton, A.; Tatlock, J.; Gonzalez, J.; Borchardt, A.; Abreo,
M.; Jewell, T.; Patel, L.; Drowns, M.; Ludlum, S.; Goble, M.; Yang, M.; Blazel, J.;
Rahavendran, R.; Skor, H.; Shi, S.; Lewis, C.; Fuhrman, S. J. Med. Chem. 2007, 50,
3969e3972.
(240 mg); mp 138 ꢀC; 1H NMR : 3.48e3.52 (2H, m, OC40H2),
d
3.65e3.68 (2H, m, OC30H2), 4.69 (1H, t, J 5.6 Hz, OH), 5.66 (2H, s,
C10H2), 7.37e7.40 (1H, m, Hp(Ph)), 7.44e7.46 (2H, m, Hm(Ph)),
7.66e7.67 (2H, m, Ho(Ph)), 8.33 (1H, s, H2), 8.48 (1H, s, H5); 13C
NMR d
: 59.9 (C40), 71.5 (C30), 80.3 (C10), 112.6 (C6), 127.7 (Cp), 128.3
(Cm), 128.7 (Co), 132.7 (Ci), 140.8 (C5), 150.5 (C3a), 152.3 (C2), 155.1
(C7); MS (ESI) m/z: 287.12 [MþH]þ; IR: 1574, 1682, 2938, 3080,
3382. Found: C, 58.61; H, 4.74; N, 19.24. C14H14N4O3 requires C,
58.73; H, 4.93; N, 19.57.
4.3. Computational details
Quantum chemical calculations were performed by employing
G09 suit11 at West Grid supercomputing center (Canada). Com-
pounds were fully optimized at B3LYP/6-31G(d,p) level. The min-
ima were confirmed by frequency calculations.
2. Sanchez, R. M.; Erhard, K.; Hardwicke, M. A.; Lin, H.; McSurdy-Freed, J.; Plant,
R.; Raha, K.; Rominger, C. M.; Schaber, M. D.; Spengler, M. D.; Moore, M. L.; Yu,
H.; Luengo, J. I.; Tedesco, R.; Rivero, R. A. Bioorg. Med. Chem. Lett. 2012, 22,
3198e3202.
4.4. Antiviral activity and cytotoxicity
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