1,2,4-Triazoloazine derivatives as a new type of herpes simplex virus inhibitors

Article abstract of DOI:10.1016/j.bioorg.2010.09.002

A new class of inhibitors of herpes simplex virus replication was found. The compounds under study are derived from condensed 1,2,4-triazolo[5,1-c][1,2, 4]triazines and 1,2,4-triazolo[1,5-a]pyrimidines, structural analogues of natural nucleic bases. Antih

Full text of DOI:10.1016/j.bioorg.2010.09.002

Bioorganic Chemistry 38 (2010) 265–270
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
1,2,4-Triazoloazine derivatives as a new type of herpes simplex virus inhibitors
S.L. Deev a,b, M.V. Yasko c, I.L. Karpenko c, A.N. Korovina c, A.L. Khandazhinskaya c, V.L. Andronova d,
G.A. Galegov d, T.S. Shestakova b, E.N. Ulomskii b, V.L. Rusinov b, O.N. Chupakhin a,b, M.K. Kukhanova c,
⇑
a Postovsky Institute of Organic Synthesis, Ural Division of the Russian Academy of Sciences, Ekaterinburg, Russia
b Ural State Technical University, Ekaterinburg, Russia
c Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Russia
d Ivanovskii Institute of Virology, Russian Academy of Medical Sciences, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2010
Available online 22 September 2010
A new class of inhibitors of herpes simplex virus replication was found. The compounds under study are
derived from condensed 1,2,4-triazolo[5,1-c][1,2,4]triazines and 1,2,4-triazolo[1,5-a]pyrimidines, struc-
tural analogues of natural nucleic bases. Antiherpetic activity and cytotoxicity of the compounds were
studied. The corresponding triphosphates of several active compounds were prepared and tested as
inhibitors of DNA synthesis catalyzed by herpes simplex virus polymerase. The potential mechanism of
their action is blocking of DNA dependent DNA polymerase, a key enzyme of viral replication.
Ó 2010 Elsevier Inc. All rights reserved.
Keywords:
Herpes simplex virus
Inhibitors
Nucleoside analogues
Triazoloazine derivatives
1. Introduction
to the drug. It is noteworthy that about 6% strains isolated from
HIV-infected patients are acyclovir-resistant. In addition to the
Herpes viruses belong to widely spread and socially dangerous
viral infections. Over a half of the world population are infected
with the virus and up to 20% exhibit signs of the disease. In addi-
tion, herpetic infections can lead to a lethal outcome in patients
with immune deficiency caused by HIV infection [1] or organ
transplantation [2]. Nucleoside analogues modified at either the
base or the ribose residue played an essential role for design of
antiherpetic agents. Guanosine acyclic analogues, acyclovir
above mentioned compounds there are some publications on the
wide antiviral activity of 6-nitro-1,2,4-triazolo[5,1-c][1,2,4]tri-
azine-7(4H)-ones [8–10]. However the antiherpetic properties of
triazoloazine derivatives were not studied.
Herein, we reported the synthesis and antiherpetic activity of
15 new derivatives of 1,2,4-triazolo[5,1-c][1,2,4]triazines and
1,2,4-triazolo[1,5-a]pyrimidines bearing an N3-alkyl fragment
with a terminal hydroxyl group linked to the azine cycle (Fig. 1).
We also showed that triphosphates of some triazoloazine deriva-
tives blocked DNA synthesis catalyzed by HSV DNA polymerase.
(Zovirax) and its L
-valine ester (ZelitrexÒ, ValtrexÒ), penciclovir
(DenavirÒ, VectavirÒ), ganciclovir (CytoveneÒ, CymeveneÒ), and
famciclovir (FamvirÒ), which is an oral prodrug of penciclovir, are
well known drugs used in medical practice [3–5]. Also, pyrimidine
derivatives modified at the nucleic base, namely, 5-iodo-20-deoxy-
uridine (HerpidÒ, IodoxeneÒ), (E)-5-(2-bromovinyl)-20-deoxyuri-
dine (ZostexÒ, HelpinÒ), and 5-trifluoromethyl-20-deoxyuridine
(VeropticÒ) have been described as antiherpetic drugs [3].
Galankevich et al. reported antiherpetic properties of tricyclic
acyclovir analogues, 3-[(2-hydroxyethoxy)methyl]-9-oxyimidazol-
o[1,2-a]purine derivatives [6,7]. When penetrating into cell,
nucleoside-based compounds pass three stages of phosphorylation
and under catalysis of herpes virus DNA polymerase are incorpo-
rated into the 30-terminus of viral DNA and terminate its synthesis.
Disadvantages of these agents are relatively high toxicity, low
bioavailability, and inevitable appearance of virus strains resistant
2. Materials and methods
2.1. Physical measurement
NMR spectra were registered on a Bruker AMX III-400 spec-
trometer with the working frequency of 400 MHz for 1H NMR
(Me4Si as the internal standard for organic solvents and sodium tri-
methylsilyl-1-propane sulfonate for D2O), and 162 MHz for 31P
NMR (with proton-phosphorus decoupling; 85% H3PO4 as the
external standard). Two-dimensional correlation experiments
(1H–13C HMBC and HSQC) and 13C NMR (100 MHz) were regis-
tered on a Bruker DRX-400 spectrometer in DMSO-d6. UV spectra
were recorded on a Shimadzu UV-2401PC spectrophotometer
(Japan). HPLC was performed on a Gilson chromatograph (France)
with UV detection. The flow rate 0.5 ml/min. Radioactivity was
measured on a SL-4000 Intertechnique counter (France).
⇑
Corresponding author. Fax: +7 (499)135 2255.
E-mail address: kukhan86@hotmail.com (M.K. Kukhanova).
0045-2068/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.09.002

266
S.L. Deev et al. / Bioorganic Chemistry 38 (2010) 265–270
10a (R = H): The yield 1.838 g (5.64 mmol), 60%; mp 146 °C; 1H
NMR (DMSO-d6) d ppm: 1.62–1.67 (m, 2H, CH2), 1.82–1.93 (m,
2H, CH2), 1.98 (s, 3H, COCH3), 4.02 (t, 2H, CH2–O), 4.28 (t, 2H,
CH2–N), 7.34–7.45 (m, 3H, C6H5), 7.66–7.69 (m, 2H, C6H5), 8.18
(s, 1H, CH(2)), 8.44 (s, 1H, CH(5)); 13C NMR (DMSO-d6) d ppm:
20.71 (CH3), 24.84 (C(20)), 25.03 (C(30)), 51.05 (C(10)), 63.34
(C(40)), 112.09 (C(6)), 127.53 (Cp), 128.24 (Cm), 128.61 (Co),
133.04 (Ci), 141.63 (C(5)), 150.33 (C(2)), 152.32 (C(3a)), 155.08
(C(7)), 170.42 (C@O). Calc. (%): C, 62.57; H, 5.56; N, 17.17.
C
17H18N4O3: Found (%): C, 62.55; H, 5.52; N, 17.18%.
10b (R = Me): The yield 2.429 g (7.14 mmol), 76%; mp 137 °C; 1H
NMR (DMSO-d6) d ppm: 1.62–1.65 (m, 2H, CH2), 1.87–1.90 (m, 2H,
CH2), 2.00 (s, 3H, COCH3), 2.40 (s, 3H, CH3), 4.04 (t, CH2), 4.23 (t, 2H,
CH2), 7.32–7.44 (m, 3H, C6H5), 7.65–7.66 (m, 2H, C6H5), 8.37 (s, 1H,
CH); 13C NMR (DMSO-d6) d ppm: 14.81 (CH3), 21.15 (CH3), 25.27
(C(20)), 25.45 (C(30)), 51.41 (C(10)), 63.80 (C(40)), 112.44 (C(6)),
127.89 (Cp), 128.65 (Cm), 128.99 (Co), 133.56 (Ci), 141.48 (C(5)),
150.94 (C(3a)), 155.17 (C(7)), 161.85 (C(2)), 170.86 (C@O); Calc.
(%): C, 63.52; H, 5.92; N, 16.46. C18H20N4O3. Found (%): C, 63.53;
H, 5.87; N, 16.55.
Fig. 1. 6-Phenyl-1,2,4-triazolo[1,5-a]pyrimidine (1) and 6-phenyl-1,2,4-triazol-
o[5,1-c][1,2,4]triazine-7-one (2–5) derivatives.
X-ray studies were performed on an automatic Xcalibur 3
diffractometer with a CCD detector (
tion, graphite monochromator) using
Monoclinic crystals a = 8.7427(6) Å, b = 10.5354(6) Å, c =
16.1262(15) Å, = 90°, b = 103.087(7)°, = 90°, Z = 4, dsub. = 1.370
g/cmꢀ3 = 0.094 mmꢀ1, V = 1446.77(19) Å3, a space group P2(1)/
x
/2h scanning, Mo K
a irradia-
a
standard procedure.
e
a
c
,
l
2.2.3. Preparation of 2-R-4-(4-hydroxybutyl)-6-phenyl-1,2,4-
triazolo[1,5-a]pyrimidine-7-ones (1a,b)
c. The structure was decoded by the direct method and specified
using SHELXS-97 and SHELXL-97 programs [11] in anisotropic (iso-
tropic for H atoms) approximation at R = 0.0368 (wR = 0.0738) for
Compound 10a,b (3.0 mmol) was added to a solution of sodium
methylate prepared from sodium (0.07 g, 3.04 mmol) and metha-
nol (30 ml). The reaction mixture was refluxed for 1 h, cooled, neu-
tralized with acetic acid and evaporated in vacuum. The products
were isolated by column chromatography on silicagel and were
eluted with ethyl acetate.
1783 reflections with I > 2
r
ðI2oÞ and GOOF 1.000. CCDC 791002
contains the supplementary crystallographic data which can be ob-
tained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
1a (R = H): The yield 0.511 g (1.8 mmol), 60%; mp 140 °C; 1H
NMR (DMSO-d6) d ppm 1.47–1.53 (m, 2H, CH2), 1.89–1.94 (m,
2H, CH2), 3.42 (q, 2H, CH2–O), 4.28 (t, 2H, CH2–N), 4.48 (t, 1H,
OH), 7.32–7.43 (m, 3H, C6H5), 7.64–7.66 (m, 2H, C6H5), 8.31 (s,
1H, CH(2)), 8.45 (s, 1H, CH(5)). 13C NMR (DMSO-d6) d ppm: 25.11
(C(20)), 29.22 (C(30)), 51.47 (C(10)), 60.22 (C(40)), 111.98 (C(6)),
127.52 (C-p), 128.25 (C-m), 128.62 (C-o), 133.03 (C-i), 141.71
(C(5)), 150.30 (C(3a)), 152.36 (C(2)), 155.06 (C(7)); Calc. (%): C,
63.37; H, 5.67; N, 19.71. C15H16N4O2 Found (%):C, 63.01; H, 5.91;
N, 19.50.
2.2. Synthesis of the compounds
2.2.1. Preparation of 2-R-6-pheny-1,2,4-triazolo[1,5-a]pyrimidine-7-
ones (6a,b)
Synthesis of 6a,b was performed similar to [12]. Briefly, ethyl
a-formyl phenyl acetate (2.28 g, 11.90 mmol) was added to a solu-
tion of 2-R-5-amino-1,2,4-triazole (11.9 mmol) in acetic acid (4 ml)
and the mixture was refluxed for 1 h and cooled. The precipitate
was filtered and dried.
1b (R = Me): The yield 0.447 g (1.6 mmol), 50%; mp 139 °C; 1H
NMR (DMSO-d6) d ppm: 1.44–1.49 (m, 2H, CH2), 1.86–1.94 (m,
2H, CH2), 2.40 (s, 3H, CH3), 3.43 (q, 2H, CH2-O), 4.23 (t, 2H, CH2–
N), 4.48 (t, 1H, OH), 7.32–7.44 (m, 3H, C6H5), 7.65–7.67 (m, 2H,
C6H5), 8.36 (s, 1H, CH). 13C NMR (DMSO-d6) d ppm: 14.39 (CH3),
25.13 (C(20)), 29.23 (C(30)), 51.40 (C(10)), 60.23 (C(40)), 111.92
(C(6)), 127.44 (Cp), 128.18 (Cm), 128.55 (Co), 133.12 (Ci), 141.08
(C(5)), 150.49 (C(3a)), 154.72 (C(7)), 161.46 (C(2)); Calc. (%): C,
64.41; H, 6.08; N, 18.78. C16H18N4O2: Found (%): C, 64.38; H,
5.98; N, 18.86.
6a (R = H): The yield 1.76 g (8.3 mmol), 70%; mp 311 °C; 1H
NMR (DMSO-d6) d 7.34–7.42 (m, 3H, C6H5), 7.64–7.66 (m, 2H,
C6H5), 8.21 (s, 1H, CH(5)), 8.21 (s, 1H, CH(2)), 13.21–13.88 (br s,
1H, NH); 13C NMR (DMSO-d6) d 111.82 (C(6)), 127.35 (Cp),
128.21 (Cm), 128.67 (Co), 133.35 (Ci), 138.79 (C(5)), 150.11
(C(3a)), 152.29 (C(2)), 155.74 (C(7)); Calc. (%): C, 62.26; H, 3.80;
N, 26.40. C11H8N4O. Found (%): C, 62.10; H, 4.00; N, 26.57%.
6b (R = Me): The yield 1.99 g (8.8 mmol), 74%; mp > 320 °C; 1H
NMR (DMSO-d6) d 2.38 (s, 3H, CH3), 7.33–7.42 (m, 3H, C6H5),
7.63–7.62(m, 2H, C6H5), 8.13 (s, 1H, CH(5)), 12.3–13.71 (br s, 1H,
NH); 13C NMR (DMSO-d6) d 14.13 (CH3), 111.88 (C(6)), 127.20
(Cp), 128.13 (Cm), 128.56 (Co), 133.51 (Ci), 138.85 (C(5)), 150.29
(C(3a)), 155.29 (C(7)), 160.74 (C(2)); Calc. (%): C, 63.71; H, 4.46;
N, 24.76%. C12H10N4O. Found (%): C, 64.01; H, 4.24; N, 24.55.
2.2.4. Preparation of monophosphates of 3a,c (3a,c-MP) and 5b
(5b-MP)
Triethylamine (0.832 mmol, 0.12 ml) was added to a solution of
1,2,4-triazole (50 mg, 0.725 mmol) in CH3CN (0.9 ml). The solution
was cooled and POCl3 (0.028 ml, 0.302 mmol) was added. The
formed precipitate was separated by centrifugation and the super-
natant was added to a solution of 3a,c or 5b (0.15 mmol) in CH3CN
(6 ml). In 40 min 50% aqueous pyridine (5 ml) was added to the
reaction mixture. The target monophosphates were isolated on a
2.2.2. Preparation of 2-R-4-(4-acetoxybutyl)-6-phenyl-1,2,4-
triazolo[1,5-a]pyrimidine-7-ones (10a,b)
A suspension of 2-R-6-phenyl-1,2,4-triazolo[1,5-a]pyrimidine-
7-one 6a,b (9.4 mmol) in 17% aqueous Na2CO3 (6 ml) was stirred
at room temperature for 0.5 h, the precipitate was filtered off, dried
and dissolved in DMF (10 ml). 4-Bromobutyl acetate (1.79 g,
9.20 mmol) was added to the resulting solution and the reaction
mixture was heated for 2 h at 100°C. The mixture was cooled,
water (250 ml) was added, the residue was filtered off and crystal-
lized from isopropanol.
DEAE cellulose column in
a gradient of aqueous NH4HCO3
(0 ? 0.3 M). Monophosphates were additionally purified by col-
umn chromatography on LiChroprep RP-18 in a gradient of meth-
anol in water (0% ? 10%).
3a-MP: The yield 21 mg (0.06 mmol), 42%, 1H NMR (D2O) d
ppm: 4.25 (q, 2H, CH2–O), 4.55 (m, 2H, CH2–N + D2O), 7.46–7.48

S.L. Deev et al. / Bioorganic Chemistry 38 (2010) 265–270
267
(m, 3H, C6H5); 7.87–7.89 (m, 2H, C6H5); 8.33 (s, 1H, CH); 31P NMR
(D2O) d, ppm: 3.32 s.
the trypan blue exclusion method. Cytotoxicity (CD50) was esti-
mated in the presence of tested compounds at the concentrations
of 0–1000 lM after 72 h incubation with uninfected cells and calcu-
3c-MP: The yield 35 mg (0.1 mmol), 61%, 1H NMR (D2O) d ppm:
2.65 (s, 3H, SMe), 4.25 (q, 2H, CH2–O), 4.55 (t, 2H, CH2–N), 7.46–
7.48 (m, 3H, C6H5); 7.87–7.89 (m, 2H, C6H5); 31P NMR (D2O) d
ppm: 2.21 s.
lated as compound concentration, at which 50% cells died [13].
2.3.3. Inhibition of recombinant HSV DNA polymerase by triphosphates
of 3a,c and 5b
5b-MP: The yield 30 mg (0.079 mmol), 52%, 1H NMR (D2O) d
ppm: 1.59–1.63 (m, 2H, CH2); 1.89–1.93 (m, 2H, CH2); 2.58 (s,
3H, Me); 3.89 (q, 2H, CH2–O), 4.28 (t, 2H, CH2–N), 7.35–7.38 (m,
3H, C6H5); 7.75–7.77 (m, 2H, C6H5); 31P NMR (D2O) d ppm: 0.91 s.
Recombinant HSV DNA polymerase was prepared as described in
[14]. The effect of the synthesized compounds on HSV DNA poly-
merase activity was measured by inhibition of [32P]dAMP incorpo-
ration into the primer-template complex:
2.2.5. Preparation of triphosphates of 3a,c (3a,c-TP) and 5b (5b-TP)
30-dGGCAGTTAAGGACATCAGAGCTCGGAACTGATGCGATTGACC/
[50-32P] dCCGTCAATTCCTGTAGTC. The reaction mixture contained
50 mM buffer HEPES, pH 7.5, 100 mM (NH4)2SO4, 6 mM MgCI2,
A
solution of monophosphates 3a,c-MP and 5b-MP
(0.057 mmol) in DMF (15 ml) and triethylamine (0.04 ml) was half
evaporated and carbonyldiimidazole (20 mg, 0.123 mmol) was
added. The mixture was stirred for 2 h and methanol (0.12 ml)
was added. The solution was stirred for another 40 min and trib-
utylammonium pyrophosphate H4P2O7 ꢁ 1,5-Bu3N (129 mg,
0.283 mmol) was added. In 24 h water was added. The product
was isolated on a DEAE cellulose column under conditions de-
scribed above. The solution was evaporated, the residue was dis-
solved in water and purified on a LiChroprep RP-18 column. The
compounds were eluted in a gradient of methanol in water
(0% ? 15%).
5 lM dNTP, 1 lCi [a-
32P]dATP (6000 Ci/mM), 10 nM primer-tem-
plate complex, and triphosphates of 3a,c or 5b at various concen-
trations. The reaction was started with addition of 2U HSV DNA
polymerase and after incubation for 30 min at 37 °C the mixture
was loaded onto DEAE filters. The filters were dried on air and
the free radioactive label was washed off with 0.4 M phosphate
buffer (pH 6). Radioactivity was measured on a scintillation coun-
ter by the Cherenkov method.
3a-TP: The yield 5 mg (0.009 mmol), 17%; 1H NMR (D2O) d ppm:
4.38 (q, 2H, CH2–O), 4.67 (t, 2H, CH2–N), 7.46–7.48 (m, 3H, C6H5);
7.85–7.88 (m, 2H, C6H5); 8.27 (s, 1H, CH); 31P NMR (D2O) d ppm:
ꢀ22.45 (t, 1P, Pb), ꢀ10.93 (d, 1P, P ), ꢀ9.73 (d, 1P, P ). UV (H2O)
3. Results and discussion
3.1. Synthesis and general properties of synthesized compounds
c
a
kmax 320 nm,
e
13,180; kmax 269 nm,
e
11,220.
Fig. 1 presents the structures of the synthesized triazolo-tri-
azine and triazolo-pyrimidine derivatives.
3c-TP: The yield 8 mg (0.016 mmol), 26%; 1H NMRP (D2O) d
ppm: 2.68 (s, 3H, SMe), 4.46 (q, 2H, CH2), 4.77 (m, 2H, CH2 + D2O),
7.54–7.56 (m, 3H, C6H5); 7.92–7.94 (m, 2H, C6H5); 31P NMR (D2O) d
ppm: ꢀ22.44 (t, 1P, Pb), ꢀ10.90 (d, 1P, P ), ꢀ9.75 (d, 1P, P ). UV
For preparation of compounds 1a,b we used alkylation of 6-
phenyl-1,2,4-triazolo[1,5-a]pyrimidine-7-ones 6a,b with bromo-
butyl acetate under basic conditions (Scheme 1). The resulting
compounds 10a,b were deacylated to give 1,2,4-triazolo[1,5-
a]pyrimidine-7-one derivatives with sodium methylate.
Proton resonances in the 1H NMR spectra of the corresponding
heterocyclic fragments and N-alkyl substituents in compounds
10a,b and 1a,b were registered. The appearance of resonances at d
4.80 ppm in the spectra of 1,2,4-triazolo[1,5-a]pyrimidine-7-ones
1a,b, were ascribed to hydroxyl groups according to the data of
NMR experiments on the deuterium exchange in the presence of
CF3COOD that unambiguously confirmed the removal of an acetyl
group.
c
a
(H2O) kmax nm kmax 323 nm,
e
13,490; kmax 251 e 26,900.
5b-TP: The yield 7 mg (0.014 mmol), 23%; 1H NMR (D2O) d
ppm: 1.72–1.77 (m, 2H, CH2), 2.02–2.08 (m, 2H, CH2); 2.51 (s,
3H, Me); 3.98 (q, 2H, 2H, CH2–O), 4.48 (t, 2H, CH2–N), 7.54–7.56
(m, 3H, C6H5); 7.88–7.90 (m, 2H, C6H5); 31P NMR (D2O) d ppm:
ꢀ22.65 (t, 1P, Pb), ꢀ10.35 (d, 1P, P ), ꢀ10.13 (d, 1P, P ). UV (H2O)
c
a
kmax nm 321,
e
12,900; kmax 263 nm, e 9,1200.
2.3. In vitro studies
2.3.1. Cell lines and viruses
The position of the N-substituent and ascription of the reso-
nances of compounds 10a,b and 1a,b in 1H and 13C NMR spectra
were performed based on the data of two two-dimensional corre-
lation experiments (2D HSQC b HMBC). The position of the alkyl
substituent at the pyrimidine cycle is confirmed by the presence
of two cross-peaks of N-CH2 proton resonances and carbon C(3a)
and C(5) resonances in 2D HMBC of 10a,b and 1a,b spectra. These
data were verified by the X-ray analysis of compound 1b (Fig. 2),
which demonstrated that azole and azine cycles were annealed
at positions [1,5-a] and formed a nearly plane bicyclic system
non-conjugated with the plane of the phenyl substituent. The tor-
sion angle between C(4)C(3) and C(6)C(11) was 38.9°. 1,2,4-Triaz-
olo[5,1-c][1,2,4]triazines (2–5) containing acyclic fragments were
prepared similar to [15].
Compounds 2a-c were synthesized by the interaction of (2-
acetoxyethoxy)methyl acetate with NH-heterocycles 20a-c
(Scheme 2).
Similarly to the synthesis of 1a,b, the key reaction in the prepa-
ration of 1,2,4-triazolo[1,5-c][1,2,4]triazines 3a–c, 4a–c, and 5a–c
was alkylation of heterocycles 7a–c with the corresponding halo-
gen-containing compounds under basic conditions (Scheme 3).
The rational for using 6a,b and 7a–c with ‘‘abnormal” bases for
the preparation of compounds 1–5 was as follows. The structure
Vero cell culture (green monkey kidney epithelial cells, ATCC No
CRL-1586) was obtained from Laboratory of tissues, Ivanovskii
Institute of Virology, Russian Academy of Medical Sciences (Mos-
cow). The acyclovir-sensitive HSV-1/L2 strain was obtained from
the State collection of viruses of Ivanovskii Institute of Virology,
Russian Academy of Medical Sciences (Moscow). The Vero cell cul-
ture was grown in Dulbecco’s modified medium (D-MEM) with 5%
fetal calf serum (PanEco, Russia) at 37 °C in a 96-well plate in the
atmosphere of 5% CO2.
2.3.2. Antiherpetic activity and cytotoxicity
The antiviral effects of the compounds were estimated from
their ability to prevent the development of the virus-induced cyto-
pathic effect (CPE) similar to the procedure described earlier [4,13].
Quantitatively the antiviral effect was expressed as ID50 (the con-
centration at which CPE was reduced by 50%). Briefly, Vero cells
placed in 96-well plates (0.8 ꢁ 106 cell/ml) were infected with the
virus with varied multiplicity of infection (0.01 or 0.1 PFU/cell)
and incubated in the Eagle’s medium supplied with the 199 med-
ium (1:1) and 2% fetal calf serum. The tested compounds at concen-
trations up to 1000
lM were added directly before infecting. After
72 h incubation the number of living cells was measured using

268
S.L. Deev et al. / Bioorganic Chemistry 38 (2010) 265–270
Scheme 1. Preparation of 4-(4-hydroxybutyl)-6-phenyl-1,2,4-triazolo[1,5-a]pyrimidine-7-ones 1a,b.
Fig. 2. Molecular structure of compound 1b.
Scheme 2. Synthesis of 4-(2-acetoxyethoxymethyl)-6-phenyl-1,2,4-triazolo[1,5-a]pyrimidine-7-ones (2a–c).
of bases in 1–5 is close to that of inosine analogues [16,17]. On the
other hand, compounds 1–5 can also be considered as pyrimidine
analogues with an annealed azole cycle. The antiviral activity of
such bicyclic pyrimidine derivatives was shown earlier. For exam-
ple, nucleoside analogues derived from imidazolo[2,1-c]pyrimi-
dine-5-ones displayed activity against hepatitis B virus [18,19].
Among 1,2,4-triazolo[1,5-c][1,2,4]triazines some compounds were
shown to be active against various strains of influenza virus A and
B [10,20], and 6-arylazolo[1,5-a]pyrimidine-7-ones were patented
as effective agents against hepatitis C virus [21]. We focused our
interest on compounds 6b and 7b,c containing phenyl residue
because introduction of the phenyl residue at position 6 of

S.L. Deev et al. / Bioorganic Chemistry 38 (2010) 265–270
269
Scheme 3. Synthesis of N-hydroxyalkyl-1,2,4-triazolo[1,5-c][1,2,4]triazines 3a–c, 4a–c, and 5a–c.
1,2,4-triazolo[5,1-c][1,2,4]triazine-7-ones or [1,2,4-triazolo[1,5-
a]pyrimidine-7-ones selectively promoted N3-alkylation of 6a,b
and 7a–c at the azine cycle [22]. Another advantage of phenyl
derivatives was their high chemical stability in both acid and
alkaline media, which was essential for the synthesis of the
corresponding triphosphates. It is noteworthy that N-alkylation of
nitro-1,2,4-triazolo[5,1-c][1,2,4]triazine-7-ones and [1,2,4-triazol-
o[1,5-a]pyrimidine-7-ones resulted in destruction of the azine cy-
cle [23]. Introduction of sugar residue at N-3 position of azine
cycle of purine was based on earlier published data [24–26]. It
was shown that 20,30-dideoxy-3-isoadenosine displayed anti-HIV
15–30 lM and demonstrated the highest selectivity index at two
tested PFU/cell.
1,2,4-Triazolo[1,5-c][1,2,4]triazines 5a,b and 1,2,4-triazolo[1,5-
a]pyrimidines 1a,b containing an N-hydroxybutyl substituent
showed similar activity, which implies that the presence or ab-
sence of the nitrogen atom in the six-membered ring does not sig-
nificantly affect antiviral properties of these series of heterocycles.
It is noteworthy that all the compounds showed low toxicity in cell
experiments. Their toxicity was comparable with that of the anti-
herpetic drugs acyclovir or ganciclovir [27] although selectivity in-
dex was lower than that of acyclovir and ganciclovir due to less
profound antiherpetic activity.
activity and some of N3,50-cyclo-4(b-
D-ribofuranosyl-vic-triazol-
o[4,5-b]pyridine-5-one derivatives were active against hepatitis C
virus. According to our data the highest biological activity displayed
the compounds containing thiomethyl or methyl residue in azole
cycle of 1,2,4-triazolo[5,1-c][1,2,4]triazine-7-ones.
3.3. Inhibition of HSV-1 DNA polymerase
One of the mechanisms of antiviral activity of nucleoside ana-
logues involves inhibition of viral DNA synthesis catalyzed by vir-
al DNA polymerase. Therefore we prepared triphosphates of 3a,c
and 5b (3a,c-TP and 5b-TP) (Scheme 4). At the first stage the cor-
responding monophosphates were obtained similarly to the
method described in [28]. The monophosphates were treated
with N,N0-carbonyldiimidazole (CDI) followed by addition of trib-
utylammonium pyrophosphate to give crude 3a,c-TP and 5b-TP.
The compounds were purified by column chromatography on
DEAE cellulose and LiChroprep RP-18 columns.
3.2. Antiherpetic activity and cytotoxicity in cell culture
Antiherpetic properties of compounds 1–5 were studied in the
Vero cell culture infected with herpes simplex virus type 1 (HSV-
1) with varied multiplicity of infection. The results are shown in
Table 1. As is seen, the structure of the compounds affected their
activity. Compounds 2 containing a 2-hydroxyethoxymethyl group
inhibited virus replication by 50% at concentrations exceeding
250 lM. At the same time for compounds 4 bearing a hydroxybu-
tene fragment the 50% inhibition was observed at concentrations
The resulting 3a,c-TP and 5b-TP were studied in cell-free exper-
iments as inhibitors of DNA synthesis catalyzed by herpes simplex
virus DNA polymerase (Table 2). As is seen, compound 3c-TP bear-
ing a methylthio group more effectively inhibited incorporation of
Table 1
[a-
32P]dAMP into the 30-terminus of the primer-template complex
Cytotoxicity and anti-HSV activity of the synthesized compounds.
than 3a-TP. These data correlate with the results on the inhibition
of virus replication in the cell culture (Table 1). Triphosphate of
antiherpetic drug acyclovir or well known inhibitor of HSV DNA
polymerase foscarnet used as controls suppressed the activity of
polymerase more effectively if compared with synthesized com-
pounds. It is noteworthy that compounds lacking a triphosphate
or an N-acyclic fragments with a terminal hydroxyl group did
not inhibit viral DNA polymerase. 50% Inhibition of the enzyme
Compound
CD50 (mM)
Multiplicity of infection
0.1 PFU/cell
0.01 PFU/cell
ID50 (mM)a
ID50 (mM)a
SI
SI
1a
1b
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
>0.8
>0.93
0.5
>0.5
>0.5
>0.5
>1
>0.5
>0.5
0.95
0.53
1.13
P0.98
P0.5
0.06
0.12
0.25
0.5
>13
>7.7
2
0.05
0.06
ND
ND
ND
>16
>15.4
ND
ND
ND
>1
0.25
0.25
0.12
0.12
0.25
0.03
0.015
0.12
0.06
0.06
>2
>2
>8.3
>4
>2
32
35
9.3
16.3
>8
was not achieved at the concentrations exceeding 800
not shown).
lM (data
ND
ND
0.06
0.03
ND
0.015
P0.007
0.07
0.03
0.03
P16.7
P16.7
ND
63
676
16
4. Conclusion
A new type of inhibitors of herpes simplex virus based on 1,2,4-
triazolo[5,1-c][1,2,4]triazine and 1,2,4-triazolo[1,5-a]pyrimidine
structures was discovered. The synthesized compounds displayed
antiherpetic activity and low cytotoxicity in cell cultures. Antiviral
properties are strongly structure dependent. Potentially, the mech-
anism of their action is blocking of DNA dependent DNA polymer-
ase, a key enzyme of viral replication.
P32
P16
CD50, cytotoxic dose, i.e., the agent concentration, at which 50% of uninfected cells
die; ID50, inhibitory dose, i.e., the agent concentration, at which the cytopathic
effect is decreased by 50%; SI, selectivity index, CD50/ID50; PFU, plaque forming unit.
ND – not determined.
a
Mean values of three independent experiments are given.

270
S.L. Deev et al. / Bioorganic Chemistry 38 (2010) 265–270
Scheme 4. Synthesis of triphosphates 3a,c-TP and 5b-TP.
[8] V.L. Rusinov, E.N. Ulomskii, O.N. Chupakhin, M.M. Zubairov, A.B. Kapustin, N.I.
Table 2
50% Inhibition of incorporation of [a-
32P]dATP
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catalyzed by HSV DNA polymerase into the
primer-template complex in the presence of
3a,c-TP, 5b-TP, foscarnet and acyclovir
triphosphate.
Compound
Concentration (l
M)a
3a-TP
3c-TP
5b-TP
Acyclovir-TP
Foscarnet
160 28
100 18
150 25
30
10
6
2
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Acknowledgments
This work was supported by Russian Federation on Basic Re-
search Grants 08-04-00552 and 09-04-12204, Presidium of Russian
Academy of Sciences on ‘‘Molecular and Cellular Biology” and State
contracts 02.740.11.0260 and 02.522.12.2011.
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