Synthesis of novel 6-azapyrimidine acyclic nucleoside analogues and antiviral evaluation

Article abstract of DOI:10.1080/15257771003781634

Acyclic nucleosides have been of considerable interest since the approval of aciclovir by the FDA to be used as an antiviral agent in the 1990s. The acyclic moieties and the bases used in the experiment were either available commercially or synthesized us

Full text of DOI:10.1080/15257771003781634

Nucleosides, Nucleotides and Nucleic Acids, 29:535–541, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257771003781634
SYNTHESIS OF NOVEL 6-AZAPYRIMIDINE ACYCLIC NUCLEOSIDE
ANALOGUES AND ANTIVIRAL EVALUATION
Malina Jasamai,1 Claire Simons,2 and Jan Balzarini3
1Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abd Aziz,
Kuala Lumpur
2Medicinal Chemistry, Welsh School of Pharmacy, Cardiff University, Cardiff,
United Kingdom
3Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
Acyclic nucleosides have been of considerable interest since the approval of aciclovir by the FDA to
2
be used as an antiviral agent in the 1990s. The acyclic moieties and the bases used in the experiment
were either available commercially or synthesized using literature methods. Vorbru¨ggen coupling
method was utilized involving reaction of persilylated heterocyclic bases with the appropriate acyclic
moiety in the presence of a Lewis acid catalyst. A series of novel 6-azapyrimidine acyclic oxosugar
nucleosides was successfully synthesized with a promising yield (more than 50%). An efficient
method of protection and deprotection was also investigated.
Keywords Antiviral; 6-azapyrimidine acyclic nucleosides; coupling; deprotection
INTRODUCTION
Since the approval of aciclovir[1] as an antiviral agent by the U.S. Food
and Drug Administration (FDA), there have been numerous publications
concerned with the synthesis of acyclic nucleoside analogues with improved
biological activity and selectivity compared with aciclovir. Aciclovir is used
in the treatment of herpes simplex virus (HSV), varicella zoster virus (VZV),
and cytomegalovirus (CMV) infections.[1] Several acyclic nucleoside ana-
logues have been approved for clinical use as antiviral agents; ganciclovir,[2]
penciclovir,[3] and valaciclovir.[4]
Synthesis of the first 6-azapyrimidine base, 6-azauracil was described by
Seibert[5] in 1947. Despite having bacteriostatic[6] and antitumour activi-
ties,[7] 6-azauracil was found to be toxic to the central nervous system.[8]
Alternatively, 6-azauridine which was synthesized later showed an absence of
Received 29 December 2009; accepted 15 March 2010.
M.J. acknowledges the Malaysian Government for research funding.
Address correspondence to Malina Jasamai, Faculty of Pharmacy, Universiti Kebangsaan Malaysia,
Jalan Raja Muda Abd Aziz, 50300, Kuala Lumpur. E-mail: malina@pharmacy.ukm.my
535

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M. Jasamai et al.
toxicity[8] and beneficial responses in some hyperplastic diseases.[9] An alkyl
substituent[10] at C-5 of nucleoside analogues, propyl group[11] in particular
was shown to exhibit significant antiviral activity against HSV.
There are two main methods employed to synthesize the acyclic moiety.
Robins et al.[12] reported a synthesis, which involved the preparation of (2-
acyloxyethoxy) methyl halides as the acyclic moiety. This was achieved by acy-
lation of the cyclic acetal, 1,3-dioxolane[13] by reacting it with either benzoyl
chloride or acetyl bromide.[13] Keyser et al.[14] on the other hand, described
the preparation of an acyclic moiety which involved the rapid reaction of 1,3-
dioxolane with trimethylsilyl iodide. The cumbersome method to synthesize
the conventional acyclic moiety is no longer required as the compound is
commercially available as the protected benzoyloxyethylcloromethylether.
The aim of this study is to synthesize novel acyclic nucleosides us-
ing commercially available acyclic moiety, which will be coupled to the 6-
azapyrimidine bases. The effective method of protection and deprotection
will also be examined.
MATERIALS AND METHODS
Chemistry
1H and 13C NMR spectra were recorded with a Brucker Avance DPX500
spectrometer operating at 500 and 125MHz, with Me4Si as internal standard.
Mass spectra were determined by the EPSRC Mass Spectrometry Centre
(Swansea, UK). Medac Ltd (Surrey, UK) performed the microanalyses.
Flash column chromatography was performed with silica gel 60 (230–400
mesh; Merck, UK) and TLC was carried out on precoated silica plates (kiesel
gel 60 F254, BDH). Melting points were determined on an electrothermal
instrument and are uncorrected. Compounds were visualized by illumina-
tion under ultraviolet (UV) light (254 nm) or by the use of vanillin stain
followed by charring on a hotplate. All solvents were dried prior to use as
described by the handbook, Purification of Laboratory Chemicals[15] and stored
˚
over 4 A molecular sieves, under nitrogen.
Synthesis of Acyclic Nucleosides
Benzoic acid 1ꢀ-(5-ethyl-6-azauracil)methoxy-ethyl ether (4)
BSA (3.50 mL, 14.07 mmol) was added to a suspension of 5-ethyl-6-
azauracil (1) (1 g, 7.03 mmol) in dry acetonitrile (20 mL) and the reaction
mixture was stirred at room temperature under nitrogen for 30 minutes. A
solution of benzoyloxyethylchloromethylether (3) (1.40 mL, 8.08 mmol) in
dry acetonitrile (20 mL) was then added. The reaction mixture was cooled
in an ice bath before TMSOTf (2.40 mL, 13.36 mmol) was added dropwise.
The reaction mixture was stirred at room temperature under nitrogen for 3

Synthesis of 6-Azapyrimidine Acyclic Nucleosides
537
hours, diluted with chloroform (150 mL) and washed with saturated aqueous
sodium bicarbonate (2 × 100 mL). The organic layer was dried (MgSO4)
and concentrated in vacuo to give yellowish syrup. Purification by column
chromatography (ethyl acetate-petroleum ether 1:1 v/v) yielded compound
4 as a thick white syrup, which gave a white solid on standing (1.25 g, 56%).
1
RF 0.62 (ethyl acetate-petroleum ether 1:2 v/v); m.p.: 80–84◦C; H NMR
(CDCl3) δ 10.59 (bs, 1, NH), 9.57 (m, 2, o-Ph), 7.44 (m, 1, p-Ph), 7.33 (m,
2, m-Ph), 5.32 (s, 2, H-1ꢁ), 4.41 (m, 2, H-4ꢁ), 3.96 (t, 2, J = 4.3 Hz, H-3ꢁ),
2.50 (q, 2, J = 7.4, CH2), 1.07 (t, 3, J = 7.3, CH3). 13C NMR (CDCl3) δ
166.68 (C O, PhCO), 156.94 (C O, C-2), 149.77 (C, C-5), 148.47 (C O,
C-4), 133.40 (CH, p-Ph), 130.16 (C, COCPh), 129.91 (CH, 2 x o-Ph), 128.66
(CH, 2 x m-Ph), 79.91(CH2, C-1ꢁ), 68.44 (CH2, C-3ꢁ), 64.37 (CH2, C-4ꢁ),
−1
22.96 (CH2), 10.60 (CH3); IRVmax/cm (NaCl, film) 1719.7 (C O), 1451.8
(C C aromatic stretch), 1275.8–1094.1 (C O) stretch; MS (ES+) m/z: 342.1
[M+Na]+; Microanalysis calculated for C15H17N3O5 (319.3164). C, 56.42%,
H, 5.37%, N, 13.16%. Found C, 56.46%, H, 5.46%, N, 12.89%.
Benzoic acid 1ꢀ-(5-propyl-6-azauracil)methoxy-ethyl ether (5)
BSA (2.20 mL, 8.96 mmol) was added to a suspension of 5-propyl-6-
azauracil (2) (0.70 g, 2.18 mmol) in dry acetonitrile (20 mL) and the reaction
mixture was stirred at room temperature under nitrogen for 30 minutes. A
solution of benzoyloxyethylchloromethylether (3) (0.90 mL, 5.15 mmol) in
dry acetonitrile (20 mL) was then added. The reaction mixture was cooled
in an ice bath before TMSOTf (1.50 mL, 8.51 mmol) was added dropwise.
The reaction mixture was stirred at room temperature under nitrogen for 3
hours, diluted with chloroform (150 mL) and washed with saturated aque-
ous sodium bicarbonate (2 × 100 mL). The organic layer was dried (MgSO4)
and concentrated in vacuo to give yellowish syrup. Purification by column
chromatography (ethyl acetate-petroleum ether 3:2 v/v) yielded compound
5 as a thick clear syrup, (0.36 g, 49%). RF 0.56 (ethyl acetate-petroleum
1
ether 1:2 v/v); H NMR (CDCl3) δ 10.23 (bs, 1, NH), 8.08 (m, 2, o-Ph),
7.59 (m, 1, p-Ph), 7.46 (m, 2, m-Ph), 5.43 (s, 2, H-1ꢁ), 4.52 (t, 2, J = 4.6 Hz,
H-4ꢁ), 4.05 (t, 2, J = 5.0 Hz, H-3ꢁ), 2.58 (t, 2, J = 7.6 Hz, CH2), 1.66 (q, 2,
J = 7.5 Hz, CH2), 0.99 (t, 3, J = 7.3 Hz, CH3).13C NMR (CDCl3) δ 166.93
(C O, PhCO), 156.94 (C O, C-2), 149.48 (C, C-5), 147.87 (C O, C-4),
133.57 (CH, p-Ph), 130.32 (C, COCPh), 130.10 (CH, 2 x o-Ph), 128.82 (CH,
2 x m-Ph), 80.05 (CH2, C-1ꢁ), 68.62 (CH2, C-3ꢁ), 64.21 (CH2, C-4ꢁ), 31.97,
−1
(CH2), 20.02 (CH2) 14.11 (CH3); IRVmax/cm (NaCl, film) 1719.9 (C O),
1451.8 (C C aromatic stretch), 1275.1–1095.6 (C O stretch); LRMS (ES+)
m/z: 168.1 [M−OCH2CH2OCOPh]+, 181.2 [M-heterocyclic base]+, 290.2
[M-propyl]+, 304.1 [M-ethyl]+, 333.3 [M]+, 356.1 [M+Na]+. LRMS (CI+)
m/z: 149.0 [M−CH2CH2OCOPh]+, 167.1 [M−CH2 heterocyclic base]+,
184.1 [M−OCH2 heterocyclic base]+, 212.1 [M−OCOPh]+, 334.2 [M+H]+.

538
M. Jasamai et al.
HRMS (ES+) m/z: Calculated mass: 351.1663 [M+NH4]+. Accurate mass:
351.1662
2-(5-Ethyl-6-azauracil)methoxy-ethanol (6)
Aqueous methylamine (25 mL) was added to compound 4 (1.09 g,
3.40 mmol) and the resulting lilac solution was heated on a steam bath
for 15 minutes. The lilac colour faded on heating. The crude product was
concentrated in vacuo. Purification by column chromatography (methanol-
dichloromethane 3:97 v/v) yielded white solids. Further purification by re-
crystallization using ethyl acetate gave compound 6 as a white solid (0.61
1
g, 83%). RF 0.32 (methanol-dichloromethane 1:9 v/v); m.p.: 91–93◦C; H
NMR (DMSO-d6) δ 12.15 (bs, 1, NH), 5.17 (s, 2, H-1ꢁ), 4.64 (bs, 1, OH), 3.55
(t, 2, J = 4.8 Hz, H-4ꢁ), 3.46 (t, 2, J = 4.4 Hz, H-3ꢁ), 2.48 (q, 2, J = 7.4 Hz,
CH2), 1.08 (t, 3, J = 7.4 Hz, CH3). 13C NMR (DMSO-d6) δ 157.19 (C O,
C-2), 149.31 (C, C-5), 147.21 (C O, C-4), 79.32 (CH2, C-1ꢁ)−, 171.39 (CH2,
C-3ꢁ), 60.43 (CH2, C-4ꢁ), 22.96 (CH2), 10.61 (CH3); IRVmax/cm (KBr, disc)
3464.1–3429.3 (O H stretch), 2984.7–2778.7 (C-H stretch), 1713.8 (C O),
1444.7 (C O stretch). MS (ES+) m/z: 238.1 [M+Na]+; Microanalysis calcu-
lated for C8H13N3O4 (215.2085). C, 44.65%, H, 6.09%, N, 19.52%. Found
C, 44.83%, H, 6.17%, N, 19.28%.
2-(5-Propyl-6-azauracil)methoxy-ethanol (7)
Aqueous methylamine (17 mL) was added to compound 5 (0.73 g, 2.18
mmol) and the resulting clear solution was heated on a steam bath for
15 minutes. The crude product was concentrated in vacuo. Purification
by column chromatography (methanol-dichloromethane 3:97 v/v) yielded
white solids. Further purification by recrystallization using ethyl acetate gave
compound 7 as a white crystalline solid (0.42 g, 84%). RF 0.38 (methanol-
dichloromethane 1:9 v/v); m.p.: 90–91◦C; 1H NMR (DMSO-d6) δ 10.13 (bs,
1, NH), 5.39, (s, 2, H-1ꢁ), 3.79 (m, 4, H-4ꢁ & H-3ꢁ), 2.82 (t, 1, J = 7.5 Hz,
OH), 2.60 (t, 2, J = 7.5 Hz, CH2), 1.68 (q, 2, J = 7.5 Hz, CH2), 1.00 (t, 3,
J = 7.3 Hz, CH3). 13C NMR (DMSO-d6) δ 156.95 (C O, C-2), 149.68 (C,
C-5), 147.80 (C O, C-4), 80.26 (CH2, C-1ꢁ), 71.74 (CH2, C-3ꢁ), 61.99 (CH2,
C-4ꢁ), 32.02, (CH2), 20.08 (CH2) 14.11 (CH3); IRVmax/cm−1 (KBr, disc) 3433.7
(O H), 2966.2–2833.1 (C-H stretch), 1704.2 (C O), 1441.6 (C O stretch);
LRMS (ES+) m/z: 252.1 [M+Na]+, 267.9 [M+K]+. LRMS (EI+) m/z: 156.2
[M-acyclic moiety]+, 169.2 [MOCH2CH2OH]+, 199.2 [M-CH2OH]+, 229.1
[M]+. LRMS (CI+) m/z: 187.2 [M-propyl]+, 216.2 [M-ethyl]+. HRMS (ES+)
m/z: Calculated mass: 247.1401 [M+NH4]+. Accurate mass: 247.1398
Antiviral Assay
CEM cells in complete medium infected with 100 µL of human im-
munodeficiency virus (HIV) type 1 and 2 were seeded at 4 × 104 cells into

Synthesis of 6-Azapyrimidine Acyclic Nucleosides
539
0.32 cm2 wells of a 96-well microplate containing 100 µL various concentra-
tions of test compounds (100 µM, 20 µM, 4 µM, and 0.8 µM). Virus stock
used in the experiment had a titre of 103.7 CCID50/mL. In the control wells,
200 µL of medium containing CEM cells and the virus with no test com-
pound was pipetted. To assess toxicity of the test compound, 100 µL of CEM
cells alone without the viruses was seeded into the wells containing 100 µL
of medium and test compound at different concentrations. The microplates
were then incubated at 37◦C for 4 days.
RESULTS AND DISCUSSION
Chemistry
The synthesis involved coupling of the heterocyclic 6-azapyrimidine bases
(compound 1 and 2) synthesized as reported by Chang[16] with commercially
available acyclic moiety (3) using the Vorbru¨ggen coupling method.[17] De-
protection of the benzoyl protecting group gave the desired acyclic nucleo-
side analogues (6 and 7) (Scheme 1).
The 6-azapyrimidine bases exist as tautomers, where the lactim form
can participate in the coupling reaction, making it necessary to temporarily
O
R
HN
O
O
N
O
O
N
R
O
i
O
Cl
HN
O
O
+
N
O
N
H
R
R
4 = C2H5
5 = C3H7
1 = C2H5
2 = C3H7
3
ii
O
R
HN
N
O
N
O
HO
R
6 = C2H5
7 = C3H7
SCHEME 1 Reagents and Conditions: i) dry acetonitrile, BSA, under nitrogen, room temperature,
TMSOTf, 3 hours; ii) aq. methylamine, steam bath, 15 minutes.

540
M. Jasamai et al.
protect these groups using bis(trimethylsilyl)acetamide (BSA) as described
in the literature.[18] Silylated 1 and 2 obtained are soluble in acetonitrile
yeilding a clear solution on completion of the reaction. The completion of
the reaction could not be monitored by thin layer chromatography (TLC) as
the RF value of the reactant (3) and the products (4 and 5) were very close.
From preliminary study, the reaction was completed after 3 hours.
The presence of the aromatic group was confirmed by the aromatic
signals at 7.33–8.11 ppm in the 1H NMR spectrum and at 128.70–133.60 ppm
in the 13C NMR spectrum. Importantly, the occurrence of the H-1 signals at
5.32–5.83 ppm in the 1H NMR spectrum and C-1 signals at 79.91–84.52 ppm
in the 13C NMR spectrum indicates that the acyclic moiety had coupled to
the respective bases forming the N -glycosidic bond.[19]
Deprotection of the benzoyl group was achieved by reacting compound
4 and 5 with methylamine on a steam bath for 15 minutes.[20] The reaction
was monitored by TLC The impurities, including N -methylbenzamide, were
removed from the crude product by column chromatography (ethyl acetate-
petroleum ether). Pure 6 and 7 were obtained by recrystallization with ethyl
acetate. The disappearance of the benzoyl protecting group in the 1H NMR
and 13C NMR spectra confirmed the presence of the desired compounds 6
and 7.
A series of novel 6-azapyrimidine acyclic nucleosides was successfully
synthesized by coupling the commercially available acyclic moiety with the
respective 6-azapyrimidine bases using the Vorbru¨ggen coupling proce-
dure.[17] An effective deprotection of the benzoyl group was achieved by
employing method described by Kelly et al.[20]
Antiviral Activity
The acyclic nucleoside analogues synthesized are neither toxic to CEM
cells at concentrations up to 100µM nor active against HIV-1 and HIV-2 virus.
The acyclic analogues (6 and 7) obtained have potential to be developed
as antiviral drug due to their non-toxic property in vitro. Further studies on
the relevant functional groups are needed to improve antiviral activity of the
nucleoside analogues synthesized.
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