Optimized synthesis of 3′-O-aminothymidine and evaluation of its oxime derivative as an anti-HIV agent

Article abstract of DOI:10.1515/hc-2015-0150

The synthesis and isolation of 3′-O-aminothymidine oximes have been optimized. Synthesized compounds were characterized by NMR and UV spectral and analytical data. A mixture of previously not reported syn and anti isomers of acetaldoximes was assessed for anti-HIV activity and the prevention of syncytia formation caused by HIV-1 infection.

Full text of DOI:10.1515/hc-2015-0150

Heterocycl. Commun. 2015; aop
Pavel N. Solyev*, Maxim V. Jasko, Tatiana A. Martynova, Ludmila B. Kalnina, Dmitry N. Nosik
and Marina K. Kukhanova
Optimized synthesis of 3′-O-aminothymidine and
evaluation of its oxime derivative as an anti-HIV
agent
DOI 10.1515/hc-2015-0150
Received July 20, 2015; accepted August 14, 2015
DNA synthesis catalyzed by reverse transcriptase if a suit-
able oxime protection is applied for the 3′-blockage, as in
typical nucleoside analogue inhibitors. Our attention was
focused on previously non-reported oxime derivatives of
Abstract: The synthesis and isolation of 3′-O-aminothy- 3′-O-aminothymidine [4]. The goal of this study was the
midine oximes have been optimized. Synthesized com- optimization of 3′-O-aminothymidine synthesis as well as
pounds were characterized by NMR and UV spectral and evaluation of anti-HIV properties of its acetaldoximes.
analytical data. A mixture of previously not reported syn
and anti isomers of acetaldoximes was assessed for anti-
HIV activity and the prevention of syncytia formation
caused by HIV-1 infection.
Results and discussion
Keywords: anti-HIV properties; Mitsunobu reaction;
nucleoside analogues; oxime derivatives; syncytium
formation.
Synthesis
Synthesis of 3′-O-aminothymidine (TONH2) is usually con-
ducted via the Mitsunobu reaction of xylothymidine with
N-hydroxyphthalimide followed by the deblocking of
the phthalimide moiety. Xylothymidine can be obtained
either by 3′-keto-derivative reduction (non-selective for
the resulting configuration) [3] or by the Mitsunobu pro-
cedure with reversible benzoic acid 3′-protection [1], but
most frequently and successfully, xylothymidine is syn-
Dedicated to the memory of the late Professor Kyoichi A. Watanabe,
a great medicinal chemist and our best friend.
Introduction
3
′-Modified nucleosides are used as inhibitors of HIV rep- thesized via 2,3′-anhydronucleoside formation [5]. Modi-
lication, and some of them have become efficient drugs. fications usually take place after the introduction of the
3
′-O-Aminothymidine is a nucleoside analogue of special 5′-protecting group, but there is also a simplified ‘one-pot’
interest as a cleavable primer extension terminator [1] and procedure [6] that was used here (Scheme 1). Thymidine
as a substrate for synthesis of antisense oligonucleotides was protected with trityl on the 5′-OH group and acti-
[
3
2, 3]. The reversible termination of oligonucleotides by vated by mesylation at 3′-OH; at this point, the solvent
′-O-aminothymidine takes place due to the -ONH frag- was evaporated, and pure intermediate 5′-O-trityl-3′-O-
2
ment capable of deamination with sodium nitrite. This mesylthymidine was crystallized from hexanes. Further
ability may be potentially extended to the termination of treatment with aqueous KOH results in formation of a
2
,3′-anhydrothymidine structure, and the use of excess of
alkali causes ring opening into 5′-O-tritylxylothymidine
1. After chromatographic purification of 1 on silica gel,
we performed the Mitsunobu procedure under typical
conditions, which proceeded smoothly. It was found
that cooling the reaction mixture is required only at the
moment the reagents are mixed, and an increase in tem-
perature until 80°C does not affect the reaction. At 80°C,
the elimination becomes a competitive reaction and
*
Corresponding author: Pavel N. Solyev, Engelhardt Institute of
Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street,
Moscow 119991, Russia, e-mail: solyev@gmail.com
Maxim V. Jasko, Tatiana A. Martynova and Marina K. Kukhanova:
Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences, 32 Vavilov Street, Moscow 119991, Russia
Ludmila B. Kalnina and Dmitry N. Nosik: Ivanovsky Institute of
Virology, Russian Ministry of Health, 16 Gamaleya Street,
Moscow 123098, Russia
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2
ꢀꢀꢀꢀꢁꢀP.N. Solyev et al.: Synthesis and anti-HIV properties of 3′-O-aminothymidine acetaldoxime
O
1) TrCl, pyridine
O
O
N-hydroxyphthalimide
2
) MsCl, pyridine
O
N
NH
3) 1 M KOH, dioxane
O
N
NH
PPh , DIAD, THF, 0°C
O
N
NH
3
HO
TrO
TrO
HO
O
82.5%
O
43.4%
O
HO
1
O
N
2
O
O
O
O
1
2
) N2H4 H2O, CH2Cl2
) CH3CHO, CH2Cl2
O
N
NH
O
N
NH
TrO
AcOH (80% aq.) HO
81.2%
O
O
71.4%
O
N
O
N
3
4
Scheme 1ꢀSynthesis of target 3′-O-aminothymidine syn/anti-acetaldoximes 4.
formation of a small amount of 5′-O-trityl-2′,3′-dideoxy- determined in the presence of the tested compounds
′,3′-didehydrothymidine can be observed. (0.01–300 μꢀ, three replicates for each dose) and were
2
The target Mitsunobu product 2 poses some difficul- assessed in 4 days. The cell concentration and viability
ties in purification on a silica gel column using the previ- were measured by the trypan blue dye exclusion calori-
ously described procedures [1–3]. Nevertheless, the earlier metric assay, and the EC and CC values were calculated.
proposed eluting system, hexane/dichloromethane/ethyl 3′-Azido-3′-deoxythymidine (AZT or zidovudine, Retrovir
50
50
®
acetate in a 3:2:2 ratio, may be used for convenient TLC from ViiV Healthcare) was used as a control. Cytotoxic-
detection [3]. We developed two-step column chroma- ity was assessed in MT-4 cells, cultured in the presence
tography purification instead. In the first step, silica gel of various doses of the compounds (0.01–1000 μꢀ) for 4
column chromatography in chloroform/ethanol system days. The data in Table 1 show that the oxime product 4
(
linear gradient of ethanol 0→5%) permitted separation possesses moderate activity against HIV-1.
of the target product contaminated with PPh O. In the
3
second step, re-purification using a fresh silica gel column
in chloroform/hexane/ethanol system in a 7:3:1.5 ratio was
conducted. This procedure allowed the isolation of pure
Syncytia formation
5
′-O-trityl-3′-O-phthalimidothymidine. Removal of the
Infection of cell culture with HIV-1 results in the forma-
tion of multinucleated giant cells or syncytia. The ability
to form syncytia may be a major contribution to the viru-
lence of HIV and may play a role in the disease progression
phthalimidyl fragment of 2 was performed using excess
hydrazine hydrate. The crude reaction mixture was then
treated with acetaldehyde. Acetaldoxime structure 3 was
the target product because it is more stable than formal-
doxime and potentially less sterically hindered than ace-
toxime, hence more likely to be a substrate for enzymes.
The trityl group was removed from the intermediate
product 3 by treatment with acetic acid without any cleav-
age of the oxime bond. Both trityl-protected 3 and depro-
tected product 4 are mixtures of syn and anti isomers that
could not be separated. Antiviral assays were carried out
on a mixture of deprotected product 4 containing syn and
anti isomers in a 1:1 ratio.
[
8, 9]. It has been shown that exposure to AZT has a sig-
nificant impact on syncytium formation and cell killing in
an in vitro assay [10]. We compared the effects of AZT and
compound 4 on syncytium development in lymphoblas-
toid cell culture MT-4 (Figure 1) according to the described
procedure [10]. Figure 1B shows cells 3 days after infection
with HIV-1, and Figure 1A shows uninfected control cells.
Table 1ꢀAntiviral activity and cytotoxicity syn+anti acetaldoximes of
3
′-O-aminothymidine.
Compoundꢁ
CC50a (ꢁμꢂ)
ꢁ EC50b (ꢁμꢂ)
Antiviral activity and toxicity
ꢃ
AZT
ꢂ
ꢂ
ꢃꢄꢅ
ꢂ>ꢂꢃꢅꢅ
ꢂ
ꢂ
ꢆꢇ
ꢅ.ꢅꢆ
Antiviral activity was determined in MT-4 cells infected
with HIV-1 (899A strain) at the multiplicity of infec-
tion 0.2–0.5 units/cell according to the previously pub-
a
Compound concentration required to reduce cell viability by 50%.
Compound concentration required to inhibit HIV replication by
b
lished procedure [7]. Antiviral activity and toxicity were 50%.
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P.N. Solyev et al.: Synthesis and anti-HIV properties of 3′-O-aminothymidine acetaldoximeꢈꢁꢀꢀꢀꢈ3
A
B
C
D
Figure 1ꢀEffects of compound 4 and AZT on syncytia formation (in 3 days after infection). (A) Control uninfected cells; (B) MT-4 cell culture
after HIV-1 infection; (C) infected cell culture growing in the presence of 0.1 μꢉ AZT; (D) infected cell culture growing in the presence of
10 μꢉ 4.
One can see that the presence of AZT (Figure 1C) or com- syringe injection was used for solutions in acetonitrile (flow rate
3
μL/min), nitrogen was applied as an inert gas, interface tempera-
pound 4 (Figure 1D) in the assays results in a significant
reduction in syncytia size. The action of both compounds
is similar, although the effective concentration of 4 was
ture was set at 180°C.
1
–1.5 orders of magnitude higher. The effects were time
Synthesis of ꢄ′-O-trityl-xylothymidine (1)
(
1–5 days) and dose dependent for AZT (0.01–1 μꢀ) and for
4
(1–150 μꢀ) (data not shown). These data correlate with
The procedure was carried out with chromatographic purification
of only small samples of the intermediate products for characteriza-
tion. A solution of thymidine (5 g, 0.02 mol) in pyridine (30 mL) was
stirred at room temperature and treated with trityl chloride (7.4 g,
anti-HIV activity of AZT and compound 4.
0
.024 mol). The mixture was stirred at 40°C for 16 h, then cooled, and
Conclusion
1
the resulting intermediate product, 5′-O-tritylthymidine, was treated
with mesyl chloride (3.2 mL, 0.045 mol). The mixture was stirred for
8
h, then quenched with 5 mL of distilled water, concentrated, and
An optimized procedure for 3′-O-aminothymidine oximes
synthesis and isolation of the intermediate products was
developed. The synthesized syn-/anti-oximes possess
anti-HIV activity and prevent cells from syncytia forma-
tion caused by HIV-1 infection.
extracted with chloroform and aqueous solution of NaHCO . The
3
organic layer was dried with Na SO , concentrated, and the residue
2
4
was crystallized at 0°C from a mixture of hexanes-chloroform. Crys-
tals of pure 5′-O-trityl-3′-O-mesylthymidine2 were separated and
1
ꢁA sample of reaction mixture was collected to isolate intermedi-
ate 5′-O-tritylthymidine. An aliquot was diluted with 1 mL of dis-
tilled water, the solution was concentrated, and the residue was
dissolved in chloroform and the solution was concentrated in vacuo.
Experimental
Diethyl azodicarboxylate (Fluka), methanesulfonyl chloride The residue was purified on a silica gel column eluting with ethanol
(
1
Acros), trityl chloride (Acros), hydrazine hydrate (Fluka), and thy- (0→5%)/chloroform system; UV: λ 267.2 nm; H NMR: δ 11.29 (s,
max
4
midine (Fluka) were purchased from the indicated suppliers. THF 1H, 3-NH), 7.48 (q, 1H, J
ꢂ=ꢂ 0.8 Hz, H-6) 7.28–7.41 (m, 15H, Tr), 6.22
ꢂ=ꢂ 6.94 Hz, H-1′), 5.30 (d, 1H, J3′-OH, 3′ ꢂ=ꢂ
6
, 5-Me
3
3
3
was distilled over sodium before use. The reaction process was (dd, 1H, J
ꢂ=ꢂ 6.74 Hz, J
1
′, 2′b
1′, 2′a
monitored using TLC on precoated Kieselgel 60F254 plates (Merck, 4.6 Hz, 3′-OH), 4.33 (m, 1H, H-3′), 3.89 (m, 1H, H-4′), 3.23–3.27 (dd,
2
3
2
Germany), and column chromatography was performed on silica 1H, J5
ꢂ=ꢂ 10.3 Hz, J
ꢂ=ꢂ 4.5 Hz, H-5′a), 3.18–3.21 (dd, 1H, J
ꢂ=ꢂ
′a, 5′b
5′a, 4′
5′a, 5′b
1
13
3
gel (40–63 μm; Merck, Germany). H and C NMR spectra were reg- 10.3 Hz, J
ꢂ=ꢂ 3.0 Hz, H-5′b), 2.13–2.28 (m, 2H, H-2′), 1.48 (d, 3H,
5′b, 4′
4
13
istered in DMSO-d on a Bruker AMX III 400 spectrometer with the
J5-Me, 6 ꢂ=ꢂ 0.8 Hz, 5-Me); C NMR: δ 163.5 (C-4), 150.3 (C-2), 143.4 (i-C
6
1
working frequency of 400 MHz for H NMR (Me Si as an internal (Tr)), 135.5 (C-6), 128.2 (m-C (Tr)), 127.8 (o-C (Tr)), 127.1 (p-C (Tr)), 109.5
4
standard) and 100.6 MHz for 1 C NMR (with carbon-proton decou- (C-5), 86.3 (C (Tr)), 85.3 (C-4′), 83.7 (C-1′), 70.4 (C-3′), 63.9 (C-5′), 39.4 (s,
3
pling, Me Si as an internal standard). UV spectra were registered on C-2′), 11.7 (s, 5-Me).
4
a Shimadzu UV-2401PC spectrophotometer in CH OH in the range 2ꢁA sample of reaction mixture was collected to isolate intermedi-
of 220–300 nm. High-resolution mass spectra (HR-ESI-MS) were ate 5′-O-trityl-3′-O-mesylthymidine. An aliquot was diluted with 1 mL
3
acquired in a positive ion mode on a Bruker maXis™ instrument; of distilled water, the solution was concentrated, the residue was
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ꢀꢀꢀꢀꢁꢀP.N. Solyev et al.: Synthesis and anti-HIV properties of 3′-O-aminothymidine acetaldoxime
dissolved in dioxane, and the residual solvent was evaporated in eluting with chloroform/hexane/ethyl acetate (7:3:1.5). Target frac-
vacuo. To a solution of 5′-O-trityl-3′-O-mesylthymidine in dioxane, a tions were concentrated in vacuo, resulting in 0.43 g (43.4%) of pure
1
1
-ꢀ KOH aqueous solution (30 mL) was added in three portions, which compound 2 as white powder; mp 109–110°C; UV: λ 270.9 nm; H
max
4
resulted in a change of the color of the mixture from yellow to red, NMR: δ 11.36 (s, 1H, 3-NH), 7.88 (m, 4H, Phth), 7.48 (q, 1H, J
ꢂ=ꢂ 1.0
6
, 5-Me
3
3
3
referring to the 5′-O-trityl-2,3′-anhydrothymidine structure formation. Hz, H-6) 7.23–7.35 (m, 15H, Tr), 6.34 (dd, 1H, J
ꢂ=ꢂ 6.0 Hz, J
ꢂ=ꢂ
1
′, 2′b
1′, 2′a
The color turned back to yellow in 12 h, af er which time the mixture 6.1 Hz, H-1′), 5.15 (m, 1H, H-3′), 4.31 (m, 1H, H-4′), 3.30–3.34 (dd, 1H,
2
3
2
was neutralized with aqueous acetic acid. The reaction mixture was
J5
ꢂ=ꢂ 10.7 Hz, J
ꢂ=ꢂ 4.2 Hz, H-5′a), 3.24–3.28 (dd, 1H, J
ꢂ=ꢂ 10.7
′a, 5′b
3
5′a, 4′
5′a, 5′b
4
concentrated and purified on a silica gel column eluting with ethanol Hz, J5
ꢂ=ꢂ 4.3 Hz, H-5′b), 2.13–2.28 (m, 2H, H-2′), 1.46 (d, 3H, J
′b, 4′
5-Me,
1
3
(0→12%)/chloroform system. Target fractions were concentrated to
ꢂ=ꢂ 1.0 Hz, 5-Me); C NMR: δ 163.4 (C-4), 163.4 (CO [Phth]), 150.2 (C-2),
6
give 8.98 g (82.5%) of pure compound 1 as a white powder; mp 231– 143.1 (i-C [Tr]), 135.3 (C-6), 134. 7 (4+5 [Phth]), 128.4 (1+2 [Phth]), 128.1
1
2
32°C; UV: λ 266.3 nm; H NMR: δ 11.24 (s, 1H, 3-NH), 7.61 (q, 1H, (m-C [Tr]), 127.8 (s, o-C [Tr]), 127.1 (p-C [Tr]), 123.3 (3+6 [Phth]), 109.8
max
4
3
J6, 5-Me ꢂ=ꢂ 1.0 Hz, H-6) 7.22–7.38 (m, 15H, Tr), 6.12 (dd, 1H, J
ꢂ=ꢂ 8.2 Hz, (C-5), 87.5 (C [Tr]), 86.6 (C-4′), 83.6 (C-1′), 81.1 (C-3′), 63.6 (C-5′), 35.6
1′, 2′a
3
3
J1′, 2′b ꢂ=ꢂ 2.2 Hz, H-1′), 5.19 (d, 1H, J
ꢂ=ꢂ 3.4 Hz, 3′-OH), 4.21 (m, 1H, (C-2′), 11.6 (5-Me).
3′-OH, 3′
2
3
H-3′), 4.09 (m, 1H, H-4′), 3.38–3.43 (dd, 1H, J
ꢂ=ꢂ 10.3 Hz, J
ꢂ=ꢂ 8.0
5
′a, 5′b
5′a, 4′
2
3
Hz, H-5′a), 3.20–3.24 (dd, 1H J
ꢂ=ꢂ 10.3 Hz, J
ꢂ=ꢂ 2.9 Hz, H-5′b),
5
′a, 5′b
5′b, 4′
2
3
2
.50–2.58 (m, 1H, H-2′a), 1.84–1.88 (dd, 1H, J
ꢂ=ꢂ 14.6 Hz, J
ꢂ=ꢂ 2.2
2
′a, 2′b
13
2′b, 1′
Synthesis of ꢄ′-O-trityl-3′-O-aminothymidine syn/anti-
acetaldoximes (3)
4
Hz, H-2′b), 1.65 (d, 3H, J
ꢂ=ꢂ 1.0 Hz, 5-Me); C NMR: δ 163.7 (C-4),
5-Me, 6
1
1
50.4 (C-2), 143.6 (i-C [Tr]), 136.7 (C-6), 128.3 (m-C [Tr]), 127.8 (o-C [Tr]),
26.9 (p-C [Tr]), 108.3 (C-5), 86.1 (C [Tr]), 84.1 (s, C-4′), 83.2 (C-1′), 68.9
(C-3′), 63.0 (C-5′), 40.7 (C-2′), 12.4 (5-Me).
A
0
solution of 5′-O-trityl-3′-phthalimidyloxythymidine (56 mg,
.088 mmol) in dichloromethane (2 mL) was cooled to 0°C and
treated dropwise with a solution of hydrazine hydrate (0.03 mL) in
dichloromethane (5 mL). The reaction mixture became cloudy as the
phthalic hydrazide formed and precipitated. Af er 2 h, the solvent
was evaporated, and the residue was treated with dichloromethane
Synthesis of ꢄ′-O-trityl-3′-phthalimidyloxythymidine (2)
A solution of 5′-O-trityl-xylothymidine (0.5 g, 1.04 mmol) in THF
(
5 mL) and acetaldehyde (0.04 mL). Af er stirring for 16 h, the mix-
(
(
15 mL) was cooled to 0°C and treated with N-hydroxyphthalimide
ture was filtered and the solution was applied to a silica gel column
0.5 g, 1.56 mmol), triphenylphosphine (0.411 g, 1.23 mmol), and
and eluted with ethanol (0→7%)/chloroform system. Target fractions
diisopropyl azodicarboxylate (0.244 mL, 1.23 mmol). Af er 6 h, the
reaction mixture was concentrated in vacuo and partitioned between
chloroform and water. Organic layer was dried with Na SO and then
were concentrated in vacuo to give 0.033 g (71.4%) of a mixture of
1
syn/anti isomers of 3 as a white powder; UV: λ 265.2 nm; H NMR:
max
2
4
4
δ 11.34 (2s, 2H, 3-NH), 7.56 and 7.54 (2q, 2ꢂ×ꢂ1H, J
ꢂ=ꢂ 1.0 Hz, H-6), 7.49
concentrated. Chromatographic purification was performed in two
steps using silica gel: first, eluting with chloroform/ethanol (95:5)
to separate a mixture of the target product and triphenylphosphine
oxide from other by-products, and second, using a new column and
6, 5-Me
3
(
q, 1H, J
ꢂ=ꢂ 5.8 Hz, CH [anti]), 7.26–7.41 (m, 30H, 2Tr), 6.95 (q, 1H,
CH, Me
3
JCH, Me ꢂ=ꢂ 5.5 Hz, CH [syn]), 6.17–6.23 (m, 2H, 2H-1′), 4.79–4.86 (m, 2H,
2
H-3′), 4.08–4.14 (m, 2H, 2H-4′), 3.24–3.34 (m, 4H, 2H-5′), 2.37–2.43 (m,
3
3
4
H, 2H-2′), 1.77 (d, 3H, J
ꢂ=ꢂ 5.5 Hz, Me [syn]), 1.76 (d, 3H, J
ꢂ=ꢂ
Me, CH
Me, CH
1
3
5
(
[
.8 Hz, Me [anti]), 1.46 (br.s, 6H, 5-Me); C NMR: δ 163.5 (C-4), 150.3
dissolved in chloroform and the solution was concentrated in vacuo.
The residue was purified on a silica gel column eluting with etha-
C-2), 149.1 and 148.8 (2s, 2CH), 143.3 (i-C [Tr]), 135.5 (C-6), 128.2 (m-C
Tr]), 127.94 (o-C [Tr]), 127.2 (p-C [Tr]), 109.8 (C-5), 86.5 (C [Tr]), 83.9 and
1
nol (0→5%)/chloroform system; mp 129–131°C; UV: λ 264.7 nm; H
max
83.8 (2s, C-4′), 82.4 and 82.2 (2s, C-1′), 82.0 and 81.6 (2s, C-3′), 64.2 and
64.1 (2s, C-5′), 36.3 and 36.0 (2s, C-2′), 14.9 (Me [anti]), 11.8 (Me [syn]),
4
NMR: δ 11.35 (s, 1H, 3-NH), 7.49 (q, 1H, J
ꢂ=ꢂ 0.8 Hz, H-6) 7.26–7.43
6
, 5-Me
3
3
(
m, 15H, Tr), 6.24 (dd, 1H, J1 ꢂ=ꢂ 6.8 Hz, J
ꢂ=ꢂ 7.4 Hz, H-1′), 5.38 (m,
+
+
′, 2′b
1′, 2′a
11.6 (5-Me). HRMS (ESI). Calcd for C H N O [M+H] , [M+Na] ): m/z
31 31 3 5
2
3
1
H, H-3′), 4.23 (m, 1H, H-4′), 3.38 (dd, 2H, J
ꢂ=ꢂ 10.5 Hz, J
ꢂ=ꢂ 4.3
5
′a, 5′a
5′a, 4′
526.2336 and 548.2156. Found: m/z 526.2332 and 548.2156.
2
Hz, H-5′a), 3.32 (dd, 2H, J
3
ꢂ=ꢂ 10.5 Hz, J
ꢂ=ꢂ 3.5 Hz, H-5′b), 3.22 (s,
5
′a, 5′b
5′b, 4′
4
13
ꢂ=ꢂ 0.8 Hz, 5-Me); C
-Me, 6
H, Me (Ms)), 2.58 (m, 2H, H-2′), 1.51 (d, 3H, J5
NMR: δ 163.4 (C-4), 150.2 (C-2), 143.2 (i-C (Tr)), 135.4 (C-6), 128.20 (m-C
Synthesis of 3′-O-aminothymidine syn/anti-acetal-
doximes (4)
(
Tr)), 127.84 (o-C (Tr)), 127.13 (p-C (Tr)), 109.87 (C-5), 86.71 (C (Tr)), 83.6
(
C-1′), 82.5 (C-4′), 80.0 (C-3′), 63.0 (C-5′), 37.4 (Me (Ms)), 36.7 (C-2′), 11.6
(
5-Me).
3
ꢁA sample of reaction mixture was collected during the reddening of A solution of 5′-O-trityl-3′-O-aminothymidine syn/anti-acetaldoximes
the solution to isolate 5′-O-trityl-2,3′-anhydrothymidine. An aliquot (0.033 g, 0.063 mmol) in 80% aqueous acetic acid (3 mL) was allowed
was treated with acetic acid to neutralize the alkali, solvents were to stand at 37°C for 16 h, then concentrated in vacuo. The residue was
then evaporated, the residue was dissolved in chloroform and the dissolved in toluene and the solution was concentrated to azeotropi-
solution was concentrated in vacuo. The residue was purified on a cally remove water. The crude product 4 was purified on a silica gel
silica gel column eluting with ethanol (0→3%)/chloroform system; column eluting with ethanol (0→20%)/chloroform system. Concen-
1
4
UV: λmax 216.0 nm, 252.0 nm; H NMR: δ 7.60 (q, 1H, J
ꢂ=ꢂ 0.9 Hz, tration of the target fractions gave 0.015 g (81.2%) of a mixture of
6, 5-Me
3
1
H-6), 7.22–7.31 (m, 15H, Tr), 5.89 (d, 1H, J1 ꢂ=ꢂ 3.7 Hz, H-1′), 5.31 (m, syn/anti-isomers of 4 as white powder; UV: λ 265.2 nm; H NMR:
′, 2′b
max
4
1
2
H, H-3′), 4.43 (m, 1H, H-4′), 3.14 (m, 2H, H-5′), 2.55–2.59 (m, 2H, H-2′a), δ 11.28 (2s, 2H, 3-NH), 7.74 (q, 1H, J
ꢂ=ꢂ 1.0 Hz, H-6 [anti]), 7.73 (q,
6, 5-Me
4
13
4
3
.43–2.50 (m, 2H, H-2′b), 1.78 (d, 3H, J
ꢂ=ꢂ 0.9 Hz, 5-Me); C NMR: 1H, J
ꢂ=ꢂ 1.0 Hz, H-6 [syn]), 7.52 (q, 1H, J
ꢂ=ꢂ 5.8 Hz, CH [anti]),
5-Me, 6
6, 5-Me
CH, Me
3
δ 170.5 (C-4), 153.2 (C-2), 143.2 (i-C (Tr)), 136.5 (C-6), 128.1 (m-C (Tr)), 6.97 (q, 1H, J
ꢂ=ꢂ 5.5 Hz, CH [syn]), 6.12–6.18 (m, 2H, 2H-1′), 5.13 (m,
CH, Me
1
(
27.8 (o-C (Tr)), 126.9 (p-C (Tr)), 116.1 (C-5), 86.7 (C-1′), 86.3 (C (Tr)), 83.4 2H, 2OH), 4.76 (m, 1H, H-3′ [syn]), 4.70 (m, 1H, H-3′ [anti]), 4.04 (m, 1H,
C-4′), 77.0 (C-3′), 62.4 (C-5′), 32.7 (C-2′), 12.9 (5-Me). H-4′ (syn]), 4.01 (m, 1H, H-4′ [anti]), 3.57–3.68 (m, 4H, H-5′), 2.16–2.33
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P.N. Solyev et al.: Synthesis and anti-HIV properties of 3′-O-aminothymidine acetaldoximeꢈꢁꢀꢀꢀꢈꢄ
3
ꢂ=ꢂ 5.5 Hz, 3J
(
m, 2ꢂ×ꢂ2H, 2ꢂ×ꢂH-2′), 1.80 (2d, 6H, J
ꢂ=ꢂ 5.8 Hz, 2Me),
[5] Horwitz, J. P.; Chua, J.; Da Rooge, M. A.; Noel, M.; Klundt, I. L.
Nucleosides. III. 1-(2′-Deoxy-3′,5′-epoxy-β-ꢊ-threo-
pentofuranosyl) thymine. J. Am. Chem. Soc. 1963, 28,
942–944.
[6] Jung, M. E.; Toyota, A. Preparation of 4′-substituted thymidines
by substitution of the thymidine 5′-esters. J. Org. Chem. 2ꢅꢅ1,
66, 2624–2635.
[7] Solyev, P. N.; Shipitsin, A. V.; Karpenko, I. L.; Nosik, D. N.;
Kalnina, L. B.; Kochetkov, S. N.; Kukhanova, M. K.; Jasko, M. V.
Synthesis and anti-HIV properties of new carbamate prodrugs
of AZT. Chem. Biol. Drug. Des. 2ꢅ12, 80, 947–952.
Me, CH
13
Me, CH
4
1
.78 (d, 3H, J
ꢂ=ꢂ 1.0 Hz, 5-Me); C NMR: δ 163.6 (C-4), 150.4 (C-2),
5-Me, 6
1
48.9 (CH [anti]), 148.6 (CH [syn]), 135.9 (C-6), 109.6 (C-5 [syn]), 109.5
(
C-5 [anti]), 84.1 (C-4′ [syn]), 83.9 (bs, C-4′ [anti]+C-1′ [syn]), 83.8 (C-1′
[
anti]), 82.5 (C-3′ [syn]), 82.1 (C-3′ [anti]), 61.7 (C-5′ [syn]), 61.7 (C-5′
anti]), 36.3 (C-2′ [syn]), 36.1 (C-2′ [anti]), 15.0 (Me [anti]), 12.2 (5-Me),
[
+
+
1
1.8 (Me [syn]). HRMS (ESI). Calcd for C H N O [M+H] and [M+Na] ):
m/z 284.1241 and 306.1060. Found: m/z 284.1241 and 306.1055.
12 17 3 5
Acknowledgments: This work was supported by the Rus-
sian Foundation for Basic Research (grants 13-04-40307-H
and 14-04-31163-mol_a).
[
8] Taleno, M.; Levy, J. A. MT-4 plaque formation can distinguish
cytopathic subtypes of the human immunodeficiency virus
(
HIV). Virology 1988, 167, 299–301.
[
9] Tersmette, M.; De Goede, R. E. Y.; Al, B. J. M.; Winkel, I. N.;
Gruters, R. A.; Cuypers, H. T.; Huisman, H. G.; Miedema, F.
Differential syncytium-inducing capacity of human immuno-
deficiency virus isolates: frequent detection of syncytium-
inducing isolates in patients with acquired immunodeficiency
syndrome (AIDS) and AIDS-related complex. J. Virol. 1988, 62,
2026–2032.
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Supplemental Material: The online version of this article
(DOI: 10.1515/hc-2015-0150) offers supplementary material,
available to authorized users.
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