1,3-Dipolar cycloaddition of alkenes to 3'-azido-3'-deoxythymidine as a route to 3'-deoxythymidin-3'-yl derivatives

Article abstract of DOI:10.1016/j.mencom.2014.06.005

1,3-Cycloaddition of acrylonitrile, acrylamide, vinyl acetate and allyl alcohol to azido group of 3'-azido-3'-deoxythymidine under mild conditions afforded the corresponding adducts which were tested as inhibitors of HIV reverse transcriptase.

Full text of DOI:10.1016/j.mencom.2014.06.005

Mendeleev
Communications
Mendeleev Commun., 2014, 24, 206–208
1,3-Dipolar cycloaddition of alkenes to 3'-azido-3'-deoxythymidine
as a route to 3'-deoxythymidin-3'-yl derivatives
Pavel N. Solyev,*a Roman A. Novikov,b Marina K. Kukhanovaa and Maxim V. Jaskoa
a V. A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russian
Federation. Fax: +7 499 135 2255; e-mail: solyev@gmail.com
b N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian
Federation
DOI: 10.1016/j.mencom.2014.06.005
1,3-Cycloaddition of acrylonitrile, acrylamide, vinyl acetate and allyl alcohol to azido group of 3'-azido-3'-deoxythymidine under
mild conditions afforded the corresponding adducts which were tested as inhibitors of HIV reverse transcriptase.
Thymidine is a nucleoside of a constant interest since the
incorporation of different substituents in its 3'-position usually
significantly changes its biological properties.1 For instance,
3'-azido-3'-deoxythymidine (AZT) is the first and still the most
applied nucleoside analogue in anti-HIV therapy. It has drawn
our attention as a substrate for the Huisgen cycloaddition of
different alkenes to azido group, resulting in derivatives with
pyrazoline, aziridine and triazole substituents at 3'-position of
3'-deoxythymidine. 3'-Triazolyl-3'-deoxythymidine derivatives
often become subjects of interest due to their biological activity.
They were extensively studied in search for reverse transcriptase
inhibitors2 but had little activity against HIV; they were found to
be effective inhibitors of human mitochondrial thymidine kinase;3
several triazole derivatives prolonged life of mice carrying Erlich
carcinoma. We concentrated our attention on obtaining under
mild chemical conditions new thymidine derivatives which could
have further application as anti-HIV drugs. Our methods require
neither catalysis with transition metals (like ‘click’-reactions of
azides with alkynes4) nor stepwise staging with specific reagents.
The target products have been obtained in moderate and good
yields.
Azido group of AZT, being a dipole, is susceptible to cyclo-
addition with dipolarophiles,5 among which acrylonitrile, acryl-
amide, vinyl acetate and allyl alcohol were selected for this study.
In pioneering articles by Gurvich6 and Huisgen,7 addition of
azide to acrylonitrile gave D2-1,2,3-triazolines, typical of further
opening of N2–N3 bond, thus, resulting in a diazo intermediate.
The nascent diazo compound is also a dipole capable of cyclo-
addition of one more acrylonitrile molecule (Scheme 1). Initially
we intended to isolate the first step triazoline product by carrying
out the reaction in a reasonable lack of the dipolarophile (0.7 equiv.)
at 37°C in methanol.† However, kinetically, the further ring
opening of 1 and the second cycloaddition proceeded faster than
the first stage of formation of 1 giving no possibility to detect 1
as a product. Moderate heating below 70°C for 0.5–5 h did not
favour the formation of aziridines 2. As a result, the only product
3a was isolated and studied, testifying that two molecules of acrylo-
nitrile were added to azido group. Nitrile fragments were not
involved in cycloaddition. This transformation was confirmed by
UV and 1H, 13C NMR spectroscopy including combination of HH
and CH correlations (COSY, HSQC, HMBC), as well as 15N NMR
and NH correlations. A product of the similar structure (3b) was
obtained by cycloaddition of AZT and acrylamide though it
required more time for the reaction.
1H, N1''H), 2.98 (m, 2H, C2''H2), 2.16 (m, 2H, C2'H2), 1.82 (s, 3H, Me).
13C NMR (DMSO-d6) d: 163.78 (s, C4), 150.40 (s, C2), 136.21 (s, C6),
121.68 and 121.65 (2s, C6''), 119.70 (s, C3''CN), 114.30 (s, C6''CN), 109.15
(s, C5), 85.04 (s, C4'), 83.68 (s, C1'), 64.19 (s, C3''), 61.36 and 61.30 (2s,
C5'), 57.86 (s, C3'), 51.83 (s, C2''), 41.28 and 41.20 (2s, C7''), 37.63 (s,
C2'), 12.23 (Me). 15N NMR (DMSO-d6) d: –4.3 (s, N5''), –113.4 (s, C6''CN),
–126.2 (s, C3''CN), –222.5 (s, N3H), –226.0 (s, N4''H), –232.7 (s, N1),
–341.2 (N1''H). UV (lmax/nm): 269.9. MS, m/z: 125.1 (4.9%, Thy), 219.3
(14.7%), 345.3 (57.6%, M–N2), 372.0 (11.7%, M–1), 408.1 (100.0%,
M+35Cl–), 410.1 (32.0%, M+37Cl–), 780.9 (65.9%, 2M+35Cl–), 782.9
(21.2%, 2M+37Cl–).
3'-{[3,5-Bis(aminocarbonyl)-4,5-dihydro-1H-pyrazol-5-yl]methyl-
amino}-3'-deoxythymidine 3b. Acrylamide (0.2 mg, 2.8 mmol) was added
to a solution of AZT (0.20 g, 0.75 mmol) in methanol (1 ml), stirred at
room temperature, and the mixture was stirred for 20 h at 37°C. The
reaction mixture was concentrated, the residue was chromatographed on
a silica gel column (20×150 mm), eluted with a linear gradient of methanol
(5% ® 20%) in chloroform. The fraction containing the target product
(a mixture of two diastereomers) was concentrated to afford 120 mg (39%)
of 3b as colourless viscous liquid, and was freeze-dried to white powder.
1H NMR (DMSO-d6) d: 11.19 (s, 1H, N3H), 7.71 and 7.72 (q, 1H, C6H,
4J6,5-Me 1.1 Hz), 7.69 and 7.66 (2s, 1H, N4''H), 7.39 and 7.24 (2s, 2H,
C6''CONH2), 7.21 and 7.01 (2s, 2H, C3''CONH2), 6.08 and 6.07 (2dd, 1H,
C1'H, 3J1',2'a 2.5 Hz, 3J1',2'b 6.2 Hz), 4.98 (m, 1H, OH), 3.66 (m, 1H, C4'H),
3.58 (m, 2H, C5'H), 3.26 (m, 1H, C4'H), 2.88 (m, 2H, C7''H), 2.69 and
2.65 (2d, 2H, H2'', 3J1'',2'' 19.7 Hz, 3J1'',2'' 19.9 Hz), 2.07 (m, 2H, H2'), 1.77
†
3'-[(3,5-Dicyano-4,5-dihydro-1H-pyrazol-5-yl)methylamino]-3'-deoxy-
thymidine 3a. Acrylonitrile (0.12 g, 2.25 mmol) was added to a solution
of AZT (0.30 g, 1.12 mmol) in methanol (3 ml), stirred at room tempera-
ture, and the mixture was stirred for 20 h at 37°C. The reaction mixture
was concentrated, and the residue was chromatographed on a silica gel
column (20×150 mm), eluted with a linear gradient of methanol (5% ® 10%)
in chloroform. The fraction containing the target product (a mixture of
two diastereomers) was concentrated to afford 100 mg (25%) of 3a as
colourless viscous liquid, and was freeze-dried to white powder. 1H NMR
(DMSO-d6) d: 11.21 (s, 1H, N3H), 9.39 (s, 1H, N4''H), 7.75 (s, 1H, C6H),
6.16 (dd, 1H, C1'H, 3J1',2'a 6.2 Hz, 3J1',2'b 6.4 Hz), 5.04 (br.s, 1H, OH), 3.74
(m, 1H, C4'H), 3.76–3.68 and 3.67–3.59 (m, 2H, C5'Ha, C5'Hb), 3.47 and
4
and 1.76 (2d, 3H, Me, J5-Me,6 1.1 Hz). 13C NMR (DMSO-d6) d: 174.99
(s, C3''CO), 163.70 (s, C6''CO), 163.58 (s, C4), 150.34 (s, C2), 144.55 and
144.46 (2s, C6''), 136.20 and 136.17 (2s, C6), 109.09 and 109.03 (2s, C5),
85.22 and 85.11 (2s, C4'), 83.70 (s, C1'), 72.59 and 72.53 (2s, C3''), 61.68
and 61.37 (2s, C5'), 57.98 and 57.74 (2s, C3'), 52.12 (s, C2''), 39.39 (s, C7''),
37.71 and 37.55 (2s, C2'), 12.16 (s, Me). UV (lmax/nm): 270.5. MS, m/z:
258.1 (9.0%), 284.1 (14.5%, M–Thy), 410.2 (24.5%, M+1), 432.2 (100.0%,
M+Na+), 444.2 (21.9%), 470.2 (7.3%), 517.3 (29.4%), 547.3 (9.8%),
568.4 (10.9%), 610.4 (126%).
2
3.30 (2d, 2H, C7''H2, J7''a,7''b 17.3 Hz), 3.41 (m, 1H, C3'H), 3.40 (br.s,
© 2014 Mendeleev Communications. All rights reserved.
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Mendeleev Commun., 2014, 24, 206–208
HO
HO
HO
HO
Thy
Thy
Thy
Thy
O
O
O
O
R
R
HN
HN
N
N
N
rapidly
[1,3] H-shift
rapidly
R = CN, CONH2
MeOH, 37 °C
R = CN, CONH2, OAc
N
N
R
N
AZT
N
N
N
N
R
R
OH
1
37 °C
O
R
R = OAc
HO
R = CN, CONH2
HO
4
3
Me
NH
2
– N2
5
rapidly
[1,3] H-shift
HO
HO
HO
6
5'
Thy
Thy
Thy
Thy
N1
O
O
O
O
O
O
1'
4'
3'
2'
NH2
N
N
N
HN1"
2"
N
HO
3"
R
R
N
HN4"
N5"
2
4
7"
5
6
6"
O
N
R
Me
NH
Thy =
3a R = CN
3b R = CONH2
O
Scheme 1
It is known that under microwave irradiation cycloadducts of
vinyl acetate and azides release AcOH affording unsubstituted
triazoles.8 In our study vinyl acetate couples with AZT‡ yielding
3'-(1,2,3-triazolo)thymidine 4 as the only product, no microwave
assistance having been applied.
Addition with poorly reactive allyl alcohol proceeded in its
excess as a solvent within 7 days.§ Two major products were
obtained: the reduction product 3'-amino-3'-deoxythymidine 5
and 3'-[(2-hydroxymethyl)aziridin-1-yl]-3'-deoxythymidine 6 (two
diastereomers in equimolar ratio). The structure of compound 6 was
confirmed by HRMS and a combination of 2D NMR techniques.
Two diastereomers are clearly observed in the 1H NMR spec-
trum of 6, which differentiate several signals of the furanose and
nucleobase (Figure 1). They appeared due to a newly formed
stereocenter at the second position of the aziridine ring. It is
possible to observe diastereomers via TLC and isolate them
separately by the silica gel column chromatography. Usually
C-monosubstituted aziridines may be observed in NMR with
the first order AMX or the second order ABX patterns for CH2
group of the cycle.9 At first sight, the situation observed for
diastereomers of 6 resembles the AMX pattern. But enrichment
with one diastereomer after chromatography dramatically simpli-
1
fies the 3''-CH2 pattern. The uncommon character of H NMR
spectrum of 6 is that here aziridine 3''-CH2 fragment reveals
itself as an AA'X degenerate system showing no Jgem in CDCl3,
while 4''-CH2OH of the aziridinyl side chain clearly indicates a
typical ABX system with geminal J 11.4 Hz and vicinal J 4.5 and
6.7 Hz. It is proper to mention that this effect may not occur if
other solvents suitable for NMR are used.10 The same effect in
small ring heterocycles has only been reported for b-hydroxy
episulfides with erythro configuration of the alcohol.11
The configuration of 3''-stereocenter was determined by the
3''-CHa shift observed in the enriched form. Compared to the
‡
3'-(1,2,3-Triazol-1-yl)-3'-deoxythymidine 4. Vinyl acetate (0.20 g,
2.3 mmol) was added to a solution ofAZT (0.20 g, 0.75 mmol) in methanol
(2 ml), stirred at room temperature, and the mixture was stirred for 7 h at
50°C. The reaction mixture was concentrated, and the residue was chromato-
graphed on a silica gel column (20×150 mm), eluted with a linear gradient
of methanol (5% ® 10%) in chloroform. The fraction containing the target
product was concentrated to afford 170 mg (77%) of 4 as colourless viscous
liquid, which was freeze-dried to white powder. 1H NMR (DMSO-d6)
d: 11.31 (s, 1H, N3H), 8.30 (d, 1H, C5''Htriazole, 3J5'',4'' 1.1 Hz), 7.81 (q, 1H,
C6H, 4J6,5-Me 1.2 Hz), 7.79 (d, 1H, C4''Htriazole, 3J4'',5'' 1.1 Hz), 6.42 (dd, 1H,
C1'H, 3J1',2' 6.7 Hz), 5.40 (m, 1H, C3'H), 5.24 (t, 1H, OH, 3J5'-OH,5' 6.7 Hz),
4.22 (m, 1H, C4'H), 3.67 (m, 2H, C5'H), 2.70 (m, 2H, C2'H), 1.81 (d, 3H,
Me, J5-Me,6 1.2 Hz). 13C NMR (DMSO-d6) d: 163.66 (s, C4), 150.38 (s,
C2), 136.18 (s, C6), 133.47 (s, C4''), 124.39 (s, C5''), 109.55 (s, C5), 84.47
(s, C4'), 84.90 (s, C1'), 60.68 (s, C5'), 58.94 (s, C3'), 37.16 (s, C2'), 12.16
(s, Me). UV (lmax/nm): 266.3. MS, m/z: 83.2 (11.4%), 279.0 (8.3%, M–N),
316.1 (21.7%, M+Na+), 332.1 (9.8%), 374.2 (15.1%), 402.3 (28.8%),
426.2 (18.1%), 432.2 (45.6%), 433.3 (10.3%), 459.0 (19.4%), 467.2
(9.2%), 487.2 (15.9%), 490.3 (22.7%), 516.3 (9.4%), 599.3 (18.0%), 609.4
(100.0%, 2M+Na+).
purified product was concentrated to afford 46 mg (19%) of 5 as colour-
less crystals, mp 160°C in agreement with reported data.15 1H NMR
3
(DMSO-d6) d: 7.76 (s, 1H, C6H), 6.08 (dd, 1H, C1'H, J1',2'a 5.4 Hz,
3J1',2'b 6.4 Hz), 4.20–4.80 (br.s, 3H, NH2 and OH), 3.65 (dd, 1H, C5'Ha,
J5'a, 4' 3.0 Hz, J5'a,5'b 11.7 Hz), 3.57 (dd, 1H, C5'Hb, J5'b,4' 3.6 Hz, J5'a,5'b
11.7 Hz), 3.53 (m, 1H, C4'H), 3.43 (m, 2H, C3'H), 2.03 (m, 2H, C2'H),
1.77 (s, 3H, Me). 13C NMR (DMSO-d6) d: 163.79 (s, C4), 150.37 (s, C2),
136.32 (s, C6), 108.91 (s, C5), 87.57 (s, C4'), 83.45 (s, C1'), 60.71 (s, C5'),
50.72 (s, C3'), 40.59 (s, C2'), 12.18 (s, Me). UV (lmax/nm): 266.8.
The fraction containing the target product 6 (eluted in 20% methanol
in chloroform) was concentrated to afford 93 mg (28%) of 6 as colour-
less viscous liquid, and was freeze-dried to white powder. 1H NMR
(DMSO-d6) d: 7.71 and 7.70 (q, 1H, C6H, 4J6,5-Me 1.2 Hz), 6.20 and 6.19
3
3
(2dd, 1H, C1'H, J1',2'a 6.0 Hz, J1',2'b 7.2 Hz), 4.99 (br.s, 1H, C5'OH),
4.65 (dd, 1H, C4''OH, J4''-OH,4''a = J4''-OH,4''b = 5.4 Hz), 3.95 and 3.86
3
3
(m, 1H, C4'H), 3.57 (m, 2H, C5'H), 3.38 (dd, 1H, C4''Ha, J4''a,2'' 4.6 Hz,
3
2J4''a,4''b 11.3 Hz), 3.18 (dd, 1H, C4''Hb, 3J4''a,2'' 6.7 Hz, 2J4''b,4''a 11.3 Hz),
2.22 (m, 1H, C3'H), 2.07 (m, 2H, C2'H), 1.77 (d, 3H, Me, 4J6,5-Me 1.2 Hz),
1.66 (m, 1H, C2''H), 1.53 and 1.51 (2d, 1H, C3''Ha, 3J3''a,2'' 3.5 and 3.6 Hz,
§
3
2
3'-Amino-3'-deoxythymidine 5 and 3'-[(2-hydroxymethyl)aziridin-1-yl]-
2J3''a,3''a' 0 Hz), 1.38 (d, C3''Ha', J3''a',2'' 6.3 Hz, J3''a',3''a 0 Hz). 13C NMR
(DMSO-d6) d: 163.74 (s, C4), 150.49 and 150.46 (2s, C2), 136.16 and 136.10
(2s, C6), 109.27 and 109.20 (2s, C5), 85.44 and 85.18 (2s, C1'), 84.06 and
83.92 (2s, C4'), 68.96 and 68.52 (2s, C3'), 63.17 and 63.14 (2s, C4''),
61.93 and 61.66 (2s, C5'), 39.83 and 39.82 (2s, C2''), 37.43 and 37.23
(2s, C2'), 30.16 and 30.07 (2s, C3''), 12.22 (s, Me). UV (lmax/nm): 267.5.
HRMS, m/z: 298.1397 (M+1), 320.1217 (M+Na+), 595.2722 (2M+1),
617.2545 (2M+Na+), 892.4047 (3M+1), 914.3870 (3M+Na+).
3'-deoxythymidine 6. A solution of AZT (0.30 g, 1.12 mmol) in allyl
alcohol (5 ml) was stirred for 96 h at 37°C. The reaction mixture was
concentrated, and the residue was chromatographed on a silica gel column
(20×150 mm), eluted with a linear gradient of methanol (5% ® 50%) in
chloroform. The fraction containing the target product 5 (eluted in 50%
methanol in chloroform) was concentrated by evaporation in vacuo and
applied on a LiChroprep RP-18 (40-63 mm, Merck). The additionally
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Mendeleev Commun., 2014, 24, 206–208
HO
be expected in the corresponding triphosphorylated forms in a
cell-free system.
(a)
5'
R:S (1:1)
O
4'
3'
N1"
H2O
The authors are grateful to Vladimir Valuev-Elliston (V. A.
Engelhardt Institute of Molecular Biology) for performing HIV-1
RT assay. This work was supported by the Russian Foundation
for Basic Research (project no. 12-04-00581).
3"-CHb
3"-CHa
HO
4"
3"
2"
4"-CHa
4'
5'
4"-CHb
Online Supplementary Materials
Supplementary data associated with this article (1H and 13C
NMR spectra for all the products, 2D NMR spectra for compounds
3a, 4 and 6; 15N NMR spectra for 3a and 6, DFT energy calcula-
tions and orbitals visualization, HIV-1 reverse transcriptase assay
procedure) can be found in the online version at doi:10.1016/
j.mencom.2014.06.005.
4.0
3.8
3.6
O
3.4
3.2
d/ppm
1.52
1.45
1.38
R (diastereomeric excess 53%)
HO
(b)
References
1 I. A. Mikhailopulo and A. I. Miroshnikov, Mendeleev Commun., 2011,
21, 57.
N
3"-CHa
3"-CHb
2 (a) K. Hirota, H. Hosono, Y. Kitade, Y. Maki, C. K. Chu, R. F. Schinazi,
H. Nakane and K. Ono, Chem. Pharm. Bull., 1990, 38, 2597; (b) D. Haebich,
W. Barth and M. Roesner, Heterocycles, 1989, 29, 2083; (c) P. Wigerinck,
A. Van Aerschot, G. Janssen, P. Claes, J. Balzarini, E. De Clercq and
P. Herdewijn, J. Med. Chem., 1990, 33, 868.
HO
∗
4"-CHa
4"-CHb
5'
4'
H2O
3 S. Van Poecke, A. Negri, F. Gago, I. Van Daele, N. Solaroli, A. Karlsson,
J. Balzarini and S. Van Calenbergh, J. Med. Chem., 2010, 53, 2902.
4 (a) W. H. Binder and C. Kluger, Curr. Org. Chem., 2006, 10, 1791;
(b) F. Amblard, J. H. Cho and R. F. Schinazi, Chem. Rev., 2009, 109,
4207.
4.0
3.8
3.6
3.4
3.2
1.52
1.45
1.38
5 S. Bräse, C. Gil, K. Knepper andV. Zimmermann, Angew. Chem. Int. Ed.,
2005, 44, 5188.
6 S. M. Gurvich and A. P. Terent’ev, in Sbornik statei po obshchei khimii
(Collected Articles on General Chemistry), Izd. AN SSSR, 1953, vol.1,
p.409 (in Russian).
d/ppm
Figure 1 1H NMR spectra of compound 6: (a) diastereomers in 1:1 ratio,
(b) enriched with 3''-R isomer.
7 R. Huisgen, G. Szeimies and L. Möbius, Chem. Ber., 1966, 99, 475.
8 S. G. Hansen and H. H. Jensen, Synlett, 2009, 3275.
9 (a) P. Baret, H. Handel and J.-L. Pierre, Org. Magn. Reson., 1974, 6, 35;
(b) S. L. Mannat, D. D. Elleman and S. J. Brois, J. Am. Chem. Soc., 1965,
87, 2220.
minor isomer this doublet is deshielded and the structure is
known to be in anti configuration.12 In addition, the conformation
of the aziridine cycle in this diastereomer can be deduced: the
4'-CH downfield position shows that it is sterically compressed
with the lone pair of aziridine nitrogen and therefore deshielded.12
Aziridine is oriented away from the furanose cycle.
Although the overall yield of the products obtained by azide
to alkene cycloadditions may be increased by the prolongation of
the reaction time and raising the temperature,7 we did not study
the relationship between the yields and conditions in this work.
It was shown earlier that nucleoside derivatives can inhibit
reverse transcriptase activity not only as chain terminators after
their triphosphorylation (like AZT) but also as mimetics of non-
nucleoside inhibitors, whose binding sites differ from that of sub-
strate.13 Bearing this in mind, we tested our 3'-thymidine deri-
vatives as inhibitors in corresponding HIV-1 RT assays. However,
they did not inhibit HIV RT at concentration up to 200 mm.
Presumably, the inhibiting activity of pyrazoline derivatives could
10 D. R. Crist, A. P. Borsetti, G. J. Jordan and C. F. Hammer, Org. Magn.
Reson., 1980, 13, 45.
11 K. D. Carlson, D. Weisleder and M. E. Daxenbichler, J. Am. Chem. Soc.,
1970, 92, 6232.
12 E. Breuer, L. Somekh and I. Ringel, Org. Mass Spectrom., 1977, 9, 328.
13 M. S. Novikov, O. N. Ivanova, A. V. Ivanov, A. A. Ozerov, V. T. Valuev-
E
lliston, K. Temburnikar, G. V. Gurskaya, S. N. Kochetkov, C. Pannecouque,
J. Balzarini and K. L. Seley-Radtke, Bioorg. Med. Chem., 2011, 19, 5794.
14 P. A. Belyakov, V. I. Kadentsev, A. O. Chizhov, N. G. Kolotyrkina, A. S.
Shashkov and V. P. Ananikov, Mendeleev Commun., 2010, 20, 125.
15 T. Sh. Lin and W. H. Prusoff, J. Med. Chem., 1978, 21, 109.
Received: 9th December 2013; Com. 13/4264
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