Synthesis of pyridine-stretched 2′-deoxynucleosides

Article abstract of DOI:10.1055/s-2002-33505

Synthesis of novel pyridine-stretched nucleoside (PSN) analogues of adenine (strA) (1), 2,6-diaminopurine (strD) (15) and hypoxanthine (strH) (17) from 4(5)-nitroimidazole has been achieved. Glycosylation of 4(5)-nitroimidazole was optimized to give consi

Full text of DOI:10.1055/s-2002-33505

LETTER
1483
Synthesis of Pyridine-stretched 2 -Deoxynucleosides
S
ynthesis of Pyri
u
s
d2 -Deox
s
ynucleosi
e
des ll Clayton,a Michael L. Davis,b William Fraser,*b Wei Li,b Christopher A. Ramsden*a
a
Lennard-Jones Laboratories, School of Chemistry and Physics, Keele University, Keele, Staffordshire, ST5 5BG, UK
Fax +44(1782)712378; E-mail: c.a.ramsden@chem.keele.ac.uk
b
Pharmaceutical Sciences Research Institute, Aston University, Aston Triangle, Birmingham, B4 7ET, UK
Fax +44(121)3590733; E-mail: w.fraser@aston.ac.uk
Received 20 June 2002
The development of TFOs for controlling gene expression
at the transcription level continues to attract attention with
much synthetic effort employed in the development of
novel base analogues for stronger triple helix formation
Abstract: Synthesis of novel pyridine-stretched nucleoside (PSN)
analogues of adenine (strA) (1), 2,6-diaminopurine (strD) (15) and
hypoxanthine (strH) (17) from 4(5)-nitroimidazole has been
achieved. Glycosylation of 4(5)-nitroimidazole was optimized to
give consistently good yields (>70%) of the desired analytically with targeted DNA.3 Replacement of thymidine and cy-
pure 5-nitro-1 - isomer 8 which on hydrogenation, C-addition of
ethoxymethylene malononitrile (EMMN) and cyclisation provides
the key intermediate 14 for PSN synthesis.
tosine by tricyclic base analogues has been shown to ei-
ther increase or decrease the stability of DNA duplexes
with the outcome very much dependant upon the precise
structural changes made. Replacement of thymidine by
Key words: 2 -deoxynucleosides, 5-aminoimidazoles, DNA, lin-
pyridoadenosine, glycosylation, nucleoside analogues
the extended thymidine analogues benzo[f]quinazoline-
2,4-dione (linear) or benzo[g]quinazoline-2,4-dione
(crescent-shaped), causes duplex destabilization4 whereas
To determine the effect of extended purine base structures
on the stability of triplex-forming oligonucleotides
(TFOs) targeted to DNA, we identified a series of pyri-
dine-stretched nucleosides (PSNs), including the 2 -
deoxyadenosine analogue 1, for potentially advantageous
incorporation into TFOs. PSNs are linear tricyclic ana-
logues of the naturally occurring purine nucleosides with
a central pyridine ring inserted between the pyrimidine
and imidazole rings.1 Benzene-stretched nucleoside ana-
logues, such as lin-benzoadenosine 2 (Figure 1), have pre-
viously been described by Leonard and co-workers2 but
their difficult synthesis hinders access to the necessary
quantities for oligomerization and study of DNA molecu-
lar recognition properties. Here we describe the synthesis
of three pyridine-stretched 2 -deoxynucleosides: stretched
2 -deoxyadenine 1 (strA), stretched 2 -deoxydiaminopu-
rine 15 (strD) and stretched 2 -deoxyhypoxanthine 17
(strH).
replacement of cytosine by linear phenothiazine and phe-
noxazine tricyclic derivatives enhances duplex stability.5
To prepare the novel 2 -deoxynucleosides 1, 15 and 17 we
selected as starting material the well known 2-deoxy-3,5-
di-O-(p-toluoyl)- -D-erythro-pentofuranosyl chloride 7,
which is prepared as the crystalline -anomer in three
steps from 2-deoxyribose.6 In previous glycosylations to
prepare ribonucleoside analogues, we employed the silver
salt of ambident nucleophile 4(5)-nitroimidazole and, aid-
ed by participation of the neighbouring 2 -substituent, ob-
tained only -anomers with the required 5-nitro-1 -
regioisomer predominating.1 Using the same conditions,
we carried out glycosylation of the silver salt 3 with the 1 -
chloro-2 -deoxysugar 7 in xylene to yield a mixture of -
anomers in which the required 5-nitro-regioisomer 8 and
the 4-nitro-regioisomer 9 were in a ratio of 1:2
(Scheme 1). The required isomer 87 was isolated in pure
crystalline form by column chromatography to produce
useable quantities of material. However, the selectivity of
this glycosylation required improvement. The outcome of
glycosylations using the chlorosugar 7 is strongly influ-
enced by choice of solvent, nucleophile and concentra-
tion.8 We repeated the glycosylation of the silver salt 3 in
acetonitrile to yield a near even distribution of all four
possible isomers (8–11, Scheme 1). Use of the lithium salt
4 under these reaction conditions resulted in an increased
yield of the -anomers (10 and 11) at the expense of a low-
er yield of each -anomer (8 and 9). Bergstrom and co-
workers9 reported that under standard sodium salt glyco-
sylation conditions,10 where the sodium salt 5 is generated
in situ using sodium hydride before addition of chlorosug-
ar 7, -anomers (8 and 9) are isolable in a 1:2 ratio. By us-
ing the preformed sodium salt 5, we slightly improved the
yield and anomeric ratio, (1:1.5) and detected the presence
of small amounts of the two -anomers (10 and 11).
NH2
NH2
N
N
N
N
N
N
N
N
N
HO
HO
O
O
HO HO
HO
2
1
Figure 1
Synlett 2002, No. 9, Print: 02 09 2002.
Art Id.1437-2096,E;2002,0,09,1483,1486,ftx,en;D13402ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

1484
R. Clayton et al.
LETTER
TolO
NM
O
+
O2N
N
Cl
TolO
3
4
5
6
M = Ag
M = Li
M = Na
M = Cs
7
i
X
N
TolO
O
Y
N
TolO
+
Y
O
N
TolO
N
X
TolO
10 X = H; Y = NO2
11 X = NO2; Y = H
8
9
X = H; Y = NO2
X = NO2; Y = H
Scheme 1 Reagents and conditions: i, THF, 62 °C, 3 h.
Figure 2 Strong NOE enhancements from NOSEY spectra: (a)
compound 8; (b) compound 9; (c) compound 11.
Glycosylation using -chlorosugar 7 probably proceeds
by an SN2 Walden inversion process at C-1 to give the -
product. Formation of the -nucleosides by a similar pro-
cess can be explained if chlorosugar 7 first anomerises.
This anomerisation occurs readily with solvents of higher
dielectric constant and is further enhanced by the presence
of lithium, silver and to a lesser extent sodium ions.8,11 We
accord with our previous work with 5-aminoimidazoles,
the EMMN gave exclusively the C-addition-elimination
product 13,12 which is associated with a single imidazole
ring proton ( = 7.83 ppm) and a primary amine signal
( = 7.72 ppm). Treatment of this 2,2-dicyanovinyl deriv-
ative 13 with methanolic NaOH under reflux (15 min) re-
sulted in simultaneous cyclisation and deprotection giving
the imidazo[4,5-b]pyridine derivative 14 (84%).13 The
structure of nitrile 14 is fully supported by its spectroscop-
ic properties. This product (14), formed in three isolation
steps from the 4(5)-nitroimidazole cesium salt 6
(Scheme 1), is a key common intermediate for the prepa-
ration of PSNs.
1
observed by H NMR that over three hours (the time for
completion of these glycosylation reactions) the chloro-
sugar 7 in d3-acetonitrile underwent 40% inversion to the
more reactive11 -anomer, compared with less than 5%
anomerisation in d8-THF. We expected, therefore, that re-
placement of acetonitrile by less polar THF would favour
formation of -anomers (8 and 9) over -anomers (10 and
11) and this led us to investigate this solvent in combina-
tion with the cesium salt 6. We reasoned that use of the
cesium salt 6 would also lead to an improvement of the
/ ratio because Cs+ is less likely to co-ordinate to the
2 -chloro substituent and enhance anomerisation of the
chlorosugar 7. Glysosylation of cesium salt 6 with chloro-
sugar 7 for three hours at 62 °C in THF gave a 71% iso-
lated yield of the required pure 5-nitro-1 - -isomer 8.
Clearly, use of the cesium salt not only improves the
anomeric ratio ( / ) but also improves the regioselectivity
(8/9) of the reaction. This preparation is amenable to
scale-up giving gram quantities of compound 8 in consis-
tent yields of at least 70%.
The imidazo[4,5-b]pyridine 14 was converted to strA (1)
without isolation of intermediates by the following proce-
dure. A suspension of compound 14 in excess diethoxy-
methyl acetate was heated under reflux (2 h). Evaporation
gave a light brown oil (O-acetylimidate), which was im-
mediately treated with methanolic ammonia (16 h). Re-
moval of the solvent gave a residue (formamidine) that
was treated with aqueous HOAc (16 h) and neutralized to
give a crystalline product. This was recrystallised from
H2O and identified as strA (1, 47%).14 The nitrile 14 was
converted to strD (15) in a single step. Guanidine hydro-
chloride was converted to the free base by stirring with
NaOMe in MeOH. One equivalent of nitrile 14 was added
and the mixture heated in a steel bomb at 145 °C for 42 h.
The solid product was filtered off and washed with H2O
and then with EtOH to yield strD (15, 45%).15 Finally, the
nitrile 14 was converted to strH (17, Scheme 2). Hydroly-
sis using H2O2/NH4OH (20 °C, 40 min) gave the amide
16, which was not isolated but, after evaporation, was im-
mediately cyclised using ethyl formate and ethanolic
NaOEt at reflux temperature (2 h). The residue crystal-
lized from H2O (adjusted to pH 6 using 2 M aq HCl) and
The identities of all four isomers (8–11) were established
by 1H and 13C NMR analysis with 5-nitro-1 - (triplet,
=
6.74 ppm) and 5-nitro-1 - (doublet, = 6.80 ppm) ano-
meric proton signals being downfield relative to those of
the 4-nitro-1 - (triplet, = 6.15ppm) and 4-nitro-1 -
(doublet, = 6.25 ppm) signals. These structural assign-
ments were confirmed by NOESY. The strong NOE en-
hancements are shown in Figure 2.
Catalytic reduction (5% Pd/C–H2–THF) of the 5-nitroim-
idazole 8 gave the amine 12 which was immediately react-
ed with ethoxymethylene malononitrile (EMMN). In
Synlett 2002, No. 9, 1483–1486 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis of Pyridine-stretched 2 -Deoxynucleosides
1485
NC
NC
References
N
N
(1) Humphries, M. J.; Ramsden, C. A. Synthesis 1999, 985.
(2) Leonard, N. J.; Sprecker, M. A.; Morrice, A. G. J. Am.
Chem. Soc. 1976, 98, 3987.
(3) Gowers, D. M.; Fox, K. R. Nucleic Acids Res. 1999, 27,
1569.
Y
H2N
N
N
TolO
TolO
ii
O
O
(4) Fox, K. R. Curr. Med. Chem. 2000, 7, 17.
(5) Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc.
1995, 117, 3873.
TolO
TolO
13
8 Y = NO2
12 Y = NH2
i
iii
(6) Hoffer, M. Chem. Ber. 1960, 93, 2777.
NH2
(7) Compound 8 (70%): Mp 162 °C (colourless solid). IR
(KBr): max = 752, 1092, 1111, 1280, 1374, 1466, 1528,
1723, 2938 cm–1. 1H NMR (CDCl3): = 2.42 (3 H, s,
ArCH3), 2.46 (3 H, s, ArCH3), 2.52 (1 H, m, 2 -CH), 3.12
(1 H, m, 2 -CH), 4.74 (3 H, m, 5 -CH2 and 4 -CH), 5.64 (1 H,
m, 3 -CH), 6.74 (1 H, t, 1 -CH), 7.24 (2 H, d, ArH), 7.30
(2 H, d, ArH), 7.84 (2 H, d, ArH), 7.97 (2 H, d, ArH), 8.05
[1 H, s, imidazole(4)-H], 8.11 [1 H, s, imidazole(2)-H].
13C NMR (CDCl3): = 21.70 (q, ArCH3), 21.77 (q, ArCH3),
40.86 (t, 2 -CH2), 63.60 (t, 5 -CH2), 74.14 (d, 4 -CH), 83.94
(d, 3 -CH), 88.99 (d, 1 -CH), 126.04 [s, Ar(4)-C], 126.18 [s,
Ar(4)-C], 129.32 (d, Ar-CH), 129.39 (d, Ar-CH), 129.55 (d,
Ar-CH), 129.81 (d, Ar-CH), 134.33 [d, imidazole(4)-CH],
138.05 [v weak s, imidazole(5)-C], 138.13 [d, imidazole(2)-
CH], 144.50 [s, Ar(1)-C], 144.70 [s, Ar(1)-C], 165.92 (s,
C=O), 166.07 (s, C=O). MS (EI): m/z (%) = 465 (2) [M+],
320 (1), 216 (28), 136 (28), 119 (100), 91 (53), 81 (93), 65
(18), 53 (14), 39 (13), 28 (13). Anal. Calcd for C24H23N3O7:
C, 61.9%; H, 4.98%; N, 9.0%. Found: C, 61.7%; H, 4.89%;
N, 8.8%.
NC
H2N
N
N
N
Y
N
N
N
iv,v
N
N
HO
HO
vi
O
O
HO
HO
O
14
1 Y = H
15 Y = NH2
vii
O
C
H2N
HN
N
N
H2N
HO
N
N
viii
N
N
N
HO
O
O
HO
(8) Štimac, A.; Kobe, J. Carbohydr. Res. 2000, 329, 317.
(9) Bergstrom, D. E.; Zhang, P.; Johnson, W. T. Nucleic Acids
Res. 1997, 25, 1935.
(10) Kazimierczuk, Z.; Cottam, H. B.; Revankar, G. R.; Robins,
R. K. J. Am. Chem. Soc. 1984, 106, 6379.
HO
16
17
Scheme 2 Reagents and conditions: i, H2, 5% Pd/C, THF, 2 h; ii,
EtOCH=C(CN)2; iii, MeOH–NaOH (aq), 90 °C, 15 min; iv,
MeCO2CH(OEt)2, reflux, 2 h; v, MeOH–NH3; vi, HN=C(NH2)2,
145 °C, 42 h; vii, H2O2/NH4OH, 20 °C, 40 min; viii, HCO2Et–EtOH–
NaOEt, reflux, 2 h.
(11) Hubbard, A. J.; Jones, A. S.; Walker, R. T. Nucleic Acids
Res. 1984, 12, 6827.
(12) Compound 13 (43%): Mp 194–196 °C (yellow needles). IR
(KBr): max = 752, 1112, 1266, 1283, 1354, 1540, 1586,
1715, 2218, 2924, 3348 cm–1. 1H NMR (d6-DMSO): = 2.39
(3 H, s, ArCH3), 2.41 (3 H, s, ArCH3), 2.72 (1 H, m, 2 -CH),
2.86 (1 H, m, 2 -CH), 4.53 (3 H, br s, 5 -CH2 and 4 -CH),
5.63 (1 H, d, 3 -CH), 6.09 (1 H, dd, 1 -CH), 7.34 (2 H, d,
ArH), 7.38 (2 H, d, ArH), 7.72 (2 H, br s, NH2), 7.79 (1 H, s,
C=CH), 7.83 [1 H, s, imidazole(2)-H], 7.86 (2 H, d, ArH),
7.95 (2 H, d, ArH). 13C NMR (d6-DMSO): = 21.14 (q,
ArCH3), 21.17 (q, ArCH3), 35.88 (t, 2 -CH2), 58.63 [s,
C(CN)2], 63.96 (t, 5 -CH2), 74.72 (d, 4 -CH), 81.69 (d, 3 -
CH), 82.81 (d, 1 -CH), 116.20 [s, imidazole(4)-C], 117.99
(s, CN), 118.56 (s, CN), 126.43 [s, 2 Ar(4)-C], 129.26 (d,
was identified as strH (17, 44%).16 All three pyridine-
stretched 2 -deoxyribonucleosides (1, 15 and 17) were
fully characterized by their spectroscopic properties
which are entirely consistent with the proposed stereo-
and regio-chemistry.14–16
In conclusion, we have described a viable synthetic route
to ‘pyridine-stretched’ nucleoside analogues via the 1 - -
2 -deoxy-5-nitroimidazole derivative 8. For 2 -deoxy nu-
cleoside analogues a substituent at position 2 cannot be
used to control the stereochemistry at the anomeric posi-
tion and we have therefore investigated reaction condi-
tions with the objective of optimising formation of the
desired stereoisomer 8. We have found that the imidazole
8 can be obtained selectively in >70% yield by using the
cesium salt of 4(5)-nitroimidazole and THF as solvent.
This product 8 is then converted in two isolation steps to
the key intermediate 14, which is then readily transformed
into tricyclic 2 -deoxynucleoside analogues.
2
Ar-CH), 129.32 (d, Ar-CH), 129.47 (d, Ar-CH), 134.13
[d, imidazole(2)-CH], 143.53 (d, C=CH), 143.84 [s, Ar(1)-
C], 144.08 [s, Ar(1)-C], 150.02 [s, imidazole(5)-C], 165.12
(s, C=O), 165.40 (s, C=O). MS (EI): m/z (%) = 511 (7) [M+],
353 (7), 159 (41), 119 (68), 91 (23), 81 (100).
(13) Compound 14 (84%): Mp 162 °C (colourless needles). IR
(KBr): max = 940, 1099, 1432, 1576, 1630, 2216, 2923,
3223, 3337 cm–1. 1H NMR (d6-DMSO): = 2.23 (1 H, m, 2 -
CH), 2.59 (1 H, m, 2 -CH), 3.52 (2 H, m, 5 -CH2), 3.83 (1 H,
d, 4 -CH), 4.37 (1 H, s, 3 -CH), 4.96 (1 H, t, 5 -OH), 5.32
(1 H, d, 3 -OH), 6.29 (1 H, t, 1 -CH), 6.79 (2 H, br s, NH2),
8.24 (1 H, s, pyridine-H), 8.42 [1 H, s, imidazole(2)-H].
13C NMR (d6-DMSO): = 39.51 (t, 2 -CH2), 61.89 (t, 5 -
CH2), 70.99 (d, 4 -CH), 82.71 (d, 3 -CH), 86.26 (s, C.CN),
87.89 (d, 1 -CH), 118.02 (s, CN), 127.11 (s, C), 134.37 [d,
imidazole(2)-CH], 142.29 (d, pyridine-CH), 148.96 (s, C),
157.84 (s, C). MS (EI): m/z (%) = 275 (84) [M+], 186 (100),
Acknowledgement
We thank Scotia Pharmaceuticals for a studentship (to RC), the
Royal Society for a grant (to WF), and the EPSRC Mass Spectro-
metry Centre, Swansea, for high-resolution mass spectra.
Synlett 2002, No. 9, 1483–1486 ISSN 0936-5214 © Thieme Stuttgart · New York

1486
R. Clayton et al.
LETTER
160 (10 0), 159 (100), 132 (35), 117 (63), 99 (32), 73 (50),
45 (39), 43 (36), 28 (26). HRMS (EI): m/z = calcd for
C12H13N5O3: 275.1018. Found: 275.1008 [M+].
5.37 (1 H, d, 3 -OH), 6.19 (2 H, br s, NH2), 6.42 (1 H, t, 1 -
CH), 7.44 (2 H, br s, NH2), 8.64 (1 H, s, 6-H), 8.83 (1 H, s,
9-H). 13C NMR (d6-DMSO): = 39.17 (t, 2 -CH2), 62.08 (t,
5 -CH2), 71.17 (d, 4 -CH), 83.84 (d, 3 -CH), 88.01 (d, 1 -
CH), 102.19 (s, C), 123.83 (d, CH), 131.33 (s, C), 145.35 (d,
CH), 150.76 (s, C), 158.27 (d, CH), 162.78 (s, C), 164.21 (s,
C). MS (FAB): m/z (%) = 318 (91) [M + H], 202 (100), 141
(15), 121 (9). HRMS-FAB: m/z calcd for C13H15N7NaO3:
340.1134. Found: 340.1129 [M+ + Na].
(14) Compound 1 (47%). Mp >280 °C (colourless needles). IR
(KBr): max = 812, 916, 1091, 1419, 1507, 1577, 1685, 3086
cm–1. 1H NMR (d6-DMSO): = 2.36 (1 H, m, 2 -CH), 2.83
(1 H, m, 2 -CH), 3.63 (2 H, m, 5 -CH2), 3.93 (1 H, m, 4 -CH),
4.49 (1 H, s, 3 -CH), 5.15 (1 H, t, 5 -OH), 5.40 (1 H, d, 3 -
OH), 6.74 (1 H, t, 1 -CH), 8.09 (2 H, br s, NH2), 8.51 (1 H,
s, 2-H), 8.92 (1 H, s, 6-H), 9.07 (1 H, s, 9-H). 13C NMR (d6-
DMSO): = 39.44 (t, 2 -CH2), 61.95 (t, 5 -CH2), 71.06 (d,
4 -CH), 83.81 (d, 3 -CH), 88.19 (d, 1 -CH), 106.43 (s, C),
123.80 (d, CH), 134.68 (s, C), 148.25 (d, CH), 151.01 (s, C),
155.73 (s, C), 157.87 (d, CH), 164.11 (s, C). MS (FAB):
m/z (%) = 303 (53) [M + H), 187 (31), 165 (30), 152 (46),
124 (36), 120 (46), 115 (35), 107 (100), 105 (43). HRMS-
FAB: m/z calcd for C13H15N6O3: 303.1206. Found:
303.1209 [M+ + H].
(16) Compound 17 (44%). Mp >280 °C (cream solid). IR (KBr):
max = 807, 1086, 1235, 1392, 1605, 1686, 2925, 3214, 3504
cm–1. 1H NMR (d6-DMSO) = 2.34 (1 H, m, 2 -CH), 2.81
(1 H, m, 2 -CH), 3.62 (2 H, m, 5 -CH2), 3.93 (1 H, d, 4 -CH),
4.49 (1 H, br s, 3 -CH), 5.10 (1 H, t, 5 -OH), 5.41 (1 H, d, 3 -
OH), 6.58 (1 H, t, 1 -CH), 8.32 (1 H, s, 2-H), 8.75 (1 H, s,
6-H), 8.95 (1 H, s, 9-H). 13C NMR (d6-DMSO): = 45.58 (t,
2 -CH2), 67.94 (t, 5 -CH2), 77.05 (d, 4 -CH), 89.99 (d, 3 -
CH), 94.28 (d, 1 -CH), 120.59 (s, C), 132.26 (d, CH), 140.99
(s, C), 153.83 (d, CH), 156.48 (s, C), 161.15 (s, C), 168.41
(s, C=O). MS (FAB): m/z (%) = 304 (11) [M + H], 216 (16),
119 (43), 114 (22), 87 (41), 82 (100). HRMS-FAB: m/z calcd
for C13H13N5O4: 304.1046. Found: 304.1041 [M + H].
(15) Compound 15 (45%). Mp >280 °C (cream solid). IR (KBr):
max = 803, 1057, 1356, 1470, 1621, 1641, 2887, 3110, 3183,
3318, 3434, 3481 cm–1. 1H NMR (d6-DMSO): = 2.23 (1 H,
m, 2 -CH), 2.81 (1 H, m, 2 -CH), 3.61 (2 H, m, 5 -CH2), 3.88
(1 H, m, 4 -CH), 4.44 (1 H, m, 3 -CH), 5.17 (1 H, t, 5 -OH),
Synlett 2002, No. 9, 1483–1486 ISSN 0936-5214 © Thieme Stuttgart · New York