The synthesis of bicyclic N4-amino-2'-deoxycytidine derivatives

Article abstract of DOI:10.1002/(SICI)1522-2675(19991110)82:11<2028::AID-HLCA2028>3.0.CO;2-E

Nucleosides which have ambivalent tautomeric properties have value in a variety of nucleic acid hybridization applications, and as mutagenic agents. We describe here synthetic studies directed to stable derivatives of this kind of nucleoside based on N

Full text of DOI:10.1002/(SICI)1522-2675(19991110)82:11<2028::AID-HLCA2028>3.0.CO;2-E

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Helvetica Chimica Acta ± Vol. 82 (1999)
The Synthesis of Bicyclic N4-Amino-2'-deoxycytidine Derivatives
1
by David Loakes, Rita Bazzanini ), and Daniel M. Brown*
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, CB22QH, UK
(Tel: (‡ 44) 1223248011; Fax: (‡ 44) 1223412178; e-mail: dmb@mrc-lmb.cam.ac.uk)
Dedicated to Prof. Dr. Frank Seela on the occasion of his 60th birthday
Nucleosides which have ambivalent tautomeric properties have value in a variety of nucleic acid
hybridization applications, and as mutagenic agents. We describe here synthetic studies directed to stable
derivatives of this kind of nucleoside based on N4-aminocytosine. Treatment of the 4-(1H-1,2,4-triazol-1-yl)-5-
(chloroethyl)pyrimidinone nucleoside derivative 5 with hydrazine leads to formation of the 6,6-bicyclic
pyrimido-pyridazin-7-one 3, and with methylhydrazine to the corresponding fixed tautomeric 1-methyl
derivative 7 (Scheme 1). If these cyclization reactions are carried out in the presence of a base, the 6-ring bicyclic
derivatives undergo rearrangement to their corresponding 5-ring pyrrolo-pyrimidin-2-one analogues
8
(Scheme 2). In the reaction of the triazolyl derivative 5 with 1-[(benzyloxy)carbonyl]-1-methylhydrazine,
spontaneous cyclization gives the 5-ring derivative 13 related to 8 rather than the open-chain product 12
(Scheme 4). Reaction of an acetylated analogue of triazolyl derivative 5 with 1,1-dimethylhydrazine gives rise to
some of the open-chain product 9, but it too cyclizes to a product that we have assigned the structure of the 6,6-
ring quaternary ammonium salt 11 (Scheme 3).
Introduction. ± Nucleosides which are capable of base-pairing with more than one
of the natural DNA/RNA bases are mutagenic. Such analogues are of use not only to
explore aspects of chemical mutagenesis [1][2], but also as tools in molecular biology.
We have extensively examined the mutagenic nucleoside 1, which behaves as both
thymidine and deoxycytidine. Its 5'-triphosphate has been used for random muta-
genesis [3][4], whilst in oligonucleotides, it has been used in primers for PCR [5] and to
study H-bonding patterns in DNA duplexes [6][7]. The analogue, however, does not
behave indiscriminately as either T or C, but has a bias towards T. For this reason, we
have for some time been examining alternative analogues that may shift this balance.
The 5,6-ring (ribo) analogue 2 was prepared [8] in the expectation that the smaller ring
size might have an effect on the tautomeric ratio. This, whilst appearing to behave more
as a cytidine analogue in its 1H-NMR spectrum, proved to be too unstable to investigate
further.
N4-Hydroxycytosine derivatives have tautomeric constants (KT) of the order of 10
with the imino (thymine-like) form predominating [9]. N4-Aminocytosine derivatives
exist predominately in the amino form, with KT of around 30 in H2O. Whilst these
compounds are highly mutagenic in vivo, N4-(alkylamino)cytosine derivatives are
significantly less mutagenic [10]. This suggests that bicyclic analogues should show
ambivalent base-pairing, but be less potent chemical mutagens. We, therefore, chose to
1
)
Present address: Consiglio Nazionale delle Recerche, I.Co.C.E.A. Area della Ricerca, Via P. Gobetti, 101,
I-40129, Bologna.

Helvetica Chimica Acta ± Vol. 82 (1999)
2029
investigate N4-amino bicyclic analogues, such as 3. The parent bicyclic compound 3
proved unstable; presumably it is susceptible to aerial oxidation. Therefore, we have
attempted to prepare alkylated derivatives in the expectation that they would be stable,
and that such compounds could then be used to investigate their ambivalent H-bonding
behaviour in oligonucleotides.
Results and Discussion. ± The 5-(2-chloroethyl)-2'-deoxyuridine derivative 4 [11]
was converted to its C4-triazolyl derivative 5, which was then subjected to a series of
reactions with various hydrazines. Thus, reaction with anhydrous hydrazine led to the
rapid displacement of the C4-triazolyl group, followed by a slower cyclization with the
5-(2-chloroethyl) group to give the bicyclic product 3 (Scheme 1). The product is rather
unstable, readily degrading to a number of products, even if stored at 48. Treatment of
the C4-triazolyl derivative 5 with methylhydrazine under similar conditions led to an
essentially single product, though in a slower reaction. Although in equivalent
displacement reactions the N-atom carrying the Me group is the more nucleophilic, we
hoped to obtain some of the desired 6 [10]. The structure of the 3',5'-di-O-acetyl rather
than 3',5'-di-O-toluoyl derivative was elucidated by NOE experiments (no NOE on
irradiation of MeN (3.14 ppm), irradiation of CH2N (t, 2.97 ppm) ! enhancement of
the exchangeable signal and NOE at CH2(4) (2.51 ppm)). From this data, we deduced
that the structure was not that of 6, but of the regioisomer 7.
It was observed that if the above cyclization reactions were carried out in the
presence of a base, a second minor product was also formed (this product was also
slowly formed on standing, particularly in solution). These minor products were rather
difficult to separate from the first-formed 6,6-bicyclic products 3 and 7. Therefore, 3 and
7 were treated with Et3N or pyridine whereupon each was converted into this second
product 8a and 8b, respectively, the methylated derivative 7 rearranging much slower
than 3 (Scheme 2). Thus, the transformation of 3 in pyridine at 508 was complete after
16 h, whereas, under the same conditions, 7 had only reacted to ca. 50%. The
rearrangement also occurred in 2,6-lutidine. The structures of 8a,b were established as
the 5-membered ring isomers from their 1H-NMR spectra. The isomer 8a was further
characterized by conversion to its crystalline hydrazone with benzaldehyde, thus
confirming the presence of the free NH2 group.
The product 8a, derived from 3, showed a s (2 H) at 4.82 ppm for an unsubstituted NH2 group. For the Me-
substituted product 8b, MeN group was a d (3.34 ppm), whilst the NH proton was a q (5.30 ppm). The MeN d
collapsed to a s in the presence of D2O.

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Helvetica Chimica Acta ± Vol. 82 (1999)
Scheme 1
Scheme 2
To investigate the formation of the 5-membered ring bicyclic analogues further, the
3',5'-di-O-acetyl-substituted analogue of 5 was reacted with 1,1-dimethylhydrazine
(Scheme 3). This was expected to give 9, which we then planned to use to examine
whether cyclization would occur leading to the 5-ring 10. Displacement of the triazolyl
group of the di-O-acetyl analogue of 5 by 1,1-dimethylhydrazine in tetrachloroethane at
1008 overnight gave the expected product 9 in low yield, besides a major product which
was polar, water-soluble, and very difficult to isolate in pure form. In an earlier

Helvetica Chimica Acta ± Vol. 82 (1999)
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Scheme 3
preliminary report [12], we suggested that this product was the quaternary ammonium
salt 11. This assignment was based on the fact that the product is water-soluble and polar:
‡
it has a mass spectrum with M at m/z 381 and a 1H-NMR spectrum corresponding to
the proposed structure 11. Isolation of the pure product has been attempted by a variety
of methods including ion-exchange and reversed-phase HPLC, but has so far eluded us
and is the subject of further work. It is anticipated that we may be able to demethylate
the purified product to give the desired bicyclic di-O-acetyl analogue of 6. Interestingly,
we have no evidence, despite attempting the reaction many times under a variety of
conditions, that ring contraction to the 5-ring analogue 10 occurred.
‡
The 1H-NMR spectrum of the salt 11 shows a s (6 H) at 3.15 ppm for Me2N , whereas for the
dimethylhydrazine product 9, there is a s (6 H) at 2.50 ppm. This is consistent with the change in chemical shift
‡
for Me2N to Me2N . Thus, the data are entirely consistent with the structure of 11 being the 6-ring quaternary
ammonium derivative.
As the methylamino residue of methylhydrazine is evidently the more nucleophilic
group [13], it was decided to use a protected methylhydrazine derivative as an
alternative route to produce the analogue 6. Once deprotected, the methylamino
residue could undergo cyclization with the 5-(chloroethyl) side chain. Thus, reaction of
the triazolyl derivative 5 with 1-[(benzyloxy)carbonyl]-1-methylhydrazine [14] gave a
product that we initially believed to be the expected compound 12 in a slow reaction
(Scheme 4). On this assumption, we reductively removed the (benzyloxy)carbonyl (Z)
group using either Pt or Pd in MeOH. However, the product that we obtained was not
the desired product but again the 5-membered-ring derivative 8b, as established by its
1H-NMR spectrum, and comparison with the product 8b obtained by ring contraction
from 7 (see above). We therefore assumed that the product had preferentially cyclized
or rearranged with N4 as the (benzyloxy)carbonyl protecting group was being removed.
Thus, the reduction was carried out in acid (AcOH or 0.1m HCl), as we hoped that
protonation of the amino group would prevent ring closure after removal of the Z
protecting group. This would then enable cyclization to occur to give the desired
regiospecific product following neutralization. However, the product 14 obtained was
again a 5-ring derivative related to 8; moreover, under these conditions, the
methylamino group had been reductively cleaved (Scheme 4).
Subsequently, we found that the intermediate (Z-protected) reaction product,
initially assigned the structure 12, was in fact the cyclized reaction product 13 (see

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Helvetica Chimica Acta ± Vol. 82 (1999)
Scheme 4
Scheme 4). This was confirmed by the MS of 13, which showed an ion at m/z 675 ([M ‡
‡
Na] ), corresponding to the loss of HCl from 12. Thus cyclization must have occurred
after displacement of the triazole moiety and prior to reduction. The open-chain
intermediate 12 was never observed in this reaction despite using a variety of
alternative reaction conditions. The formation of the 5-membered ring bicyclic product
is surprising. In this connection, when the triazole 5 was treated with ammonia, the
cytidine derivative 15 was formed which did not undergo cyclization (Scheme 5).
Neither did the N-hydroxycytidine derivative 16 [15] cyclize to give the corresponding
bicyclic product. The presence of the Z group at the methylhydrazine renders it
particularly unreactive, both in terms of the initial nucleophilic displacement of the C4-
triazolyl group and towards further cyclization.
Scheme 5
It is of interest to speculate on the nature of the formation of the bicyclic compound
13. We have previously experienced the fact that the chloroethyl side chain is
particularly unreactive towards nucleophilic displacement reactions, except for intra-
molecular cyclization. Therefore, the formation of 13 probably does not arise by first
displacing chloride, followed by cyclization. However, the cyclization of the chloro-
ethyl group to form a bicyclic compound is, nevertheless, a slow reaction, compared
to the nucleophilic displacement of the C4-triazolyl group. Although the Z-protect-
ed hydrazine is particularly unreactive, it appears that, once the initial reaction to
displace the triazole moiety has occurred, cyclization with the chloroethyl group is
spontaneous.

Helvetica Chimica Acta ± Vol. 82 (1999)
2033
Lastly, we turn to the question of the mechanism whereby the 6,6-ring hydrazine
derivatives of type 7 rearrange to 5,6-ring isomers, carrying an N4-amino function in the
cytosine moiety (see Scheme 2). The evidence is strong that the first-formed products
are the 6,6-ring bicyclic compounds of type 7 and result from a displacement first of the
triazolyl residue of 5 followed by ring closure. Rearrangement is base-catalysed and
occurs in the cases where the N4-amino N-atom carries a proton. Two mechanisms
suggest themselves (Scheme 6). In the first, an intramolecular displacement at C(4) by
the N4-amino group occurs, via a strained transition entity 17. Alternatively, if the base
is acting as a nucleophile, an intermediate 18 may be postulated which would lead in a
second displacement reaction to the thermodynamically and kinetically favoured 5-
membered ring product. The fact that the rearrangement occurs in 2,6-lutidine strongly
suggests that the first mechanism is the more likely route.
Scheme 6
Experimental Part
General. Unless otherwise stated, reactions were worked up as follows: After removal of the solvent, the
product was dissolved in CHCl3 and washed with aq. sodium hydrogen carbonate soln. The combined org.
fractions were dried (Na2SO4) and evaporated. TLC: pre-coated F254 silica gel plates. Column chromatography
(CC): Merck silica gel 60 or reversed-phase column LiChroprep RP-18 (Merck). M.p.: Gallenkamp melting
point apparatus; uncorrected. UV Spectra: Perkin-Elmer-Lambda-2 spectrophotometer; in 10% MeOH/H2O
1
unless otherwise stated; lmax (e) in nm. H-NMR Spectra: Bruker DRX 300; in (D6)DMSO, unless otherwise
stated; d in ppm, J values in Hz. NOE Experiments: Bruker-AMX-500 spectrometer. Mass spectra: Kratos
MS890; in m/z (rel. %).
5-(2-Chloroethyl) 1-[2-deoxy-(3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl]-4-(1H-1,2,4-triazol-1-yl)pyrimidin-
1(1H)-one (5). To a suspension of 1H-1,2,4-triazole (4.72 g, 68.3 mmol) in dry MeCN (100 ml) at 08,
phosphoric trichloride (1.27 ml, 13.7 mmol) was added dropwise, and the mixture was stirred at 08 for 15 min.
After this time, dry Et3N (11.5 ml, 82 mmol) was added and the mixture stirred at 08 for a further 20 min, then at
r.t. for 10 min. A soln. of 5-(2-chloroethyl)-1-[2-deoxy-3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl]pyrimidine-2,4-
(1H,3H)-dione [11] (2.4 g, 4.5 mmol) in dry MeCN (10 ml) and DMF (10 ml) was then added dropwise with
vigorous stirring. The mixture was kept under Ar at r.t. overnight and then evaporated. The residue was
dissolved in CHCl3, the soln. washed with aq. NaHCO3 soln., dried, and evaporated, and the orange syrup
purified by CC (silica gel, AcOEt/hexane 1:1): 2.27 g (86%) of 5. White solid. UV: 323 (5200), 242 (33200),
min. 216 (14200). M.p. 155 ± 1578. 1H-NMR: 2.28 (s, MeC6H4); 2.39 (s, MeC6H4); 2.59 ± 2.68 (m, 1 H C(2'));
2.87 ± 3.17 (m, 1H C(2'), CH2CH2Cl); 3.57 (t, J ˆ 7.1, CH2CH2Cl2); 4.56 ± 4.74 (m, H C(4'), 2 H C(5'));
5.62 ± 5.65 (m, H C(3')); 6.28 (t, J ˆ 6.5,
H
C(1')); 7.22, 7.36, 7.76, 7.93 (4 d, 8 arom. H (Tol)); 8.37
‡
(s, H C(6)); 8.40 (s, CH(triazole)); 9.36 (s, CH (triazole)). FAB-MS: 578.8 ([M ‡ H] ). HR-MS: 578.18268
([M ‡ H] , C29H29N5O635Cl ; calc. 578.18066; deviation 3.50 ppm).
‡
‡

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Helvetica Chimica Acta ± Vol. 82 (1999)
6-[2-Deoxy-3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl]-2,3,4,6-tetrahydropyrimido[4,5-c][1,2]pyridazin-7(1H)-
one (3). To a soln. of 5 (0.25 g, 0.43 mmol) in dry MeCN (10 ml), anh. hydrazine (20 ml, 0.64 mmol) was added,
and the soln. was stirred at r.t. for 2 h. The soln. was concentrated and chromatographed (5% MeOH/CHCl3):
0.19 g (87%) of 3. White solid. UV: 278 (10900), 244 (33300), min. 269 and 218; pH 1: 274 (10600), 240 (31850),
min. 269 and 233; pH 12: 274 (10500), 240 (31750), min. 268 and 233. 1H-NMR: 2.37 (s, MeC6H4); 2.38
(s, MeC6H4); 2.43 ± 2.63 (m, CH2, 2 H C(2')); 3.32 (br. s, NH); 3.49 ± 3.69 (m, CH2N); 4.44 ± 4.48
(m, H C(4')); 4.50 ± 4.63 (m, 2 H C(5')); 5.56 (br. s, H C(3')); 6.28 ± 6.37 (m,H C(1')); 7.10 (s, H C(5));
‡
7.31 ± 7.36 (m, 4 arom. H (Tol)); 7.86 ± 8.31 (m, 4arom. H (Tol)); 9.64 (br. s, NH). FAB-MS: 505.9 ([M ‡ H] ).
‡
‡
HR-MS: 505.20813 ([M ‡ H] , C27H29N4O6 ; calc. 505.20871; deviation 1.10 ppm).
6-[2-Deoxy-3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl]-2,3,4,6-tetrahydro-1-methylpyrimido[4,5-c][1,2]pyrida-
zin-7(1H)-one (7). To a soln. of 5 (0.25 g, 0.43 mmol) in MeCN (10 ml), methylhydrazine (35 ml, 0.66 mmol)
was added and the soln. stirred at r.t. for 8 h. The soln. was evaporated and the residue chromatographed (5%
MeOH/CHCl3): 0.19g (85%) of 7. Off-white foam. UV 285 (12100), 243 (38700), min. 218 and 273; pH 1: 303
(14950), 244 (41700), min. 216 and 269; pH 12: 285 (13200), 239 (37600), min 229 and 266. 1H-NMR: 2.22 ± 2.32
(m, 2H C(4)); 2.37 (s, MeC6H4); 2.38 (s, MeC6H4); 2.37 ± 2.54 (m, 2H C(2')); 2.88 (m, CH2N); 3.12 (s, MeN);
4.46 ± 4.48 (m, H C(4')); 4.51 ± 4.65 (m, 2 H C(5')); 5.52 (t, J ˆ 6.9, NH); 5.56 ± 5.59 (m, H C(3')); 6.33 (t, J ˆ
6.3, H C(1')); 7.31 ± 7.37 (m, 4 arom. H (Tol)); 7.34 (s, H C(5)); 7.86-7.92 (m, 4 arom. H (Tol)). FAB-MS:
‡
‡
‡
519.8 ([M ‡ H] ). HR-MS: 519.22802 ([M ‡ H] , C28H31N4O6 ; calc. 519.22437; deviation 7.0 ppm).
In the same manner the 3',5'-di-O-acetyl instead of 3',5'-di-O-toluoyl derivative was prepared. White foam.
UV: 292, 229, min. 250; pH 1: 305, min. 252. 1H-NMR: 2.06 (s, Ac); 2.07 (s, Ac); 2.24 ± 2.30 (m, 2 H C(2')); 2.51
(m, 2 H C(4)); 2.97 (t, J ˆ 5.7, CH2N); 3.14 (s, MeN); 4.12 ± 4.17 (m, H C(4')); 4.22 ± 4.25 (m, 2 H C(5'));
5.15 ± 5.19 (m, H C(3')); 5.59 (t, J ˆ 5.7, NH); 6.24 (t, J ˆ 6.7, H C(1')); 7.38 (s, H C(5)). EI-MS: 366 ([M ‡
‡
‡
‡
H] ). HR-MS: 366.1572 ([M ‡ H] , C16H22N4O6 ; deviation 3.2 ppm).
7-Amino-3-[2-deoxy-3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl]-3,5,6,7-tetrahydro-2H-pyrrolo[2,3-d]pyrimidin-
2-one (8a). A soln. of 3 (200 mg, 0.6 mmol) in MeCN (10 ml) and Et3N (1 ml) was heated under reflux
overnight. Alternatively, a soln. of 3 was stirred in pyridine at r.t. overnight. After evaporation, the product was
chromatographed (5% MeOH/CHCl3): 146 mg (73%) of 8a. White foam. UV: 283 (10400), 245 (32000), min.
270 and 215; pH 1: 286 (11100), 244 (29800), min. 270 and 215. 1H-NMR: 2.37 (s, MeC6H4); 2.38 (s, MeC6H4);
2.44 ± 2.58 (m, 2 H C(2'), CH2); 3.58 (t, J ˆ 7.5, CH2N); 4.22 ± 4.23 (m, H C(4')); 4.50 ± 4.63 (m, 2 H C(5'));
4.82 (s, NH2); 5.54 ± 5.56 (m, H C(3')); 6.36 (t, J ˆ 7.8, H C(1')); 7.27 (s, H C(4)); 7.32 ± 7.36 (m, 4 arom. H
‡
‡
(Tol)); 7.86 ± 7.91 (m,4 arom. H (Tol)). FAB-MS: 505.9 ([M ‡ H] ), 527.9 ([M ‡ Na] ). HR-MS: 527.18983
‡
‡
([M ‡ Na] , C27H28N4O6Na ; calc. 527.19067; deviation 1.60 ppm).
Benzaldehyde Hydrazone of 8a: After treatment of 8a with benzaldehyde in CH2Cl2, the soln. was
evaporated and the residue chromatographed (2% MeOH/CHCl3): pale yellow solid which recrystallized from
EtOH. M.p. 200 ± 2028. UV: 327 (23900), 240 (21200), min. 272 and 222; pH 1: 334 (17800), 247 (19600), min.
1
296 and 226; pH 12: 328 (24200), 240 (21000), min. 272. H-NMR: 2.35 (s, MeC6H4); 2.39 (s, MeC6H4); 2.43 ±
2.63 (m, 2 H C(2')); 2.71 ± 2.88 (m, CH2); 3.94 ± 4.00 (m, CH2(6)), 4.51 ± 4.58 (m, H C(4')); 4.59 ± 4.67
(m, 2 H C(5')); 5.57 ± 5.60 (m, H C(3')); 6.36 (t, J ˆ 7.5, H C(1')); 7.31 ± 7.37 (m, 4 arom. H (Tol)); 7.40 ±
7.47 (3 H, m, 3 arom. H (Ph)); 7.63 (s, H C(4)); 7.74 (1 H, s, 1 arom. H (Ph)); 7.77 (s, NˆCH); 7.90
‡
(s, 1 arom. H(Ph)); 7.86 ± 7.95 (m, 4 arom. H (Tol)). FAB-MS: 593.24 ([M ‡ H] ). HR-MS: 593.23926 ([M ‡
‡
‡
H] , C34H33N4O6 ; calc. 593.23999; deviation 1.20 ppm).
3-[2-Deoxy-3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl]-3,5,6,7-tetrahydro-7-(methylamino)-2H-pyrrolo[2,3-
d]pyrimidin-2-one (8b) (Method A). A soln. of 7 (150 mg, 0.29 mmol) in MeCN (10 ml) and Et3N (0.5 ml) was
heated under reflux overnight. After evaporation, the product was chromatographed (5% MeOH/CHCl3) to
give a white solid which was recrystallized from EtOH: 124 mg (83%) of 8b. UV: 283 (10800), 244 (31900), min.
269 and 217; pH 1: 293 (12800), 244 (32900), min. 268 and 216; pH 12: 283 (11900), 240 (31700), min. 265 and
229. M.p. 175 ± 1768. 1H-NMR: 2.37 (s, MeC6H4); 2.38 (s, MeC6H4); 2.49 ± 2.65 (m, 2 H C(2'), CH2(5)); 3.34
(d, MeN, s after D2O wash); 3.59 (t, J ˆ 7.5, CH2(6)); 4.43 ± 4.63 (m, H C(4'), 2 H C(5')); 5.30 (q, J ˆ 5.7,
exchangeable NH); 5.50 ± 5.60 (m, H C(3')); 6.34 (t, J ˆ 7.6, H C(1')); 7.31 ± 7.36 (m,H C(4), 4 arom. H
‡
‡
(Tol)); 7.85 ± 7.91 (m, 4 arom. H (Tol)). FAB-MS: 519.3 ([M ‡ H] ). HR-MS: 519.22380 ([M ‡ H] ,
‡
C28H31N4O6 ; calc. 519.22437; deviation 1.10 ppm). Anal. calc. for C28H30N4O6: C 64.9, H 5.8, N 10.8; found:
C 64.93, H 5.87, N 10.80.
6-(3,5-Di-O-acetyl-2-deoxy-b-d-ribofuranosyl)-1,2,3,4,6,7-hexahydro-2,2-dimethyl-7-oxopyrimido[4,5-
c][1,2]pyridazinium (11). To a soln. of the 3',5'-di-O-acetyl-substituted analog of 5 [16] (1 g, 1.7 mmol) in
tetrachloroethane (25 ml), 1,1-dimethylhydrazine (0.4 ml, 5.3 mmol) was added and the soln. heated at 1008
overnight (TLC: two main products, one with Rf 0). The product was extracted (H2O/CHCl3), and each of the

Helvetica Chimica Acta ± Vol. 82 (1999)
2035
two layers was evaporated. The org. layer was chromatographed (5% MeOH/CHCl3) to give an off-white foam,
which was characterized as 5-(2-chloroethyl)-1-(3,5-di-O-acetyl-2-deoxy-b-d-ribofuranosyl)-N4-(dimethylami-
1
no)cytosine (9; 0.32 g, 33%). Off-white foam. H-NMR: 2.06 (s, Ac); 2.07 (s, Ac), 2.22 ± 2.36 (m, 2 H C(2'));
2.50 (s, Me2N); 2.95 ± 3.05 (m, CH2); 3.14 (s, CH2Cl); 4.12 ± 4.19 (m, H C(4')); 4.23 ± 4.25 (m, 2 H C(5'));
‡
5.14 ± 5.19 (m, H C(3')); 5.59 (br. s, NH); 6.24 (t, J ˆ 6.7, H C(1')); 7.38 (s, H C(6)). EI-MS: 417 (M ).
The aq. layer was evaporated and purified by CC (reversed-phase C-18 silica gel, H2O ! 25% MeOH/
1
H2O): 0.28 g (31%) of 11. Brown foam. H-NMR: 2.06 (s, Ac); 2.07 (s, Ac); 2.12 ± 2.32 (m, 2 H C(2')); 2.79
‡
(t, J ˆ 5.3, CH2); 3.15 (s, Me2N ); 3.44 (t, J ˆ 5.6, CH2N); 4.06 ± 4.10 (m,H C(4')); 4.20 ± 4.27 (m,2 H C(5'));
‡
5.14 ± 5.16 (m, H C(3')); 6.24 ± 6.30 (m, H C(1')); 7.23 (s, H C(6)); 8.24 (s, NH). EI-MS: 381 (M ).
7-{[(Benzyloxy)carbonyl]methylamino}-3-[2-deoxy-3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl]-3,5,6,7-tetrahy-
dro-2H-pyrrolo[2,3-d]pyrimidin-2-one (13). To a soln. of 5 (0.5 g, 0.865 mmol) in tetrachloroethane (10 ml), 1-
[(benzyloxy)carbonyl]-1-methylhydrazine [14] (0.38 g, 2.14 mmol) was added and the soln. heated at 858
overnight. After dilution with CHCl3 and workup as described, the product was chromatographed (AcOEt/
hexane/MeOH 1:1:0 ! 7: 3 : 0.1): 0.52 g (97%) of 13. Off-white solid. UV: 283 (10400), 241 (34800), min. 267
and 219; pH 1: 300 (9900), 245 (24200), min. 269. 1H-NMR: 2.37 (s, MeC6H4); 2.39 (s, MeC6H4); 2.53 ± 2.72
(m, 2 H C(2'), CH2(5)); 3.08 (s, MeN); 3.60 ± 3.82 (m, CH2(6)); 4.48 ± 4.63 (m,H C(4')); 2 H C(5')); 5.09 ±
5.14 (m, CH2); 5.56 ± 5.58 (m, H C(3')); 6.33 (t, J ˆ 7.3,
H C(1')); 7.22 ± 7.39 (m, 8 arom. H); 7.56
(s, H C(4)); 7.84 ± 7.92 (m, 5 arom. H). FAB-MS: 675.2448 ([M ‡ Na] ). HR-MS: 675.2448 ([M ‡ Na] ,
‡
‡
‡
C36H36N4O8Na ; calc. 652.2431; deviation 2.50 ppm).
Pyrrolopyrimidinone 8b (Method B). To a soln. of 13 (0.4 g, 0.6 mmol) in dry MeOH (20 ml), 10% Pd/C
(or PtO2) catalyst (50 mg) was added and the soln. stirred under H2 for 2 h. The suspension was filtered through
Celite and the cake washed with MeOH. The solvent was evaporated and the residue chromatographed (5%
MeOH/CHCl3): 0.25 g (79%) of 8b. White solid. For data, see above (Method A).
3-[2-Deoxy-3,5-di-O-(p-toluoyl)-b-d-2-ribofuranosyl]-3,5,6,7-tetrahydro-2H-pyrrolo[2,3-d]pyrimidin-2-one
(14). To a soln. of 13 (0.4 g, 0.6 mmol) in AcOH (20 ml), Adamꢀs catalyst (50 mg) was added and the soln.
stirred under H2 for 4 h. Workup and FC as described for 8b (Method B) gave 16 (0.13 g, 43%). White solid. UV:
1
277 (8400), 244 (32800), min. 265 and 216; pH 1; 285 (9500), 244 (31600), min. 269 and 220. H-NMR: 2.37
(s, MeC6H4); 2.38 (s, MeC6H4); 2.42 ± 2.49 (m, 2 H C(2')); 2.61 ± 2.69 (m, CH2); 3.49 (t, J ˆ 7.8, CH2(6)); 4.43 ±
4.44 (m, H C(4')); 4.50 ± 4.63 (m, 2 H C(5')); 5.55 ± 5.57 (m, H C(3')); 6.34 (t, J ˆ 7.9, H C(1')); 7.32 ± 7.37
‡
(m, H C(6), 4 arom. H (Tol)); 7.86 ± 7.92 (m, 4 arom. H (Tol)); 7.99 (s, NH). FAB-MS: 490.7 ([M ‡ H] ).
‡
‡
HR-MS: 490.19792 ([M ‡ H] , C27H28N3O6 ; calc. 490.19781; deviation 0.20 ppm).
5-(2-Chloroethyl)-1-[2-deoxy-3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl]cytosine (15). A soln. of 5 (0.25 g,
0.43 mmol) in ammonia-saturated dioxane (10 ml) was stirred at r.t. overnight. The soln. was evaporated and the
product chromatographed (5% MeOH/CHCl3) to give a white solid. Attempts to recrystallize resulted in gel
formation: 0.22 g (97%) of 15. UV: 248 (23000), 272 (sh), min. 232; pH 1: 285 (11000), 245 (29300), min. 220
and 268. 1H-NMR: 2.37 (s, MeC6H4); 2.39 (s, MeC6H4); 2.44 ± 2.57 (m, 2 H C(2'), CH2); 3.53 (t, J ˆ 7, CH2Cl);
4.47 ± 4.64 (m, H C(4'), 2 H C(5')); 5.57 ± 5.59 (m, H C(3')); 6.31 (t, J ˆ 7.6, H C(1')); 7.09 (br. s, NH2);
7.30 ± 7.36 (m, 4 arom. H (Tol)); 7.47 (s,H C(6)); 7.85 ± 7.92 (m, 4 arom. H (Tol)). FAB-MS: 526.9 ([M ‡
H] ), 548.9 ([M ‡ Na] ). HR-MS: 548.15620 ([M ‡ Na] , C27H28N3O635ClNa ; calc. 548.15643; deviation
‡
‡
‡
‡
0.40 ppm).
We thank Dr. David Neuhaus for the NOE experiments, Dr. Mao Jun Guo for helpful discussions, and
Nycomed Amersham plc for financial assistance.
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Received August 20, 1999