Synthesis and biological activity of N4-methyl-5-azacytidines

Article abstract of DOI:10.1135/cccc19980713

Protected N⁴-methyl and N⁴,N⁴-dimethyl derivatives of 5-azacytidine 3 and 4 were prepared by selective aminolysis of benzoylated 4-methoxy-1-(β-D-ribofuranosyl)-1,3,5-triazin-2(1H)-one 5, by glycosylation of silylated N⁴-methyl-or N⁴,N⁴-dimethyl-5-azacytosines 7 and 8 with 2,3,5-tri-O-benzoyl-α,β-D-ribofuranosyl chloride (11) or by several modifications of the isocyanate method. By the isocyanate approach, also the α-D anomer of protected N⁴-methyl-5-azacytidine 17 was obtained as a minor product. The protected dimethyl derivative 4 was also obtained by the reaction of isobiuret 22 with dimethylformamide dimethyl acetal. The free nucleosides 1 and 2 were obtained either by aminolysis of the free methoxy nucleoside 23 with methylamine or dimethylamine, respectively, or by methanolysis or ammonolysis of the corresponding benzoyl derivatives 3 and 4. The free α-D anomer 24 was obtained by methanolysis of its tribenzoate 17. Nucleosides 1 and 2 exhibited a lower antibacterial, antitumor and antiviral activity than the unsubstituted 5-azacytidine.

Full text of DOI:10.1135/cccc19980713

N4-Methyl-5-azacytidines
713
SYNTHESIS AND BIOLOGICAL ACTIVITY OF N4-METHYL-5-AZACYTIDINES
1
2,
Naeem B. HANNA*, Milena MASOJIDKOVA and Alois PISKALA **
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10 Prague 6, Czech Republic; e-mail: 1 saman@uochb.cas.cz, 2 piskala@uochb.cas.cz
Received December 9, 1997
Accepted April 9, 1998
Protected N4-methyl and N4,N4-dimethyl derivatives of 5-azacytidine 3 and 4 were prepared by selective
aminolysis of benzoylated 4-methoxy-1-(β-D-ribofuranosyl)-1,3,5-triazin-2(1H)-one 5, by glycosyl-
ation of silylated N4-methyl- or N4,N4-dimethyl-5-azacytosines 7 and 8 with 2,3,5-tri-O-benzoyl-α,β-
D-ribofuranosyl chloride (11) or by several modifications of the isocyanate method. By the isocyanate
approach, also the α-D anomer of protected N4-methyl-5-azacytidine 17 was obtained as a minor pro-
duct. The protected dimethyl derivative 4 was also obtained by the reaction of isobiuret 22 with di-
methylformamide dimethyl acetal. The free nucleosides 1 and 2 were obtained either by aminolysis
of the free methoxy nucleoside 23 with methylamine or dimethylamine, respectively, or by methano-
lysis or ammonolysis of the corresponding benzoyl derivatives 3 and 4. The free α-D anomer 24 was
obtained by methanolysis of its tribenzoate 17. Nucleosides 1 and 2 exhibited a lower antibacterial,
antitumor and antiviral activity than the unsubstituted 5-azacytidine.
Key words: 5-Azacytidines; 1,3,5-Triazines; 5-Azapyrimidines; Nucleosides; Antitumor activity.
5-Azacytidine1 (Azacitidine) and 2′-deoxy-5-azacytidine2 (Decitabine) are used in clini-
cal treatment of acute leukemia3,4. Both agents are potent hypomethylating agents and
are used as experimental tools in molecular and cell biology for induction of gene
expression and cell differentiation5. Wide spectrum of biological activity of the men-
tioned 5-azacytosine nucleosides stimulated our interest in substituted congeners of
these compounds. Previously, some 6-substituted 5-azacytidines6–8 were prepared in
our laboratory and their biological activity was described. In continuation of the study
of substituted 5-azacytosine nucleosides, we were interested in N4-methyl-5-azacy-
tidine (1) and N4,N4-dimethyl-5-azacytidine (2). In this paper, we wish to present the
preparation and biological activity of these nucleosides.
Benzoylated N4-methyl- and N4,N4-dimethyl-5-azacytidines, 3 and 4, have been pre-
pared in 44 and 79% yields by short treatment of 4-methoxy-1-(2,3,5-tri-O-benzoyl-β-
* Present address: Beckman, 2500 Harbor Blvd., Fullerton, CA 92634-3100, U.S.A.
**The author to whom correspondence should be addressed.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

714
Hanna, Masojidkova, Piskala:
D-ribofuranosyl)-1,3,5-triazin-2(1H)-one9 (5) with methylamine and dimethylamine, re-
spectively, in methanol (Scheme 1). The dimethyl derivative 4 was also prepared in
66% yield using 4-methylsulfanyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1,3,5-
triazin-2(1H)-one10 (6) as intermediate. However, the best method for the preparation of
nucleosides 3 and 4 consists in ribosylation of silylated N4-methyl- and N4,N4-dimethyl-
5-azacytosines 7 and 8 (prepared by the reaction of the respective bases11 9 and 10 with
hexamethyldisilazane at elevated temperature) with 2,3,5-tri-O-benzoyl-α,β-D-ribofu-
ranosyl chloride6 (11) in acetonitrile. Additionally to high yields (almost 90%), this
reaction was highly stereoselective (no corresponding α-anomers were detected).
Lower yields (12–24%) of methyl derivatives 3 and 4 were obtained by various modi-
fications of the isocyanate method which proved to be convenient for the preparation of
6-substituted 5-azacytosine nucleosides6–8 or for the preparation of 5-azacytosine and
MeNR
MeNR
BzO
O
Cl
HMDS
N
N
N
N
+
BzO OBz
N
H
O
N
OSiMe
3
XMe
N
11
9, R = H
10, R = Me
7, R = H
8, R = Me
N
N
O
RO
2
O
1
MeNR
N
R O
O
N
O
N
OR
RO
1
1
N
O
O
OR
R O
N
1
5, R = Bz; X = O
6, R = Bz; X = S
23, R = H ; X = O
R O
1
2
N
17, R = Bz; R = H
2
1
2
MeNR
1
1
24, R = R = H
OR
R O
DMF
DMA
OMe
1
2
1, R = R = H
H N
2
N
1
2
2, R = H; R = Me
HN
O
1
2
3, R = Bz; R = H
BzO
O
1
2
2
4, R = Bz; R = Me
MeNR
HN
HN
1
N
R
OBz
BzO
22
Me
2
O
N
O
C O
BzO
BzO
O
N
R
+
NH
1
NH
2
R
1
OBz
14
BzO
BzO OBz
1
2
12, R = R = H
15, R = R = H
1
2
1
2
13, R = H; R = Me
16, R = H; R = Me
1
2
1
2
18, R = CHO; R = H
20, R = CHO; R = H
1
2
1
2
19, R = CHO; R = Me
21, R = CHO; R = Me
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

N4-Methyl-5-azacytidines
715
its methyl derivatives11. Addition of N-methylguanidine (12) or N,N-dimethylguanidine
(13) to 2,3,5-tri-O-benzoyl-β-D-ribofuranosyl isocyanate6 (14) afforded carbamoylgua-
nidines 15 and 16, respectively. Cyclocondensation of these unisolated intermediates
with triethyl orthoformate or dimethylformamide dimethyl acetal gave the respective
nucleosides 3 and 4. In the case of the methyl derivative 3, its α-D anomer 17 was also
isolated in a 5% yield. Comparable results were obtained in a modification of this
approach. Formyl derivatives of methyl- and dimethylguanidine 18 and 19 (prepared by
reaction of the respective guanidines 12 and 13 with ethyl formate) were added to the
isocyanate 14 and the unisolated formylcarbamoylguanidines 20 and 21 were cyclized by
the treatment of a mixture of chlorotrimethylsilane and triethylamine or N,O-bis(tri-
methylsilyl)acetamide to the respective methyl derivatives 3 and 4. The dimethyl deri-
vative 4 was eventually obtained in a 45% yield by cyclocondensation of
4-methyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)isobiuret9 (22) with dimethylform-
amide dimethyl acetal in benzene. Free nucleosides 1 and 2 were obtained in good
yields by methanolysis or ammonolysis of the respective benzoyl derivatives 3 and 4 or
by reaction of 4-methoxy-1-(β-D-ribofuranosyl)-1,3,5-triazin-2(1H)-one12 (23) with
methylamine and dimethylamine, respectively, in methanol. Methanolysis of benzoyl-
ated α-D anomer 17 gave the free nucleoside 24 in a 65% yield.
The assignment of anomeric configuration in compounds 1–4, 17 and 24 was in-
ferred from the chemical shifts of H-1′ protons on the basis of a well known rule and
the sign of Cotton effect in CD spectra of the free nucleosides 1, 2 and 24. The β-nucleo-
sides 1 and 2 exhibited positive B2u Cotton effects as 5-azacytidine and the α-anomer
24 showed a negative Cotton effect as the α-anomer of 5-azacytidine. The β-configura-
tion of the nucleosides 1 and 2 follows moreower from their formation by aminolysis
of the methoxy nucleoside 23 which is converted by ammonolysis to the known 5-aza-
cytidine1.
Similarly to unsubstituted 5-azacytidine13, the CD spectra of methyl derivatives 1
and 2 exhibited intensive positive B2u Cotton effects at about 250 nm. By contrast, the
α-D anomer 24 showed a negative Cotton effect nearly at the same wavelength as the
respective β-D anomers 1 and 2. The magnitude of the negative band of 24 is much
larger than that of the positive bands of 1 and 2. The position of the extremes in CD
spectra does not correspond to the maxima in UV spectra exactly. This fact can be
explained by superposition of the B2u with the B1u band. The latter band is obviously of
a smaller magnitude and the same sign as the more intensive B2u band. The sign of the
B
2u Cotton effects of nucleosides 1, 2 and 24 has the same direction as the sign of B2u
Cotton effects of the analogous pyrimidine nucleosides14–16. This indicates an anti con-
formation around the C–N glycosyl bond for N4-substituted methyl derivatives of 5-aza-
cytidine 1, 2 and 24.
Nucleosides 1 and 2 were tested for their antibacterial activity using a culture of E. coli B
growing on a mineral medium with glucose17. N4-Methyl-5-azacytidine (1) inhibited
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

716
Hanna, Masojidkova, Piskala:
the growth of bacteria to the extent of 17% at 4 µM concentration while N4,N4-di-
methyl-5-azacytidine (2) was much less active (15% growth inhibition at 400 µM con-
centration). By contrast, the unsubstituted 5-azacytidine inhibited the growth of E. coli B
to the extent of 50% even at 1 µM concentration18. N4-Methyl-5-azacytidine (1) and
N4,N4-dimethyl-5-azacytidine (2) were also tested for their ability to inhibit the growth
of four tumor cell lines in vitro: L1210 murine lymphocytic leukemia, WI-L2 human
B-lymphoblastic leukemia, CCRF-CEM human T-lymphoblastic leukemia, and
LoVo/L, a human colon carcinoma. N4-Methyl-5-azacytidine (1) produced 29, 34 and
39% inhibition of growth of WI-L2, CCRF-CEM and LoVo/L, respectively, at 100 µM
concentrations but was inactive in inhibiting L1210 at the same concentration. N4,N4-Dimethyl-
5-azacytidine (2) inhibited to the extent of 29% the growth of LoVo/L at 100 µM concen-
tration but did not inhibit growth of other tumor cell lines at the same concentration.
These results indicate a lower antitumor activity of nucleosides 1 and 2 in comparison
with unsubstituted 5-azacytidine3. N4-Methyl-5-azacytidine (1) and N4,N4-dimethyl-5-
azacytidine (2) exhibited no in vitro antiviral activity against HSV-1, HSV-2, adeno-
viruses, rhinoviruses, influenza and parainfluenza viruses at ≤1 µM concentration.
N4-Methyl-5-azacytidine (1) reduced rhinovirus 1-A cytopathology in HeLa cells by
50% at 100 µM concentration but was inactive against other viruses. In this connection,
it is worth mentioning that unsubstituted 5-azacytidine and 2′-deoxy-5-azacytidine
were active against HIV-1 even at 1 µM concentrations19. Eventually, it is of interest to
note that nucleosides 1 and 2 exhibited a lower potential carcinogenity than 5-azacy-
tidine20 or 6-methyl-5-azacytidine21 when estimated by a polarographic method22–24
Nucleosides 1 and 2 have also shown interesting antisecretory effects25.
.
The high biological activity of 5-azacytidine is based on its structural and conforma-
tional resemblance with cytidine which enables its incorporation into nucleic acids and
subsequent covalent addition of mercapto groups of enzymes to the reactive double
bond in the 5,6 position of the 1,3,5-triazine ring26. The CD spectra of methyl deriva-
tives 1 and 2 indicate an anti conformation around the C–N glycosyl bond of these
nucleosides similarly to unsubstituted 5-azacytidine. However, substitution of hydrogen
atoms on the amino group of 5-azacytidine by methyl groups prevents, predominantly
for sterical reasons, the incorporation into nucleic acids and this fact may explain the
lower activity of these nucleosides in comparison with the unsubstituted 5-azacytidine.
EXPERIMENTAL
Melting points were determined on a Kofler block and are uncorrected. Unless stated otherwise, the
solutions were evaporated at 35 °C/2.5 kPa and analytical samples were dried at 40 Pa (room tem-
perature). Thin-layer chromatography (TLC) was performed on Silufol UV 254 plates (Kavalier, Vo-
tice, Czech Republic) in solvent systems chloroform–methanol (98 : 2, I) and butan-1-ol–acetic
acid–water (5 : 2 : 3, II). The spots were detected visually in UV light (254 nm). Column chromato-
graphy was performed with silica gel according to Pitra (Service Laboratories of this Institute). UV
spectra were measured on a Unicam SP 8000 spectrophotometer (Pye Unicam, Cambridge, U.K.) in
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

N4-Methyl-5-azacytidines
717
buffer solutions of ionic strength 0.01 prepared according to Perrin27, λ are given in nm and ε in m2 mol–1.
CD spectra were recorded on a Roussel–Jouan/II dichrographe. Optical rotations were registered on
a Perkin–Elmer polarimeter, type 141 MCA at 22 °C. 1H NMR spectra of the nucleosides 1 and 2
were measured on a Varian XL-200 instrument (200 MHz) in D2O with sodium 3-(trimethylsilyl)-1-
propanesulfonate (DSS) as internal standard. 1H NMR spectra of the nucleosides 3, 4, 17 and 24
were measured on a Varian UNITY 500 instrument at 500 MHz in hexadeuteriodimethyl sulfoxide
with the solvent signal as the internal reference [δ(1H) = 2.50 ppm]. The chemical shifts (δ) are given
in ppm and coupling constants (J) in Hz. The mass spectra (m/z) were measured on a ZAB-EQ (VG
Analytical Ltd, Manchester, U.K.) spectrometer using the FAB technique (ionization by Xe, acceler-
ating voltage 8 kV), matrices glycerol and thioglycerol. Stationary cultivation of E. coli B was per-
formed at 37 °C in mineral medium with glucose17. The tested compounds were added before
inoculation and the growth of bacteria was measured 16 h later.
N4-Methyl-2′,3′,5′-tri-O-benzoyl-5-azacytidine (3) and Its α-D Anomer 17
Method A. A mixture of the blocked methoxy nucleoside9 5 (0.572 g, 1 mmol), methanol (7 ml)
and a 13% solution of dry methylamine in methanol (1.5 ml) was stirred at room temperature for 5 min
and crystallization of the product induced by scratching. The mixture was chilled to –15 °C (30 min),
the product filtered off with suction and recrystallized from methanol to yield 0.250 g (44%) of 3,
m.p. 245 °C (dec.), RF 0.60 (I), [α]D –60.2 (c 0.20, DMF). UV spectrum (MeOH), λmax (log ε): 260,
inflexion (4.22), 229 (4.63), 203 (4.38). 1H NMR spectrum: 8.42 s, 1 H (H-6); 8.25 q, 1 H,
J(NH,CH3) = 4.9 (NH); 8.00 d, 2 H (arom.); 7.89 t, 2 H (arom.); 7.64 m, 3 H (arom.); 7.50–7.40 m,
6 H (arom.); 6.06–6.00 m, 3 H (H-1′, H-2′, H-3′); 4.74 m, 1 H (H-4′); 4.69 dd, 1 H, J(5′a,4′) = 3.9,
J(gem.) =12.0 (H-5′a); 4.62 dd, 1 H, J(5′b,4′) = 5.9, J(gem.) = 12.0 (H-5′b); 2.77 d, 3 H, J(CH3,NH) =
4.9 (N-CH3). Mass spectrum: 571 (MH+). For C30H26N4O8 (570.3) calculated: 63.15% C, 4.59% H,
9.82% N; found: 63.45% C, 4.73% H, 9.90% N.
Method B. A mixture of N4-methyl-5-azacytosine11 9 (1.68 g, 13 mmol), hexamethyldisilazane (5 ml)
and ammonium sulfate (0.01 g) was refluxed on an oil bath (160–170 °C) for 5 h. The mixture was
then evaporated at 50–60 °C (bath temperature), the semicrystalline residue coevaporated with to-
luene (20 ml) and dried in vacuo at 50 °C for 4 h to give 2.7 g of crude silylated N4-methyl-5-aza-
cytosine 7. A mixture of crude 7 (2.7 g), acetonitrile (15 ml) and tri-O-benzoyl-α,β-D-ribofuranosyl
chloride 11 (prepared from 5.30 g, 10.4 mmol of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose28 by
the known procedure6) was kept overnight at room temperature. The crystalline precipitate was filtered
off by suction and washed with acetonitrile to yield 3.0 g (portion A) of crude 3, m.p. 240–242 °C
(dec.). The mother liquor was evaporated, the residue dissolved in chloroform (50 ml), the solution
washed with ice-cold 5% solution of sodium hydrogencarbonate (100 ml), dried (anhydrous sodium
sulfate) and evaporated. The residue was dissolved in 1,2-dichloroethane (10 ml) and petroleum ether
(10 ml) was added. The mixture was allowed to stand overnight to yield 2.6 g (portion B) of 3, m.p.
243–244 °C (dec.). Both portions (5.6 g) were combined and recrystallized from a mixture of 1,2-di-
chloroethane–petroleum ether to afford 5.20 g (88%, based on starting 1-O-acetyl-2,3,5-tri-O-ben-
zoyl-β-D-ribofuranose) of 3, m.p. 243–245 °C (dec.) without depression on admixture with a sample
of 3 prepared by method A.
Method C. To a solution of N-methylguanidine hydrochloride (0.220 g, 2 mmol) in methanol (10 ml),
methanolic 1 M NaOMe (2 ml) was added and the mixture diluted with benzene (10 ml). Sodium
chloride was filtered off with suction, the filtrate evaporated and dried in vacuo to give N-methylgua-
nidine (12). To a stirred mixture of 12 (2 mmol), a solution of crude isocyanate 14 (prepared from
1.008 g, 2 mmol of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribose28 by a known procedure6) was added
dropwise at room temperature. The mixture was stirred for 1 h, the small insoluble portion removed
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

718
Hanna, Masojidkova, Piskala:
by filtration through a layer of celite and evaporated. A solution of the residue in chloroform (10 ml)
was washed with water (10 ml), dried (anhydrous sodium sulfate) and evaporated to give the crude
carbamoylguanidine 15. A solution of crude 15 (2 mmol) in triethyl orthoformate (10 ml) was heated
(bath temperature 115 °C) in a distillation apparatus in a stream of nitrogen for 2.5 h. The mixture
was evaporated and chromatographed on a column of silica gel (50 g). Elution was performed with
toluene–ethyl acetate (100 : 0 – 0 : 100, v/v). The more mobile portion was crystallized from meth-
anol to yield 0.140 g (12%) of 3, m.p. 243–245 °C (dec.) without depression on admixture with the
sample prepared by method A. The less mobile portion gave, on crystallization from methanol and
acetonitrile, 0.060 g (5%) of the α-D anomer 17, m.p. 238–239 °C (dec.), RF 0.50 (I), [α]D –58.4 (c 0.12,
CHCl3). 1H NMR spectrum: 8.63 s, 1 H (H-6); 8.20 d, 2 H (arom.); 8.12 q, 1 H, J(NH,CH3) = 4.9 (NH);
7.83 d, 2 H (arom.); 7.78 d, 2 H (arom.); 7.70–7.59 m, 3 H (arom.); 7.53 t, 2 H (arom.); 7.45 t, 2 H
(arom.); 7.39 t, 2 H (arom.); 6.59 d, 1 H, J(1′,2′) = 4.9 (H-1′); 6.05 t, 1 H, J(2′,1′) = J(2′,3′) = 5.0
(H-2′); 5.98 dd, 1 H, J(3′,2′) = 5.2, J(3′4′) = 6.1 (H-3′); 5.22 m, 1 H (H-4′); 4.66 dd, 1 H, J(5′a,4′) =
3.9, J(gem.) = 12.0 (H-5′a); 4.59 dd, 1 H, J(5′b,4′) = 4.9, J(gem.) = 12.0 (H-5′b); 2.68 d, 3 H,
J(CH3,NH) = 4.9 (N-CH3). Mass spectrum: 571 (MH+). For C30H26N4O8 (570.3) calculated: 63.15% C,
4.59% H, 9.82% N; found: 63.36% C, 4.66% H, 10.10% N.
Method D. To a solution of crude amidinourea 15 prepared by method C from crude isocyanate 14
(2 mmol) in benzene (5 ml), dimethylformamide dimethyl acetal (0.5 ml) was added. The solution
was kept overnight at room temperature and the precipitate filtered off with suction to give 0.180 g
(portion A) of 3, m.p. 241–244 °C (dec.). The mother liquor was evaporated and the residue chroma-
tographed in the way indicated under method C to yield 0.120 g (portion B) of 3, m.p. 241–244 °C
(dec.) (methanol) and 0.030 g (2.6%) of the α-D anomer 17, m.p. 238–239 °C (dec.) (acetonitrile).
Both portions of the β-D anomer 3 were combined and recrystallized from methanol to give 0.250 g
(22%) of pure 3, m.p. 243–245 °C (dec.). Both products were identical with the respective samples
of 3 and 17 prepared by method C (mixed m.p., TLC).
Method E. A solution of N-methylguanidine (12) (prepared from 0.220 g, 2 mmol of its hydro-
chloride as given under method C) in a mixture of ethanol (1 ml) and ethyl formate (0.17 ml, 2.2 mmol)
was kept at room temperature for 1 h, evaporated and the residue dried in vacuo for 2 h to give crude
1-formyl-3-methylguanidine (18). To a solution of crude 18 (2 mmol) in acetonitrile (5 ml), a solu-
tion of crude isocyanate 14 (prepared from 1.008 g, 2 mmol of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-
ribofuranose28 by a known procedure6) in acetonitrile (10 ml) was added dropwise at room
temperature with stirring. The solution was treated with chlorotrimethylsilane (1.5 ml) and triethy-
lamine (1.5 ml) and allowed to stand at room temperature for 30 min. The mixture was diluted with
benzene (20 ml), the crystals of triethylamine hydrochloride were filtered off with suction, the filtrate
was evaporated and the residue chromatographed on a column of silica gel (25 g). Elution was per-
formed as given under method C. The major portion was crystallized from a mixture of 1,2-dichlo-
roethane–petroleum ether to yield 0.180 g (16%) of 3, m.p. 242–244 °C (dec.) without depression on
admixture with the sample prepared by method A.
N4,N4-Dimethyl-2′,3′,5′-tri-O-benzoyl-5-azacytidine (4)
Method A. A mixture of blocked methoxy nucleoside9 5 (0.572 g, 1 mmol) and a 7% solution of
dry dimethylamine in methanol (2.5 ml) was stirred at room temperature for 5 min. The mixture was
kept at 0 °C for 30 min and the product filtered off by suction to afford 0.460 g (79%) of 4, m.p.
216–219 °C. The sample for analysis was recrystallized from acetonitrile [m.p. 220 °C (dec.)], RF
0.73 (I), [α]D –61.9 (c 0.45, DMF). UV spectrum (MeOH), λmax (log ε): 265, inflexion (4.05), 226
(4.69), 204 (4.39). 1H NMR spectrum: 8.52 s, 1 H (H-6); 7.99 d, 2 H (arom.); 7.88 t, 4 H (arom.);
7.64 m, 3 H (arom.); 7.50–7.41 m, 6 H (arom.); 6.04–6.00 m, 3 H (H-1′, H-2′, H-3′); 4.75 td, 1 H,
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

N4-Methyl-5-azacytidines
719
J(4′,5′a) = 3.9, J(4′,3′) ≈ J(4′,5′a) ≈ 6.1 (H-4′); 4.69 dd, 1 H, J(5′a,4′) = 3.9, J(gem.) = 12.0 (H-5′a);
4.62 dd, 1 H, J(5′b,4′) = 5.9, J(gem.) = 12.0 (H-5′b); 3.17 s, 3 H (N-CH3); 3.08 s, 3 H (N-CH3).
Mass spectrum: 585 (MH+). For C31H28N4O8 (584.3) calculated: 63.69% C, 4.83% H, 9.58% N;
found: 63.93% C, 4.92% H, 9.81% N.
Method B. The blocked methylsulfanyl nucleoside10 6 (0.588 g, 1 mmol) was treated in analogy to
method A with a 7% solution of dry dimethylamine in methanol (2.5 ml) to afford 0.386 g (66%) of 4,
m.p. 216–218 °C (dec.) without depression on admixture with the sample prepared by method A.
Method C. N4,N4-Dimethyl-5-azacytosine11 10 (1.812 g, 13 mmol) was silylated and the syrupy
silylated base 8 (2.65 g) ribosylated in analogy to the preparation of 3 (method B). The product deposited
from the reaction mixture was filtered off by suction to give 5.02 g (portion A) of the nucleoside 4
m.p. 211–214 °C (dec.). The mother liquor was worked up in analogy to the preparation of 3
(method B) and the syrupy product crystallized from a mixture of 1,2-dichloroethane–petroleum ether
(1 : 1) to afford 0.35 g (portion B) of 4, m.p. 211–214 °C (dec.). Both portions (5.37 g) were com-
bined and recrystallized from a mixture of 1,2-dichloroethane–petroleum ether (1 : 1) to yield 5.0 g
(86%, based on starting 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose) of 4, m.p. 216–219 °C
(dec.) without depression on admixture with the sample prepared by method A.
Method D. A solution of crude isocyanate 14 (prepared from 1.008 g, 2 mmol of 1-O-acetyl-2,3,5-
tri-O-benzoyl-β-D-ribofuranose28 by a known procedure6) in acetone (5 ml) was added dropwise at
room temperature to a magnetically stirred mixture of 1,1-dimethylguanidine (13) [prepared from
0.248 g, 2 mmol of its hydrochloride as described for 12 (see preparation of 3, method C)] and
acetone (5 ml). The mixture was allowed to stand for 1 h and evaporated to yield the crude carba-
moylguanidine 16 (2 mmol) which was further worked up and reacted with orthoformate in analogy
to the preparation of 3 (method C) to yield 0.140 g (12%) of 4, m.p. 216–219 °C (dec.) without
depression on admixture with the sample prepared by method A.
Method E. To a solution of crude carbamoylguanidine 16 (2 mmol) prepared by method D in ben-
zene (5 ml), dimethylformamide dimethyl acetal (0.5 ml, 3.3 mmol) was added. The solution was
kept overnight at room temperature and worked up in analogy to the preparation of 3 (method D) to
give 0.280 g (24%) of 4, m.p. 216–219 °C (dec.) without depression on admixture with the sample
prepared by method A.
Method F. A mixture of blocked isobiuret9 22 (0.561 g, 1 mmol), benzene (5 ml) and dimethylform-
amide dimethyl acetal (0.13 ml) was stirred at room temperature for 10 days. The precipitate was
filtered off by suction to yield 0.264 g (45%) of 4, m.p. 216–219 °C (dec.) without depression on
admixture with the sample prepared by method A.
4-Methoxy-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1,3,5-triazin-2(1H)-one (5)
A mixture of blocked methylsulfanyl nucleoside 6 (0.588 g, 1 mmol) and a 3.7% solution of dry
methylamine in methanol (5 ml) was stirred at room temperature for 5 min. The mixture was kept in
the refrigerator (–20 °C) for 30 min and the precipitate filtered off by suction to yield crude 5, m.p.
150–155 °C (dec.). Recrystallization of the crude product (0.240 g) from ethanol afforded 0.200 g
(35%) of the pure methoxy derivative 5, m.p. 161–163 °C (dec.) without depression on admixture
with an authentic sample9, [α]D –23.3 (c 0.6, DMF). Mass spectrum: 572 (MH+). For C30H25N3O9
(571.3) calculated: 63.05% C, 4.41% H, 7.35% N; found: 63.27% C, 4.32% H, 7.61% N.
N4-Methyl-5-azacytidine (1)
Method A. A mixture of the free methoxy nucleoside12 23 (0.259 g, 1 mmol) and a 7% solution of
dry methylamine in methanol (2 ml) was stirred at room temperature for 1.5 h and the crystals fil-
tered off by suction to yield 0.20 g of crude 1, m.p. 149–151 °C (dec.). The crude product was re-
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

720
Hanna, Masojidkova, Piskala:
crystallized from methanol and dried at 110 °C/40 Pa for 8 h to give 0.164 g (64%) of pure 1, m.p.
155–157 °C (dec.), RF 0.24 (II), [α]D +34.5 (c 0.5, H2O). UV spectrum (MeOH), λmax (log ε): 250
1
(3.87), 213 (4.22). CD spectrum (H2O), λmax ([Θ]max): 249 (+7 800). H NMR spectrum: 2.89 s, 3 H
(N4-CH3); 8.45 s, 1 H (H-6); 3.88 m, 2 H (2 × H-5′); 4.00–4.50 m, 3 H (H-2′ + H-3′ + H-4′); 5.78 d,
1 H, J(1′,2′) = 3.0 (H-1′). Mass spectrum: 258 (M+). For C9H14N4O5 (258.2) calculated: 41.86% C,
5.46% H, 21.70% N; found: 41.89% C, 5.76% H, 21.83% N.
Method B. A mixture of blocked nucleoside 3 (0.571 g, 1 mmol), methanol (4 ml) and methanolic
1 M NaOMe (0.2 ml) was stirred at room temperature for 30 min. The mixture was kept overnight in
the refrigerator (–20 °C) and then acidified with acetic acid (0.05 ml) to yield 0.226 g of crude 1,
m.p. 144–147 °C (dec.). The crude product was recrystallized from methanol and dried at 110 °C/40 Pa
for 8 h to give 0.191 g (74%) of pure 1, m.p. 155–157 °C (dec.) without depression on admixture
with the sample prepared by method A.
N4,N4-Dimethyl-5-azacytidine (2)
Method A. A mixture of the free methoxy nucleoside12 23 (0.259 g, 1 mmol) and a 7% solution of
dry dimethylamine in methanol (2 ml) was stirred at room temperature for 3 min. The mixture was
kept for 45 min at room temperature, evaporated and coevaporated with methanol (5 ml). The crys-
talline residue was triturated with ethanol (1 ml) and the slurry kept overnight in the refrigerator (–20 °C) to
afford 0.220 g of crude 2, m.p. 127–129 °C (dec.). Recrystallization of the crude product from ethanol
gave 0.193 g (71%) of pure 2, m.p. 128–130 °C (dec.), RF 0.25 (II), [α]D +24.1 (c 0.5, H2O). UV
spectrum, λmax (log ε): (MeOH), 258 (3.89), 219 (4.24); (pH 2.32), 262 (3.88), 211 (4.11); (pH 6.94),
258 (3.89), 217 (4.31); (pH 10.93), 257 (3.85), 218 (4.25). CD spectrum (H2O), λmax ([Θ]max): 248 (+6 450).
1H NMR spectrum: 3.12 s, 3 H (N4′-CH3); 3.21 s, 3 H (N4′′-CH3); 3.88 m, 2 H (2 × H-5′); 4.09 m,
1 H (H-4′); 4.24 m, 1 H (H-3′); 4.49 dd, 1 H, J(2′,1′) = 3.0, J(2′,3′) = 5.0 (H-2′); 5.78 d, 1 H, J(1′,2′) =
3.0 (1′-H). Mass spectrum: 273 (MH+). For C10H16N4O5 (272.2) calculated: 44.12% C, 5.92% H,
20.58% N; found: 44.31% C, 5.96% H, 20.48% N.
Method B. A mixture of the tribenzoate 4 (0.585 g, 1 mmol), methanol (4 ml) and methanolic 1 M
NaOMe (0.2 ml) was stirred at room temperature for 1 h. The solution was kept overnight, acidified
with acetic acid (0.05 ml) and applied onto a column of Amberlite IRC-50 [H+] ion exchange resin
(10 ml) prepared in methanol. The column was washed with methanol (100 ml) and the effluent
evaporated. The residue was crystallized from ethanol to yield 0.196 g (72%) of 2, m.p. 128–130 °C
(dec.) without depression on admixture with the sample prepared by method A.
Method C. A mixture of the tribenzoate 4 (0.585 g, 1 mmol) and a 7 M solution of dry ammonia
in methanol (20 ml) was stirred at room temperature for 3 days. The solution was evaporated, the
residue triturated with ether (20 ml) and crystallized from ethanol to yield 0.152 g (56%) of 2, m.p.
128–130 °C (dec.) without depression on admixture with the sample prepared by method A.
Method D. A mixture of the tribenzoate 4 (0.585 g, 1 mmol) and a 7% solution of dry dimethyl-
amine in methanol (15 ml) was stirred at room temperature for 3 days. The solution was evaporated,
the residue triturated with ether (10 ml) and crystallized from ethanol to give 0.190 g (70%) of 2,
m.p. 128–130 °C (dec.) without depression on admixture with the sample prepared by method A.
4-Methylamino-1-(α-D-ribofuranosyl)-1,3,5-triazin-2(1H)-one (24)
A mixture of the blocked α-D anomer 17 (0.035 g, 0.06 mmol), methanol (0.5 ml) and methanolic 1 M
NaOMe (0.02 ml) was magnetically stirred for 20 min and kept overnight at room temperature. The
crystalline product was filtered off with suction to yield 0.010 g (65%) of the free nucleoside 24,
m.p. 241–243 °C (dec.), RF 0.24 (II). UV spectrum, λmax (log ε): (MeOH), 252 (3.94), 213 (4.25);
(pH 2.36), 257 (3.48), 226 (3.50); (pH 6.86), 252 (3.84), 213 (4.22); (pH 10.96), 227 (4.32). CD
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

N4-Methyl-5-azacytidines
721
1
spectrum (pH 6.86), λmax ([Θ]max): 248 (–16 570), 226 sh (–10 650). H NMR spectrum: 8.46 s, 1 H
(H-6); 7.97 q, 1 H, J(NH,CH3) = 4.9 (NH); 6.03 d, 1 H, J(1′,2′) = 4.2 (H-1′); 5.41 br, 1 H (OH);
5.22 br, 1 H (OH); 4.82 br t, 1 H (OH); 4.10 t, 1 H, J(2′,1′) = J(2′,3′) = 4.4 (H-2′); 4.06 dd, 1 H,
J(3′,2′) = 4.6, J(3′,4′) = 6.8 (H-4′); 4.01 ddd, 1 H, J(4′,5′a) =2.7, J(4′,5′b) = 4.2, J(4′,3′) = 6.8 (H-4′);
3.60 dd, 1 H, J(5′a,4′) = 2.7, J(gem.) = 12.2 (H-5′a); 3.42 dd, 1 H, J(5′b,4′) = 4.2, J(gem.) = 12.2
(H-5′b); 2.73 d, 3 H, J(CH3,NH) = 4.9 (N-CH3). Mass spectrum: 259 (MH+). For C9H16N4O5 (258.2)
calculated: 41.86% C, 5.46% H, 21.70% N; found: 41.63% C, 5.34% H, 21.42% N.
The authors are indebted to Mrs I. Krizkova for measurement of UV spectra, to Mrs Z. Ledvinova for
determination of optical rotations, to Mrs A. Strejckova and Mrs Y. Cerna for technical assistance, to
the late Dr I. Fric for interpretation and measurement of CD spectra, to Dr A. Cihak for estimation of
antibacterial effects, to the staff of the Analytical Laboratory (Dr V. Pechanec, Head) for elemental
analyses and the staff of the Central Laboratory of Mass Spectrometry (Dr K. Ubik, Head) for mass
spectral data. The antitumor and antiviral effects were tested by one of the authors (N. B. H.) during his
stay in the laboratory of Prof. R. K. Robins (Nucleic Acids Research Institute, Costa Mesa, U.S.A.).
REFERENCES
1. Piskala A., Sorm F.: Collect. Czech. Chem. Commun. 1964, 29, 2060.
2. Piskala A., Sorm F. in: Nucleic Acid Chemistry (L. B. Townsend and R. S. Tipson, Eds), Part 1,
p. 443. Wiley, New York 1978.
3. Vesely J., Cihak A.: Pharmacol. Ther. A2 1978, 813.
4. Rivard G. E., Momparler R. L., Demers J., Benoit P., Raymond R., Lin K.-T., Momparler L. F.:
Leukemia Res. 1981, 5, 453.
5. Riggs A. D., Jones P. A.: Adv. Cancer Res. 1983, 40, 1.
6. Piskala A., Hanna N. B., Zajicek J., Cihak A.: Collect. Czech. Chem. Commun. 1989, 54, 2502.
7. Hanna N. B., Zajicek J., Piskala A.: Nucleosides Nucleotides 1997, 16, 129.
8. Hanna N. B., Masojidkova M., Fiedler P., Piskala A.: Collect. Czech. Chem. Commun. 1998, 63,
222.
9. Piskala A., Ulrich M., Sorm F.: Czech. 152,016; Chem. Abstr. 1974, 81, 105914.
10. Piskala A., Ulrich M., Sorm F.: Czech. 152,017; Chem. Abstr. 1974, 81, 105915.
11. Piskala A.: Collect. Czech. Chem. Commun. 1967, 32, 3966.
12. Sorm F., Piskala A.: Ger. Offen. 1,922,702; Chem. Abstr. 1970, 72, 79427.
13. Ref.2, p. 435.
14. Rogers G. T., Ulbricht T. L. V.: Eur. J. Biochem. 1971, 22, 457.
15. Miles D. W., Inskeep W. H., Robins M. J., Winkley M. W., Robins R. K., Eyring H.: J. Am.
Chem. Soc. 1970, 92, 3872.
16. Pullman B., Berthod A.: FEBS Lett. 1972, 20, 341.
17. Cihak A., Sorm F.: Collect. Czech. Chem. Commun. 1965, 30, 2091.
18. Sorm F., Piskala A., Cihak A., Vesely J.: Experientia 1964, 20, 202.
19. Bouchard J., Walker M. C., Leclerc J. M., Lapointe N., Beaulieu R., Thibodeau L.: Antimicrob.
Agents Chemother. 1990, 34, 306.
20. Novotny L., Vachalkova A., Piskala A.: Collect. Czech. Chem. Commun. 1994, 59, 1691.
21. Novotny L., Vachalkova A., Piskala A.: Collect. Czech. Chem. Commun. 1995, 60, 1469.
22. Novotny L., Vachalkova A.: Neoplasma 1990, 37, 377.
23. Novotny L., Vachalkova A.: Neoplasma 1990, 37, 555.
24. Novotny L., Vachalkova A.: Neoplasma 1991, 38, 223.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

722
Hanna, Masojidkova, Piskala:
25. Cihak A., Piskala A., Korbova L., Cizkova J., Kucerova V.: Experientia 1986, 42, 32.
26. Kees U. R., Avramis V. I.: Anti-Cancer Drugs 1995, 6, 303.
27. Perrin D. D.: Aust. J. Chem. 1963, 16, 572.
28. Recondo E. F., Rinderknecht H.: Helv. Chim. Acta 1959, 42, 1171.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)