Synthesis of some 6-substituted 5-azacytidines

Article abstract of DOI:10.1135/cccc19980222

Protected 6-substituted benzyl, phenyl and chloromethyl derivatives of 5-azacytidine 8-10 have been prepared by addition of phenylacetyl-(2), benzoyl-(3) or (chloroacetyl)guanidine (4) to 2,3,5-tri-O-benzoyl-β-D-ribosyl isocyanate (1) and subsequent silylation-mediated cyclization of the obtained acyl(carbamoyl)guanidines 5-7. 4-Amino-6-phenyl-1,3,5-triazin-2(1H)-one (12) was obtained by condensation of carbamoylguanidine (13) with methyl benzoate in presence of methanolic sodium methoxide or by condensation of 13 with triethyl orthobenzoate in N,N-dimethylformamide. Stannic chloride catalyzed condensation of silylated 6-phenyl derivative 11 with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribose (14) in 1,2-dichloroethane afforded a 1.2 : 1 mixture of N¹ and N³ nucleosides 9 and 15, respectively. Methanolysis of the protected compounds 8-10 and 15 gave the respective free nucleosides 16-19. The latter compounds inhibited the growth of bacteria E. coli B to a much lower extent than the unsubstituted 5-azacytidine.

Full text of DOI:10.1135/cccc19980222

222
Hanna, Masojidkova, Fiedler, Piskala:
SYNTHESIS OF SOME 6-SUBSTITUTED 5-AZACYTIDINES
1
2,
Naeem B. HANNA*, Milena MASOJIDKOVA , Pavel FIEDLER 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 April 7, 1997
Accepted November 27, 1997
Protected 6-substituted benzyl, phenyl and chloromethyl derivatives of 5-azacytidine 8–10 have been
prepared by addition of phenylacetyl- (2), benzoyl- (3) or (chloroacetyl)guanidine (4) to 2,3,5-tri-O-
benzoyl-β-D-ribosyl isocyanate (1) and subsequent silylation-mediated cyclization of the obtained
acyl(carbamoyl)guanidines 5–7. 4-Amino-6-phenyl-1,3,5-triazin-2(1H)-one (12) was obtained by con-
densation of carbamoylguanidine (13) with methyl benzoate in presence of methanolic sodium methoxide or
by condensation of 13 with triethyl orthobenzoate in N,N-dimethylformamide. Stannic chloride cata-
lyzed condensation of silylated 6-phenyl derivative 11 with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribose (14) in
1,2-dichloroethane afforded a 1.2 : 1 mixture of N1 and N3 nucleosides 9 and 15, respectively. Methano-
lysis of the protected compounds 8–10 and 15 gave the respective free nucleosides 16–19. The latter
compounds inhibited the growth of bacteria E. coli B to a much lower extent than the unsubstituted
5-azacytidine.
Key words: 5-Azapyrimidines; 1,3,5-Triazines; Nucleosides; Circular dichroism; Antibacterial activity.
In continuation of our study of 5-azapyrimidine nucleosides, we were also interested in
the synthesis and biological activity of 6-substituted 5-azacytidines. The synthesis of
6-methyl-5-azacytidine was described in a preceding paper1. In this communication we
wish to give an account of our further studies in this field. The isocyanate method
which proved to be convenient for the synthesis of 6-methyl-5-azacytidine1 has been
used for the preparation of 6-substituted benzyl, phenyl and chloromethyl derivatives of
5-azacytidine. Reaction of 2,3,5-tri-O-benzoyl-β-D-ribosyl isocyanate (1) with phenyl-
acetyl- (2), benzoyl- (3) and (chloroacetyl)guanidine (4) afforded the respective
acyl(carbamoyl)guanidines 5–7. Silylation-mediated cyclization of these intermediates
gave the respective protected 6-substituted 5-azacytidines 8–10 (Scheme 1). As silyla-
tion agents were used bis(trimethylsilyl)acetamide, bis(trimethylsilyl)trifluoroacet-
amide or a mixture of chlorotrimethylsilane and triethylamine. This procedure is
* 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)

6-Substituted 5-Azacytidines
223
unambiguous in respect to the position of the ribosyl group and also to the anomeric
configuration. The structure of the products was confirmed by elemental analysis, mass
spectra and spectral data on comparison with the analogous blocked 6-methyl-5-azacy-
tidine1.
RO
O
NH
2
N=C=O
NH
2
O
1
N
N
R
HN
O
N
1
OR
N
O
RO
R
HN
O
RO
RO
O
1, R = Bz
+
OR
RO
R
OR
RO
NH
2
1
C
NH
R COHN
1
R
Bz
Bz
Bz
H
C H CH
8
9
1
6
5
2
1
R
R
R
C H
6
5
Bz
Bz
Bz
C H CH
2
3
4
5
C H CH
6
5
2
6
5
2
CH Cl
10
16
17
18
2
C H
6
6
7
C H
6
5
5
C H CH
6
5
5
2
CH Cl
CH Cl
2
2
H
C H
6
H
CH Cl
2
SCHEME 1
Protected 6-phenyl-5-azacytidine 9 was also obtained by direct glycosylation of the
silylated base 11 (Scheme 2). The starting 4-amino-6-phenyl-1,3,5-triazin-2(1H)-one (12)
was obtained either by condensation of carbamoylguanidine (13) with methyl benzoate
in presence of sodium methoxide in methanol or by condensation of carbamoylgua-
nidine (13) with triethyl orthobenzoate in DMF. Stannic chloride-catalyzed condensa-
tion of the silylated 6-phenyl derivative 11 (prepared by treatment of the base 12 with
hexamethyldisilazane at elevated temperature) with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-
D-ribose (14) in 1,2-dichloroethane (reaction time 30 min) gave a 1.2 : 1 mixture of N1
and N3 nucleosides 9 and 15, respectively. The separation of the positional isomers was
carried out by column chromatography on silica gel. Crystallization of the N3 derivative
15 from a mixture of ethanol and ethyl acetate gave a lower-melting 1 : 1 adduct with
ethyl acetate. The solvent was removed by heating at 140 °C. The compound changed
at its melting point to the higher-melting unsolvated form. In the infrared spectra of the
solvate of 15 with ethyl acetate, the typical bands of ethyl acetate were found besides
the bands of 15. This finding indicates that the solvent does not form covalent or hy-
drogen bonds with 15. The structure of the N1 nucleoside 9 was proved on identification
with the product obtained by cyclization of benzoyl(carbamoyl)guanidine (6) and the
structure of the N3 derivative 15 was inferred from infrared spectra by comparison with
the analogous 6-methyl derivative1.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

224
Hanna, Masojidkova, Fiedler, Piskala:
Methanolysis of the protected nucleosides 8–10 and 15 with sodium methoxide in
methanol afforded the respective free nucleosides 16–19. 6-Chloromethyl-5-azacytidine
(18) was obtained in the form of a very hygroscopic amorphous dihydrate which hydro-
lyzed similarly to 5-azacytidine2, rather rapidly on storage at room temperature.
NH
NH
2
NHSiMe
N
2
3
BzOMe / NaOMe
or C H C(OEt)
HMDS
H N
2
N
N
N
N
6
5
3
H N
O
C H
6
N
H
O
2
C H
6
N
OSiMe
3
5
5
13
12
11
C H
6
5
BzO
14
OAc
N
N
N
O
H N
2
O
RO
O
OBz
SnCl
BzO
15, R = Bz
19, R = H
9
+
4
OR
RO
SCHEME 2
The free 6-substituted 5-azacytidines 16–18 and the N3 isomer 19 were tested for
their antibacterial activity using a culture E. coli B growing on a mineral medium with
glucose. Similarly to 6-methyl-5-azacytidine1 (85% inhibition of growth at 1 000 µg/ml
concentration), all of these nucleosides exhibited a much lower degree of growth in-
hibition in comparison with the parent nucleoside 5-azacytidine3 (50% inhibition of
growth at 0.25 µg/ml concentration) and also in comparison with 6-amino-5-azacy-
tidine4 (46% inhibition of growth at 100 µg/ml concentration). The highest degree of
growth inhibition exhibited 6-chloromethyl-5-azacytidine (18) (39% at 100 µg/ml con-
centration), 6-benzyl-5-azacytidine (16) was less active (18% at 1 000 µg/ml concentra-
tion) and 6-phenyl-5-azacytidine (17) was not active at all even at 1 000 µg/ml
concentration. However, the N3 isomer of the latter nucleoside 19 inhibited the growth
of bacteria E. coli B to the extent of 32% at 100 µg/ml concentration. By contrast, the
N3 isomer of 6-methyl-5-azacytidine1 was inactive even at 1 000 µg/ml concentration.
Eventually it is of interest to note that 6-methyl-5-azacytidine1 exhibited a higher
potential carcinogenity5 than 5-azacytidine6,7 when estimated by a polarographic
method8–10. The 6-substituted 5-azacytidines described in this paper are also tested for
their potential carcinogenity by the same method. The results of this study will be
published later.
CD spectra of nucleosides 16 and 17 are very similar to those of 6-methyl-5-azacy-
tidine1 and indicate a syn conformation aroung the C–N glycosyl bond of 6-substituted
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

6-Substituted 5-Azacytidines
225
5-azacytidines. By contrast, unsubstituted 5-azacytidine1 is a typical anti nucleoside.
The high biological activity of 5-azacytidine is based on its structural and conforma-
tional resemblance to 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 ring11. Due to remarkable changes in mole-
cular conformation, 6-subtituted 5-azacytidines 16–18 are obviously not able to be in-
corporated into nucleic acids. Moreover, the substituents in the position 6 prevent
covalent interactions of the double bond in the position 5,6 of the triazine ring with
enzymes. These facts explain the dramatic differences in biological activity of 6-sub-
stituted 5-azacytidines 16–18 and the unsubstituted 5-azacytidine.
EXPERIMENTAL
The 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
temperature). Thin-layer chromatography (TLC) was performed on Silufol UV 254 plates (Kavalier,
Votice, Czech Republic) in solvent systems A: chloroform–methanol (98 : 2) and B: butan-1-ol–
acetic acid–water (5 : 2 : 3). The spots were detected visually in UV light (254 nm). Column chro-
matography was performed with silica gel according to Pitra (Service Laboratories of the Institute).
1H NMR spectra were measured on a Varian XL-200 instrument (200 MHz) in hexadeuteriodi-
methyl sulfoxide and referenced to the signal of solvent (δ(1H) 2.50), chemical shifts (δ) are given
in ppm and coupling constants (J) in Hz. UV spectra were measured on a Unicam SP 8000 spectro-
photometer (Pye Unicam, Cambridge, U.K.) in buffer solutions of ionic strength 0.01 prepared ac-
cording to Perrin12, 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 polarimeter Perkin–Elmer, type
141 MCA at 22 °C. The IR spectra were recorded on a Zeiss UR-20 instrument, the wavenumbers
are given in cm–1. The mass spectra (m/z) were measured on a ZAB-EQ (VG Analytical Ltd, Man-
chester, U.K.) spectrometer using the FAB technique (ionization by Xe, accelerating voltage 8 kV),
matrices glycerol or thioglycerol. Stationary cultivation of Escherichia coli B was performed at 37 °C in
mineral medium with glucose13. The tested compounds were added before inoculation and the growth
of bacteria was measured 16 h later.
4-Amino-6-phenyl-1,3,5-triazin-2(1H)-one (12)
Method A. A solution of carbamoylguanidine14 (13) (10.2 g, 0.10 mol) in methanolic 1 M NaOMe
(100 ml) was treated with methyl benzoate (15 ml, 0.12 mol) and the mixture refluxed for 7 h at 100 °C
(bath temperature). The mixture was evaporated, the residue dissolved in water (100 ml), the solution
acidified with acetic acid (7 ml) and the precipitate filtered off with suction to give 9.5 g (50%) of
the compound 12, m.p. 330–333 °C (dec.); the sample for analysis was recrystallized from ethanol-
water (1 : 1) and dried at 120 °C/30 Pa for 3 h, m.p. 333–335 °C (dec.), ref.15 m.p. 334–335 °C
(dec.), RF 0.73 (B). UV spectrum, λmax (log ε): (MeOH), 252 (4.34), 210 (4.78); (pH 2.26), 276
1
(4,34), 226 (4.18); (pH 6.94), 260 (4.23), 203 (4.75). Mass spectrum: 189 (MH+). H NMR spectrum:
11.60 br, 1 H (NH); 7.50 br, 2 H (NH2); 8.17 m, 2 H (arom.); 7.60–7.42 m, 3 H (arom.). For
C9H8N4O (188.2) calculated: 57.44% C, 4.28% H, 29.77% N; found: 57.47% C, 4.19% H, 29.57% N.
Method B. A mixture of carbamoylguanidine14 (13) (0.408 g, 4 mmol), DMF (3 ml) and triethyl
orthobenzoate (3 ml) was heated for 1.5 h at 145–150 °C (bath temperature). The mixture was evapo-
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

226
Hanna, Masojidkova, Fiedler, Piskala:
rated in vacuo at 55–60 °C (bath temperature), the residue triturated with ether (10 ml) and crystal-
lized from water to give 0.35 g (46%) of 12, m.p. 328–331 °C (dec.). The recrystallized product
melted at 333–335 °C (dec.) undepressed with the sample prepared by method A.
2-Trimethylsilylamino-4-trimethylsilyloxy-6-phenyl-1,3,5-triazine (11)
A mixture of triazine 12 (3.44 g, 18 mmol), hexamethyldisilazane (8 ml) and ammonium sulfate
(0.05 g) was refluxed at 160–165 °C (bath temperature) for 4 h. The excess of hexamethyldisilazane
was evaporated in vacuo at 55–60 °C (bath temperature). The residue was coevaporated with toluene
(10 ml), dried in vacuo and minced to a powder which was dried again at 50 °C in vacuo for 30 min.
The crude product was used in glycosylation without any further purification. Yield, 5.9 g (98%) of
compound 11, m.p. 110–115 °C (dec.). IR spectrum (Nujol): ν(ring)(triazine) 1 559 s, 1 540 s, sh, 1 515 m;
ν(ring)(phenyl) 1 594 s, 1 491 m; δ rock (CH3) asym. 847 s, 873 m, sh, sym. 750 w, sh, 760 w;
ν(Si–C) asym. 682 w, sym. 623 w, 650 w. For C18H24N4OSi2 (332.6) calculated: 54.17% C, 7.27% H,
16.84% N; found: 53.81% C, 7.10% H, 17.05% N.
N-(Benzoylcarbamimidoyl)-N′-(2,3,5-tri-O-benzoyl-β-D-ribosyl)urea (6)
A solution of crude isocyanate 1 (prepared by a known procedure4 from 1.008 g, 2 mmol of blocked
ribose16 14) in dry acetone (10 ml) was added dropwise at room temperature to a magnetically stirred
mixture of benzoylguanidine17 (3) (0.326 g, 2 mmol) and dry acetone (5 ml). The mixture was stirred
for 30 min, evaporated and the residue treated with toluene (20 ml). The small insoluble portion was
filtered off, the clear filtrate evaporated to give crude acyl(carbamoyl)guanidine 6 which was used in
the next step without purification. An analytical sample was obtaine by chromatography of the product
from a parallel experiment on a column of silica gel (30 g). Elution was performed with toluene–ethyl
acetate (100 : 0–80 : 20, v/v). The major portion was dried at 60 °C/40 Pa for 2 h to give 1.0 g
(77%) of the pure 6 as a chromatographically homogeneous solid foam; RF 0.61 (A), [α]D –50 ° (c 0.1,
CHCl3). IR spectrum (CHCl3): ν(NH) 3 384 m; ν(C=O) 1 727 vs (benzoate), 1 690 m (amide), 1 657 s
(urea); ν(C=N) 1 620 m; ν(ring) arom. 1 603 m, 1 584 m (benzoyl), amide II bound 1 563 m, amide II
free 1 525 m, sh. UV spectrum (MeOH), λmax (log ε): 260, inflexion (4.42), 230 (4.80), 210 (4.66).
1
Mass spectrum: 651 (MH+). H NMR spectrum: 10.60 br, 1 H (NH); 9.20 br, 1 H (NH); 8.70 br, 1 H
(NH2); 8.55 br, 1 H (NH2); 8.10–7.80 m, 8 H (arom.); 7.70–7.35 m, 12 H (arom.); 5.81–5.60 m, 3 H
(H-1′ + H-2′ + H-3′); 4.63–4.46 m, 3 H (H-4′ + 2 × H-5′). For C35H30N4O9 (650.7) calculated:
64.61% C, 4.65% H, 8.61% N; found: 64.63% C, 4.60% H, 8.43% N.
2′,3′,5′-Tri-O-benzoyl-6-phenyl-5-azacytidine (9)
A solution of crude acyl(carbamoyl)guanidine 6 (prepared from 1 mmol of 14 via crude 1) in aceto-
nitrile (5 ml) was treated with chlorotrimethylsilane (1 ml, 7.9 mmol) and triethylamine (1 ml, 7.3 mmol).
The mixture was kept for 30 min at room temperature, diluted with benzene (20 ml), the insoluble
triethylammonium chloride filtered off with suction and the filtrate evaporated. The residue was dried
in vacuo for 30 min, dissolved in ethanol (4 ml) and allowed to stand overnight at room temperature.
The precipitate was filtered off with suction and applied on a column of silica gel (30 g). Elution was
performed with toluene–ethyl acetate (100 : 0 – 0 : 100, v/v). The major portion was crystallized from
ethanol–petroleum ether and recrystallized from ethanol to afford 0.253 g (43%) of compound 9, m.p.
118–120 °C (dec.), RF 0.31 (A), [α]D +61° (c 0.21, CHCl3). IR spectrum (CHCl3): ν(NH2) 3 543 w, 3 423 m;
ν(C=O) 1 729 m, 1 715 s, sh, 1 695 m, sh; ν(C=N) 1 621 s; δ(NH2) 1 649 s, sh. UV spectrum
1
(MeOH), λmax (log ε): 265, inflexion (4.41), 229 (4.78), 210 (4.73). Mass spectrum: 633 (MH+). H NMR
spectrum: 7.88 br, 1 H (NH2); 7.81 br, 1 H (NH2); 7.98 m, 2 H (arom.); 7.78–6.90 m, 4 H (arom.);
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

6-Substituted 5-Azacytidines
227
7.66–7.50 m, 6 H (arom.); 7.50–7.27 m, 8 H (arom.); 6.15 m, 2 H (H-2′ + H-3′); 5.66 d, 1 H, J(1′,2′)
= 1.2 (H-1′); 4.64–4.50 m, 3 H (H-4′ + 2 × H-5′). For C35H28N4O8 (632.6) calculated: 66.45% C,
4.46% H, 8.86% N; found: 66.39% C, 4.67% H, 8.67% N.
In a parallel experiment a solution of purified acyl(carbamoyl)guanidine 6 (0.325 g, 0.5 mmol) in
acetonitrile (4 ml) was treated with bis(trimethylsilyl)trifluoroacetamide (1.2 ml, 4.5 mmol), kept
overnight at room temperature, evaporated and the residue crystallized from ethanol. Recrystalliza-
tion of the product from the same solvent afforded 0.237 g (75%) of 9, m.p. 118–120 °C (dec.), that
was in all respects identical with the above mentioned product.
Ribosylation of the Silylated Base 11
A stirred mixture of silylated base 11 (0.997 g, 3 mmol) and blocked ribose16 14 (1.51 g, 3 mmol) in
1,2-dichloroethane (15 ml) was treated dropwise at 0 °C with a solution of stannic chloride (0.51 ml,
4 mmol) in 1,2-dichloroethane (5 ml). The solution was kept for 30 min at room temperature and
cautiously shaken with an icecold 5% solution of sodium hydrogencarbonate (100 ml). The mixture
was extracted with chloroform (100 ml), filtered through a layer of cellite, the organic layer dried
(anhydrous sodium sulfate) and evaporated. The residue was applied on a column of silica gel (50 g)
packed in toluene. Elution was performed with toluene–ethyl acetate (75 : 25 – 0 : 100, v/v). Fractions
containing the more mobile portion were collected, evaporated and the residue triturated with ethanol
to give 0.64 g (34%) of the N3 nucleoside 15 m.p. 225–228 °C (dec.), RF 0.60 (A), [α]D –57.2° (c 0.20,
CHCl3), IR spectrum (CHCl3): ν(NH2) 3 494 m, 3 373 s; ν(C=O) 1 729 vs, 1 714 s, sh, 1 695 s, sh;
ν(C=N) 1 560 s; δ(NH2) 1 613 s. UV spectrum (MeOH), λmax (log ε): (MeOH), 261 (4.53), 229
1
(4.72), 212 (4.71). Mass spectrum: 633 (MH+). H NMR spectrum: 8.46 br, 2 H (NH2); 8.26 m, 2 H
(arom.); 8.02–7.82 m, 6 H (arom.); 7.70–7.36 m, 12 H (arom.); 6.22 br s, 1 H (H-1′); 6.10 m, 2 H
(H-2′ + H-3′); 4.75–4.55 m, 3 H (H-4′ + 2 × H-5′). For C35H28N4O8 (632.6) calculated: 66.45% C,
4.46% H, 8.86% N; found: 66.16% C, 4.35% H, 8.82% N. Recrystallization of the product from
ethanol–ethyl acetate afforded the solvate of 15 with ethyl acetate, m.p. 198–200 °C (resolidification)
and 220–224 °C (dec.); at 130 °C the sample changed (release of ethyl acetate). For C35H28N4O8
C4H8O2 (720.7) calculated: 64.99% C, 5.03% H, 7.77% N; found 64.83% C, 4.98% H, 7.54% N. The
IR spectra of the solvated sample exhibited besides of the bands of nucleoside 15 also typical bands
of ethyl acetate at 1 045 (m) and 1 376 (m) cm–1. Drying of the solvate in vacuo at 140 °C for 5 h
afforded the lower melting modification of nucleoside 15, m.p. 198–200 °C (resolidification) and
220–224 °C (dec.), without any changes at 130 °C. For C35H28N4O (632.6) calculated: 66.45% C,
4.46% H, 8.86% N; found 66.16% C, 4.35% H, 8.82% N.
Fractions containing the less mobile isomer were collected, evaporated and the residue crystallized
from ethanol to give 0.759 g (40%) of nucleoside 9, m.p. 118–120 °C (dec.) underpressed with a
sample of 9 prepared by cyclization of 6.
6-Phenyl-5-azacytidine (17)
A mixture of blocked nucleoside 9 (0.316 g, 0.5 mmol), methanol (1.5 ml) and methanolic 1
M
NaOMe (0.15 ml) was stirred at room temperature for 2.5 h and kept overnight. The solution was
acidified with acetic acid (0.02 ml) and decationized on a column of Amberlite IRC-50[H+] ion ex-
change resin (10 ml) which was prepared in methanol. The column was washed with methanol (100 ml),
the effluent evaporated, the residue dissolved in isopropyl alcohol (1 ml), the solution precipitated
with ether, the amorphous solid filtered off and crystallized from isopropyl alcohol to yield 0.102 g
(64%) of nucleoside 17, m.p. 200–201 °C (dec.), RF 0.54 (B), [α]D –1.0° (c 0.1, H2O). The sample
for analysis was dried at 80 °C/40 Pa for 2 h. UV spectrum, λmax (log ε): (MeOH), 260 (4.21), 210
(4.69); (1 M HCl), 277 (4.22), 208 (4.40); (pH 2.26), 277, inflexion (4.10), 248 (4.35), 227, inflexion
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

228
Hanna, Masojidkova, Fiedler, Piskala:
(4.25); (pH 6.94), 248 (4.28); (pH 10.84), 254 (4.42), 218 (4.34). CD spectrum (pH 6.86), λmax
([Θ]max): 268 (+720), 234 (–360). Mass spectrum: 321 (MH+). For C14H16N4O5 (320.3) calculated:
52.50% C, 5.04% H, 17.49% N; found: 52.21% C, 4.92% H, 17.77% N.
4-Amino-6-phenyl-3-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one (19)
A mixture of blocked nucleoside 15 (0.070 g, 0.11 mmol), methanol (1 ml) and methanolic 1 M NaOMe
(0.1 ml) was magnetically stirred at room temperature for 3 h. The solution was acidified with acetic
acid (0.01 ml) and decationized in analogy to the above preparation of 17. The residue after evapora-
tion of the methanolic effluent from the column was crystallized from isopropyl alcohol (1 ml) to
give 0.030 g (86%) of the free nucleoside 19, m.p. 200–202 °C (dec.), RF 0.54 (B), [α]D +38.7° (c 0.07,
H2O). UV spectrum, λmax (log ε): (MeOH), 263 (4.33), 212 (4.39); (pH 2.36), 279 (4.29); (pH 6.86),
263 (4.38), 210 (4.35); (pH 10.96), 259 (4.27), 216 (4.14). The sample for analysis was dried at 80 °C/40 Pa
for 2 h. For C14H16N4O5 (320.3) calculated: 52.50% C, 5.04% H, 17.49% N; found: 52.64% C, 5.07% H,
17.75% N.
2′,3′,5′-Tri-O-benzoyl-6-benzyl-5-azacytidine (8)
A solution of crude isocyanate 1 prepared by a known procedure4 from 1.008 g, 2 mmol of blocked
ribose16 14) in dry acetone (10 ml) was added dropwise at room temperature to stirred mixture of
(phenylacetyl)guanidine17 (2) (0.334 g, 2 mmol) and dry acetone (5 ml). The mixture was stirred for
30 min and worked up in analogy to the preparation of 6 to yield the crude syrupy acyl(carbamoyl)gua-
nidine 5. A solution of crude 5 (2 mmol) in acetonitrile (10 ml) was treated with chlorotrimethyl-
silane (1.7 ml, 13 mmol) and triethylamine (1.7 ml, 12 mmol), the mixture kept for 30 min at room
temperature and the mixture worked up in analogy to the preparation of 9. The crude product was
crystallized from ethanol (4 ml) and recrystallized from methanol–1,2-dichloroethane (2 : 1) to give
0.970 g (70%) of nucleoside 8, m.p. 223–226 °C (dec.), RF 0.35 (A), [α]D +10.1° (c 0.51, CHCl3).
IR spectrum (CHCl3); ν(NH2) 3 545 m, 3 427 m; ν(C=O) 1 728 s, 1 714 s, sh, 1 694 m, sh; ν(C=N)
1 621 s; δ(NH2) 1 651 m, sh. UV spectrum (MeOH), λmax (log ε): 265, inflexion (4.41), 229 (4.73),
1
210 (4.69). Mass spectrum: 647 (MH+). H NMR spectrum: 7.95 m, 2 H (arom. + NH2); 7.80 m, 17 H
(arom. + NH2); 7.00 m, 3 H (arom. + NH2); 6.23 d, 1 H, J(1′,2′) = 1.8 (H-1′); 6.06 br t, 1 H, J = 7.0
(H-3′); 5.98 dd, 1 H, J(2′,1′) = 1.8, J(2′,3′) = 6.4 (H-2′); 4.65–4.45 m, 3 H (H-4′ + 2 × H-5′); 4.25 d,
1 H (CH2-Ph); 4.03 d, 1 H, J(gem) = 15.6 (CH2-Ph). For C36H30N4O8 (646.7) calculated: 66.87% C,
4.56% H, 8.66% N; found: 66.79% C, 4.57% H, 8.36% N.
6-Benzyl-5-azacytidine (16)
A mixture of blocked nucleoside 8 (0.20 g, 0.31 mmol), methanol (2 ml) and methanolic 1 M NaOMe
(0.1 ml) was stirred at room temperature for 4 h and the solution worked up in analogy to the prep-
aration of 17. A solution of the crude product in methanol (2 ml) was precipitated with ether and the
amorphous solid crystallized from ethanol to give 0.080 g (77%) of free nucleoside 16, m.p. 206–208 °C
(dec.), RF 0.71 (B), [α]D –33.8° (c 0.1, DMF). UV spectrum, λmax (log ε): (MeOH), 240, inflexion
(3.95), 209 (4.32); (0.1 M HCl), 245, inflexion (3.44), 209 (4.46); (pH 2.32), 222 (4.05); (pH 6.94),
239, inflexion (3.94); (pH 10.93), 221 (4.46). CD spectrum (pH 6.93), λmax ([Θ]max): 271 (+50), 237
(–210). For C15H18N4O5 (334.3) calculated: 53.89% C, 5.43% H, 16.76% N; found: 53.77% C, 5.36% H,
17.00% N.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

6-Substituted 5-Azacytidines
229
2′,3′,5′-Tri-O-benzoyl-6-chloromethyl-5-azacytidine (10)
A solution of crude isocyanate 1 (prepared by a known procedure4 from 1.008 g, 2 mmol of blocked
ribose16 14) in dry acetone (10 ml) was added dropwise at room temperature to a stirred mixture of
(chloroacetyl)guanidine17 (4) (0.271 g, 2 mmol) and dry acetone (5 ml). The mixture was stirred for
30 min and worked up in analogy to the preparation of 6 to yield the crude syrupy acyl(carba-
moyl)guanidine. A solution of crude 7 (2 mmol) in acetonitrile (10 ml) was treated with chlorotri-
methylsilane (1.7 ml, 13 mmol) and triethylamine (1.7 ml, 12 mmol), the mixture kept for 30 min at
room temperature and worked up in analogy to the preparation of 9. The crude product was crystal-
lized from ethanol (2 ml) and recrystallized from 1,2-dichloroethane–methanol (2 : 1) to afford 0.726 g
(60%) of nucleoside 10, m.p. 217–218 °C (dec.), RF 0.39 (A), [α]D +10.5° (c 0.21, CHCl3). IR spec-
trum (CHCl3): ν(NH2) 3 539 m, 3 422 m; ν(C=O) 1 724 s; ν(C=N) 1 630 vs; δ(NH2) 1 656 m, sh.
1
UV spectrum (MeOH), λmax (log ε): 266, inflexion (4.43), 230 (4.76), 210 (4.64). H NMR spectrum:
8.05–7.80 m, 8 H (arom. + NH2); 7.60 m, 3 H (arom. + NH2); 7.50–7.30 m, 6 H (arom. + NH2); 6.13 d,
1 H, J(1′,2′) = 1.5 (H-1′); 6.07 m, 2 H (H-2′ + H-3′); 4.79 d, 1 H (CH2-Cl); 4.67 d, 1 H, J(gem) = 13.8
(CH2-Cl); 4.70–4.50 m, 3 H (H-4′ + 2 × H-5′). For C30H26ClN4O8 (605.1) calculated: 59.86% C,
4.10% H, 9.32% N, 5.98% Cl; found: 59.55% C, 4.17% H, 9.26% N, 5.86% Cl.
6-Chloromethyl-5-azacytidine (18)
A mixture of protected nucleoside 10 (0.20 g, 3 mmol), methanol (2 ml) and methanolic 1 M NaOMe
(0.1 ml) was stirred at room temperature for 3 h and worked up in analogy to the preparation of 16.
A solution of the crude product in methanol (2 ml) was precipitated with ether and the amorphous
powder dried for 3 h at 80 °C/40 Pa to give 0.064 g (65%) of the dihydrate of 18, m.p. 77–80 °C,
RF 0.41 (B), [α]D +24.4° (c 0.1, DMF). For C9H13ClN4O5 2 H2O (328.7) calculated: 33.89% C,
5.21% H, 17.05% N, 10.76% Cl; found: 33.62% C, 5.00% H, 16.86% N, 10.43% Cl. The very hy-
groscopic product hydrolyzed rapidly on storage at room temperature for several days.
The authors are indebted to Mrs I. Krizkova for measurement of UV spectra, to the late Dr I. Fric for
measurement and interpretation of CD spectra, to Mrs Z. Ledvinova for determination of optical rota-
tions, to Mrs A. Strejckova and Mrs Y. Cerna for technical assistance, 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.
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