Synthesis of N4-amino and N4-hydroxy derivatives of 5-azacytidine. A facile rearrangement of the N4-amino derivative to 5-(3-β-D-ribofuranosylureido)-1H-1,2,4-triazole

Article abstract of DOI:10.1135/cccc20040905

Treatment of methoxyribosyltriazinone 4 with hydrazine in methanol afforded crude 4-hydrazino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one (N ⁴-amino-5-azacytidine) (2), which rearranged rapidly to isomeric 5-ribosylureidotriazole 6. The rearrangement proceeds easily also in water solutions of 2. Alkaline hydrolysis of 6 gave a mixture of 5-ureidotriazole 7 and 5-aminotriazole 8. Acid hydrolysis of 6 afforded only 7. This compound was also prepared by thermal rearrangement of 5-amino-1-carbamoyltriazole 9 or on reaction of cyano(formyl)guanidine 10 with hydrazine hydrochloride. Treatment of benzoylated methoxyribosyltriazinone 4a with hydrazine in methanol gave only the rearranged product 6a. Reaction of tribenzoylribosyl isocyanate 12 with aminotriazole 8 gave 1-triazolecarboxamidotribenzoylribose 13, which afforded by methanolysis oxazoloribofuranose 14 and aminotriazole 8. Compound 14 was also obtained by methanolysis of blocked ribosylcarbamate 16. Reaction of methoxyribosyltriazinone 4 with hydroxylamine in methanol afforded 4-hydroxylamino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one (N ⁴-hydroxy-5-azacytidine) (3), which on the action of excess hydroxylamine yielded 1-cyano-1-hydroxy-5-β-D-ribofuranosylisobiuret (19). Treatment of methoxy-1,3,5-triazinone 18 with a solution of hydroxylamine in methanol gave 4-hydroxylamino-1-methyl-1,3,5-triazin-2(1H)-one (N ⁴-hydroxy1-methyl-5-azacytosine) (17). Heating of cyano(formyl)guanidine 10 with hydroxylamine hydrochloride in water lead to the formation of triuret (21). The mechanisms of the reactions of methoxyribosyltriazinone 4 with hydrazine and hydroxylamine are discussed. Compounds 2, 6 and 19 exhibited no significant antibacterial or cytostatic activity.

Full text of DOI:10.1135/cccc20040905

N4-Amino and N4-Hydroxy Derivatives of 5-Azacytidine
905
SYNTHESIS OF N4-AMINO AND N4-HYDROXY DERIVATIVES OF
5-AZACYTIDINE. A FACILE REARRANGEMENT OF THE N4-AMINO
DERIVATIVE TO 5-(3-β-D-RIBOFURANOSYLUREIDO)-1H-1,2,4-TRIAZOLE
1,
2
Alois PÍSKALA *, Naeem B. HANNA, Milena MASOJÍDKOVÁ , Pavel FIEDLER and
3
Ivan VOTRUBA
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
2
166 10 Prague 6, Czech Republic; e-mail: piskala@uochb.cas.cz, masojidkova@uochb.cas.cz,
votruba@uochb.cas.cz
3
Received Novem ber 12, 2003
Accepted February 22, 2004
Treatm en t of m eth oxyribosyltriazin on e 4 with h ydrazin e in m eth an ol afforded crude
4-h ydrazin o-1-β-D-ribofuran osyl-1,3,5-triazin -2(1H)-on e (N4-am in o-5-azacytidin e) (2), wh ich
rearran ged rapidly to isom eric 5-ribosylureidotriazole 6. Th e rearran gem en t proceeds easily
also in water solution s of 2. Alkalin e h ydrolysis of 6 gave a m ixture of 5-ureidotriazole 7
an d 5-am in otriazole 8. Acid h ydrolysis of 6 afforded on ly 7. Th is com poun d was also pre-
pared by th erm al rearran gem en t of 5-am in o-1-carbam oyltriazole 9 or on reaction of cya-
n o(form yl)guan idin e 10 with h ydrazin e h ydroch loride. Treatm en t of ben zoylated m eth oxy-
ribosyltriazin on e 4a with h ydrazin e in m eth an ol gave on ly th e rearran ged product 6a. Reac-
tion of triben zoylribosyl isocyan ate 12 with am in otriazole 8 gave 1-triazolecarboxam ido-
triben zoylribose 13, wh ich afforded by m eth an olysis oxazoloribofuran ose 14 an d am in o-
triazole 8. Com poun d 14 was also obtain ed by m eth an olysis of blocked ribosylcarbam ate 16.
Reaction of m eth oxyribosyltriazin on e
4 with h ydroxylam in e in m eth an ol afforded
4-h ydroxylam in o-1-β-D-ribofuran osyl-1,3,5-triazin -2(1H)-on e (N4-h ydroxy-5-azacytidin e) (3),
wh ich on th e action of excess h ydroxylam in e yielded 1-cyan o-1-h ydroxy-5-β-D-ribofuran o-
sylisobiuret (19). Treatm en t of m eth oxy-1,3,5-triazin on e 18 with a solution of h ydroxyl-
am in e in m eth an ol gave 4-h ydroxylam in o-1-m eth yl-1,3,5-triazin -2(1H)-on e (N4-h ydroxy-
1-m eth yl-5-azacytosin e) (17). Heatin g of cyan o(form yl)guan idin e 10 with h ydroxylam in e
h ydroch loride in water lead to th e form ation of triuret (21). Th e m ech an ism s of th e reac-
tion s of m eth oxyribosyltriazin on e 4 with h ydrazin e an d h ydroxylam in e are discussed. Com -
poun ds 2, 6 an d 19 exh ibited n o sign ifican t an tibacterial or cytostatic activity.
Keyw ord s: 1,3,5-Triazin es; 1,2,4-Triazoles; Nucleosides; Ureas; Hydrazon es; Cleavage; An ti-
bacterial activity.
In con tin uation of our study of th e n ucleoside an tibiotic 5-azacytidin e1-4
(1) (Ch art 1) an d its N4-substituted derivatives5,6, we were also in terested in
th e syn th esis an d biological activity of 4-h ydrazin o-1-β-D-ribofuran osyl-
1,3,5-triazin -2(1H)-on e (N4-am in o-5-azacytidin e) (2) (Sch em e 1) an d
Collect. Czech. Chem. Commun. (Vol. 69) (2004)
doi:10.1135/cccc20040905

906
Pískala et al.:
4-h ydroxylam in o-1-β-D-ribofuran osyl-1,3,5-triazin -2(1H)-on e (N4-h ydroxy-
5-azacytidin e) (3) (Sch em e 3). N4-Meth yl derivatives5 of 5-azacytidin e,
wh ich exh ibited an tibacterial activity, h ave been prepared in good yields by
am in olysis of th e well available 4-m eth oxy-1-β-D-ribofuran osyl-1,3,5-tri-
azin -2(1H)-on e7 (4) (Ch art 1). For th is reason we h ave used an an alogous
approach for th e preparation of n ucleosides 2 an d 3. In th is com m un ica-
tion we wish to discuss plausible m ech an ism s for th e form ation of n ucleo-
sides 2 an d 3 from th e m eth oxy n ucleoside 4 with h ydrazin e an d h ydroxyl-
am in e, respectively.
X
X
N
N
N
N
N
O
N
O
RO
Me
O
5, X = NHNH2
17, X = NHOH
18, X = OMe
OR OR
1, R = H, X = NH2
4, R = H, X = OMe
4a, R = Bz, X = OMe
CHART 1
Treatm en t of m eth oxy n ucleoside 4 with a solution of water-free h ydra-
zin e in m eth an ol at room tem perature for 4 m in gave crude h ydrazin e de-
rivative 2 (Sch em e 1), wh ich precipitated upon addition of eth er. Th e struc-
ture of 2 was proved by com parison of its UV spectra with th ose of th e
kn own structurally related 4-h ydrazin o-1-m eth yl-1,3,5-triazin -2(1H)-on e8
(N4-am in o-1-m eth yl-5-azacytosin e) (5) (Ch art 1). 1H NMR spectrum of
crude 2, wh ich was n ot well resolvable, in dicated th e presen ce of th ree spe-
cies. We assum e th e presen ce of 2, th e rearran ged product 6 an d possibly a
rin g-open ed in term ediate of th e rearran gem en t. All attem pts to purify
crude 2 by recrystallization or ch rom atograph y were n ot successful. On ly
th e rearran ged product 6 was isolated. Prolon ged treatm en t of 4 with h y-
drazin e in m eth an ol afforded pure crystallin e 5-(3-β-D-ribofuran osyl-
ureido)-1H-1,2,4-triazole (6) in an excellen t yield (Sch em e 1). Th e tran sfor-
m ation of 2 to 6 proceeded also rapidly in water solution s of crude 2 even
at room tem perature or very slowly on boilin g of crude 2 in m eth an ol.
Th e structure of th e rearran ged product 6 was in ferred from spectral
data an d was con firm ed by alkalin e h ydrolysis, wh ich afforded a m ixture
of 5-ureido-1H-1,2,4-triazole (7) an d th e well kn own 5-am in o-1H-1,2,4-tri-
azole9 (8). Com poun ds 7 an d 8 were isolated as picrates. Th e picrate of
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

N4-Amino and N4-Hydroxy Derivatives of 5-Azacytidine
907
am in otriazole 8 was also previously described10. On prolon gation of th e al-
kalin e h ydrolysis on ly am in otriazole 8 was detected in th e reaction m ix-
ture. Acid h ydrolysis of ribosylureidotriazole 6 gave exclusively ureidotri-
azole 7, h owever in a rath er low yield. Both types of h ydrolysis were accom -
pan ied by cleavage of th e C–N glycoside bon d so th at on ly th e aglycon es
were isolated. Ureidotriazole 7 was also prepared in low yield by th erm al re-
arran gem en t of th e kn own 5-am in o-1-carbam oyl-1H-1,2,4-triazole11 (9) or
on h eatin g of h ydrazin e h ydroch loride with 1-cyan o-3-form ylguan idin e12
(10) in water (Sch em e 1).
NHNH2
OMe
N
NHNH2
N N
CHO
N
N
H
N
N
O
N
O
HN
O
RO
HO
HO
O
O
O
NH2NH2
H2O
HN
MeOH
OR OR
OH OH
OH OH
2
11
4, R = H
4a, R = Bz
N
N
N
NH2NH2
MeOH
NaOH
HN
HN
HN
N
HN
H2N
NaOH
-ribose
-HOCN
N
N
H2N
O
8
HN
O
RO
7
O
NH2NH2.HCl
OR OR
O
N
NH
H2N
H2N
N
6, R = H
6a, R = Bz
MeONa
NC
CHO
N
N
H
N
H
9
10
SCHEME 1
Th e m ech an ism of th e rearran gem en t of N4-am in oazacytidin e 2 to
5-ribosylureidotriazole 6 in water solution s could be explain ed as follows.
Due to th e low electron den sity in position 6 of th e triazin e rin g, covalen t
addition of water to th e C=N double bon d in position s 5 an d 6 takes place.
Th e labile covalen t h ydrate is tran sform ed by a prototropic rearran gem en t
to th e rin g-open ed N-form yl derivative 11 (Sch em e 1), wh ich cyclizes to
ribosylureidotriazole 6. Th e reason for th is rearran gem en t is obviously th e
m uch lower stability of th e 1,3,5-triazin e in com parison with th e
1,2,4-triazole rin g. Th e reaction of m eth oxy n ucleoside 4 with h ydrazin e in
m eth an ol affords N4-am in o-5-azacytidin e (2) as th e prim ary product, wh ich
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

908
Pískala et al.:
rearran ged, eith er by th e action of traces of water in th e depicted m an n er
or, m ore probably, th e triazin e rin g open s by th e action of h ydrazin e an d
th e triazole rin g is form ed via a form am idrazon e in term ediate. Th e rear-
ran gem en t of th e crude am in oazacytidin e 2 in m eth an ol proceeds very
slowly, because th e addition of m eth an ol to th e C=N double bon d in posi-
tion 5 an d 6 an d subsequen t open in g of th e triazin e rin g proceeds n ot so
easy as with water or h ydrazin e. Con sequen tly, closin g to th e triazole is
slow. Also, we can n ot exclude th at th e rearran gem en t is m ediated by traces
of water presen t in th e solven t.
Th e reaction of triben zoyl derivative 4a (Ch art 1) with h ydrazin e in
m eth an ol afforded on ly th e rearran ged product 6a (Sch em e 1). From th e
m oth er liquor, a secon d portion of 6a was obtain ed, wh ich con tain ed also
som e α-an om er (α/β = 1:4). Th is result in dicates th at th e triazin e rin g of th e
blocked n ucleoside 4a is m ore reactive to h ydrazin e or water th an th e free
n ucleoside 4. Th e yield of n ucleoside 6a was rath er low due to partial
deben zoylation of th e ribose m oiety, wh ich occurs durin g th e reaction . Th e
structure of com poun d 6a was in ferred from spectral data an d was also con -
firm ed by m eth an olysis, wh ich gave th e free rearran ged product 6. An alter-
n ative preparation of th e blocked ribosylureidotriazole 6a on con den sation
of 2,3,5-tri-O-ben zoyl-β-D-ribosyl isocyan ate (12) with am in otriazole 8 an d
subsequen t rearran gem en t of th e blocked 5-am in o-1-(ribosylcarbam oyl)-
1H-1,2,4-triazole 13 failed (Sch em e 2). Th e prim arily form ed in term ediate
13 did n ot rearran ge like th e un substituted carbam oyl derivative 9. In th is
con n ection it sh ould be m en tion ed th at an an alogous situation was ob-
O
N
N
NCO
HN
N
BzO
BzO
8
O
O
H2N
OBz OBz
OBz OBz
12
13
EtOH
MeONa
–8
HO
NHCOOMe
O
NHCOOEt
MeONa
HO
BzO
NH
O
O
OH
O
OH OH
OBz OBz
O
14
15
16
SCHEME 2
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

N4-Amino and N4-Hydroxy Derivatives of 5-Azacytidine
909
served in th e case of 1-(m eth ylcarbam oyl)-5-am in o-1H-1,2,4-triazole13
.
Meth an olysis of th e blocked n ucleoside 13, in stead of givin g th e respective
free n ucleoside, afforded am in otriazole9 8, isolated as picrate10, an d
oxazoloribofuran ose12 14, wh ich were both iden tified by com parison with
auth en tic sam ples. Th e form ation of oxazoloribofuran ose 14 proceeds obvi-
ously via m eth yl N-β-D-ribofuran osylcarbam ate (15), wh ich un dergoes
an om erization an d cyclization to yield th e fin al product 14. To prove th is
assum ption we prepared ribofuran osooxazole 14 by m eth an olysis of th e
blocked ribosylureth an e 16, wh ich was prepared by a kn own procedure
from isocyan ate 12 an d eth an ol14.
4-Hydroxylam in o-1-β-D-ribofuran osyl-1,3,5-triazin -2(1H)-on e (N4-h ydro-
xy-5-azacytidin e) (3) was obtain ed by reaction of m eth oxy n ucleoside 4
with a sm all excess of h ydroxylam in e in m eth an ol (Sch em e 3). Th e product
was isolated as am orph ous m on oh ydrate by colum n ch rom atograph y on
silica gel. Th e h igh ly h ygroscopic product was ch aracterized by com parison
of its UV spectra with th ose of th e structurally related 4-h ydroxylam in o-
1-m eth yl-1,3,5-triazin -2(1H)-on e (N4-h ydroxy-1-m eth yl-5-azacytosin e) (17),
wh ich was prepared by reaction of 4-m eth oxy-1-m eth yl-1,3,5-triazin -
2(1H)-on e15 (18) with a solution of h ydroxylam in e in m eth an ol (Ch art 1).
NMR spectra of 17 in dicate th e prevalen ce of th e h ydroxyim in o over th e
h ydroxyam in o tautom eric form in solution . Prolon ged action of a large
excess of h ydroxylam in e on m eth oxy n ucleoside 4 afforded 1-cyan o-
1-h ydroxy-5-β-D-ribofuran osylisobiuret (19) (Sch em e 3). Th e structure of
th is rin g-open ed product was in ferred from elem en tal an alysis, m ass spec-
trum , IR spectrum (exh ibitin g ch aracteristic ban ds of th e cyan o group, C=N
bon d an d also of th e N–O bon d), an d even tually from 1H an d 13C NMR
spectra. Th e form ation of 19 can be explain ed by h ydrolytic open in g of th e
triazin e rin g, in an alogy to th e above m en tion ed N4-am in oazacytidin e 2,
yieldin g th e form yl derivative 20a, followed by tran sform ylation to 20b,
h ydrolysis of th e im in o group an d form ation of oxim e 20c, wh ich elim i-
n ates water to yield th e fin al product 19. Th is course of th e reaction was
also supported by th e form ation of triuret (21) by th e action of h ydroxyl-
am in e h ydroch loride on 1-cyan o-3-form ylguan idin e (10) (Sch em e 3). Th e
fin al product 21 was obviously form ed via oxim e 22 an d 1,3-dicyan o-
guan idin e (23).
Com poun ds 2, th e rearran ged product 6 an d th e decom position product
19 were tested for an tibacterial activity again st Escherichia coli. N4-Am in o-
5-azacytidin e (2) exh ibited on ly an 87% in h ibition of growth at 1 m g/m l
con cen tration an d th e product 19 h as sh own a 100% growth in h ibition un -
der th e sam e con dition s. Both com poun ds were in active at lower con cen -
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

910
Pískala et al.:
tration s. Th e rearran ged product 6 was com pletely in active even at ≤1 m g/m l
con cen tration s. Also in vitro in h ibition of cell growth with com poun ds 2, 6
an d 19 was evaluated in th e followin g cell cultures: h um an T-lym ph o-
blastoid CCRF-CEM cell lin e (ATCC CCL 119), h um an prom yeolocytic leu-
kem ia HL-60 (ATCC CCL 240) cells, h um an cervix carcin om a HeLa S3 cells
(ATCC CCL 2.2) an d m ouse leukem ia L1210 cells (ATCC CCL 219). Non e of
th e m en tion ed com poun ds exh ibited an y con siderable activity at 10 µM
con cen tration s.
HO
H
OMe
N
NHOH
N
N
N
OHC
N
N
NH
N
O
N
O
HN
O
HO
HO
HO
O
O
H2O
O
NH2OH
MeOH
OH OH
OH OH
OH OH
3
4
20a
HO CHO
N
HO CH=NOH
N
HO
CN
N
N
HN
HN
NH
O
NH
O
H
O
O
NH
HN
O
HO
HO
HO
O
O
O
NH2OH
H2O
- H2O
OH OH
OH OH
OH OH
20b
19
20c
NH
NH
NC
CN
NC
N
CH=NOH
NH2OH.HCl
H2O
-H2O
N
H
N
H
10
N
H
H
22
23
H2O
O
O
O
H2N
N
H
N
H
NH2
21
SCHEME 3
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N4-Amino and N4-Hydroxy Derivatives of 5-Azacytidine
911
EXPERIMENTAL
Meltin g poin ts were taken on a h eated m icroscope stage (Kofler block) an d are un corrected.
Un less stated oth erwise, th e solution s were evaporated at 35 °C/2.5 kPa an d an alytical sam -
ples were dried at 40 Pa (room tem perature). Th in -layer ch rom atograph y (TLC) was per-
form ed on Silufol UV 254 plates (Kavalier, Votice, Czech Republic) in solven t system s A,
ch loroform –m eth an ol (98:2), or B, butan -1-ol–acetic acid–water (5:2:3). Th e spots of th e
studied com poun ds were detected eith er in UV ligh t (254 n m ) (D1) or with n in h ydrin (D2).
Colum n ch rom atograph y was perform ed with silica gel accordin g to Pitra (Service Labora-
tories of th is In stitute). IR spectra were recorded on a IR spectrom eter Zeiss UR-20 an d on a
FTIR spectrom eter Bruker IFS 55 in KBr pellets, Nujol or DMSO solution s. UV spectra were
m easured on a Un icam SP 8000 spectrom eter (Pye–Un icam , Cam bridge, U.K.) in buffer solu-
tion s of ion ic stren gth 0.01 prepared accordin g to Perrin 16, λ are given in n m an d ε in
m 2 m ol–1. Optical rotation s were recorded on a polarim eter Perkin –Elm er, type 141 at 22 °C
an d are given in deg cm 3 g–1 dm –1 1H an d 13C NMR spectra were m easured at 500 an d
.
125.7 MHz on a Varian Un ity 500 in strum en t in DMSO-d6 with th e solven t sign al as th e in -
tern al referen ce (δ(1H) 2.5 an d δ(13C) 39.70 ppm ). Th e ch em ical sh ifts (δ-scale) are given in
ppm an d couplin g con stan ts (J) in Hz. Th e m ass spectra (m/z (% rel. in t.)) were m easured on
a ZAB-EQ (Microm ass, Man ch ester, U.K.) spectrom eter usin g a FAB ion ization (bom bardin g
gas Xe at 8 kV). A m ixture of glycerol an d th ioglycerol (1:3, v/v) was used as m atrix; sam -
ples were dissolved in m eth an ol or water. Station ary cultivation of Escherichia coli was per-
form ed at 37 °C in a m in eral m edium with glucose17. Th e tested com poun ds were added be-
fore in oculation an d bacterial growth was m easured 16 h later.
4-Hydrazin o-1-β-D-ribofuran osyl-1,3,5-triazin -2(1H)on e (2)
A solution of m eth oxytriazin on e7 4 (0.259 g, 1 m m ol) in m eth an ol (20 m l) was treated with
a 0.5 M solution of water-free h ydrazin e in m eth an ol (3.0 m l, 1.5 m m ol) an d th e m ixture
kept at room tem perature for 4 m in . Th e solution was precipitated with eth er (20 m l), th e
product filtered off by suction an d wash ed with eth er to yield 0.20 g (72%) of th e crude
m on oh ydrate of 2, m .p. 105–115 °C (dec.), RF 0.47 (B, D1, D2), [α]D –22.7 (c 0.20, water). All
attem pts to purify th e sam ple by recrystallization or colum n ch rom atograph y were un suc-
cessful, on ly th e rearran ged product 6 was isolated. UV, λm ax (log ε), pH 2.33: 258 (3.53),
223 (3.76); pH 7.05: 211 (4.16) (rearran gem en t to 6); pH 10.99: 216 (4.01) (rearran gem en t to
6). MS: 260 (10) [M + H]+, 128 (7) [M + H]+ – aglycon e. For C8H13N5O5·H2O (277.2) calcu-
lated: 34.66% C, 5.45% H, 25.56% N; foun d: 34.38% C, 5.15% H, 25.16% N.
4-Hydrazin o-1-m eth yl-1,3,5-triazin -2(1H)-on e (5)
Th is com poun d was prepared by a kn own 8 procedure. UV, λm ax (log ε), pH 2.30: 258 (3.76),
223 (3.65); pH 7.03: 250 (3.75), 209 (4.11); pH 10.97: 252 (3.84), 216 (4.03). 1H NMR: 9.05
br s, 1 H (NH); 8.175 s, 1 H (H-6); 3.20 s, 3 H (CH3); 4.41 br s, 2 H (NH2). 13C NMR: 154.595
(C-2), 165.21 (C-4), 158.535 (C-6), 34.005 (CH3). MS: 142 (4) [M + H]+.
5-(3-β-D-Ribofuran osylureido)-1H-1,2,4-triazole (6)
Method A. A m ixture of m eth oxytriazin on e7 4 (0.259 g, 1 m m ol) an d a 0.5 M solution of
water-free h ydrazin e in m eth an ol (3.0 m l, 1.5 m m ol) was stirred at room tem perature for
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

912
Pískala et al.:
20 m in . Th e crystals were filtered off by suction to give 0.23 g (89%) of triazole 6, m .p.
202–205 °C (dec.), recrystallization from water raised th e m eltin g poin t to 206–209 °C (dec.),
RF 0.48 (B, D2), [α]D –62.2 (c 0.50, water). UV, λm ax (log ε), MeOH: 220 (3.99); pH 0: 213
(4.04); pH 7.0: 213 (4.09). IR (Nujol): 3389 m , 3370 m , 3294 s, 3207 s, 3110 m , 3045 m
[ν(OH, NH)]; 1701 [ν(C=O)]. 1H NMR: 13.10 br, 1 H, 9.80 br s, 1 H an d 7.90 br, 1 H (3 ×
NH); 7.90 br, 1 H (H-3); 5.08 br s, 1 H, 4.93 br s, 1 H an d 4.78 br s, 1 H (3 × OH); 5.23 dd,
1 H, J(1′,2′) = 5.6, J(1′,NH) = 9.4 (H-1′); 3.72 t, 1 H, J(2′,1′) = J(2′,3′) = 5.5 (H-2′); 3.85 dd,
1 H, J(3′,4′) = 3.9, J(3′,2′) = 5.4 (H-3′); 3.68 br q, 1 H, J = 3.9 (2 ×) an d 4.4 (H-4′); 3.45 dd,
1 H, J(5′a,4′) = 3.9, J(gem ) = 11.7 (H-5′a); 3.39 dd, 1 H, J(5′b,4′) = 4.4, J(gem ) = 11.7 (H-5′b).
13C NMR: 147.0 br (2 × C) (C-3 an d C-5), 153.72 (C=O), 84.515 (C-1′), 74.55 (C-2′), 70.56
(C-3′), 83.45 (C-4′), 62.06 (C-5′). MS: 260 (1) [M + H]+, 128 (3) [M + H]+ – aglycon e. For
C8H13N5O5 (259.2) calculated: 37.07% C, 5.05% H, 27.02% N; foun d: 37.15% C, 5.12% H,
26.75% N.
Method B. A solution of N4-am in o derivative 2 (29 m g, 0.1 m m ol) in h ot water (1.5 m l)
was kept after coolin g at room tem perature for 3 h an d evaporated. Th e residue was
triturated with eth an ol an d th e solid recrystallized from a m ixture of m eth an ol an d water to
yield 15 m g (52%) of 6, m .p. 204–205 °C (dec.) with out depression on adm ixture of a sam -
ple prepared by m eth od A.
Method C. A m ixture of 2 (29 m g, 0.1 m m ol) an d m eth an ol (5 m l) was refluxed for 24 h .
Th e crystals were filtered off by suction an d recrystallized from a m ixture of m eth an ol an d
water to afford 17 m g (57%) of 6, m .p. 203–205 °C (dec.) with out depression on adm ixture
of a sam ple prepared by m eth od A.
Method D. A solution of blocked n ucleoside 6a (28.5 m g, 0.05 m m ol) in m eth an olic 0.2 M
NaOMe (0.5 m l) was kept at room tem perature for 90 m in . Th e solution was treated with
acetic acid (0.01 m l) an d applied on to a colum n of Am berlite IRC [H+] ion exch an ge resin
(10 m l, packed in m eth an ol). Th e colum n was eluted with m eth an ol (100 m l), th e eluate
evaporated an d th e residue co-evaporated with water (3 × 5 m l). Crystallization of th e resi-
due from eth an ol afforded 5 m g (38%) of com poun d 6, m .p. 202–205 °C with out depression
on adm ixture of a sam ple prepared by m eth od A.
Alkalin e Hydrolysis of 6
A solution of ribosylureidotriazole 6 (0.259 g, 1 m m ol) in 2 M KOH (2 m l) was refluxed for
90 m in . Th e solution was applied on to a colum n of Am berlite IRC-50 [H+] ion exch an ge
resin (30 m l, packed in m eth an ol) an d th e colum n eluted with m eth an ol (150 m l). Th e
eluate was evaporated, th e residue boiled with 96% eth an ol (3 m l), th e solution filtered an d
th e clear filtrate precipitated with a solution of picric acid (0.120 g) in eth an ol (2 m l). Th e
m ixture was kept at room tem perature for 1 h , th e yellow crystals filtered off with suction
an d wash ed with eth an ol to yield 0.125 g (35%) of th e picrate of ureidotriazole 7, m .p.
218–220 °C (dec.), RF 0.51 (B, D2, 7) an d 0.90 (B, D1, picric acid). Th e an alytical sam ple was
recrystallized from m eth an ol, m .p. 220–222 °C (dec.). UV, λm ax (log ε), MeOH: 356 (4.16),
240 (in fl.) (4.11), 213 (4.45). 1H NMR: 10.59 br s, 1 H, 9.0 br, 2 H, 6.83 br s, 2 H (3 × NH +
NH2); 8.70 s, 1 H (triazole H-3); 8.59 s, 2 H (picrate H-3 + H-5). 13C NMR: 160.95 (triazole
C-5), 141.37 (triazole C-3), 153.67 (ureido C=O), 148.43 (picrate C-1), 142.04 (picrate C-2),
125.44 (picrate C-3), 124.67 (picrate C-4). MS: 128 (1) [M7 + H]+, 85 (12) [M8 + H]+. For
C3H5N5O·C6H3N3O7 (356.2) calculated: 30.34% C, 2.26% H, 31.46% N; foun d: 30.62% C,
2.34% H, 31.78% N.
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N4-Amino and N4-Hydroxy Derivatives of 5-Azacytidine
913
Th e m oth er liquor after isolation of th e picrate of 7 was evaporated an d th e residue crys-
tallized from eth an ol (2 m l) to afford 0.045 g (14%) of th e picrate of am in otriazole 8, m .p.
228–232 °C (dec.). An an alytical sam ple was recrystallized from water, m .p. 233–236 °C
(dec.) with out depression on adm ixture of an auth en tic10 sam ple, RF 0.48 (B, D2, 8) an d
0.90 (B, D1, picric acid). UV, λm ax (log ε), MeOH: 355 (4.15), 240 (4.13), 211 (4.37). 1H NMR:
8.31 s, 1 H (H-3); 8.00 br, 4 H (N1-H + N2-H + NH2); 8.59 s, 2 H (picrate H-3 + H-5).
13C NMR: 160.99 (C-5), 139.54 (C-3), 151.02 (picrate C-1), 142.04 (picrate C-2), 125.38
(picrate C-3), 124.37 (picrate C-4). MS: 85 (12) [M8 + H]+. For C2H4N4O·C6H3N3O7 (313.2)
calculated: 30.68% C, 2.26% H, 31.31% N; foun d: 30.97% C, 2.15% H, 31.51% N.
On prolon gation of th e tim e of h ydrolysis to 2 h , 22% of th e picrate of 7 an d 41% of th e
picrate of 8 were obtain ed by th e sam e workin g-up procedure. Prolon gation of th e tim e of
h ydrolysis to 8 h led to com plete h ydrolysis of th e in term ediate 7. TLC in dicated on ly th e
presen ce of th e fin al product 8.
Acid Hydrolysis of 6
A solution of ribosylureidotriazole 6 (0.259 g, 1 m m ol) in 10% h ydroch loric acid (6 m l) was
h eated at 100 °C (bath tem perature) for 1 h an d evaporated. Th e residue was triturated with
m eth an ol (10 m l), a sm all in soluble portion filtered off an d th e filtrate applied on to a col-
um n of Am berlite IR-45 [OH–] (40 m l, packed in m eth an ol). Th e colum n was eluted with
m eth an ol (200 m l), th e eluate filtered with ch arcoal an d evaporated. Th e residue was dis-
solved in eth an ol (6 m l) an d, after addition of two drops of water an d filtration of th e solu-
tion (to rem ove sligh t turbidity), a solution of picric acid (0.240 g) in eth an ol (6 m l) was
added. Con cen tration of th e solution gave 0.14 g of crude picrate of 7, m .p. 195–210 °C
(dec.). Recrystallization of th e crude m aterial from m eth an ol afforded in two portion s 0.10 g
(28%) of th e picrate of 7, m .p. 217–219 °C (dec.) with out depression on adm ixture with th e
sam ple prepared by alkalin e h ydrolysis.
5-Ureido-1H-1,2,4-triazole (7)
Method A. A solution of th e picrate of 7 (0.178 g, 0.5 m m ol) in m eth an ol (60 m l) was ap-
plied on to a colum n of Am berlite IR-45 [OH–] (60 m l, packed in m eth an ol). Th e colum n was
wash ed with m eth an ol (600 m l) an d th e eluate evaporated. Th e residue was crystallized
from 9:1 eth an ol–water to yield 0.050 g (78%) of ureidotriazole 7; th e com poun d decom -
posed at 245–250 °C with out m eltin g up to 350 °C, RF 0.51 (B, D2). UV, λm ax (log ε), pH
2.30: 222 (3.81); pH 6.90: 210 (4.07); pH 10.8: 218 (3.92). MS: 128 (100) [M + H]+. For
C3H5N5O (127.1) calculated: 28.35% C, 3.97% H, 55.10% N; foun d: 28.56% C, 3.90% H,
55.12% N.
Method B. 5-Am in o-1-carbam oyltriazole11 9 (0.381 g, 3 m m ol) was h eated at 180 °C for
10 m in . Th e startin g com poun d m elted an d th en resolidified. Th e crude product was pow-
dered an d extracted with boilin g 9:1 eth an ol–water (50 m l). Th e in soluble portion was fil-
tered off after coolin g an d th e filtrate evaporated. A solution of th e residue in 9:1 eth an ol–
water (5 m l) was treated with a solution of picric acid (0.12 g) in eth an ol (2 m l), th e yellow
precipitate filtered off with suction an d wash ed with eth an ol to afford 0.07 g (7%) of th e
picrate of 7, m .p. 218–220 °C (dec.) with out depression on adm ixture of a sam ple obtain ed
from h ydrolysis of ribosylureidotriazole 6. Th e free ureidotriazole 7 was obtain ed from th e
picrate usin g m eth od A. Yield 0.018 g (5%, based on 9), th e product decom posed at 245–
250 °C with out depression on adm ixture of a sam ple prepared by m eth od A.
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914
Pískala et al.:
Method C. A m ixture of cyan o(form yl)guan idin e 10 (0.448 g, 4 m m ol), h ydrazin e h ydro-
ch loride (0.274 g, 4 m m ol) an d water (2 m l) was refluxed for 20 m in . Th e m ixture was
cooled to 0 °C, th e crystals were filtered off with suction an d wash ed with ice water an d
m eth an ol to give 0.11 g of th e crude h ydroch loride of ureidotriazole 7 con tam in ated with
th e free base. 1H NMR: 10.46 br s, 1 H, 9.15 br, 2 H an d 6.92 br s, 2 H (3 × NH + NH2);
8.30 s, 1 H (H-3). 13C NMR: 150.16 (C-5), 143.47 (C-3), 154.36 (C=O). MS: 128 (100) [M +
H]+. Th e free base 7 was obtain ed by m eth od A. Yield 0.055 g (11%), th e product decom -
posed at 245–250 °C with out depression on adm ixture of a sam ple prepared by m eth od A.
5-[3-(2,3,5-Tri-O-ben zoyl-β-D-ribosyl)ureido]-1H-1,2,4-triazole (6a)
A m ixture of th e blocked m eth oxy n ucleoside7 4a (1.143 g, 2 m m ol) an d m eth an ol (50 m l)
was sh ortly h eated to dissolve th e startin g com poun d. Th e solution , after coolin g to room
tem perature, was treated with 0.5 M water-free h ydrazin e in m eth an ol (6 m l). Th e product
started to deposit from solution alm ost im m ediately. Th e m ixture was kept overn igh t at
room tem perature an d th e precipitate filtered off with suction to give 0.220 g of crude 6a,
m .p. 182–190 °C (dec.). Recrystallization of th e crude m aterial from m eth an ol–ch loroform
afforded 0.170 g (15%) of pure 6a, m .p. 195–200 °C (dec.), RF 0.51 (A, D1), [α]D –52.6
(c 0.20, DMF). 1H NMR: 13.30 br, 1 H, 10.05 br, 1 H an d 8.00 br, 1 H (3 × NH); 7.87 br s,
1 H (H-3); 8.03 d, 2 H, 7.88 d, 4 H, 7.67 t, 1 H, 7.64 t, 2 H, 7.51 t, 2 H an d 7.45 t, 4 H
(arom .); 5.82 dd, 1 H, J(1′,2′) = 5.7, J(1′,NH) = 9.0 (H-1′); 5.79 dd, 1 H, J(2′,3′) = 4.9, J(2′,1′) =
5.7 (H-2′); 5.68 br t, 1 H, J(3′,2′) = J(3′,4′) = 4.8 (H-3′); 4.60 m , 2 H (H-4′ + H-5′a); 4.55 dd,
1 H, J(5′b,4′) = 5.6, J(gem .) = 12.6 (H-5′b). 13C NMR: 151.80 br (C-5); 146.50 br (C-3); 153.58
(ureido C=O); 165.68, 164.89 an d 164.87 (3 × ben zoyl C=O); 134.08, 133.97, 133.71, 129.47
(2 × C); 129.45 (4 × C); 128.98 (2 × C); 128.94 (2 × C); 128.91 (2 × C); 128.84 an d 128.654
(2 × C) (arom .); 83.44 (C-1′); 73.835 (C-2′); 71.33 (C-3′); 77.86 (C-4′); 64.41 (C-5′). MS: 572
(8) [M + H]+, 445 (5) [M]+ of th e triben zoylribosyl ion . For C29H25N5O8 (571.6) calculated:
60.94% C, 4.41% H, 12.25% N; foun d: 60.95% C, 4.35% H, 12.04% N.
Th e m oth er liquor of th e crude product was evaporated an d applied on to a colum n of sil-
ica gel (50 g), prepared in ch loroform . Th e colum n was eluted with 250-m l portion s of ch lo-
roform con tain in g 5, 10, 20 an d 40% of m eth an ol. Th e fraction s con tain in g th e m ajor por-
tion of th e product were evaporated an d th e residue (0.4 g) was crystallized from a m ixture
of ch loroform –m eth an ol to yield an oth er portion of 6a (0.040 g, 3.5%). Evaporation of th e
m oth er liquor an d crystallization of th e residue from m eth an ol gave 0.050 g (4.3%) of a
m ixture of 6a an d its α-an om er, m .p. 162–166 °C (dec.), [α]D –33.6 (c 0.21, DMF). 1H NMR:
10.10 br an d 8.0 br (NH); 8.05–7.40 m , 15 H (arom .); β-an om er: 5.82 dd, 1 H, J = 5.4 an d
9.6 (H-1′); 5.79 dd, 1 H, J = 5.4 an d 5.6 (H-2′); 5.68 br t, 1 H, J = 5.5 (H-3′); 4.60 m , 2 H an d
4.54 m , 1 H (H-4′ + 2 × H-5′); α-an om er: 6.21 dd, 1 H, J = 4.5 an d 9.2 (H-1′); 6.02 m , 2 H
(H-2′ + H-3′) (α/β = 1:4). 13C NMR, β-an om er: 83.45 (C-1′), 73.85 (C-2′), 71.345 (C-3′), 77.87
(C-4′), 64.41 (C-5′); α-an om er: 80.19 (C-1′), 73.96 (C-2′), 72.31 (C-3′), 78.03 (C-4′), 64.45
(C-5′); th e oth er sign als were n ot distin guish ed. MS: 572 (3) [M + H]+, 445 (7) [M]+ of th e
triben zoylribosyl ion . For C29H25N5O8 (571.6) calculated: 60.94% C, 4.41% H, 12.25% N;
foun d: 60.62% C, 4.39% H, 11.90% N.
5-Am in o-1-[(2,3,5-tri-O-ben zoyl-β-D-ribosyl)carbam oyl]-1H-1,2,4-triazole (13)
A solution of crude isocyan ate 12 (prepared from 5 m m ol of 1-O-acetyl-2,3,5-tri-O-ben zoyl-
β-D-ribose18 by a kn own procedure19) in toluen e (25 m l) was added dropwise at room tem -
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

N4-Amino and N4-Hydroxy Derivatives of 5-Azacytidine
915
perature to a stirred m ixture of am in otriazole 8 (0.42 g, 5 m m ol) in dry aceton e (10 m l). Th e
m ixture was stirred at room tem perature for 1 h an d th en evaporated. A solution of th e resi-
due in ch loroform (50 m l) was wash ed with water (3 × 10 m l), dried (an h ydrous MgSO4),
evaporated an d th e syrupy residue applied on to a colum n of silica gel (50 g). Th e colum n
was eluted successively with 250 m l portion s of toluen e, m ixtures of toluen e with 10, 30
an d 50% of eth yl acetate an d eth yl acetate. Th e elution was followed by TLC in solven t sys-
tem A. Fraction s con tain in g th e m ajor product were collected, evaporated an d th e residue
dried at 80 °C/40 Pa to yield 1.5 g (52%) of th e blocked n ucleoside 13 as a foam , RF 0.24
(A, D1), [α]D –32.1 (c 0.20, ch loroform ). UV, λm ax (log ε), MeOH: 229 (4.73), 208 (4.57).
1H NMR: 9.60 d, 1 H, J(NH,1′) = 9.0 (NH); 7.32 br s, 2 H (NH2); 7.64 s, 1 H (H-3); 8.03 d,
2 H, 7.91 d, 2 H, 7.84 d, 2 H, 7.65 m , 3 H, 7.50 t, 2 H, 7.47 t, 2 H an d 7.42 t, 2 H (arom .);
5.83 dd, 1 H, J(1′,2′) = 4.0, J(1′,NH) = 9.0 (H-1′); 5.92 m , 2 H (H-2′ + H-3′); 4.62 m , 2 H (H-4′ +
H-5′a); 4.53 dd, 1 H, J(5′b,4′) = 6.0, J(gem .) = 12.9 (H-5′b). 13C NMR: 165.70, 164.86 an d
164.80 (ben zoyl C=O); 134.05, 133.94, 133.66, 129.57 (2 × C); 129.48 (2 × C); 129.42 (2 × C);
128.99 (2 × C); 128.86 (4 × C); 128.80 an d 128.72 (2 × C) (arom .); 157.15 (C-5); 150.52
(C-3); 151.14 (carbam oyl C=O); 83.815 (C-1′); 73.77 (C-2′); 70,90 (C-3′); 78.02 (C-4′); 63.96
(C-5′). MS: 572 (75) [M + H]+, 445 (100) [M]+ of th e triben zoylribosyl ion . For C29H25N5O8
(571.6) calculated: 60.94% C, 4.41% H, 12.25% N; foun d: 61.15% C, 4.25% H, 11.98% N.
4,5-Dih ydro-(1,2-dideoxy-α-D-ribofuran oso[1,2-d]-1,3-oxazol-2-on e (14)
Method A. A solution of th e blocked n ucleoside 13 (0.285 g, 0.5 m m ol) in 1 M NaOMe
(2.5 m l) was kept at room tem perature for 3 h . Th e solution was acidified with acetic acid
(0.2 m l) an d applied on to a colum n of Am berlite IRC-50 [H+] ion exch an ge resin (30 m l),
wh ich was prepared in m eth an ol. Th e colum n was wash ed with m eth an ol (200 m l) an d th e
eluen t evaporated. Th e residue was crystallized from eth an ol (3 m l) to afford 0.045 g (51%)
of th e oxazolon e 14, m .p. 165–167 °C with out depression on adm ixture of an auth en tic
sam ple12, RF 0.82 (B, D2), [α]D +111.3 (c 0.108, water). UV, λm ax (log ε), MeOH: 209 (3.77).
1H NMR: 8.60 br s, 1 H (NH); 5.52 d, 1 H, J(1,2) = 5.3 (H-1); 4.78 t, 1 H, J(2,1) ≈ J(2,3) = 5.4
(H-2); 3.78 br dt, 1 H, J(3,2) ≈ J(3,OH) = 6.0, J(3,4) = 9.3 (H-3); 5.38 d, 1 H, J(OH,3) = 6.5
(3-OH); 3.59 ddd, 1 H, J(4,5a) = 2.0, J(4,5b) = 5.3, J(4,3) = 9.3 (H-4); 3.66 ddd, 1 H, J(5a,4) =
2.0, J(5a,OH) = 5.0, J(gem ) = 12.2 (H-5a); 3.42 dt, 1 H, J(5b,4) ≈ J(5b,OH) = 5.0, J(gem ) = 12.2
(H-5b); 4.73 br t, 1 H, J(OH,5) = 5.2 (5-OH). 13C NMR spectra: 158.28 (C=O), 84.73 (C-1),
78.73 (C-2 or C-4), 70.54 (C-3), 78.94 (C-4 or C-2), 60.15 (C-5). MS: 176 (100) [M + H]+. For
C6H9NO5 (175.1) calculated: 41.14% C, 5.18% H, 8.00% N; foun d: 41.35% C, 5.25% H,
8.23% N.
Th e m oth er liquor was evaporated an d a solution of th e residue in eth an ol (2 m l) was
treated with a solution of picric acid (0.12 g) in eth an ol (2 m l) to deposit im m ediately th e
picrate of am in otriazole 8. Yield 0.125 g (80%), m .p. 233–236 °C (dec.) with out depression
on adm ixture of an auth en tic sam ple10, RF 0.48 (B, D2, 8) an d 0.90 (B, D1, picric acid). For
C2H4N4·C6H3N3O7 (313.2) calculated: 30.68% C, 2.26% H, 31.31% N; foun d: 30.97% C,
2.31% H, 31.60% N.
Method B. A solution of (triben zoylribosyl)carbam ate14 16 (0.266 g, 0.5 m m ol) in 1
M
NaOMe (2.5 m l) was allowed to stan d at room tem perature for 1.5 h . Th e solution was
decation ized with Am berlite IRC-50 [H+], as described by m eth od A. Th e eluen t was evapo-
rated, th e residue co-evaporated with water (3 × 5 m l) an d crystallized from eth an ol (1 m l)
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916
Pískala et al.:
with seedin g to yield 0.038 g (43%) of com poun d 14, m .p. 165–167 °C with out depression
on adm ixture of an auth en tic sam ple12
.
4-Hydroxylam in o-1-m eth yl-1,3,5-triazin -2(1H)-on e (17)
A solution of 4-m eth oxy-1-m eth yl-1,3,5-triazin -2-on e15 (18; 0.070 g, 0.5 m m ol) in
1 M
h ydroxylam in e in m eth an ol (6 m l) was allowed to stan d at room tem perature for 1.5 h . Th e
deposited crystals were filtered off with suction to give 0.05 g (70%) of com poun d 17, m .p.
224–226 °C (dec.), RF 0.54 (B, D1). UV, λm ax (log ε), MeOH: 275 (4.43), 216 (4.66); pH 2.30:
265 (3.91), 227 (3.93); pH 6.90: 275 (3.45), 217 (4.11); pH 10.80: 254 (3.89), 225 (3.91). IR
(KBr): 3307 m , 3095 m , br [ν(OH,NH)]; 1724 s, 1714 s, sh [ν(C=O)]; 1660 s, 1645 s, sh
[ν(C=N)]. IR (DMSO): 1726 s, 1717 s, sh [ν(C=O)]; 1659 s (asym m etric) [ν(C=N)]. 1H NMR:
9.90 br, 1 H an d 9.82 br, 1 H (NH, OH); 7.72 s, 1 H (H-6); 3.105 s, 3 H (CH3). 13C NMR:
151.58 br, 2 C (C-2 an d C-4); 149.24 br (C-6); 32.30 (CH3). MS: 143 (6) [M + H]+. For
C4H6N4O2 (142.1) calculated: 33.81% C, 4.26% H, 39.42% N; foun d: 33.79% C, 4.20% H,
39.69% N.
4-Hydroxylam in o-1-β-D-ribofuran osyl-1,3,5-triazin -2(1H)-on e (3)
A solution of m eth oxytriazin on e7 4 (0.259 g, 1 m m ol) in 1 M h ydroxylam in e in m eth an ol
(3 m l) was kept at room tem perature overn igh t. Th e deposited crystals were filtered off with
suction to yield 0.04 g (14%) of th e decom position product 19, m .p. 215–219 °C (dec.). Th e
m oth er liquor was evaporated an d th e residue applied on to a colum n of silica gel (10 g),
wh ich was prepared in ch loroform . Th e colum n was eluted with 50 m l portion s of ch loro-
form con tain in g 10, 20, 30 an d 40% of m eth an ol. Th e fraction s con tain in g m ajor portion s
of th e product were collected, evaporated an d th e residue dissolved in a 2:8 m ixture of
m eth an ol–isopropyl alcoh ol (3 m l). Th e deposited am orph ous h ygroscopic solid was filtered
off with suction to afford 0.10 g (36%) of th e m on oh ydrate of 3, m .p. 110–120 °C (dec.),
RF 0.37 (B, D1). UV, λm ax (log ε), MeOH: 280 (3.34), 215 (3.96); pH 2.30: 268 (3.34), 225
(3.89); pH 7.03: 250 (in fl.) (3.81), 217 (4.00) (partly decom poses). For C8H12N4O6·H2O
(278.2) calculated: 34.54% C, 5.07% H, 20.14% N; foun d: 34.84% C, 4.78% H, 19.89% N. Th e
product was very h ygroscopic an d decom posed rapidly on stan din g at room tem perature.
1-Cyan o-1-h ydroxy-5-β-D-ribofuran osylisobiuret (19)
A solution of m eth oxytriazin on e7 4 (0.259 g, 1 m m ol) in 1 M h ydroxylam in e in m eth an ol
(12 m l) was kept at room tem perature overn igh t to deposit 0.170 g (61.5%) of com poun d
19, m .p. 213–218 °C (dec.), RF 0.34 (B, D1), [α]D –31.4 (c 0.15, water). UV, λm ax (log ε),
MeOH: 220 (4.22); pH 2.3: 226 (4.02); pH 7.0: 221 (4.28); pH 11.0: 221 (4.30). IR (KBr):
3348 s, 3315 s, 3235 s [ν(OH,NH)]; 2195 s, ≈2170 m , sh [ν(C≡N)]; 1673 vs (am ide I); 1632 vs
[ν(C=N)]; 1547 vs (tran s-am ide II); 1479 s [β(NOH)]; 1336 s, 1322 s [am ide III, ν(C–N)];
1089 vs, 1040 s [ν(C–O)]; 920 s [ν(N–O)]; 774 m , 766 m [γ(NOH)]; 592 m , br (am ide VI). IR
(DMSO): ≈3490 vs, sh , 3435 vs, ≈3285 s, sh [ν(NH,OH)]; 2166 s [ν(C≡N)]; 1690 vs (am ide I);
1627 vs [ν(C=N)]; 1538 vs (tran s-am ide II); 1323 s [am ide III, ν(C≡N)]; 900 w [ν(N–O)];
772 m [γ(NOH)]. 1H NMR: 9.27 d, 1 H, J(NH,1′) = 9.5 (5-NH); 8.33 s, 1 H (3-NH); 5.08 br s,
1 H, 4.93 br s, 1 H an d 4.76 br t, 1 H, J ≈ 5.0 (3 × ribosyl OH); 5.17 dd, 1 H, J(1′,2′) = 5.6,
J(1′,NH) = 9.5 (H-1′); 3.65 t, 1 H, J(2′,1′) = J(2′,3′) = 5.5 (H-2′); 3.79 dd, 1 H, J(3′,4′) = 4.0,
J(3′,2′) = 5.4 (H-3′); 3.62 br q, J = 4.0 (2 ×), J = 5.0 (H-4′); 3.40 m , 2 H (2 × H-5′); + AcOD:
Collect. Czech. Chem. Commun. (Vol. 69) (2004)

N4-Amino and N4-Hydroxy Derivatives of 5-Azacytidine
917
3.42 dd, 1 H, J(5′a,4′) = 4.1, J(gem ) = 11.6 (H-5′a); 3.36 dd, 1 H, J(5′b,4′) = 4.9, J(gem ) = 11.6
(H-5′b). Th e reson an ce sign al of th e N-OH proton , wh ich appeared as a very broad sin glet,
was n ot evaluated. 13C NMR: 163.08 (C=N), 155.33 (C=O), 121.245 (C≡N), 83.955 (C-1′),
74.48 (C-2′), 70.60 (C-3′), 83.56 (C-4′), 62.36 (C-5′). MS: 279 (18) [M + 3 H]+, 277 (3) [M +
H]+, 147 (10) [M + 3 H]+ – aglycon e of th e dih ydro derivative, 145 (6) [M + H]+ – aglycon e.
For C8H12N4O6 (276.2) calculated: 34.79% C, 4.38% H, 20.28% N; foun d: 34.50% C, 4.09% H,
19.99% N.
Triuret (21)
A m ixture of cyan o(form yl)guan idin e12 10 (0.112 g, 1 m m ol), h ydroxylam in e h ydroch loride
(0.070 g, 1 m m ol) an d water (2 m l) was refluxed for 30 m in . Th e m ixture was cooled an d
th e deposited crystals filtered off with suction to yield 0.050 g (34%) of triuret (21), m .p. >
350 °C (dec.), RF 0.76 (B, D2). UV, λm ax (log ε), MeOH: 208 (4.01); pH 2.36: 220 (2.98);
pH 6.86: 206 (4.12); pH 10.96: 227 (4.22). 1H NMR: 9.67 br s, 2 H (2 × NH); 7.23 br s, 2 H
an d 6.95 br s, 2 H (2 × NH2). 13C NMR: 154.23 (2 × C), 153.01 (3 × C=O). MS: 147 (2) [M +
H]+. For C3H6N4O3 (146.1) calculated: 24.66% C, 4.14% H, 38.35% N; foun d: 24.84% C,
4.12% H, 38.61% N.
The authors are indebted to Ms I. Křížková for measurement of UV spectra, Ms Z. Ledvinová for de-
termination of optical rotations, to Ms Y. Černá for technical assistance, to Dr A. Čihák 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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