Synthesis of N4-alkyl-5-azacytidines and their base-pairing with carbamoylguanidines - A contribution to explanation of the mutagenicity of 2′-deoxy-5-azacytidine

Article abstract of DOI:10.1135/cccc20030711

A series of N⁴-alkyl-5-azacytidines 3a-3h were prepared by treatment of the 4-methoxy analogue 4 with the respective amines. In the case of propyl-, butyl-, sec-butyl-, benzyl- or furfurylamine, aggregates of azacytidines 3a-3e with their hydrolytic products 5a-5e were isolated. Similar aggregates were obtained with 1-methyl-5-azacytosine (6) and 2-(methylcathamoyl)guanidine (7). Compound 7 was prepared by the reaction of guanidine with methyl isocyanate; the reaction of 2 or 3 equivalents gave the di- or tricarbamoyl derivatives 11 and 12, respectively. Cyclization of 7 and 11 with DMF dimethyl acetal afforded azacytosines 6 and 13, respectively. Aggregates of guanosine with 5-azacytosine nucleosides 1, 2 and 15 or of 5-aza-5,6-dihydrocytosine nucleosides 16 and 17 with 5-azacytidine (1) and its 2′-deoxy congener 2 have been prepared by co-crystallization of the respective pairs of nucleosides. The anomers of (deoxyribosylcarbamoyl)guanidine 20a and 20b have been prepared by hydrolysis of the deoxy nucleoside 2. An aggregate of the picrate (8a) of (ribosylcarbamoyl)guanidine 8 with cytidine (9) has been obtained by co-crystallization of both components. Reaction of the methoxy nucleoside 4 with tert-butylamine gave, by contrast to the above mentioned amines, the α-anomer of O-methylribosylisobiuret 22, which was cyclized by DMF dimethyl acetal to the α-anomer of N⁴,N⁴-dimethyl-5-azacytidine 24. The connection of the base-pairing ability of carbamoylguanidines with the mutagenicity of 2′-deoxy-5-azacytidine (2) as well as the mechanism of inhibition of DNA methyltransferase by this nucleoside analogue is discussed. In contrast to the unsubstituted 5-azacytidine (1) or its N⁴-methyl derivatives, none of the N⁴-alkyl derivatives exhibited any antibacterial or antitumor activity at 100 μg/ml or 10 μmol/l concentrations, respectively.

Full text of DOI:10.1135/cccc20030711

Mutagenicity of 2′-Deoxy-5-azacytidine
711
SYNTHESIS OF N4-ALKYL-5-AZACYTIDINES AND THEIR BASE-
PAIRING WITH CARBAMOYLGUANIDINES – A CONTRIBUTION TO
EXPLANATION OF THE MUTAGENICITY OF 2′-DEOXY-5-AZACYTIDINE
1,
2
Alois PÍSKALA *, Naeem B. HANNA, Milena MASOJÍDKOVÁ, Miroslav OTMAR ,
3
Pavel FIEDLER and Karel UBIK
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: piskala@uochb.cas.cz,
2
3
otmar@uochb.cas.cz, ubik@uochb.cas.cz
Received Novem ber 14, 2002
Accepted February 23, 2003
A series of N4-alkyl-5-azacytidin es 3a–3h were prepared by treatm en t of th e 4-m eth oxy an a-
logue 4 with th e respective am in es. In th e case of propyl-, butyl-, sec-butyl-, ben zyl- or
furfurylam in e, aggregates of azacytidin es 3a–3e with th eir h ydrolytic products 5a–5e were
isolated. Sim ilar aggregates were obtain ed with 1-m eth yl-5-azacytosin e (6) an d 2-(m eth yl-
carbam oyl)guan idin e (7). Com poun d 7 was prepared by th e reaction of guan idin e with
m eth yl isocyan ate; th e reaction of 2 or 3 equivalen ts gave th e di- or tricarbam oyl derivatives
11 an d 12, respectively. Cyclization of 7 an d 11 with DMF dim eth yl acetal afforded aza-
cytosin es 6 an d 13, respectively. Aggregates of guan osin e with 5-azacytosin e n ucleosides 1, 2
an d 15 or of 5-aza-5,6-dih ydrocytosin e n ucleosides 16 an d 17 with 5-azacytidin e (1) an d its
2′-deoxy con gen er 2 h ave been prepared by co-crystallization of th e respective pairs of
n ucleosides. Th e an om ers of (deoxyribosylcarbam oyl)guan idin e 20a an d 20b h ave been pre-
pared by h ydrolysis of th e deoxy n ucleoside 2. An aggregate of th e picrate (8a) of (ribosyl-
carbam oyl)guan idin e 8 with cytidin e (9) h as been obtain ed by co-crystallization of both
com pon en ts. Reaction of th e m eth oxy n ucleoside 4 with tert-butylam in e gave, by con trast
to th e above m en tion ed am in es, th e α-an om er of O-m eth ylribosylisobiuret 22, wh ich was
cyclized by DMF dim eth yl acetal to th e α-an om er of N4,N4-dim eth yl-5-azacytidin e 24. Th e
con n ection of th e base-pairin g ability of carbam oylguan idin es with th e m utagen icity of
2′-deoxy-5-azacytidin e (2) as well as th e m ech an ism of in h ibition of DNA m eth yltran sferase
by th is n ucleoside an alogue is discussed. In con trast to th e un substituted 5-azacytidin e (1)
or its N4-m eth yl derivatives, n on e of th e N4-alkyl derivatives exh ibited an y an tibacterial or
an titum or activity at 100 µg/m l or 10 µm ol/l con cen tration s, respectively.
Keyw ord s: 1,3,5-Triazin es; 5-Azapyrim idin es; Nucleosides; 5-Aza-2′-deoxycytidin e; Cytostatic
activity; Decitabin e; An titum or drugs; Mutagen esis; Self assem bly; Nucleobase pairs.
Th e n ucleoside an tibiotic 5-azacytidin e1–5 (1, azacitidin e) an d 2′-deoxy-
5-azacytidin e6–8 (2, decitabin e) are active drugs for th e th erapy of acute leu-
kem ia9–13. 2′-Deoxy-5-azacytidin e (2) h as also sh own clin ical activity
Collect. Czech. Chem. Commun. (Vol. 68) (2003)
doi:10.1135/cccc20030711

712
Pískala et al.:
again st m etastatic lun g can cer14 an d h orm on e-in depen den t prostate can-
cer15. 5-Azacytidin e (1) is ph osph orylated an d in corporated16 in to RNA
an d, after deoxygen ation , also in to DNA. 5-Aza-2′-deoxycytidin e (2) is in -
corporated16 in to DNA. 5-Azacytosin e-con tain in g DNA 17–20 is a poten t in -
h ibitor of DNA m eth yltran sferase due to a covalen t in teraction 21,22 of th e
th iol (SH) group in th e active site of th e en zym e with th e labile 5-aza-
cytosin e rin g23,24. Hyperm eth ylation of DNA is a ch aracteristic ph en om e-
n on of tum or cells, wh ich allows in h ibition of tum or suppressor gen es, an d
h en ce tum or progression . Association of tum or progression with h yper-
m eth ylation h as been docum en ted in several solid tum ors as well as in
acute leukem ia an d ch ron ic m yelogen ous leukem ia18–20. It h as been sh own
th at 1 an d particularly 2, wh ich is th e m ost poten t h ypom eth ylatin g agen t
so far available, in duce cellular differen tiation 25 an d en h an ced expression of
gen es in volved in tum or suppression , im m un ogen icity, an d program m ed
cell death 14,15
.
In con n ection with structure–activity relation sh ip in vestigation s in n u-
cleosides related to 5-azacytidin e (1), we prepared a series of N4-alkyl-5-aza-
cytidin es. Recen tly, we described N4-m eth yl-5-azacytidin es possessin g an
an tibacterial activity26. In th is paper, we are presen tin g th e preparation of
oth er N4-alkyl-5-azacytidin es an d th eir ability to form aggregates with cor-
respon din g carbam oylguan idin es obtain ed by th eir subsequen t h ydrolysis.
Som e of th ese results were form erly in cluded in con feren ce proceedin gs27.
RESULTS AND DISCUSSION
N4-Alkyl-5-azacytidin es 3a–3h were prepared by treatm en t of th e 4-m eth -
oxy an alogue28 4 with th e respective am in es in m eth an ol (Sch em e 1). In
th e case of propyl-, butyl-, sec-butyl-, ben zyl- or furfurylam in e, aggregates
of com poun ds 3a–3e with th eir h ydrolytic products 5a–5e were isolated.
Th e com plexes are crystallin e with sh arp m eltin g poin ts. Th eir elem en tal
an alyses in dicate th e 1:1 ratio of th e com pon en ts. We suppose a possible
an alogy with th e reversed Watson –Crick base pair guan in e–cytosin e (G:C)
with th e expectation th at th e arran gem en t of th e com pon en ts in th e aggre-
gates could be very close to th e bin din g m otif of type I. Neverth eless, due
to th e flexibility an d poten tial existen ce of several tautom eric form s of
carbam oylguan idin es 5a–5e, th ere are m ore ways h ow to arrange H-bonds
in th e aggregates. In aggregates I, we assum e th e ability of carbam oyl-
guan idin es to exist n ot on ly in th e m ore stable diam in o form A, wh ich
could com prise an in tram olecular h ydrogen bon d between th e free am in o
group and the carbonyl group, but also in the less stable am ino-im ino form B,
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
713
NR1R2
N
OCH3
N
NH2
N
a, R1 = Pr, R2 = H
b, R1 = Bu, R2 = H
c, R1 = i-Bu, R2 = H
d, R1 = Bn, R2 = H
e, R1 = furfuryl, R2 = H
f, R1 = i-Pr, R2 = H
g, R1 = R2 = Bu
(i)
N
N
N
N
O
N
O
N
R
O
Rf
Rf
1, R = Rf
2, R = dRf
15, R = Araf
4
3a-3h
h, R1 = 2-(imidazol-4-yl)ethyl, R2 = H
19, R = Bn3Araf
(ii)
NR1R2
NR1R2
HN
NH
H2N
N
(i) R1R2NH, MeOH; (ii) H2O.
HN
Rf
O
HN
Rf
O
B-form
A-form
5a-5h
Rf
R1
O
NH
Rf
NH
R1
NH
O
NH
HN
HN
NH
R1
N
N
H2N
N
HN
HN
NH
N
O
HN
Rf
O
Rf
R1
3a-3e
5a-5e
(B-form)
I
5a-5e
(A-form)
5a-5e
(B-form)
V
R1
N
H
N
O
H
NH
Rf
R1
H
N
NH
O
HN
N
N
H
Rf
H2N
N
HN
N
HN
N
O
R1
Rf
HN
Rf
O
HN
R1
3a-3e
5a-5e
(A-form)
Ia
5a-5e
(A-form)
5a-5e
(A-form)
Va
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

714
Pískala et al.:
wh ich could associate with 5-azacytosin es th rough a tritopic H-bon d in ter-
action , sin ce con siderable stabilization is expected th rough th e form ation
of an aggregate in volvin g th ree h ydrogen bon ds. However, due to th e pro-
posed in tram olecular H-bon d between on e am in o group an d th e carbon yl
group of carbam oylguan idin es, we can n ot exclude also a ditopic H-bon d in -
teraction of type Ia. Wh ile th is base-pair geom etry h as two prim ary attrac-
tive h ydrogen bon ds an d two attractive secon dary in teraction s, th e bin din g
m otif I h as th ree prim ary attractive h ydrogen bon ds, two repulsive an d two
attractive secon dary in teraction s.
On th e con trary, n o associates were isolated wh en isopropylam in e,
dibutylam in e or h istam in e was used for th e am in olysis of 4. Alth ough , th e
respective n on -associated products 3f–3h were obtain ed in h igh yields, th e
degradation products 5f–5h were also detected in m in or am oun ts in th e re-
action m ixture. Moreover, in th e case of th e dibutyl derivative 3g, th e for-
m ation of an aggregate of type I or Ia is precluded due to th e absen ce of
h ydrogen atom on on e of th e am in o groups. Th e ratio of 5-azacytidin es
an d carbam oylguan idin es depen ds on reaction con dition s, especially on
th e con cen tration of th e am in e, th e reaction tim e an d also on th e relative
stability to h ydrolysis an d/or am in olysis, an d on th e solubility of th e re-
spective 5-azacytidin e. By colum n ch rom atograph y on silica gel or
Am berlite IRC [H+], th e aggregates are destroyed an d th e in dividual com po-
n en ts 3a–3h an d 5a–5h can be isolated. Both com pon en ts of th e aggregates
are also well separable by TLC on silica gel. Th e carbam oylguan idin es
5a–5h , wh ich are n ot an om erically stable, were obtain ed after ch rom atog-
raph y as a m ixture of α- an d β-an om ers of ribofuran osyl an d ribopyran osyl
derivatives. In th is way, th e in dividual com pon en ts of th e aggregates of 3b
an d 3d with th e respective carbam oylguan idin es 5b an d 5d h ave been sepa-
rated. Com poun ds 3b an d 3d were isolated as in dividual crystallin e com -
poun ds. However, th e carbam oylguan idin e com pon en t 5b, wh ich was after
isolation ch aracterized as a crystallin e picrate, con tain s th e α-furan osyl as
well as α- an d β-pyran osyl isom ers (β-fur:α-fur:β-pyr:α-pyr = 5:2:1:1.5). Th e
aggregates of 5-azacytidin es with carbam oylguan idin es are also form ed by
co-crystallization of 1:1 m ixtures of th e in dividual com pon en ts from m eth -
an ol or m ixtures of m eth an ol an d aceton itrile. By th is procedure, aggre-
gates of 3b an d 3d with th e respective carbam oylguan idin es 5b an d 5d
were prepared. Com poun ds 5b an d 5d were obtain ed by h ydrolysis of th e
respective 5-azacytidin es 3b an d 3d with 1 M am m on ia solution in water.
In an attem pt to prepare 1-m eth yl-5-azacytosin e (6) by con den sation of
2-(m eth ylcarbam oyl)guan idin e (7) with eth yl form ate in m eth an ol
(Sch em e 2), a sh arp m eltin g 1:2 aggregate of com poun d 6 with th e startin g
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
715
com poun d 7 was obtain ed. It is of in terest to n ote th at 1:1 an d 2:1 aggre-
gates of 6 with 7, prepared on evaporation of water solution s of th e respec-
tive m ixtures of 6 an d 7, exh ibited alm ost th e sam e sh arp m eltin g poin ts as
th e 1:2 aggregate. We suggest for th e 1:1 aggregate of 6 with 7 a sim ilar ar-
ran gem en t of h ydrogen bon ds as sh own in bin din g m otifs I or Ia of th e
ribosyl derivatives. Th e structures of th e 1:2 or 2:1 associates of 6 with 7
h ave n ot been studied. Th e form ation of aggregates between proton ated
carbam oylguan idin es an d 5-azacytosin es was observed in case of th e sh arp-
m eltin g 1:1 aggregate of 1-m eth yl-5-azacytosin e (6) with 2-(m eth ylcarba-
O
O
NH2
NH2
N
(i)
(i)
(i)
HN
H2N
HN
NH
O
HN
HN
NH
H2N
NH
10
H2N
CH3
CH3
N
HN
N
HN
O
CH3
(ii)
CH3
O
HN
O
NH2
7
11
CH3
CH3
12
N
N
(ii)
(iii)
N
O
O
CH3
HN
NH
6·7
CH3
6
N
N
(i) CH3NCO; (ii) DMF DMA; (iii) HCOOEt
N
O
CH3
CH3
13
H
N
H
O
NH
NH
NH
NH
H
N
O
NH
CH3
H
HN
HN
N
N
N
NH
X
X
N
O
H
H N
N
O
H
H
CH3
CH3
6
7a
6
7a
II
IIa
X = picrate
X
R1
H
NH2
N
N
O
N
H2N
N
R2
HN
HN
O
R
Rf
7b, R = R1= R2 = H, X = Cl
7c, R = R1 = R2 = CH3, X = Cl
8a, R = Rf, R1 = R2 = H, X = picrate
8
SCHEME 2
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

716
Pískala et al.:
m oyl)guan idin ium picrate (7a), wh ich was obtain ed by co-crystallization of
both com pon en ts in m eth an ol. We assum e in th is case th e existen ce of
bin din g m otifs II or IIa in th e crystal lattice. Th e preparation of an an alo-
gous com plex of 5-azacytidin e (1) with picrate 8a was n ot successful due to
th e extrem ely low solubility of 1 in m eth an ol. However, a sim ilar co-
crystallization of cytidin e (9) with picrate 8a from eth yl acetate gave th e
sh arp-m eltin g aggregate.
Com poun d 7 was obtain ed on reaction of guan idin e (10) with 1 equiva-
len t of m eth yl isocyan ate in aceton e. Wh en 2 or 3 equivalen ts of m eth yl
isocyan ate were used, th e respective di- or trisubstituted com poun ds 11 an d
12 were obtain ed. Con den sation 29 of 7 with DMF dim eth yl acetal gave
n on -associated 1-m eth yl-5-azacytosin e (6) an d th e sam e reaction of th e di-
substituted derivative 11 afforded 1-m eth yl-4-(3-m eth ylureido)-1,3,5-triazin -
2(1H)-on e (13).
In order to exten d our kn owledge about th e com plem en tary base-pairin g
of 5-azacytosin e n ucleosides, we prepared aggregates by crystallization of
1:1 m ixtures of guan osin e with 5-azacytosin e n ucleosides 1, 2 an d 1-β-D-
arabin ofuran osyl-5-azacytosin e30 (15), wh ich could com prise th e H-bon d
m otifs III (Sch em e 3), an alogous to th e n orm al Watson –Crick base pair
N
R
N
H
N
H
N
H
H
N
N
O
NH
Rf
O
H
NH
HN
HN
N
H
N
HN
HN
N
N
R
O
N
R
O
14
1, R = Rf
2, R = dRf
1, R = Rf
2, R = dRf 17, R = dRf (B-form)
16, R = Rf (B-form)
15, R = β-D-arabinofuranosyl
III
IV
R
H
N
H
NH2
Cl
NH
H
O
N
H
NH
HN
HN
HN
HN
N
H
N
R
O
H
H
N
R
O
H
16, R = Rf (A-form)
17, R =dRf (A-form)
18, R = 2,3,5-tri-O-
-benzyl-β-D-arabino-
furanosyl (A-form)
16, (B-form)
17, (B-form)
18, (B-form)
18a, R = 2,3,5-tri-O-benzyl-
-β-D-arabinofuranosyl
VI
SCHEME 3
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
717
C:G. Also aggregates of 1 an d 2 with th e respective dih ydro derivatives31 16
an d 17, wh ich could in volve a tritopic H-bon d m otif IV with th e tauto-
m eric B-form of th e dih ydro derivatives, h ave been prepared. For a spectral
study of th e base-pairin g ability of 5-aza-5,6-dih ydrocytosin e n ucleosides,
th e triben zyl derivative 18, wh ich was isolated as its h ydroch loride 18a,
was prepared by reduction of th e kn own 1-(2,3,5-tri-O-ben zyl-β-D-arabin o-
furan osyl)-5-azacytosin e30 (19).
Alth ough th e kin etics of h ydrolysis of 2′-deoxy-5-azacytidin e (2) to
2-[(2-deoxy-β-D-erythro-pen tofuran osyl)carbam oyl]guan idin e (20b ) was
studied by an alytical m eth ods24 an d th e h ydrolytic product 20b was con -
sidered in m an y papers, its preparation h as n ot been described as yet. We
prepared a m ixture of an om ers of carbam oylguan idin es 20a an d 20b in
an alogy to th e ribosyl23 derivative by h ydrolysis of th e deoxy n ucleoside 2
with 1 M am m on ia in water (Sch em e 4). Th e basic h ydrolytic products 20a
an d 20b were isolated in h igh yields as carbam oylguan idin ium form ates
21a an d 21b (con tam in ated with about 10% of th e pyran osyl isom ers 21c
an d 21d ). A m ixture of free bases 20a–20d was liberated from th eir salts us-
in g a basic ion exch an ge resin (Dowex 2X8) an d an om eric carbam oyl-
guan idin es 20a an d 20b (α:β = 1:1) were obtain ed on crystallization from
m eth an ol. Isolation of an y isom er of 20 in pure state was im possible due to
spon tan eous an om erization an d rin g rearragem en t durin g th e procedure.
An attem pt to prepare an aggregate of 2′-deoxy-5-azacytidin e (2) with a
m ixture of an om ers of carbam oylguan idin es 20a an d 20b (α:β = 1:1) by
co-crystallization of both com pon en ts from m eth an ol was n ot successful
due to a very low solubility of 2 in th is solven t. However, FAB-MS (vide in-
fra) of a m ixture of 2 with 20a an d 20b, in dicates th e ability of th ese com -
pon en ts to form a 1:1 aggregate.
NH2
N
NH2
HCOO
(i)
(ii)
H2N
H2N
NH
2
HN
O
HN
O
R
R
20a-20d
21a-21d
a, R = 2-deoxy-α -D-erythro-pentofuranosyl
b, R = 2-deoxy-β-D-erythro-pentofuranosyl
c, R = 2-deoxy-α -D-erythro-pentopyranosyl
d, R = 2-deoxy-β-D-erythro-pentopyranosyl
(i) NH4OH; (ii) HCOOH
SCHEME 4
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

718
Pískala et al.:
Wh en tert-butylam in e was used for am in olysis of th e m eth oxy n ucleoside
4, th e displacem en t proceeded m uch m ore slowly because of steric h in -
dran ce; even in boilin g m eth an ol, cleavage of th e triazin e rin g followed by
subsequen t an om erization occurred m ain ly un der th e form ation of
1-α-D-ribofuran osyl-4-O-m eth ylisobiuret (22) (Sch em e 5). Th e α-D con figu-
ration of 22 was in ferred from th e opposite sign of th e Cotton effect in CD
spectra in com parison with th e kn own β-D-ribofuran osylisobiuret1 23,
wh ich was used as an in term ediate for th e first syn th esis of 5-azacytidin e
(1); an im proved preparation of 23 is described in th e Experim en tal. Th e
m on oh ydrate of com poun d 23 crystallizes well, wh ich facilitates its isola-
tion even from a com plex reaction m ixture. However, it is n ot con ven ien t,
in con trast to its syrupy an h ydrous form , as an in term ediate for th e syn th e-
sis of 5-azacytidin e because of th e occurren ce of h ydrolytic side reaction s in
th e con den sation step with orth oform ate1.
R
OCH3
(i)
(ii)
N
N
H2N
N
4
N
O
HN
O
α-Rf
R
22, R = α−Rf
23, R = 2,3,5-tri-O-acetyl-
-β-Rf
24, R = NMe2
25, R = OMe
(i) t-BuNH2; (ii) DMF dimethyl acetal
SCHEME 5
Th e an om eric con figuration of isobiuret 22 was also con firm ed by its
con den sation with DMF dim eth yl acetal in m eth an ol to 4-(dim eth yl-
am in o)-1-α-D-ribofuran osyl-1,3,5-triazin -2(1H)-on e (24). Th e α-D con figura-
tion of com poun ds 22 an d 24 follows also from com parison of 1H NMR
spectra of th ese com poun ds an d th e β-D an om er 23. Th e form ation of 24
can be explain ed by subsequen t reaction of th e prim arily form ed
4-m eth oxy-1-α-D-ribofuran osyl-1,3,5-triazin -2(1H)-on e (25) with dim eth yl-
am in e released durin g con den sation of ribosylisobiuret 22 with DMF
dim eth yl acetal.
Mass Spectrometry
Th e first eviden ce on th e n ature of th e com plexes of carbam oylguan idin es
with 5-azacytosin es cam e from fast-atom -bom bardm en t m ass spectrom etric
(FAB-MS) experim en ts, wh ose outstan din g suitability for detection of n on -
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
719
covalen t H-bon d-m ediated com plexes was described in th e literature32–38
.
Th e com position of th ese associates was proved by MS in th e cases of th e
n on -h ygroscopic aggregates of 5-azacytidin es 3d an d 3e with th e respective
carbam oylguan idin es 5d an d 5e. In th e spectrum , both types of m olecular
peaks – of th e in dividual com pon en ts an d of th eir associates – were presen t.
Even th e peaks of th e dim ers of in dividual com pon en ts 3d an d 3e or 5d
an d 5e as well as th e peaks attributable to th e associates of ben zyl- or
furfurylguan idin e (form ed by fragm en tation of carbam oylguan idin es 5d
an d 5e) with th e respective 5-azacytidin es 3d an d 3e, respectively, an d also
associates of ben zyl- or furfurylguan idin e with th e respective carbam oyl-
guan idin es 5d an d 5e were detected. Th e form ation of n on -covalen t dim ers
of carbam oylguan idin es could be explain ed by association of th eir tauto-
m eric A- an d B-form s th rough a tritopic H-bon d m otif V (Sch em e 1). A
sim ilar situation was form erly observed in th e case of 2-am in opyrim idin -4-
on e (isocytosin e)39,40, cyclic ben zyliden e derivatives of carbam oylguan idin e
(6-ben zyl-5,6-dih ydro-5-azacytosin e)41 an d som e oth er poten tially tauto-
m eric com poun ds (for recen t reviews on H-bon din g in n on -covalen t
syn th esis, see refs42,43). However, also in th ese cases, th e self-association
of carbam oylguan idin es 5d an d 5e by com plem en tary h ydrogen bon ds as
sh own in th e m otif Va (Sch em e 1) or by an an alogous don or-don or–
acceptor-acceptor (DD–AA) dim erization of carbam oylguan idin es in th e
A-form with out an in tram olecular h ydrogen bon d could be con sidered. In
th e cases of dim ers of 5-azacytidin es an d of th eir associates with guan id-
in es, we assum e th e in volvem en t of proton ated species of th ese com -
poun ds, wh ich could serve as proton don ors an d form com plem en tary
associates with th e respective n eutral m olecules of 5-azacytidin es or
carbam oylguan idin es as acceptors. Due to th e isolation of un proton ated ag-
gregates, we assum e th at th e associates of 5-azacytidin es with carbam oyl-
guan idin es or th e dim ers of carbam oylguan idin es are also form ed between
th e n eutral m olecules.
FAB-MS of th e 1:2 aggregate of 6 with 7 h as sh own , besides peaks of th e
in dividual com pon en ts, also peaks attributable to th eir 1:1, 1:2 an d 2:1 as-
sociates. Moreover, peaks of dim ers of 6 an d 7 an d th e trim er of 7 as well as
peaks of guan idin e an d m eth yl isocyan ate form ed by fragm en tation of 7
an d also peaks of th e aggregates of guan idin e with 6 an d 7 were observed.
Th e in ten sities of th e peaks of th e aggregates are depen den t on con cen tra-
tion of th e sam ple in th e m atrix. Wh en a forty-tim es-diluted sam ple of th e
1:1 aggregate of 6 with 7 was applied, n o associates were detected at all.
Th is result stron gly in dicates th at th e form ation of th ese aggregates is based
on h ydrogen bon din g an d n ot on covalen t in teraction s, wh ich could be
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

720
Pískala et al.:
also taken in to con sideration . For th e 1:1 aggregate of 6 with 7 we assum e
assem bly m odes an alogous to I or Ia an d for th e dim er of 7 assem bly m odes
an alogous to V or Va. Th e structure of th e h eterotrim ers of 6 with 7 or th e
h om otrim er of 7 was n ot clarified. Because th e peaks of th ese aggregates
were m uch sm aller th an th e peaks of th e dim eric structures, we assum e th at
th e th ird com pon en t of th e h etero- or h om otrim ers is boun d to th e dim ers
by a weaker H-bon d in teraction . In th e case of th e dim er of 6 an d th e aggre-
gates with guan idin e, we assum e th e in volvem en t of proton ated species as
in th e case of th e above m en tion ed ribosyl derivatives. In FAB-MS of th e
(m eth ylcarbam oyl)guan idin ium picrate (7a), on ly a n egligible peak of a
dim er of 7 was observed. However, a 1:1 m ixture of 1-m eth yl-5-azacytosin e
(6) an d picrate 7a or th e aggregate of both com pon en ts exh ibited, in addi-
tion to th e m olecular peaks of th e in dividual com pon en ts, also peaks of th e
associate of 6 with 7 an d th e dim er of 6. An alogous results were obtain ed in
th e case of th e aggregate of cytidin e (9) with picrate 8a. Th ese facts con firm
th at n on -covalen t dim eric structures could be form ed also by aggregation
of proton ated with un proton ated m olecules. We suggest for th e aggregate
6·7a th e bin din g m odes II or IIa. An an alogous base-pairin g could be for-
m ulated also for th e aggregate 9·8a. Th e m eth yl derivative 6 is at lower pH
proton ated at position 3 of th e triazin e rin g, sim ilarly to cytosin e, an d
could be able to form com plem en tary H-bon ds with th e n eutral m olecule
of 6. In th is con n ection , it is of in terest to n ote th at proton ated cytosin e is
kn own to be in volved in th e Hoogsteen m ode of base pairin g44 or in som e
C+:C m ism atch base pairs in volvin g proton ated cytosin e45. FAB-MS of com -
poun ds 11–13 in dicated also th e presen ce of dim eric structures. Despite
m an y possibilities of th e location of h ydrogen bon ds, we assum e a sim ilar
arran gem en t to th at in 2-ureidopyrim idin -4(1H)-on e derivatives46.
In th e cases of aggregates III or IV, in addition to th e m olecular peaks of
th e in dividual com pon en ts, also th e peaks of th eir associates were observed
by FAB-MS. Addition ally, th e peaks correspon din g to th e dim ers of dih ydro
derivatives 16 or 17 an d dim ers of 1, 2 or 15 were observed. Sim ilarly,
dih ydro derivative 18 exh ibited, in addition to th e m olecular peak, also a
peak of th e dim er. For th e dim ers of dih ydro derivatives 16–18 we assum e
th e existen ce of a tritopic H-bon d in teraction between th e tautom eric
form s A an d B as sh own in th e bin din g m otif VI. As m en tion ed above, ag-
gregates an alogous to VI were con sidered to exist in th e crystallin e state of
som e 6-ben zyl-5,6-dih ydro-5-azacytosin es41.
Alth ough th e preparation of an aggregate of 2′-deoxy-5-azacytidin e (2)
with a m ixture of 20a an d 20b was n ot successful, FAB-MS of a 1:1 m ixture
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
721
of 2 with 20a an d 20b h as sh own th e associate of 2 with 20a or 20b, th e
dim ers of 20a or 20b an d th e dim er of 2.
1H NMR Spectroscopy
In solubility of 5-azacytidin es an d th eir n on covalen t h eterodim ers in CDCl3
an d th e com petitive h ydrogen bon din g with th e solven t oxygen in
DMSO-d6 are respon sible for alm ost n o eviden ce of th e n ature of th ese ag-
gregates from 1H NMR spectra. As th e on ly in dication of h ydrogen bon din g
in th ese aggregates can be con sidered a down field sh ifted sign al of on e NH
proton in th e picrates of 5a, 7a an d 8a, wh ich can be assign ed to in tra-
m olecular h ydrogen bon ds persistin g even in th is stron g h ydrogen bon d
acceptor solven t. Sim ilar results were obtain ed with un substituted
carbam oylguan idin ium ch loride (7b ) an d 1,1-dim eth yl-2-(m eth ylcarba-
m oyl)guan idin ium ch loride (7c), wh ich were prepared earlier in con n ection
with an oth er project29. Also th e down field sh ifts of NH proton s in com -
poun d 11 an d 12 in dicate th e existen ce of in tram olecular h ydrogen bon ds.
13C NMR Spectroscopy
For th e aforem en tion ed reason , also 13C NMR spectra, m easured in
DMSO-d6, h ave n ot sh own an y h ydrogen bon din g in th e aggregates of 6
with 7 or 7a. However, th e broad sign als of two carbon yl carbon s in th e
spectra of com poun ds 11 an d 12 could be con sidered as an eviden ce for th e
existen ce of two in tram olecular h ydrogen bon ds.
IR Spectroscopy
IR spectra of th e aggregates of 3d an d 3e with 5d an d 5e, respectively, in
Nujol suspen sion s or KBr pellets sh owed th e existen ce of stron g h ydrogen
bon ds in th eir crystal lattice. In th is con n ection it sh ould be m en tion ed
th at a specific h ydrogen bon din g between 1,6-dim eth yl-5-azacytosin e an d
2′,3′,5′-tri-O-acetylguan osin e was described in th e literature47. Th e auth ors
assum ed th is com plex would h ave a sim ilar structure as th e G:C base pair in
DNA. We suggest th e existen ce of H-bon d-m ediated aggregates of type I or
Ia in th e solid state wh ich could m oreover associate by h ydrogen bon ds
in to exten ded supram olecular arrays. Th e m ode of association of type I is
obviously very stable an d h as been sh own to exist in crystallin e com plexes
m odeled on th e G:C base pair48
.
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

722
Pískala et al.:
However, IR spectra of th e 1:2 aggregate of 6 with 7 in KBr pellets in di-
cate th e existen ce of m ore th an th ree differen t H-bon ds. We assum e a sim i-
lar arran gem en t of th e m olecules in th e solid state to th at in th e afore-
m en tion ed aggregates of th e ribosyl derivatives 3 with 5. IR spectra of 7 in
KBr pellets in dicate th e presen ce of th e diam in o form but n ot th e co-
existen ce of th e am in o with th e im in o form , an d a sim pler arran gem en t of
th e m olecules in th e crystal lattice in com parison with its aggregate with 6.
IR spectra of com poun ds 11–13 in KBr pellets exh ibited also th e presen ce of
in term olecular h ydrogen bon ds; h owever, th ere are n o in dication s to as-
sum e th eir arran gem en t.
IR spectra of th e dih ydro derivative 18 in ch loroform exh ibited νas(NH2)
ban ds at 3484 an d 3392 cm –1, in dicatin g th e presen ce of 1:1 aggregates
(on e h ydrogen atom of an am in o group in volved in a h ydrogen bon d, pre-
sum ably H–N–H···O=C), as well as a ban d at 3324 cm –1, attributable to h ig-
h er associates (a fully boun d am in o group). Th e ν(C=O) ban d at 1724 cm –1
suggests th e presen ce of a partial positive ch arge on th e un saturated n itro-
gen atom in position 3 of th e 5,6-dih ydro-5-azacytosin e rin g, wh ich could
be expected because of th e existen ce of an N–H···N=C h ydrogen bon d in
th e aggregate VI. Th e form ation of th e aggregate VI was m oreover sup-
ported by deuteration experim en ts (see Experim en tal). Sim ilar results were
also obtain ed from IR spectra of 18 m easured in tetrach lorom eth an e an d
aceton itrile.
CD Spectroscopy
CD spectra of N4-substituted 5-azacytidin es 3b an d 3f–3h exh ibited in ten -
sive B2u Cotton effects at 250 n m sim ilarly to th e N4-substituted m eth yl
derivatives of 5-azacytidin e26 an d also to th e un substituted 5-azacytidin e5.
Th is in dicates an an ti con form ation aroun d th e C–N glycosyl bon d of th ese
n ucleosides.
Biological Activity
N4-Substituted 5-azacytidin es 3b, 3d an d 3f-3h as well as th e aggregates of
3a–3e with 5a–5e were tested for th eir an tibacterial activity usin g a culture
of E. coli B growin g on a m in eral m edium with glucose49. Non e of th ese
com poun ds exh ibited an y an tibacterial activity at ≤100 µg/m l con cen tra-
tion s. Also in vitro in h ibition of cell growth with com poun ds 3d , 3f–3h , th e
aggregates 3d ·5d , 3e·5e an d th e m ixture of h ydrolytic products 21a–21d
was evaluated in th e followin g cell cultures: h um an T-lym ph oblastoid
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
723
CCRF-CEM cell lin e (ATCC CCL 119), h um an prom yelocytic leukem 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.
Potential Carcinogenicity
Even tually it is of in terest to n ote th at N4-substituted 5-azacytidin es 3d , 3f
an d 3g exh ibited a m uch lower poten tial carcin ogen icity th an un sub-
stituted 5-azacytidin e50,51 or 6-m eth yl-5-azacytidin e52, wh en estim ated by a
polarograph ic m eth od53–55
.
A Remark to the Mutagenicity of 2′-Deoxy-5-azacytidine (2) and Its Mecha-
nism of Inhibition of DNA Methyltransferase
In absen ce of X-ray crystallograph ic data, we were n ot able to clarify th e
exact location of th e h ydrogen bon ds in th e aforem en tion ed associates;
h owever, th e form ation of aggregates of type I or II suggests th at, in respect
of h ydrogen -bon din g ability, carbam oylguan idin es or carbam oylguan idin -
ium salts could m im ic guan in e an d bin d cytosin e. Un proton ated carba-
m oylguan idin e in th e A-form with an in tram olecular h ydrogen bon d could
in our opin ion , also form DA–AD associates sim ilar to aden in e; in th e
A-form with out an in tram olecular h ydrogen bon d it could m im ic cytosin e
sim ilarly to 5-azacytosin e. We assum e carbam oylguan idin e could beh ave as
a un iversal base with a h igh affin ity to cytosin e. Th e above m en tion ed ag-
gregates represen t in fact m odels for a possible pairin g of carbam oyl-
guan idin e in corporated in to DNA with cytosin e. As we foun d form erly,
5-azacytosin e is able to form a pair with guan in e, alth ough less stable th an
cytosin e56, an d in corporation of 5-azacytosin e n ucleosides in to n ucleic ac-
ids is followed by rapid h ydrolytic cleavage of th e base, wh ich can ch an ge
gen etic in form ation 23. Recen tly, it was publish ed by Jackson -Grusby et al.57
th at th e h ydrolysis of 5-azacytosin e to carbam oylguan idin e in 5-aza-
cytosin e-con tain in g DNA m ay really be a reason for m utagen icity of
2′-deoxy-5-azacytidin e (2). Its h ydrolysis to th e carbam oylguan idin e 20b
could proceed via th e covalen t adduct 26 of th e SH group of m eth yl-
tran sferase to th e h igh ly reactive double bon d in position s 5 an d 6 of th e
triazin e rin g in 2 an d th e rin g-open ed in term ediate 27 (Sch em e 6). An
en zym e-in depen den t way via th e covalen t h ydrate 28 an d th e form yl deriv-
ative 29 is also possible. In both cases, th e 5-azacytosin e rin g fun ction s as a
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

724
Pískala et al.:
form ylatin g agen t affordin g th e S-form yl derivative of m eth yltran sferase in
th e first case an d in th e secon d case form ic acid, wh ich form s salt 21b with
th e stron gly basic product 20b.
NH2
N
NH2
N
N
(i)
HN
20b
MT
S
MT
S
HN
O
N
O
H
H
dRf
dRf
26
(iii)
27
(ii)
NH2
N
NH2
N
HN
HN
OHC
(iv)
2
HO
N
O
HN
O
H
dRf
dRf
29
28
(v)
N
NH2
N
O
H3C
MT
H3C
H
(vi), (vii)
N
NH
SAM = S-adenosylmethionine
MT = DNA methyltransferase
S
N
O
N
O
H
H
dRf
dRf
30
31
(i) H2O, - OHCS-MT; (ii) HS-MT; (iii) H2O, - HCOOH; (iv) H2O; (v) –S-MT.SAM+; (vi) reduction,
- HS-MT; (vii) deamination
SCHEME 6
Mutation s described in th e paper57 con sist predom in an tly in C:G → G:C
tran sversion s; n everth eless, C:G → T:A an d C:G → A:T tran sition s were also
observed. Th e C:G → G:C tran sversion is explain ed by th e ability of th e
aglycon e of carbam oylguan idin e 20b form ed by th e aforem en tion ed
en zym e-assisted or spon tan eous h ydrolysis of th e 1,3,5-triazin e rin g of
5-azacytosin e-con tain in g DNA, to m im ic guan in e. Th e auth ors assum e th e
existen ce of an in tram olecular H-bon d in 20b an alogous to th e A-form of
th e substituted derivatives 5a–5h , wh ich would be able to bin d cytosin e
th rough two am in o groups, sim ilarly to th e bin din g m otif Ia. Th is pair
would be isosteric with th e n atural G:C pair in th e m in or groove. Th e C:G →
T:A tran sition is explain ed by th e form ation of abasic sites on th e basis of
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
725
th e kn own A-rule an d th e C:G → A:T tran sition h as n ot been clarified at all
by th is auth ors. In con trast to th is paper, we assum e th e in volvem en t of
carbam oylguan idin ium ion in th e form ation of a pair with cytosin e. Th e
C:G → A:T tran sition could be explain ed, in our opin ion , by th e aforem en -
tion ed ability of n eutral carbam oylguan idin e in th e tautom eric A-form with
an in tram olecular H-bon d to m im ic aden in e. Also th e C:G → T:A tran sition
n eed n ot be explain ed on ly by th e form ation of abasic sites, as proposed in
th e m en tion ed paper57, but also by a partial m eth ylation at N-5 of th e cova-
len t adduct 26 with form ation of th e N5-m eth yl derivative 30, followed by
reductive cleavage of th e C–S bon d an d deam in ation of th is in term ediate
affordin g 5,6-dih ydro-5-azath ym idin e (31), wh ich could m im ic th ym in e.
Th is idea is also supported by th e fact th at th e kn own an tibiotic 5,6-di-
h ydro-5-azath ym idin e (31) was isolated from th e culture filtrates of
Streptomyces platensis var. clarensis58,59 an d th at 5-azacytidin e (1) is pro-
duced by Streptoverticillium ladakanus var. ladakanus3,4. Moreover, m eth yl-
ation at N-5 in presen ce of S-aden osylm eth ion in e was m ore recen tly
docum en ted by Gabbara an d Bh agwat60. Th e putative bin din g m odes of th e
aglycon es of carbam oylguan idin e 20b, carbam oylguan idin ium form ate 21b
an d dih ydro-5-azath ym idin e 31 with th ym in e, cytosin e an d aden in e, re-
spectively, are sh own in Sch em e 7.
CH3
H2N
N
H
O
N
H
N
H
O
H
N
HN
dRf
H
H
H
N
NH
N
N
N
H
H
N
O
H
O
H
H
O
N
HCOO
Cytosine
dRf
Thymine
21b
20b
H
O
N
O
H
H
N
H3C
H
H
N
N
NH
N
N
N
dRf
Adenine
31
SCHEME 7
Th e earlier studies21,22 of th e in h ibition of DNA m eth yltran sferase by
5-azacytosin e-con tain in g DNA proposed two m ech an ism s for covalen t
bon d form ation with m eth ylase. Th e first con sists in th e reaction of a
n ucleoph ilic catalyst, presum ably a th iol (SH) group of th e en zym e, with
th e h igh ly reactive carbon -6 un der form ation of th e 5,6-dih ydrotriazin e 26,
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

726
Pískala et al.:
wh ich could be slowly reversible. Th e secon d m ech an ism is based on th e
ability of 5-azacytosin e n ucleosides to fun ction as form ylatin g agen ts. Th e
in itially form ed covalen t adduct 26 could un dergo a reversible prototropic
rearran gem en t to form th e rin g-open ed in term ediate 27, wh ich could h y-
drolyze irreversibly to yield an in active form yl derivative of th e m eth ylase
an d th e h ydrolytic product 20b. Because it was kn own 61 th at 5-azacytosin e-
con tain in g DNA does n ot require S-aden osylm eth ion in e to in h ibit th e
m eth ylase, San ti et al.21,22 con cluded th at m eth ylation at N-5 of th e adduct
26 n eed n ot be in volved. In our opin ion , th e in itially form ed adduct 26 is
n ot stable en ough to explain th e in h ibition of DNA m eth ylase by
2′-deoxy-5-azacytidin e (2). It is well kn own 62,63 th at covalen t adducts of
5-azacytosin e n ucleosides are detectable on ly in stron gly acidic m edia, in
wh ich th e 5-azacytosin e rin g is proton ated in position 3. Ben jam in 62 h as
sh own th at covalen t adducts of 5-azacytosin e n ucleosides, in n eutral m edia
presen t in n egligible, n ot detectable am oun ts, are extrem ely un stable bein g
rapidly tran sform ed eith er by elim in ation back to th e respective 5-aza-
cytosin e n ucleoside an d th e free n ucleoph ile or by a prototropic rear-
ren gem en t to a rin g-open ed product, wh ich could be h ydrolyzed to th e
respective form ylated n ucleoph ile an d th e correspon din g glycosyl deriva-
tive of carbam oylguan idin e. In our opin ion , DNA m eth ylase is in activated
in absen ce of S-aden osylm eth ion in e by form ylation of th e catalytic th iol
group of th e en zym e via 26 an d 27 as proposed earlier by San ti et al.21,22
an d Jackson -Grusby et al.57 or in presen ce of S-aden osylm eth ion in e also by
m eth ylation of th e labile in term ediate 26 at N-5 to form a tigh tly boun d
covalen t adduct 30 as foun d by Gabbara an d Bh agwat60. Th e free active
DNA m eth ylase could be released from its form yl derivative by h ydrolysis
with th e form ation of form ic acid an d from th e relatively stable adduct 30
on th e action of reductive en zym es with th e form ation of 2′-deoxy-
5,6-dih ydro-5-m eth yl-5-azacytidin e, wh ich could be even tually deam in ated
to 5,6-dih ydro-5-azath ym idin e (31) as m en tion ed above. Th e form ation of
th e stable dih ydro derivative 31 was n ot taken in to con sideration in an y of
th e previous studies on th e m ech an ism of in h ibition of DNA m eth ylase by
2′-deoxy-5-azacytidin e (2). Gabbara an d Bh agwat60 h ave speculated th at th e
m eth ylated covalen t adduct 30 could elim in ate on h eatin g th e m eth ylase
with restoration of th e double bon d between N-5 an d C-6 to form a
5-m eth yltriazin ium cation . However, th ey h ave n ot ruled out th e possibil-
ity of oth er rearran gem en ts of th e triazin e rin g60. In our experien ce, th e pu-
tative triazin ium cation would be very un stable un der n eutral con dition s
an d would react with water to a 6-h ydroxy derivative, wh ich could un dergo
a Dim roth -type rearran gem en t to give N4-m eth yl-5-azacytosin e. However,
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
727
th ere h as been n o eviden ce so far, wh ich would substan tiate th e form ation
of a 5-m eth yltriazin ium cation or an y subsequen t products.
CONCLUSION
Th e h igh biological activity of 5-azacytidin e (1) an d 2′-deoxy-5-azacytidin e
(2) is based on th eir structural an d con form ation al resem blan ce with
cytidin e an d 2′-deoxycytidin e, wh ich en ables th eir in corporation in to n u-
cleic acids an d subsequen t covalen t in teraction of th e reactive double bon d
in th e 5,6 position of th e 1,3,5-triazin e rin g with regulatory protein s. Th e
CD spectra of N4-substituted 5-azacytidin es in dicate an an ti con form ation
aroun d th e C–N glycosyl bon d of th ese n ucleosides sim ilarly to un sub-
stituted 5-azacytidin e. However, substitution of h ydrogen atom s on th e
am in o group of 5-azacytidin e by th e bulky alkyl groups preven ts (predom i-
n an tly because of steric h in dran ce) th eir in corporation in to n ucleic acids.
Th is is probably th e m ain reason for th e low biological activity in com pari-
son with th e N4-m eth yl-5-azacytidin es an d especially with th e un sub-
stituted 5-azacytidin e (1). Th e form ation of aggregates of carbam oyl-
guan idin es or th eir proton ated form s with 5-azacytosin es or cytosin e,
wh ich represen t in fact m odels for th e base-pairin g ability of carbam oyl-
guan idin e in corporated in to DNA, is in agreem en t with th e observed57 C:G
→ G:C tran sversion caused by 2′-deoxy-5-azacytidin e (2). Th e C:G → T:A
tran sition , wh ich was also observed57 in th e m utation al spectrum of 2,
could be explain ed by m eth ylation at N-5 of 5-azacytosin e-con tain in g DNA
an d subsequen t tran sform ation to 5,6-dih ydro-5-azath ym in e-con tain in g
DNA. Th is idea is supported by th e m icrobial production 58 of 5,6-dih ydro-
5-azath ym idin e (31) an d by a m ore recen t in vestigation of Gabbara an d
Bh agwat60, wh o h ave docum en ted m eth ylation at N-5 of 5-azacytosin e-
con tain in g DNA. Th e form ation of th e stable dih ydro derivative 31 h as n ot
been taken in to con sideration in an y of th e earlier studies on th e m ech a-
n ism of in h ibition of DNA m eth ylase by 2′-deoxy-5-azacytidin e (2).
EXPERIMENTAL
Meltin g poin ts were taken on a h eated m icroscope stage (Kofler block) an d are n ot cor-
rected. Un less stated oth erwise, th e solution s were evaporated at 35 °C/2.5 kPa an d an alyti-
cal sam ples were dried at 40 Pa (room tem perature). Th in -layer ch rom atograph y (TLC) was
perform ed on Silufol UV 254 plates (Kavalier, Votice, Czech Republic) in th e followin g sol-
ven t system s: A ch loroform –m eth an ol (98:2), B butan -1-ol–acetic acid–water (5:2:3), C eth yl
acetate–aceton e–eth an ol–10 m M p h osp h ate bu ffer p H 7.0 (4:1:1:1) an d D acetic acid –
water–m eth an ol–p rop an -1-ol–ch loroform (1:2:3:4:5). Th e sp ots of 5-azacytid in es 3a–3h
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728
Pískala et al.:
were detected visually in UV ligh t (254 n m ) (D1,) th e spots of carbam oylguan idin es 5a–5h
wh ich are n ot visible in UV ligh t were detected with th e sodium n itroprusside–potassium
ferricyan ide reagen t (oran ge spots) (D2) or with n in h ydrin (D3) an d th e spot of isobiuret 22
was detected by h eatin g (D4). Colum n ch rom atograph y was perform ed with silica gel ac-
cordin g to Pitra (Service Laboratories of th is In stitute). IR spectra (ν in cm –1) were recorded
on a FTIR spectrom eter Bruker IFS 88 in th e region 3800–400 cm –1 in KBr pellets or in th e
in dicated solven t. UV spectra were m easured on a Un icam SP 8000 spectroph otom eter
(Pye–Un icam , Cam bridge, En glan d) in buffer solution s of ion ic stren gth 0.01 prepared ac-
cordin g to Perrin 64, λ are given in n m an d ε in m 2 m ol–1. CD spectra were recorded on a
Roussel–Jouan /II dich rograph e. Optical rotation s were registered on
a polarim eter
Perkin –Elm er, type 141 MCA 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) 2.50 an d δ(13C) 39.70 ppm , respec-
tively). For th e m easurem en t of 1H NMR in D2O, DSS as in tern al stan dard was used. Th e
ch em ical sh ifts (δ) are given in ppm an d couplin g con stan ts (J) in Hz. Mass spectra (m/z (%
rel. in t.)) were m easured on a ZAB-EQ m ass spectrom eter (Microm ass, Man ch ester, U.K.) us-
in 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 a m atrix an d sam ples were dissolved in m eth an ol or water. Elem en tal
an alysis was provided on a Perkin –Elm er CHN An alyser 2400, Series II Sys (Perkin –Elm er,
Norwolk (CT), U.S.A.). Station ary cultivation of Escherichia coli B was perform ed at 37 °C in a
m in eral m edium with glucose49. Th e tested com poun ds were added before in oculation an d
th e growth of bacteria was m easured 16 h later. Th e ratios of th e in dividual com pon en ts in
aggregates of type III, V an d VI as well as th e ratios of isom ers in com poun ds 20a–20d an d
21a–21d were estim ated by 1H NMR spectra m easured in D2O.
N4-Substituted 5-Azacytidin es 3b, 3d , 3f–3h an d Aggregates of 5-Azacytidin es 3a–3e with
Carbam oylguan idin es 5a–5e. Gen eral Procedure
A m ixture of m eth oxytriazin on e28 4 (0.259 g, 1 m m ol), m eth an ol an d th e respective am in e
was stirred at room tem perature. Th e solution was evaporated, th e residue co-evaporated
with m eth an ol (4 × 10 m l) an d crystallized from an appropriate solven t or worked up by
colum n ch rom atograph y (vide infra).
N4-Isopropyl-5-azacytidin e (3f)
Meth an ol (6 m l), isopropylam in e (1 m l), reaction tim e 1 h . Yield: 0.223 g (78%) of th e
n ucleoside 3f, m .p. 172–173 °C (dec.) (i-PrOH), RF (B) 0.82 (D1), [α]D +27.5 (c 0.5, water).
UV, λm ax (log ε): (MeOH), 255 (3.97), 213 (4.38); (pH 2.46), 258 (3.79); (pH 6.93) 250,
in flexion (3.84), 213 (4.23); (pH 11.1), 227 (4.40). CD (pH 6.93), λm ax ([Θ]m ax): 250 (+9450).
1H NMR: 8.52 s, 1 H (H-6); 7.99 d, 1 H, J(NH,CH) = 8.1 (NH); 5.66 d, 1 H, J(1′,2′) = 3.9
(H-1′); 5.41 d, 1 H, J(OH,2′) = 5.4 (OH); 5.10 t, 1 H, J(OH,5′) = 5.1 (OH); 5.01 d, 1 H,
J(OH,3′) = 5.8 (OH); 4.08 ddd, 1 H, J(2′,1′) = 3.9, J(2′,3′) = 5.0 J(2′,OH) = 5.4 (H-2′); 4.00 td,
1 H, J(3′,2′) = 5.0, J(3′,4′) = J(3′,OH) = 5.7 (H-3′); 3.97 dsept, 1 H, J(CH,CH3) = 6.6, J(CH,NH) =
8.1 (N-CH); 3.84 dt, 1 H, J(4′,5′) = 3.0, J(4′,3′) = 5.6 (H-4′); 3.68 ddd, 1 H, J(5′a,4′) = 2.9,
J(5′a,OH) = 4.9, J(gem ) = 12.0 (H-5′a); 3.55 ddd, 1 H, J(5′b,4′) = 3.2, J(5′b,OH) = 5.2, J(gem ) =
12.0 (H-5′b); 1.115 d, 3 H an d 1.11 d, 3 H, J(CH3,CH) = 6.6 (CH3). 13C NMR: 162.86 (C-4),
155.57 (C-6), 153.41 (C-2), 89.34 (C-1′), 84.445 (C-4′), 74.01 (C-2′), 69.17 (C-3′), 60.32
(C-5′), 42.20 (N-CH), 22.00 (2 × CH3). FAB-MS: 595 (1) [2 M3f + Na]+, 573 (4) [2 M3f + H]+,
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Mutagenicity of 2′-Deoxy-5-azacytidine
729
309 (5) [M3f + Na]+, 287 (18) [M3f + H]+, 155 (100) [aglycon e3f + H]+. For C11H18N4O5
(286.3) calculated: 46.15% C, 6.34% H, 19.57% N; foun d: 46.34% C, 6.55% H, 19.30% N.
N4-Butyl-5-azacytidin e (3b)
Meth an ol (5 m l), butylam in e (0.2 m l, 2 m m ol), reaction tim e 4 h . A solution of th e syrupy
crude product in m eth an ol (5 m l) was applied on to a colum n of Am berlite IRC-50 [H+] ion
exch an ge resin (10 m l), wh ich was prepared in m eth an ol. Th e colum n was eluted with
m eth an ol (100 m l) an d th e effluen t evaporated. A solution of th e residue in aceton itrile
(3 m l) was kept at room tem perature for 3 days to yield 0.141 g (47%) of th e n ucleoside 3b,
m .p. 86–88 °C, RF (B) 0.72 (D1), [α]D +25.8 (c 0.1, water). UV, λm ax (log ε): (MeOH), 252
(3.88), 213 (4.26); (pH 2.30), 258 (3.87), 220 (3.92); (pH 6.95), 252 (3.90), 213 (4.27); (pH
10.92), 250, in flexion (3.76), 226 (4.17). CD (pH 7.00), λm ax ([Θ]m ax): 250 (+8970). For
C
12H20N4O5 (300.3) calculated: 47.99% C, 6.71% H, 18.66% N; foun d: 47.69% C, 6.65% H,
18.63% N. Nucleoside 3b was h ygroscopic an d decom posed on storage to h ydrolytic prod-
ucts.
In a sim ultan eous experim en t, to a solution of th e crude product in m eth an ol (5 m l)
silica gel (1 g) was added an d th e m ixture evaporated to a powder, wh ich was applied on to
a colum n of silica gel (10 g) prepared in ch loroform . Th e colum n was eluted with ch loro-
form –m eth an ol (100:0–85:15, v/v). Fraction s con tain in g th e product were collected, evapo-
rated an d th e residue crystallized from aceton itrile to give 0.150 g (50%) of n ucleoside 3b,
m .p. 86–88 °C with out depression on adm ixture with a sam ple prepared by th e isolation
procedure m en tion ed above.
N4,N4-Dibutyl-5-azacytidin e (3g)
Meth an ol (2.5 m l), dibutylam in e (0.34 m l, 2 m m ol), reaction tim e 5 days. Yield: 0.182 g
(51%) of n ucleoside 3g, m .p. 125–127 °C (aceton itrile), RF (B) 0.47 (D1), [α]D +8.4 (c 0.2, wa-
ter). UV, λm ax (log ε): (MeOH), 261 (3.96), 219 (4.30); (pH 2.30), 268 (3.92), 225 (4.10); (pH
7.03), 263 (3.90); (pH 11.0), 263 (3.96), 221 (4.28). CD (pH 7.00), λm ax ([Θ]m ax): 250 (+6990).
1H NMR: 8.64 s, 1 H (H-6); 5.68 d, 1 H, J(1′,2′) = 3.9 (H-1′); 5.43 d, 1 H, J(OH,2′) = 5.1 (OH);
5.12 dd, 1 H, J(OH,5′) = 4.9 an d 5.1 (OH); 5.03 d, 1 H, J(OH,3′) = 5.8 (OH); 4.10 td, 1 H,
J(2′,1′) = 3.9, J(2′3′) = J(2′,OH) = 5.0 (H-2′); 4.02 td, 1 H, J(3′,2′) = 5.0, J(3′,4′) = J(3′,OH) = 5.6
(H-3′); 3.85 dt, 1 H, J(4′,5′) = 3.0, J(4′,3′) = 5.6 (H-4′); 3.69 ddd, 1 H, J(5′a,4′) = 2.9, J(5′a,OH) =
4.9, J(gem ) = 12.2 (H-5′a); 3.56 ddd, 1 H, J(5′b,4′) = 3.2, J(5′b,OH) = 5.1, J(gem ) = 12.2
(H-5′b); 3.52 m , 2 H an d 3.43 m , 2 H (N-CH2); 1.52 m , 4 H an d 1.27 m , 4 H (C-CH2); 0.90 t,
3 H an d 0.885 t, 3 H, J(CH3,CH2) = 7.3 (CH3). 13C NMR: 162.76 (C-4), 155.79 (C-6), 153.02
(C-2), 89.37 (C-1′), 84.48 (C-4′), 74.09 (C-2′), 69.17 (C-3′), 60.30 (C-5′), 47.15 an d 46.83
(N-CH2), 30.225, 29.095, 19.84 an d 19.69 (C-CH2), 14.01 an d 13.96 (CH3). FAB-MS: 379 (2)
[M3g + Na]+, 357 (15) [M3g + H]+, 225 (100) [aglycon e3g + H]+. For C16H28N4O5 (356.4) calcu-
lated: 53.92% C, 7.92% H, 15.72% N; foun d: 53.73% C, 7.85% H, 16.02% N.
N4-Ben zyl-5-azacytidin e (3d )
Meth an ol (2 m l), ben zylam in e (0.22 m l, 2 m m ol), reaction tim e 3 h . Th e crude product was
triturated with eth er (5 m l) an d crystallized from m eth an ol (1 m l). Recrystallization from
m eth an ol gave 0.224 g (67%) of pure n ucleoside 3d , m .p. 168–170 °C (dec.), RF (B) 0.71
(D1), [α]D +23.9 (c 0.1, water). UV, λm ax (log ε): (MeOH), 252 (3.99), 213 (4.37); (pH 2.30),
252 (3.89), 222 (4.01); (pH 7.03), 250 (3.97), 212 (4.37); (pH 10.97), 229 (4.34), 217 (4.24).
1H NMR: 8.60 t, 1 H, J(NH, CH2) = 6.3 (NH); 8.59 s, 1 H (H-6); 7.35–7.20 m , 5 H (arom .);
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730
Pískala et al.:
5.66 d, 1 H, J(1′,2′) = 3.7 (H-1′); 5.42 d, 1 H, J(OH,2′) = 5.4 (OH); 5.12 t, 1 H, J(OH,5′) = 5.1
(OH); 5.01 d, 1 H, J(OH,3′) = 5.8 (OH); 4.44 d, 2 H, J(CH2,NH) = 6.3 (arom .-CH2); 4.09 ddd,
1 H, J(2′,1′) = 3.7, J(2′,3′) = 5.0, J(2′,OH) = 5.4 (H-2′); 4.01 td, 1 H, J(3′,2′) = 5.0, J(3′,4′) =
J(3′,OH) = 5.6 (H-3′); 3.85 dt, 1 H, J(4′,5′) = 3.1, J(4′,3′) = 5.6 (H-4′); 3.69 ddd, 1 H, J(5′a,4′) =
2.9, J(5′a,OH) = 5.1, J(gem ) = 12.2 (H-5′a); 3.50 ddd, 1 H, J(5′b,4′) = 3.2, J(5′b,OH) = 5.1,
J(gem ) = 12.2 (H-5′b). 13C NMR: 164.13 (C-4), 155.95 (C-6), 153.37 (C-2), 138.98, 128.505,
127.38 an d 127.07 (C-arom ), 89.46 (C-1′), 84.43 (C-4′), 74.04 (C-2′), 69.10 (C-3′), 60.25
(C-5′), 43.71 (N-CH2). FAB-MS: 691 (<1) [2 M3d + Na]+, 669 (2) [2 M3d + H]+, 357 (2) [M3d
+
Na]+, 335 (42) [M3d + H]+, 203 (100) [aglycon e3d + H]+. For C15H18N4O5 (334.3) calculated:
53.89% C, 5.43% H, 16.76% N; foun d: 53.82% C, 5.42% H, 16.90% N.
N4-[2-(Im idazol-4-yl)eth yl]-5-azacytidin e (3h )
Meth an ol (2 m l), h istam in e (4 m m ol, prepared from 0.736 g of its dih ydroch loride by th e
action of 8 m l of 1 M NaOMe, dilution with 6 m l ben zen e, rem oval of sodium ch loride by
filtration an d evaporation of th e filtrate), reaction tim e 2.5 h . Th e deposited n ucleoside 3h
was filtered off with suction . A solution of th e crude product (0.220 g, m .p. 175–182 °C
(dec.)) in m eth an ol was evaporated. Durin g evaporation th e less soluble an d h igh er m eltin g
m odification of th e product was form ed. Recrystallization from m eth an ol gave 0.142 g
(42%) of pure n ucleoside 3h , m .p. 230–232 °C (dec.), RF (B) 0.51 (D1), [α]D +23.8 (c 0.1, wa-
ter). UV, λm ax (log ε): (MeOH), 252 (3.91), 213 (4.33); (pH 2.30), 250 (3.92), 222 (4.09); (pH
7.03), 250 (3.91), 212 (4.35); (pH 10.97), 226 (4.37). CD (pH 6.86): λm ax ([Θ]m ax): 250
(+10 650). 1H NMR: 11.80 brs, 1 H (NH); 8.54 s, 1 H (H-6); 8.14 t, 1 H, J(NH,CH2) = 5.6
(NH); 7.52 s, 1 H an d 6.81 brs, 1 H (CH-im idazole); 5.67 d, 1 H, J(1′,2′) = 3.9 (H-1′); 5.44 d,
1 H, J(OH,2′) = 5.1 (OH); 5.12 t, 1 H, J(OH,5′) = 5.0 (OH); 5.03 d, 1 H, J(OH,3′) = 5.6 (OH);
4.08 td, 1 H, J(2′,1′) = 3.9, J(2′,3′) = J(2′,OH) = 5.0 (H-2′); 4.00 td, 1 H, J(3′,2′) = 4.9, J(3′,4′) =
J(3′,OH) = 5.6 (H-3′); 3.85 dt, 1 H, J(4′,5′) = 3.0, J(4′,3′) = 5.6 (H-4′); 3.69 ddd, 1 H, J(5′a,4′) =
2.9, J(5′a,OH) = 5.0, J(gem ) = 12.2 (H-5′a); 3.55 ddd, 1 H, J(5′b,4′) = 3.2, J(5′b, OH) = 5.0,
J(gem ) = 12.2 (H-5′b); 3.43 m , 2 H (N-CH2); 2.73 brt, 2 H, J(CH2,CH2) = 7.5 (CH2-im idazole).
13C NMR: 163.795 (C-4), 155.64 (C-6), 153.395 (C-2), 134.89 (CH-im idazole), 132.79
(C-im idazole), 89.39 (C-1′), 84.43 (C-4′), 74.05 (C-2′), 69.115 (C-3′), 60.27 (C-5′), 40.63
(N-CH2), 29.74 (CH2). FAB-MS: 699 (<1) [2 M3h + Na]+, 677 (2) [2 M3h + H]+, 361 (3) [M3h
+
Na]+, 339 (40) [M3h + H]+, 207 (100) [aglycon e3h + H]+. For C13H18N6O5 (338.3) calculated:
46.15% C, 5.36% H, 24.85% N; foun d: 46.13% C, 5.44% H, 25.12% N.
In a sim ultan eous experim en t, in wh ich on ly 2 m m ol of h istam in e were used, a lower
m eltin g m odification of n ucleoside 3h was obtain ed. Recrystallization of th e crude product
from m eth an ol gave 0.092 g (27%) of pure n ucleoside 3h , m .p. 193–197 °C (dec.). Accordin g
to TLC an d UV spectra, th e product was iden tical with th e h igh er-m eltin g m odification .
Aggregate of N4-Propyl-5-azacytidin e (3a) with N1-Propyl-N2-(β-D-ribofuran osylcarbam oyl)-
gu an idin e (5a )
Meth an ol (2 m l), propylam in e (0.16 m l, 2 m m ol), reaction tim e 2 h . Yield: 0.126 g (45%) of
th e aggregate, m .p. 134–136 °C (dec.) (aceton itrile), RF (B) 0.62 (D1, 3a) an d 0.43 (D2, 5a),
[α]D –25.6 (c 0.1, water). UV, λm ax (log ε): (MeOH), 258, in flexion (3.90), 226 (4.51); (pH
2.30), 257 (3.83), 223 (3.86); (pH 7.03), 250 (3.88); (pH 10.97), 227 (4.68). For C21H38N8O10
(562.6) calculated: 44.83% C, 6.81% H, 19.92% N; foun d: 44.85% C, 6.83% H, 20.14% N.
Th e aggregate of 3a with 5a was h ygroscopic an d decom posed on storage to products of h y-
drolysis.
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Mutagenicity of 2′-Deoxy-5-azacytidine
731
Aggregate of N4-Butyl-5-azacytidin e (3b) with N1-Butyl-N2-(β-D-ribofuran osylcarbam oyl)-
guan idin e (5b)
Meth an ol (5 m l), butylam in e (0.7 m l, 7 m m ol), reaction tim e 40 m in . Yield: 0.148 g (50%)
of th e aggregate, m .p. 130–132 °C (dec.) (aceton itrile), RF (B) 0.72 (D1, 3b) an d 0.55 (D2, 5b),
[α]D –22.1 (c 0.5, water). UV, λm ax (log ε): (MeOH), 255 (3.68), 227 (4.25); (pH 2.46), 258
(3.85), 219 (4.56); (pH 6.93), 255, in flexion (3.98); (pH 11.1), 228 (4.72). For
C
23H42N8O10·1/2 H2O (590.7) calculated: 46.07% C, 7.23% H, 18.69% N; foun d: 46.05% C,
7.25% H, 18.63% N. Th e aggregate 3b with 5b was h ygroscopic an d decom posed on storage
to products of h ydrolysis.
In a sim ultan eous experim en t, a solution of crude 3b (prepared from 1 m m ol of 4 as de-
scribed above) in 1 M am m on ia (5 m l) was kept at room tem perature for 1 h , evaporated an d
co-evaporated with eth an ol (3 × 5 m l) to give crude carbam oylguan idin e 5b. A solution of
th is product in m eth an ol (5 m l) was treated with a solution of crude 3b (prepared from
1 m m ol of 4 by th e above m en tion ed procedure) in m eth an ol (5 m l). Evaporation of th is
m ixture an d crystallization of th e residue from aceton itrile gave 0.408 g (69%) of th e aggre-
gate of 3b with 5b, m .p. 130–132 °C (dec.) with out depression on adm ixture with th e sam -
ple described above.
Separation of th e Aggregate of 3b with 5b
Method A. A solution of th e aggregate of 3b with 5b (0.118 g, 0.2 m m ol) in m eth an ol
(2 m l) was applied on to a colum n of Am berlite IRC 50 [H+] ion exch an ge resin (5 m l),
wh ich was prepared in m eth an ol. Th e colum n was eluted with m eth an ol (50 m l), th e efflu-
en t evaporated an d th e residue crystallized from aceton itrile to afford 0.024 g (40%) of 3b,
m .p. 86–88 °C (dec.), with out depression on adm ixture with a sam ple of th e product de-
scribed above. Th e colum n was furth er eluted with a 9:1 m ixture of m eth an ol–acetic acid
(50 m l) an d th e effluen t evaporated. A solution of th e residue in eth an ol (0.5 m l) was
treated with a solution of picric acid (0.025 g) in eth an ol (0.5 m l) to yield 0.025 g (24%) of
th e picrate of N1-butyl-N2-(ribosylcarbam oyl)guan idin e (5b an d isom ers), m .p. 142–147 °C
(dec.), RF (B) 0.58 (D2, 5b an d isom ers) an d 0.90 (D1, picric acid). 1H NMR: 9.40 s, 1 H, 8.88
br s, 1 H, 8.47 br s, 1 H an d 8.38 br, 1 H (guan idin ium NH); 7.94 br s, 1 H (carbam oyl NH);
8.58 s, 2 H (picrate CH); 5.13 br t, J ≈ 6.0 [+ DOAc: 5.13 d, J(1′,2′) = 4.8] (H-1′ of 5b); 5.42 dd,
J(1′,2′) = 4.9, J(1′,NH) = 9.0 [+ DOAc: 5.42 d, J(1′,2′) = 4.9] (H-1′ of th e α-furan osyl isom er);
5.01 dd, J(1′,2′) = 3.2, J(1′,NH) = 8.5 [+ DOAc: 5.00 d, J(1′,2′) = 3.4] (H-1′ of th e α-pyran osyl
isom er); 4.84 br t, J ≈ 8.0 [+ DOAc: 4.84 d, J(1′,2′) = 8.8] (H-1′ of th e β-pyran osyl isom er) (5b
(β-fur):α-fur:α-pyr:β-pyr ≈ 5:2:1.5:1). Th e oth er sign als were n ot resolvable. FAB-MS: 291 (42)
[M5b an d isom ers + H]+, 116 (20) [M + H]+ of butylguan idin e. For C11H22N4O5·C6H3N3O7
(519.4) calculated: 39.29% C, 4.84% H, 18.87% N; foun d: 39.00% C, 4.55% H, 18.78% N.
Method B. A solution of th e aggregate of 3b with 5b (0.118 g, 0.2 m m ol) in m eth an ol
(5 m l) was worked up by colum n ch rom atograph y on silica gel (10 g) in an alogy to th e
preparation of 3b. Th e fraction s con tain in g 3b were collected, evaporated an d th e residue
crystallized from aceton itrile to yield 0.024 g (40%) of 3b, m .p. 86–88 °C (dec.) with out de-
pression on adm ixture with a sam ple of th e product prepared by m eth od A. Th e very polar
carbam oylguan idin e 5b was n ot eluted from th e colum n .
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Pískala et al.:
Aggregate of N4-sec-Butyl-5-azacytidin e (3c) with N1-sec-Butyl-N2-(β-D-ribofuran osyl-
carbam oyl)guan idin e (5c)
Meth an ol (2 m l), sec-butylam in e (0.2 m l, 2 m m ol), reaction tim e 4 h . Th e crude product was
applied on to a colum n of Am berlite IRC-50 [H+] ion exch an ge resin (10 m l), wh ich was pre-
pared in m eth an ol. Th e colum n was eluted with m eth an ol (100 m l), th e effluen t evaporated
an d th e residue crystallized from aceton itrile (3 weeks, room tem perature) to yield 0.074 g
(25%) of th e aggregate of 3c with 5c, m .p. 130–132 °C (dec.), RF (B) 0.72 (D1, 3c) an d 0.52
(D2, 5c), [α]D –1.5 (c 0.1, water). UV, λm ax (log ε): (MeOH), 255, in flexion (3.98), 226 (4.53);
(pH 2.30), 258 (3.85), 219 (4.02); (pH 7.03), 250, in flexion (3.90); (pH 10.07), 227 (4.68). For
C
23H42N8O10·1/2H2O (590.7) calculated: 46.07% C, 7.23% H, 18.69% N; foun d: 46.06% C,
7.14% H, 18.95% N. Th e aggregate of 3c with 5c was h ygroscopic an d decom posed on stor-
age to products of h ydrolysis.
Aggregate of N4-Ben zyl-5-azacytidin e (3d) with N1-Ben zyl-N2-(β-D-ribofuran osylcarbam oyl)-
gu an idin e (5d )
Meth an ol (2 m l), ben zylam in e 0.22 m l, 2 m m ol), reaction tim e 5 h . Yield: 0.059 g (18%) of
th e aggregate, m .p. 136–139 °C (dec.) (aceton itrile), RF (B) 0.71 (D1, 3d ) an d 0.57 (D2, 5d ),
[α]D –27.4 (c 0.1, water). UV, λm ax (log ε): (MeOH), 255, in flexion (3.95), 228 (4.57); (pH
2.30), 255 (3.95), 219 (4.25); (pH 7.03), 255, in flexion (3.96), 225, in flexion (4.44); (pH
10.97), 228 (4.69). IR (KBr): 3512 m [ν(OH)]; 3408 s [ν(NH) free, urea]; 3363 s [ν(NH), im in o
group]; 3275 s, br, 3240 s, br, sh [ν(NH) bon ded]; 1757 m , 1678 s, 1655 s [ν(C=O) + β(NH),
triazin e]; 1621 vs [am ide I, urea, ν(C=N), triazin e]; 1598 s [ν(rin g), ph en yl]; 1576 s, 1560 s,
sh [am ide II, urea + ν(C=N)]; 1515 s, sh , 1497 s [ν(C=N), ν(C–N), triazin e + ν(rin g), ph en yl];
1288 s (am ide III, urea); 796 s (rin g breath in g, triazin e); 600 m [γ(NH)]. FAB-MS: 669 (<1)
[2 M3d + H]+, 659 (1) [M3d + M5d + H]+, 649 (<1) [2 M5d + H]+, 484 (2) [M3d + ben zyl-
guan idin e + H]+, 474 (2) [M5d + ben zylguan idin e + H]+, 357 (2) [M3d + Na]+, 347 (3) [M5d
+
Na]+, 335 (4) [M3d + H]+ , 325 (16) [M5d + H]+, 203 (12) [aglycon e3d + H]+, 150 (100) [M +
H]+ of ben zylguan idin e. For C29H38N8O10 (658.7) calculated: 52.87% C, 5.81% H, 17.02% N;
foun d: 52.69% C, 5.75% H, 17.30% N.
In a sim ultan eous experim en t, a solution of crude 3d (prepared from 1 m m ol of 4 by th e
aforem en tion ed procedure) in 1 M am m on ia (6 m l) was kept at room tem perature for 1 h ,
evaporated an d th e residue co-evaporated with eth an ol (3 × 5 m l). Th e crude carbam oyl-
guan idin e 5d th us obtain ed was treated with a solution of crude 3d (prepared by th e above
m en tion ed procedure from 1 m m ol of 4) in m eth an ol (5 m l). Th e m ixture was evaporated
an d th e residue crystallized from aceton itrile–m eth an ol (95:5, v/v) to give 0.382 g (58%) of
th e aggregate of 3d with 5d , m .p. 136–139 °C (dec.) with out depression on adm ixture with a
sam ple prepared by m eth od A.
Aggregate of N4-Furfuryl-5-azacytidin e (3e) with N1-Furfuryl-N2-(β-D-ribofuran osylcarbam oyl)-
guan idin e (5e)
Meth an ol (2 m l), furfurylam in e (0.18 m l, 2 m m ol), reaction tim e 7 h . Yield: 0.150 g (47%)
of th e aggregate, m .p. 141–143 °C (dec.) (aceton itrile–m eth an ol 9:1, v/v), RF (B) 0.68 (D1, 3e)
an d 0.61 (D2, 5e), [α]D –19.7 (c 0.5, water). UV, λm ax (log ε): (MeOH), 260 (3.63), 225 (4.36);
(pH 2.30), 254 (3.64), 222 (4.06); (pH 7.03), 253 (3.70), 215 (4.31); (pH 10.97), 228 (4.54),
215 (4.38). IR (KBr): 3526 m , sh [ν(OH)]; 3410 s, br, sh [ν(NH) free, urea]; 3368 s [ν(NH),
im in o group]; 3275 s, br, sh [ν(NH) bon ded]; 1749 w, sh , 1730 w, sh , 1678 s, 1655 s [ν(C=O)
+ β(NH), triazin e]; 1623 vs [am ide I, urea, ν(C=N), triazin e]; 1571 s, 1563 s [am ide II +
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Mutagenicity of 2′-Deoxy-5-azacytidine
733
ν(C=N)]; 1512 m , sh , 1502 s [ν(C=N), ν(C–N) + ν(rin g), furan ]; 1302 m , 1294 m (am ide III,
urea); 1011 m (rin g breath in g, furan ); 798 w (rin g breath in g, triazin e); 599 w [γ(NH)].
FAB-MS: 649 (1) [2 M3e + H]+, 639 (4) [M3e + M5e + H]+, 629 (2) [2 M5e + H]+, 464 (<1) [M3e
+
furfurylguan idin e + H]+, 454 (<1) [M5e + furfurylguan idin e + H]+, 325 (17) [M3e + H]+, 315
(47) [M5e + H]+, 193 (35) [aglycon e3e + H]+, 140 (15) [M + H]+ of furfurylguan idin e. For
C
25H34N8O12 (638.6) calculated: 47.02% C, 5.36% H, 17.55% N; foun d: 47.09% C, 5.38% H,
17.51% N.
2-(Meth ylcarbam oyl)guan idin e (7)
A m ixture of guan idin e h ydroch loride (3.84 g, 40 m m ol) an d 1 M sodium eth oxide in eth a-
n ol (40 m l) was stirred at room tem perature for 15 m in an d th e in soluble sodium ch loride
filtered off with suction . Th e filtrate was evaporated at 35 °C (bath tem perature) an d th e res-
idue dried for 1 h in vacuum to give crude syrupy guan idin e. To a stirred m ixture of th is
product with dry aceton e (40 m l) a solution of m eth yl isocyan ate (2.4 m l, 40 m m ol) in dry
aceton e (20 m l) was added dropwise at 0 °C. Th e m ixture was th en stirred at room tem pera-
ture for 2 h an d kept overn igh t in a refrigerator. Th e crystallin e precipitate was filtered off
with suction to give 3.50 g (75%) of 7, m .p. 132–135 °C, RF (B) 0.28 (D2). Recrystallization
of th e un ique crude product from eth an ol raised th e m eltin g poin t to 137–139 °C. UV, λm ax
(log ε): (EtOH), 225 (4.21); (pH 11.15), 225 (4.38). IR (KBr): 3403 s [ν(NH) free tran s am ide];
3365 s [νas(NH2)]; 3266 s, 3123 m , sh [νs(NH2) bon ded]; 1730 m , sh [ν(C=0)]; 1668 s (am ide I);
1611 s, 1597 s [βs(NH2)]; 1552 s [ν(C=N)]; 1496 vs, br (am ide II); 1463 s, br [δ+as(Me)]; 1408 s
[δs(Me)]; 1302 s (am ide III); 1153 m [δ−as(Me) + ν(C=O)]; 1092 m [βas(NH2)]; 1062 w, br, sh
[ν(N–Me)]; 665 m , vbr, sh (am ide V); 588 m , br, 565 m , br [γs(NH2)]; 527 m [δ(N–C–N),
urea]. 1H NMR: 6.56 br s, 4 H (guan idin e NH); 6.04 br q, 1 H, J(NH,CH3) = 4.4 (carbam oyl
NH); 2.49 d, 3 H, J(CH3,NH) = 4.4 (CH3). 13C NMR: 166.19 (C=O), 160.92 (C=N), 26.53
(CH3). FAB-MS: 349 (6) [3 M7 + H]+, 255 (8) [2 M7 + Na]+, 233 (100) [2 M7 + H]+, 176 (7) [M7
+
M10 + H]+, 139 (14) [M7 + Na]+, 117 (100) [M7 + H]+, 60 (14) [M10 + H]+, 57 (5) M• + of
m eth yl isocyan ate. For C3H8N4O (116.1) calculated: 31.03% C, 6.94% H, 48.26% N; foun d:
31.20% C, 7.02% H, 48.54% N. Crystallization of a m ixture of 7 (0.058 g, 0.5 m m ol) an d
picric acid (0.115 g, 0.5 m m ol) from eth an ol–water gave 0.156 g (90%) of 2-(m eth yl-
carbam oyl)guan idin ium picrate (7a), m .p. 235–236 °C (dec.). IR (Nujol): 3449 m , 3439 s,
3404 m [νas(NH2), ν(NH) free]; 3375 m , sh , 3323 m , 3294 m [ν(NH) bon ded]; 3245 m , sh ,
3215 m , 3183 m [νs(NH2)]; 1744 s [ν(C=O), CO–NH+=]; 1687 s, 1653 s [βs(NH2)]; 1629 s, sh ,
1610 s, br [ν(C=N), ν(rin g) of th e picrate]; 1567 s, 1559 s [νas(NO2)]; 1505 m , sh , 1497 s
(am ide II); 1430 m [ν(rin g) of th e picrate]; 1416 m [δs(Me)]; 1364 s [ν(C–N)]; 1339 s, 1320 s
[νs(NO2)]; 1057 w, br [ν(N–Me)]; 743 m , 726 m [γs(NO2)]; 568 m , 559 m [γs(NH2)]; 526 m
[δ(N–C–N), urea]. 1H NMR: 9.65 br s, 1 H an d 8.01 br s, 4 H (guan idin ium NH); 7.36 br q, 1 H,
J(NH,CH3) = 4.5 (carbam oyl NH); 2.645 d, 3 H, J(CH3,NH) = 4.5 (CH3); 8.60 s, 2 H (picrate
CH). 13C NMR: 161.01 (C=O), 155.345 (N-C), 153.88, 142.03, 125.41 an d 124.41 (picrate C),
26.27 (CH3). FAB-MS: 117 (35) [M7 + H]+, 60 (12) [M10 + H]+, 57 (26) M• + of m eth yl
isocyan ate. For C3H8N4O·C6H3N3O7 (345.2) calculated: 31.31% C, 3.21% H, 28.40% N;
foun d: 31.60% C, 3.00% H, 28.16% N.
1-Meth yl-5-azacytosin e (6)
A solution of 2-(m eth ylcarbam oyl)guan idin e (7) (0.116 g, 1 m m ol) in a m ixture of m eth an ol
(1.5 m l) an d dim eth ylform am ide dim eth yl acetal (0.13 m l, 1 m m ol) was kept at room tem -
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734
Pískala et al.:
perature for 3 days an d th e precipitate filtered off with suction to give 0.083 g (66%) of 6,
m .p. 258–260 °C (dec.) with out depression on adm ixture with an auth en tic sam ple65, RF (B)
0.33 (D1). UV, λm ax (log ε): (MeOH), 209 (4.02), 243 (3.72); (pH 6.94), 201 (4.36), 245 (3.68);
(pH 10.93), 214 (3.89), 245 (3.70). IR (KBr): 3500 m , br, sh , 3408 m , br [νas(NH2)]; 3340 s,
br, sh , 3303 s, br, 3249 s, br, 3183 s, br, 3045 m , br, sh [νs(NH2)]; 1757 m , sh , 1734 s, sh ,
1707 s, br, sh , 1681 s, br, 1668 s, vbr, sh [ν(C=O) + βs(NH2)]; 1616 s, 1513 s, 1487 s, br, 1354 s
[ν(C=N), ν(C–N)]; 1123 s [βs(NH2)]; 795 s (rin g breath in g); 711 w, vbr [γs(NH2)]. 1H NMR:
8.24 s, 1 H (H-6); 7.33 brs, 1 H an d 7.31 brs, 1 H (NH2); 3.19 s, 3 H (CH3). 13C NMR: 166.87
(C-4), 159.65 (C-6), 154.755 (C-2), 34.20 (CH3). FAB-MS: 275 (17) [2 M6 + Na]+, 253 (28)
[2 M6 + H]+, 149 (27) [M6 + Na]+, 127 (100) [M6 + H]+.
Aggregates of 1-Meth yl-5-azacytosin e (6) with 2-(Meth ylcarbam oyl)guan idin e (7)
Method A. A solution of 2-(m eth ylcarbam oyl)guan idin e (7) (0.464 g, 4 m m ol) in a m ixture
of m eth an ol (4 m l) an d eth yl form ate (0.5 m l) was kept at room tem perature for 5 days an d
crystallization of th e product in duced by scratch in g. Th e precipitate was filtered off with
suction to give 0.165 g (32%) of th e m on oh ydrate of a 1:2 aggregate of 6 an d 7, m .p.
166–167 °C (dec.), RF (D) 0.48 (D3, 7) an d 0.55 (D1, 6). UV, λm ax (log ε): (MeOH), 211 (4.33),
225 (4.40); (pH 2.50), 254 (3.64); (pH 7.06), 243, in flexion (3.56), 225 (3.80); (pH 11.15),
225 (4.57). IR (KBr): 3505 m [νs(NH2) free]; 3381 s, 3330 s, sh , 3290 s, br, sh [νas(NH2),
ν(NH) bon ded]; 3219 s, 3080 m , br, sh [νs(NH2)]; 1706 s [ν(C=O) triazin e]; 1673 s, 1663 s, sh
[βs(NH2), triazin e + am ide I free, urea]; 1628 s [am ide I bon ded, urea + ν(C=N), triazin e];
1604 s, 1595 s, sh [βs(NH2), ν(C=N), guan idin e]; 1552 s, br, sh (am ide II bon ded, urea); 1500
vs, br [am ide II free, urea, ν(C=N), triazin e]; 1462 s, sh , 1445 s, sh [δ+as(Me)]; 1410 s [δs(Me),
urea]; 1365 m [δs(Me), triazin e]; 1310 s, br (am ide III, urea); 1121 m [βs(NH2), triazin e];
1086 m [δ−as(Me)]; 791 s (rin g breath in g, triazin e); 714 m , vbr, [γs(NH2), triazin e]; 650 m , vbr,
sh (am ide V, urea); 607 m [δ(C=O), triazin e]; 544 m , br [δ(N–C–N), urea]. 1H NMR: 8.24 s, 1 H
(H-6 of 6); 7.31 br s, 2 H (NH2 of 6); 6.52 br, 8 H (4 × NH2 of 7); 5.98 br s, 2 H (2 × NH of
7); 3.20 s, 3 H (CH3 of 6); 2.49 d, 6 H, J(CH3,NH) = 4.6 (2 × CH3 of 7) (6:7 = 1:2). 13C NMR:
166.84 (C-4 of 6), 166.22 (C=O of 7), 160.91 (C=N of 7), 159.63 (C-6 of 6), 154.73 (C-2 of 6),
34.18 (CH3 of 6), 26.52 (CH3 of 7). FAB-MS: 369 (7) [2 M6 + M7 + H]+, 359 (5) [M6 + 2 M7
+
H]+, 349 (3) [3 M7 + H]+, 275 (2) [2 M6 + Na]+, 265 (3) [M6 + M7 + Na]+, 255 (4) [2 M7 + Na]+,
253 (5) [2 M6 + H]+, 243 (100) [M6 + M7 + H]+, 233 (57) [2 M7 + H]+, 186 (10) [M6 + M10 + H]+,
176 (7) [M7 + M10 + H]+, 149 (8) [M6 + Na]+, 139 (7) [M7 + Na]+, 127 (79) [M6 + H]+, 117
(100) [M7 + H]+, 60 (20) [M10 + H]+, 57 (7) M• + of m eth yl isocyan ate. For C4H6N4O·
(C3H8N4O)2·H2O (376.4) calculated: 31.91% C, 6.42% H, 44.66% N; foun d: 31.95% C,
6.56% H, 44.32% N.
Method B. A solution of a m ixture of 6 (21.5 m g) an d 7 (20 m g) in water was evaporated
an d th e residue dried to con stan t weigh t to give th e m on oh ydrate of a 1:1 aggregate of 6
with 7, m .p. 163–165 °C (dec.). FAB-MS: 253 (1) [2 M6 + H]+, 243 (8) [M6 + M7 + H]+, 233 (4)
[2 M7 + H]+, 127 (29) [M6 + H]+, 117 (100) [M7 +H]+, 60 (22) [M10 + H]+, 57 (12) M• + of
m eth yl isocyan ate. Wh en th e sam e solution of a sam ple of th e 1:1 aggregate of 6 with 7 was
diluted with a forty tim es larger volum e of water, n o aggregates h ave been detected at all.
For C4H6N4O·C3H8N4O·H2O (260.2) calculated: 32.31% C, 6.20% H, 43.07% N; foun d:
32.02% C, 6.38% H, 43.00% N.
Method C. A solution of a m ixture of 6 (21.5 m g) an d 7 (10 m g) in water was evaporated
an d th e residue dried to con stan t weigh t to give th e dih ydrate of a 2:1 aggregate of 6 with
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Mutagenicity of 2′-Deoxy-5-azacytidine
735
7, m .p. 162–163 °C (dec.). For (C4H6N4O)2·C3H8N4O·2H2O (404.2) calculated: 32.68% C,
5.98% H, 41.58% N; foun d: 32.40% C, 6.12% H, 41.73% N.
Aggregate of 1-Meth yl-5-azacytosin e (6) with 2-(Meth ylcarbam oyl)guan idin ium
Picrate (7a)
A m ixture of 6 (25.2 m g, 0.2 m m ol) an d th e picrate 7a (69 m g, 0.2 m m ol) was dissolved in
boilin g m eth an ol an d th e solution evaporated. Th e residue (m .p. 205–208 °C (dec.)) was
recrystallized from m eth an ol to give 60 m g (64%) of th e aggregate, m .p. 211–213 °C (dec.),
RF (D) 0.48 (D3, 7); 0.55 (D1, 6) an d 0.78 (D1, picric acid). 1H NMR: 8.24 s, 1 H (H-6 of 6);
7.34 br s, 2 H (NH2 of 6); 3.20 s, 3 H (CH3 of 6); 8.59 s, 2 H (picrate CH); 9.67 br s, 1 H an d
8.03 br s, 4 H (guan idin ium NH of 7a); 7.36 br t, 1 H, J(NH,CH3) = 4.5 (carbam oyl NH of
7a); 2.65 d, 3 H, J(CH3,NH) = 4.5 (m eth ylcarbam oyl CH3 of 7a) (6:7a ≈ 1:1, based on th e sig-
n als of th e m eth yl groups). 13C NMR: 166.83 (C-4 of 6), 161.025 (C=O of 7a), 159.63 (C-6 of
6), 155.36 (C-N of 7a), 154.80 (C-2 of 6), 153.92, 142.04, 125.43 an d 124.41 (picrate C),
34.17 (CH3 of 6), 26.29 (CH3 of 7a). FAB-MS: 253 (<1) [2 M6 + H]+, 243 (5) [M6 + M7 + H]+,
233 (1) [2 M7 + H]+, 127 (27) [M6 + H]+, 117 (100) [M7 + H]+, 60 (67) [M10 + H]+, 57 (7) M• +
of m eth yl isocyan ate. For C4H6N4O·C3H8N4O·C6H3N3O7 (471.3) calculated: 33.13% C,
3.64% H, 32.69% N; foun d: 32.99% C, 3.66% H, 32.39% N.
2-(β-D-Ribofuran osylcarbam oyl)guan idin ium Picrate (8a) an d Its α-D An om er
Th e product was prepared by a kn own procedure23 1H NMR: 8.59 s, 2 H (ben zen e CH); 9.58
.
br s, 1 H an d 8.10 br s, 4 H (guan idin ium NH); β-D an om er: 7.96 d, 1 H, J(NH,1′) = 8.1
(carbam oyl NH); 5.13 br dd, 1 H, J(1′,2′) = 4.7 (H-1′); 3.87 br t, 1 H, J(2′,3′) = 4.4 (H-2′); 3.77
br t, 1 H, J(3′,4′) = 4.0 (H-3′); 3.71 br q, 1 H (H-4′); 3.46 dd, 1 H, J(5′a,4′) = 4.1, J(gem ) = 11.8
(H-5′a); 3.40 dd, 1 H, J(5′b,4′) = 4.3, J(gem ) = 11.8 (H-5′b); 5.13 br s, 1 H, 4.99 br s, 1 H an d
4.78 br s, 1 H (OH); α-D an om er: 7.69 d, 1 H, J(NH,1′) = 9.1 (carbam oyl NH); 5.42 dd, 1 H,
J(1′,2′) = 4.6, J(1′,NH) = 9.1 (H-1′); 3.98 br q, 1 H, J(2′,3′) = 4.6 (H-2′); 3.89 br t, 1 H, J(3′,4′) ≈
4.5 (H-3′); 3.74 (covered), 1 H (H-4′); 3.47 dd, 1 H, J(5′a,4′) = 4.0 , J(gem ) = 12.0 (H-5′a);
3.36 dd, 1 H, J(5′b,4′) = 4.6, J(gem ) = 12.0 (H-5′b); 5.47 br s, 1 H, J ≈ 4.0, 4.80 br s, 1 H an d
4.70 br s, 1 H (OH) (β:α ≈ 6.5:1, based on th e sign als of H-1′). FAB-MS: 235 (12) [M8 + H]+,
60 (100) [M10 + H]+.
2-Carbam oylguan idin ium Ch loride (7b) an d
1,1-Dim eth yl-2-(m eth ylcarbam oyl)guan idin ium Ch loride (7c)
For th e preparation of guan idin ium ch lorides 7b an d 7c, see ref.29 1H NMR of 7b: 10.42 br s,
.
1 H an d 8.21 br s, 4 H (guan idin ium NH); 7.20 br s, 2 H (carbam oyl NH); on dilution (1:4)
th e followin g sign als of th e NH proton s were observed: 10.45 br s, 1 H an d 8.23 br s, 4 H
(guan idin ium NH); 7.22 br s, 2 H (carbam oyl NH). 1H NMR of 7c: 10.36 s, 1 H, 9.16 br, 1 H
an d 8.47 br, 1 H (guan idin ium NH); 8.46 br q, 1 H, J(NH,CH3) = 4.6 (m eth ylcarbam oyl NH);
3.10 br s, 6 H (2 × guan idin ium CH3); 2.68 d, 3 H, J(CH3,NH) = 4.6 (m eth ylcarbam oyl CH3);
on dilution th e followin g sign als of th e NH proton s were observed: 10.30 s, 1 H, 9.16 br, 1 H
an d 8.45 br, 1 H (guan idin ium NH); 8.38 br q, 1 H (m eth ylcarbam oyl NH).
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

736
Pískala et al.:
Aggregate of Cytidin e (9) with 2-(β-D-Ribofuran osylcarbam oyl)guan idin ium Picrate (8a)
an d Its α-D An om er
A m ixture of cytidin e (9) (48.6 m g, 0.2 m m ol) an d 2-(β-D-ribofuran osylcarbam oyl)-
guan idin ium picrate (8a) (92.6 m g, 0.2 m m ol) was dissolved in boilin g m eth an ol an d th e
solution evaporated. Crystallization of th e residue (m .p. 96–98 °C) from eth yl acetate gave
85 m g (60%) of th e aggregate, m .p. 94–96 °C, RF (D) 0.29 (D3, 8); 0.36 (D1, 9) an d 0.78 (D1,
picric acid). 1H NMR: 7.42 br s, 1 H an d 7.28 br s, 1 H (NH2 of 9); 10.00 br, 1 H an d 8.26 br s,
4 H (guan idin ium NH of 8a); 7.92 d, 1 H (carbam oyl NH of β-8a); 7.65 d, 1 H (carbam oyl
NH of α-8a) (9:8a = 1.2:1 an d β-8a:α-8a ≈ 3.2:1, based on th e sign als of H-1′ of th e ribosyl
group); on dilution (1:4) the following signals of the NH protons were observed: 7.29 br s, 1 H
an d 7.18 br s, 1 H (NH2 of 9); 10.00 br, 1 H an d 8.15 br s, 4 H (guan idin ium NH of 8a); 7.93 d,
1 H, J = 7.8 (carbam oyl NH of β-8a); 7.66 d, 1 H, J = 9.0 (carbam oyl NH of α-8a); th e sign als
of th e oth er proton s were n ot evaluated in th is m easurem en t. FAB-MS: 487 (5) [2 M9 + H]+,
478 (15) [M8 + M9 + H]+, 244 (100) [M9 + H]+, 235 (95) [M8 + H]+. For C9H13N3O7·
C7H14N4O5·C6H3N3O7 (706.5) calculated: 37.40% C, 4.28% H, 19.82% N; foun d: 37.58% C,
4.51% H, 18.70% N.
1,2-Bis(m eth ylcarbam oyl)guan idin e (11)
To a stirred m ixture of guan idin e, (prepared from guan idin e h ydroch loride (1.92 g, 20 m m ol)
in an alogy to th e preparation of 7) in dry aceton e (20 m l), a solution of m eth yl isocyan ate
(2.4 m l, 40 m m ol) in dry aceton e (10 m l) was added dropwise at 0 °C. Th e m ixture was th en
stirred at room tem perature for 2 h an d kept overn igh t in a refrigerator. Th e crystals were
filtered off with suction to give 2.80 g (81%) of 11, m .p. 174–176 °C (dec.). Recrystallization
from eth an ol raised th e m eltin g poin t to 179–181 °C (dec.), RF 0.36 (B, D2). UV, λm ax (log ε):
(EtOH), 233 (4.27); (0.1 M HCl), 214 (4.24); (pH 9.53), 209 (4.09), 216 (4.06), 234 (4.33). IR
(KBr): 3405 m , sh , 3388 m , 3349 s, 3333 s, sh , 3291 m , 3113 w, br, sh [ν(NH), νas(NH2),
νs(NH2)]; 1700 m , 1680 s, 1638 vs (am ide I); 1601 m , sh [βs(NH2)]; 1565 s, br, 1537 s, br
(am ide II); 1472 m , sh , 1451 m [δ+as(Me) + ν(C=O)]; 1403 m [δs(Me)]; 1303 s, 1268 s (am ide
III); 1171 w, 1159 m [δ−as(Me) + ν(C=O)]; 526 m , br [δ(N–C–N)]. 1H NMR: 8.90 br s, 1 H,
8.60 br s, 1 H, 7.70 br s, 1 H, 7.40 br s, 1 H an d 7.00 br s, 1 H (5 × NH); 2.60 br d, 6 H,
J(CH3,NH) = 4.4 (2 × CH3). 13C NMR: 157.42 br an d 156.38 br (2 × C=O), 156.21 (C=N),
26.70 br an d 26.17 (2 × CH3). FAB-MS: 347 (4) [2 M11 + H]+, 174 (100) [M11 + H]+, 117 (25)
[M7 + H] +, 60 (36) [M10 + H]+, 57 (14) M• + of m eth yl isocyan ate. For C5H11N5O2 (173.2) cal-
culated: 34.68% C, 6.40% H, 40.44% N; foun d: 34.93% C, 6.58% H, 40.67% N.
1,2,3-Tris(m eth ylcarbam oyl)guan idin e (12)
To a stirred m ixture of guan idin e (prepared from guan idin e h ydroch loride (0.96, 10 m m ol)
in an alogy to th e preparation of 7) in dry aceton e, a solution of m eth yl isocyan ate (1.8 m l,
30 m m ol) in dry aceton e (5 m l) was added dropwise at 0 °C. Th e m ixture was th en stirred at
room tem perature for 3 h . Durin g th is tim e, th e volum in ous crystallin e precipitate dis-
solved. Th e clear solution was filtered off with suction to rem ove a sm all in soluble portion ,
kept overn igh t in a refrigerator an d th e crystals were filtered off with suction to give 1.40 g
of 12, m .p. 185–190 °C (resolidification ). Evaporation of th e m oth er liquor an d crystallization
of th e residue from aceton e (3 m l) gave a secon d crop of 12. Overall yield: 1.90 g (82%).
Recrystallization of th e un ique crude product from eth an ol raised th e m eltin g poin t to
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
737
191–194 °C (resolidification ), RF 0.50 (B, D1). UV, λm ax (log ε): (EtOH), 224 (4.30), 233
(4.27), 239 (4.29); (0.1 M HCl), 228 (4.41); (pH 2.40), 231 (4.27); (pH 6.88), 234 (4.27); (pH
11.15), 231 (4.10). IR (KBr): 3398 m , br, 3327 s, br, sh , 3286 s, 3223 s, br, 3102 m , br
[ν(NH)]; 1718 s, 1707 s, 1695 s, 1670 s, 1623 s, br [am ide I, ν(C=N)]; 1568–1520 s, br (am ide
II); 1470 m , 1447 m , br [δ+as(Me)]; 1410 s, 1401 s [δs(Me)]; 1320 s, br, 1250 s, br (am ide III);
1174 m , 1158 m [ν(C=O) + δ−as(Me)]; 690 m , 678 m (am ide V). 1H NMR: 11.98 br s, 1 H,
10.78 br s, 1 H, 9.14 br s, 1 H, 8.18 br s, 1 H an d 7.76 br q, 1 H, J(NH,CH3) = 4.5 (5 × NH);
2.72 br s, 3 H, 2.60 br s, 3 H an d 2.64 d, 3 H, J(CH3,NH) = 4.5 (3 × CH3). 13C NMR: 162.93
(C=N), 154.685 br, 153.03 br an d 151.41 (3 × C=O), 26.535 br, 26.15 br an d 26.01 (3 × CH3).
FAB-MS: 461 (3) [2 M12 + H]+, 231 (100) [M12 + H]+, 174 (22) [M11 + H]+, 117 (24) [M7 + H]+,
60 (46) [M10 + H]+, 57 (26) M• + of m eth yl isocyan ate. For C7H14N6O3 (230.2) calculated:
36.52% C, 6.13% H, 36.51% N; foun d: 36.80% C, 6.19% H, 36.26% N.
1-Meth yl-4-(3-m eth ylureido)-1,3,5-triazin -2(1H)-on e (13)
A m ixture of 1,2-bis(m eth ylcarbam oyl)guan idin e (11) (0.173 g, 1 m m ol), m eth an ol (2 m l)
an d dim eth ylform am ide dim eth yl acetal (0.13 m l, 1 m m ol) was stirred at room tem perature
for 24 h . Th e precipitate was filtered off with suction to give 0.152 g (83%) of 13, m .p.
241–243 °C (dec.), RF 0.45 (B, D1). UV, λm ax (log ε): (MeOH), 211 (4.03), 239 (4.03); (pH
2.50), 242 (4.04); (pH 7.06), 238 (4.06); (pH 11.15), 228 (4.07). IR (KBr): 3372 m , sh ,
3305 m , 3244 m , 3213 m , sh , 3143 m , br, 3100 m , sh [ν(NH) bon ded]; 3065 m , 3045 m , sh
[ν(C–H)]; 3010 m , 2988 m [νas(Me)]; 2890 w, sh , 2858 w, sh [νs(Me)]; 1713 s, 1685 vs, br
[ν(C=O), triazin e + am ide I, free urea]; 1630 vs, 1614 s, sh [am ide I, bon ded urea + ν(C=N),
triazin e]; 1569 m , sh , 1545 s (am ide II, bon ded urea); 1524 vs, 1498 vs [ν(C=N), triazin e];
1477 s, 1453 m , sh [δ+as(Me)]; 1426 s [ν(C–N)]; 1410 m , 1403 s, 1373 m , 1364 w, sh [δs(Me),
urea + triazin e]; 1298 vs, 1251 m (am ide III, urea); 1077 m [δ−as(Me), triazin e]; 796 m (rin g
breath in g, triazin e); 651 w, br (am ide V, urea); 537 w [δ(N–C–N), urea). 1H NMR: 10.09 br s,
1 H (ureido NH in position 1); 8.90 br q, 1 H, J(NH,CH3) = 4.6 (ureido NH in position 3);
8.51 s, 1 H (H-6); 3.30 s, 3 H (triazin e CH3); 2.77 d, 3 H, J(CH3,NH) = 4.6 (ureido CH3).
FAB-MS: 367 (4) [2 M13 + H]+, 184 (45) [M13 + H]+, 127 (74) [M6 + H]+, 57 (100) M• + of
m eth yl isocyan ate. For C6H9N5O2 (183.2) calculated: 39.34% C, 4.95% H, 38.23% N; foun d:
39.63% C, 5.11% H, 38.09% N.
Aggregates of Guan osin e (14) with 5-Azacytidin e (1), 2′-Deoxy-5-azacytidin e (2) an d
1-β-D-Arabin ofuran osyl-5-azacytosin e (15)
Method A. A m ixture of 1 (0.120 g) an d 14 (0.140 g) was crystallized from water–aceton e
(1:1, vv) (3 m l) to give 0.147 g of a 1:2 aggregate of 1 with 14, m .p. 214–218 °C (dec.).
FAB-MS: 528 (6) [M1 + M14 + H]+, 489 (5) [2 M1 + H]+, 284 (100) [M14 + H]+, 245 (84) [M1
H]+, 152 (100) [aglycon e14 + H]+, 113 (100) [aglycon e1 + H]+.
+
Method B. A m ixture of 2 (0.110 g) an d 14 (0.140 g) was crystallized from m eth an ol–water
(98:2, v/v) (10 m l) to give 0.143 g of a 1:1 aggregate of 2 with 14, m .p. 141–143 °C (dec.).
FAB-MS: 512 (4) [M2 + M14 + H]+, 457 (2) [2 M2 + H]+, 284 (34) [M14 + H]+, 229 (12) [M2
H]+, 152 (52) [aglycon e14 + H]+, 113 (45) [aglycon e2 + H]+.
+
Method C. A m ixture of 15 (0.050 g) an d 14 (0.060 g) was crystallized from m eth an ol to
give 0.044 g of a 1:1 aggregate of 15 with 14, m .p. 219–223 °C (dec.). FAB-MS: 528 (1) [M14
+
M15 + H]+, 489 (<1) [2 M15 + H]+, 284 (13) [M14 + H]+, 245 (5) [M15 + H]+, 152 (35)
[aglycon e14 + H]+, 113 (9) [aglycon e15 + H]+.
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

738
Pískala et al.:
Aggregates of 5-Azacytidin e (1) an d 2′-Deoxy-5-azacytidin e (2) with th e Respective
Dih ydro Derivatives 16 an d 17
Method A. A m ixture of 1 (0.120 g) an d 16 (0.120) was crystallized from water–aceton e
(1:1, v/v) (4 m l) to give 0.145 g of a 1:1 aggregate of 1 with 16, m .p. 205–207 °C (dec.).
FAB-MS: 493 (4) [2 M16 + H]+, 491 (7) [M1 + M16 + H]+, 489 (5) [2 M1 + H]+, 247 (100) [M16
H]+, 245 (53) [M1 + H]+, 115 (45) [aglycon e16 + H]+, 113 (50) [aglycon e1 + H]+.
+
Method B. A m ixture of 2 (0.115 g) an d 17 (0.115 g) was crystallized from m eth an ol (10 m l)
to give a 1:1 aggregate of 2 with 17, m .p. 182–190 °C (dec.). FAB-MS: 461 (6) [2 M17 + H]+,
459 (5) [M2 + M17 + H]+, 457 (1) [2 M2 + H]+, 231 (58) [M17 + H]+, 229 (8) [M2 + H]+, 115 (25)
[aglycon e17 +H]+, 113 (28) [aglycon e2 + H]+.
1-(2,3,5-Tri-O-ben zyl-β-D-arabin ofuran osyl)-5,6-dih ydro-5-azacytosin e (18)
A m ixture of 19 (0.514 g, 1 m m ol), acetic acid (5 m l), 2,2-dim eth oxypropan e (2.5 m l) an d
zin c powder (0.65 g) was stirred at room tem perature for 5 h an d th e in soluble m aterial
filtered off with suction . Th e filtrate was diluted with water an d extracted with ch loroform
(3 × 20 m l). Th e organ ic ph ase was sh aken with a 5% solution of NaHCO3, dried (an h ydrous
MgSO4) an d evaporated. Th e residue was dissolved in m eth an olic h ydrogen ch loride (38 m g
HCl/5 m l), con cen trated to a sm aller volum e an d precipitated with eth er (15 m l). Th e
precipitate crystallized slowly to give the crude product, which was recrystallized three tim es from
ben zen e (4 m l) to yield 0.400 g (72%) of th e pure h ydroch loride of 18a, m .p. 154–156 °C
(dec.), [α]D –22.8 (c 0.1, CH2Cl2). 1H NMR (CDCl3): 10.30 br, 1 H an d 9.25 br, 1 H (2 × NH);
8.20 br, 2 H (NH2); 7.36–7.15 m , 15 H (arom .); 6.01 d, 1 H, J(1′,2′) = 5.2 (H-1′); 4.82 d, 1 H
an d 4.59 d, 1 H, J(gem ) = 10.36 (2 × H-6); 4.53 d, 1 H an d 4.44 d, 1 H, J(gem ) = 12.0
(O-ben zyl); 4.50 d, 1 H an d 4.48 d, 1 H, J(gem ) = 12.0 (O-ben zyl); 4.42 d, 1 H an d 4.38 d, 1 H,
J(gem ) = 11.5 (O-ben zyl); 4.10 dd, 1 H, J(2′,3′) = 3.7, J(2′,1′) = 5.2 (H-2′); 3.96 q, 1 H, J(4′,3′) =
J(4′,5) = 4.9 (H-4′); 3.93 dd, 1 H, J(3′,2′) = 3.7, J(3′,4′) = 4.9 (H-3′); 3.56 d, 2 H, J(H5′,4′) = 4.9
(2 × H-5′). For C29H32N4O5·HCl (553.0) calculated: 62.98% C, 5.83% H, 6.41% Cl, 10.13% N;
foun d: 62.80% C, 5.92% H, 6.43% Cl, 10.18% N. A m ixture of h ydroch loride 18a (0.055 g,
0.1 m m ol), m eth an ol (1 m l) an d 1 M NaOMe (0.1 m l) was diluted with eth yl acetate (5 m l)
an d th e m ixture wash ed with a saturated solution of sodium ch loride (3 × 5 m l). Th e or-
gan ic ph ase was dried (an h ydrous MgSO4) an d evaporated to give 0.045 g of th e un ique free
base 18 as a syrup, RF (A) 0.58 (D1). 1H NMR (CDCl3): th e sign als of NH an d NH2 groups
were n ot detectable; 7.34–7.20 m , 15 H (arom .); 6.14 d, 1 H, J(1′,2′) = 5.6 (H-1′); 4.72 d, 1 H
an d 4.60 d, 1 H, J(gem ) = 9.8 (2 × H-6); 4.53 d, 1 H an d 4.46 d, 1 H, J(gem ) = 12.0 (O-ben zyl);
4.51 d an d 4.48 d, J(gem ) = 11.5 (O-ben zyl); 4.50 d, 1 H an d 4.38 d, 1 H, J(gem ) = 11.5
(O-ben zyl); 4.12 dd, 1 H, J(3′,2′) = 4.3, J(3′,4′) = 5.4 (H-3′); 3.95 dd, 1 H, J(2′,3′) = 4.3, J(2′,1′) =
5.6 (H-2′); 3.89 br q, 1 H (H-4′); 3.59 dd, 1 H, J(5′a,4′) = 4.4, J(gem ) = 10.7 (H-5′a); 3.55 dd, 1 H,
J(5′b,4′) = 4.8, J(gem ) = 10.7 (H-5′b). IR (CHCl3): 3484 w, 3392 w, br, sh , 3324 m , br
[νas(NH2)]; 3174 m , br [νs(NH2)]; 1724 s [ν(C=O)] (on deuteration sh ifted down by 1–2 cm –1);
1702 s, 1669 s, sh [ν(C=O) + βs(NH2)] (on deuteration in CCl4 a pron oun ced sh ift down an d
th e lower ban d disappears); 1653 s [βs(NH2)] (on deuteration in CCl4 th e ban d disappears);
1613 s [ν(C=N) + β(NH)] (on deuteration in CCl4 an d in aceton itrile a pron oun ced sh ift up);
1548 s [ν(C=N) + β(NH) + ν(C–N)] (on deuteration a sh oulder appeared on th e down side of
th e ban d); 1281 s (ν(C–N)]. A very sim ilar IR h as been obtain ed also in CCl4. IR (MeCN):
3459 m , 3356 m , vbr [νas(NH2)]; 3191 m , br [νs(NH2)]; 1724 s [ν(C=O)]; 1702 vs, 1680 s
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

Mutagenicity of 2′-Deoxy-5-azacytidine
739
[ν(C=O) + βs(NH2)]; 1656 s, 1651 s, 1638 s [βs(NH2)]; 1620 vs, 1551 s [ν(C=N) + β(NH)]; 1729 s
[ν(C–N)]. FAB-MS: 1033 (3) [2 M18 + H]+, 517 (100) [M18 + H]+.
An om eric 2-(D-erythro-2-Deoxypen tosylcarbam oyl)guan idin ium Form ates (21a–21d )
A m ixture of 2′-deoxy-5-azacytidin e (2) (2.28 g, 10 m m ol) an d 1 M am on ia (30 m l) was
stirred at room tem perature for 2 h , th e solution evaporated an d th e residue co-evaporated
with eth an ol (1 × 50 m l). Th e residue was dissolved in eth an ol (20 m l), acidified with form ic
acid (1 m l) an d kept overn igh t at room tem perature. Th e crystallin e product was filtered off
by suction to give 2.41
g (91%) of a m ixture of guan idin ium form ates 21a –21d
(21a:21b:21c:21d = 9.9:7.1:2.7:1), m .p. 149–154 °C (dec.), [α]D +13.9 (c 0.1, 1 M am m on ia).
UV, λm ax (log ε): (pH 2.30), 217 (3.44); (pH 6.95), 221 (3.85); (pH 10.92), 223 (4.38). 1H NMR
(D2O): 8.46 s 1 H (form ate CH); α-D-furan osyl derivative 21a: 5.77 dd, 1 H, J = 3.5 an d 7.0
(H-1′); 2.49 dt, 1 H, J = 6.3, 7.1 an d 14.2 (H-2′a); 1.96 dt, 1 H, J = 3.0 (2×) an d 14.2 (H-2′b);
4.37 dt, 1 H, J = 2.9 (2×) an d 6.3 (H-3′); 4.10 br q, 1 H, J(average) = 4.5 (H-4′); 3.65 dd, 1 H,
J = 3.8 an d 12.4 (H-5′a); 3.59 dd, 1 H, J = 5.2 an d 12.4 (H-5b); β-D-furan osyl derivative 21b:
5.78 t, J = 7.0 (H-1′); 2.26 ddd, 1 H, J = 2.9, 6.2 an d 14.0 (H-2′a); 2.14 dt, 1 H, J = 6.7, 7.2
an d 14.0 (H-2′b); 4.39 dt, 1 H, J = 2.7 (2×) an d 6.5 (H-3′); 3.96 td, 1 H, J = 2.8 an d 4.5 (2×)
(H-4′); 3.67 dd, 1 H, J = 4.5 an d 12.0 ((H-5′a); 3.62 dd, 1 H, J = 4.2 an d 12.0 (H-5′b);
α-D-pyran osyl derivative 21c: 5.04 dd, 1 H, J = 2.0 an d 10.0 (H-1′); 1.96, 1 H (H-2′a, covered
by th e H-2′b sign al of 21a); 1.87 br q, 1 H, J = 11.5 (2×) an d 12.0 (H-2′b); 4.00 ddd, J = 3.4,
4.3 an d 11.0 (H-3′); 3.91 dd, 1 H, J = 3.2 an d 12.6 (H-5′a); 3.68 dd, 1 H, J = 1.0 an d 12.6
(H-5′b); β-D-pyran osyl derivative 21d : 5.34 dd, 1 H, J = 2.8 an d 8.1 (H-1′); 2.09 ddd, 1 H, J =
3.0, 6.4 an d 13.9 (H-2′a); 1.89 ddd, 1 H, J = 3.2, 8.4 an d 13.9 (H-2′b); 4.16 dt, 1 H, J = 3.2
(2×) an d 6.4 (H-3′); 3.84 m , 3 H (H-4′, H-5′a an d H-5′b). 1H NMR (DMSO-d6): 8.35 s, 1 H
(form ate CH); 8.25 br, 5 H (guan idin ium NH); 7.63 d, 1 H (carbam oyl NH); th e sign als of
th e oth er proton s were n ot evaluated. For C8H16N4O6 (264.2) calculated: 36.36% C, 6.10%
H, 21.20% N; foun d: 36.30% C, 6.26% H, 21.30% N.
An om eric 2-(D-erythro-2-Deoxypen tofuran osylcarbam oyl)guan idin es (20a an d 20b)
A solution of a m ixture of 21a–21d (2.642 g, 10 m m ol) in water (50 m l) was applied on to a
colum n of Dowex 2X8 (50 m l) an d th e colum n eluted with water (1200 m l). Evaporation of
th e eluate, co-evaporation of th e residue with eth an ol an d crystallization from eth an ol gave
a m ixture of 20a–20d (2.16 g, m .p. 124–133 °C). Recrystallization of th e crude product from
m eth an ol (10 m l) gave 1.545 g (70.8%) of a m ixture of 20a an d 20b (20a:20b = 1:1), m .p.
155–162 °C. 1H NMR (D2O): α-D an om er 20a: 5.70 brm , 1 H (H-1′); 2.48 dt, 1 H, J = 6.6, 6.9
an d 13.9 (H-2′a); 1.90 dt, 1 H, J = 3.5 (2×) an d 13.9 (H-2′b); 4.31 dt, 1 H, J = 4.1 (2×) an d 6.4
(H-3′); 4.01 dt, 1 H, J = 4.0 (2×) an d 4.7 (H-4′); 3.68–3.58 m , 2 H (H-5′); β-D an om er 20b:
5.75 brm , 1 H (H-1′); 2.18 ddd, 1 H, J = 2.3, 6.0 an d 13.9 (H-2′a); 2.07 ddd, 1 H, J = 6.7, 7.2
an d 13.9 (H-2′b); 4.33 dt, 1 H, J = 2.9 (2×) an d 6.5 (H-3′); 3.89 td, 1 H, J = 2.7 an d 4.8 (2×)
(H-4′); 3.68–3.58 m , 2 H (H-5′). FAB-MS: 437 (9) [2 M20a
+ H]+, 219 (87) [M20a
+
an d 20b
an d 20b
H]+, 60 (25) [M10 + H]+. For C7H14N4O4 (218.2) calculated: 38.53% C, 6.47% H, 25.68% N;
foun d: 38.49% C, 6.63% H, 25.46% N.
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

740
Pískala et al.:
Mass spectrum of a 1:1 Mixture of 2′-Deoxy-5-azacytidin e (2) an d th e An om eric
2-(D-erythro-2-Deoxypen tofuran osylcarbam oyl)guan idin es (20a an d 20b)
A m ixture of 2 (5 m g) an d an d a 1:1 m ixture of 20a an d 20b (5 m g) was powdered in an
agate m ortar an d th e sam ple used for m easurem en t. FAB-MS: 479 (5) [2 M2 + Na]+, 469 (4)
[M2 + M20a
+ Na]+, 459 (2) [2 M20a
+ Na]+, 457 (7) [2 M2 + H]+, 447 (14) [M2
+
+
or 20b
or 20b
M20a
+ H]+, 437 (12) [2 M20a
+ H]+, 251 (11) [M2 + Na]+, 241 (13) [M20a
or 20b
or 20b or 20b
Na]+, 229 (14) [M2 + H]+, 219 (100) [M20a
+ H]+, 113 (79) [aglycon e2 + H]+, 103 (10)
or 20b
[aglycon e20a
+H]+, 60 (30) [M10 + H]+.
or 20b
4-O-Meth yl-1-(α-D-ribofuran osyl)isobiuret (22)
A m ixture of m eth oxytriazin on e28 4 (0.259 g, 1 m m ol), m eth an ol (7 m l) an d tert-butylam in e
(0.7 m l) was stirred at room tem perature for 26 h . Th e solution was refluxed for 2 h , kept at
room tem perature overn igh t an d evaporated. Th e residue was co-evaporated with eth an ol
(3 × 5 m l) an d crystallized from th e sam e solven t to yield 0.105 g of isobiuret 22, m .p.
158–160 °C (dec.). Recrystallization of th e crude product from eth an ol afforded 0.080 g
(32%) of pure isobiuret 22, m .p. 170–172 °C (dec.), RF 0.58 (C, D4), [α]D +123.8 (c 0.3, water).
UV (MeOH), λm ax (log ε): 220 (4.27). CD (pH 6.93), λm ax ([Θ]m ax): 223 (+10 170). IR (Nujol):
3390 s, 3296 s, 3234 m , sh [ν(OH, NH2, NH)]; 1665 s (am ide I); 1631 m , sh [ν(C=N)].
1H NMR: 8.10 br s, 1 H an d 7.70 br s, 1 H (NH2); 6.40 d, 1 H, J(NH,1′) = 9.8 (NH); 5.40 dd, 1 H,
J(1′,2′) = 4.9, J(1′,NH) = 9.8 (H-1′); 5.25 d, 1 H, J(OH,2′) = 5.0 (OH); 5.08 d, 1 H, J(OH,3′) = 5.5
(OH); 4.66 t, 1 H, J(OH,5′) = 5.6 (OH); 3.91 q, J(2′,1′) = J(2′,OH) = J(2′,3′) = 5.0 (H-2′);
3.85 dt, 1 H, J(3′,2′) = J(3′,4′) = 5.0, J(3′,OH) = 5.5 (H-3′); 3.70 td, 1 H, J(4′,5′a) = 3.7, J(4′,5′b) =
J(4′,3′) = 4.9 (H-4′); 3.65 s, 3 H (OCH3); 3.42 ddd, 1 H, J(5′a,4′) = 3.7, J(5′a,OH) = 5.2, J(gem ) =
12.0 (H-5′a); 3.34 ddd, 1 H, J(5′b,4′) = 4.9, J(5′b,OH) = 5.8, J(gem ) = 12.0 (H-5′b). 13C NMR:
163.20 (C=N), 163.04 (C=O), 82.79 (C-1′), 80.64 (C-4′), 71.44 (C-2′), 70.05 (C-3′), 61.93
(C-5′). FAB-MS: 250 (45) [M22 + H]+. For C8H15N3O6 (249.2) calculated: 38.56% C, 6.07% H,
16.86% N, 12.45% OCH3; foun d: 38.67% C, 6.12% H, 16.85% N, 12.37% OCH3.
4-O-Meth yl-1-(2,3,5-tri-O-acetyl-β-D-ribofuran osyl)isobiuret (23)
A stirred m ixture of powdered 1,2,3,5-tetra-O-acetyl-β-D-ribofuran ose (3.18 g, 10 m m ol) an d
dry toluen e (50 m l) was saturated with dry h ydrogen ch loride for 4 h . Th e solution was kept
at room tem perature overn igh t with exclusion of m oisture, evaporated an d th e residue
co-evaporated with toluen e (4 × 30 m l) to give syrupy 2,3,5-tri-O-acetyl-D-ribofuran osyl ch lo-
ride. A stirred m ixture of th is product with dry toluen e (50 m l) an d silver cyan ate (5.25 g,
35 m m ol) was h eated with in 30 m in to 100 °C an d kept at th is tem perature for 1 h . Th e
silver salts were filtered off an d th e filtrate evaporated to give syrupy 2,3,5-tri-O-acetyl-
β-D-ribofuran osyl isocyan ate. A solution of th is product in dry aceton e (20 m l) was added
dropwise at 0 °C with exclusion of m oisture to a stirred solution of fresh ly prepared
O-m eth ylisourea (0.66g, 9 m m ol) in aceton e (10 m l). Th e reaction m ixture was kept at room
tem perature for 30 m in an d evaporated to give crude 23 as th ick syrup. A solution of th is
product in ben zen e (30 m l) was sh aken with water (30 m l), kept at room tem perature for
1 h an d th e crystallin e precipitate filtered off with suction to give 3.16 g (80%) of th e
m on oh ydrate of 23, m .p. 70–76 °C, RF 0.92 (B, D3), [α]D –190 (c 0.35, DMF). UV (EtOH),
λm ax (log ε): 221 (4.25). CD (MeOH–H2O, 9:1), λm ax ([Θ]m ax): 225 (–3450). 1H NMR: 8.10 br,
1 H an d 7.70 br, 1 H (NH2); 7.87 d, 1 H, J(NH,1′) = 9.9 (NH); 5.42 dd, 1 H, J(1′,2′) = 6.0,
Collect. Czech. Chem. Commun. (Vol. 68) (2003)