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

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 e^1–5(1, azacitidin e) an d 2′-deoxy-

5-azacytidin e^6–8(2, decitabin e) are active drugs for th e th erapy of acute leu-

kem ia^9–13. 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 cer¹⁴an d h orm on e-in depen den t prostate can-

cer¹⁵. 5-Azacytidin e (1) is ph osph orylated an d in corporated¹⁶in to RNA

an d, after deoxygen ation , also in to DNA. 5-Aza-2′-deoxycytidin e (2) is in -

corporated¹⁶in to DNA. 5-Azacytosin e-con tain in g DNA ^17–20is a poten t in -

h ibitor of DNA m eth yltran sferase due to a covalen t in teraction ^21,22of 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 g^23,24. 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 ia^18–20. 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 ²⁵an 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 alogue²⁸4 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

O

N

O

N

R

O

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

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

NH

R¹

N

H₂N

N

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

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

NH₂

N

(i)

HN

H₂N

HN

NH

O

HN

NH

H₂N

NH

10

H₂N

CH₃

N

HN

N

HN

O

CH₃

(ii)

CH₃

O

HN

O

NH₂

7

11

CH₃

12

N

(ii)

(iii)

N

O

CH₃

HN

NH

6·7

CH₃

6

N

(i) CH₃NCO; (ii) DMF DMA; (iii) HCOOEt

N

O

CH₃

13

H

N

H

O

NH

H

N

O

NH

CH₃

H

HN

N

NH

X

N

O

H

H N

N

O

H

CH₃

6

7a

6

7a

II

IIa

X = picrate

X

R¹

H

NH₂

N

O

N

H₂N

N

R²

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 ²⁹of 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

N

O

NH

Rf

O

H

NH

HN

N

H

N

HN

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

N

H

N

R

O

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 derivatives³¹16

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 ods²⁴an 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 ribosyl²³derivative 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

NH

2

HN

O

HN

O

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 osylisobiuret¹23,

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

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 literature^32–38

.

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)⁴¹an 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 refs^42,43). 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 g⁴⁴or 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-d₆are 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 ν_as(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 B_2uCotton effects at 250 n m sim ilarly to th e N⁴-substituted m eth yl

derivatives of 5-azacytidin e²⁶an 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 e^50,51or 6-m eth yl-5-azacytidin e⁵², wh en estim ated by a

polarograph ic m eth od^53–55

.

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

(i)

HN

20b

MT

S

MT

S

HN

O

N

O

H

dRf

26

(iii)

27

(ii)

NH₂

N

NH₂

N

HN

OHC

(iv)

2

HO

N

O

HN

O

H

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

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 paper⁵⁷con 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. clarensis^58,59an d th at 5-azacytidin e (1) is pro-

duced by Streptoverticillium ladakanus var. ladakanus^3,4. 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

N

NH

N

H

N

O

H

O

H

O

N

HCOO

Cytosine

dRf

Thymine

21b

20b

H

O

N

O

H

N

H₃C

H

N

NH

N

dRf

Adenine

31

SCHEME 7

Th e earlier studies^21,22of 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 ⁶¹th 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.^21,22con 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 ^62,63th 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 ⁶²h 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.⁵⁷or 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 agwat⁶⁰h 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 observed⁵⁷C: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 observed⁵⁷in 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 ⁵⁸of 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

Collect. Czech. Chem. Commun. (Vol. 68) (2003)

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 ^–1in 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 ²m 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 ³g^–1dm ^{–1 1}H 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 e²⁸4 (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), R_F(B) 0.82 (D₁), [α]_D+27.5 (c 0.5, water).

UV, λ_{m 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), λ_{m 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 M_3f+ Na]⁺, 573 (4) [2 M_3f+ H]⁺,

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Mutagenicity of 2′-Deoxy-5-azacytidine

729

309 (5) [M_3f+ Na]⁺, 287 (18) [M_3f+ H]⁺, 155 (100) [aglycon e_3f+ 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, R_F(B) 0.72 (D₁), [α]_D+25.8 (c 0.1, water). UV, λ_{m 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), λ_{m 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), R_F(B) 0.47 (D₁), [α]_D+8.4 (c 0.2, wa-

ter). UV, λ_{m 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), λ_{m 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)

[M_3g+ Na]⁺, 357 (15) [M_3g+ H]⁺, 225 (100) [aglycon e_3g+ 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.), R_F(B) 0.71

(D₁), [α]_D+23.9 (c 0.1, water). UV, λ_{m 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 M_3d+ Na]⁺, 669 (2) [2 M_3d+ H]⁺, 357 (2) [M_3d

+

Na]⁺, 335 (42) [M_3d+ H]⁺, 203 (100) [aglycon e_3d+ 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.), R_F(B) 0.51 (D₁), [α]_D+23.8 (c 0.1, wa-

ter). UV, λ_{m 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): λ_{m 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 M_3h+ Na]⁺, 677 (2) [2 M_3h+ H]⁺, 361 (3) [M_3h

+

Na]⁺, 339 (40) [M_3h+ H]⁺, 207 (100) [aglycon e_3h+ 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), R_F(B) 0.62 (D₁, 3a) an d 0.43 (D₂, 5a),

[α]_D–25.6 (c 0.1, water). UV, λ_{m 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), R_F(B) 0.72 (D₁, 3b) an d 0.55 (D₂, 5b),

[α]_D–22.1 (c 0.5, water). UV, λ_{m 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.), R_F(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)

[M_5ban 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 .

Collect. Czech. Chem. Commun. (Vol. 68) (2003)

732

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.), R_F(B) 0.72 (D₁, 3c) an d 0.52

(D₂, 5c), [α]_D–1.5 (c 0.1, water). UV, λ_{m 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), R_F(B) 0.71 (D₁, 3d ) an d 0.57 (D₂, 5d ),

[α]_D–27.4 (c 0.1, water). UV, λ_{m 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 M_3d+ H]⁺, 659 (1) [M_3d+ M_5d+ H]⁺, 649 (<1) [2 M_5d+ H]⁺, 484 (2) [M_3d+ ben zyl-

guan idin e + H]⁺, 474 (2) [M_5d+ ben zylguan idin e + H]⁺, 357 (2) [M_3d+ Na]⁺, 347 (3) [M_5d

+

Na]⁺, 335 (4) [M_3d+ H]⁺, 325 (16) [M_5d+ H]⁺, 203 (12) [aglycon e_3d+ 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), R_F(B) 0.68 (D₁, 3e)

an d 0.61 (D₂, 5e), [α]_D–19.7 (c 0.5, water). UV, λ_{m 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 +

Collect. Czech. Chem. Commun. (Vol. 68) (2003)

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 M_3e+ H]⁺, 639 (4) [M_3e+ M_5e+ H]⁺, 629 (2) [2 M_5e+ H]⁺, 464 (<1) [M_3e

+

furfurylguan idin e + H]⁺, 454 (<1) [M_5e+ furfurylguan idin e + H]⁺, 325 (17) [M_3e+ H]⁺, 315

(47) [M_5e+ H]⁺, 193 (35) [aglycon e_3e+ 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, R_F(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, λ_{m 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 [ν_as(NH₂)]; 3266 s, 3123 m , sh [ν_s(NH₂) bon ded]; 1730 m , sh [ν(C=0)]; 1668 s (am ide I);

1611 s, 1597 s [β_s(NH₂)]; 1552 s [ν(C=N)]; 1496 vs, br (am ide II); 1463 s, br [δ⁺_as(Me)]; 1408 s

[δ_s(Me)]; 1302 s (am ide III); 1153 m [δ⁻_as(Me) + ν(C=O)]; 1092 m [β_as(NH₂)]; 1062 w, br, sh

[ν(N–Me)]; 665 m , vbr, sh (am ide V); 588 m , br, 565 m , br [γ_s(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 [ν_as(NH₂), ν(NH) free]; 3375 m , sh , 3323 m , 3294 m [ν(NH) bon ded]; 3245 m , sh ,

3215 m , 3183 m [ν_s(NH₂)]; 1744 s [ν(C=O), CO–NH⁺=]; 1687 s, 1653 s [β_s(NH₂)]; 1629 s, sh ,

1610 s, br [ν(C=N), ν(rin g) of th e picrate]; 1567 s, 1559 s [ν_as(NO₂)]; 1505 m , sh , 1497 s

(am ide II); 1430 m [ν(rin g) of th e picrate]; 1416 m [δ_s(Me)]; 1364 s [ν(C–N)]; 1339 s, 1320 s

[ν_s(NO₂)]; 1057 w, br [ν(N–Me)]; 743 m , 726 m [γ_s(NO₂)]; 568 m , 559 m [γ_s(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 -

Collect. Czech. Chem. Commun. (Vol. 68) (2003)

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, R_F(B)

0.33 (D₁). UV, λ_{m 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 [ν_as(NH₂)]; 3340 s,

br, sh , 3303 s, br, 3249 s, br, 3183 s, br, 3045 m , br, sh [ν_s(NH₂)]; 1757 m , sh , 1734 s, sh ,

1707 s, br, sh , 1681 s, br, 1668 s, vbr, sh [ν(C=O) + β_s(NH₂)]; 1616 s, 1513 s, 1487 s, br, 1354 s

[ν(C=N), ν(C–N)]; 1123 s [β_s(NH₂)]; 795 s (rin g breath in g); 711 w, vbr [γ_s(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.), R_F(D) 0.48 (D₃, 7) an d 0.55 (D₁, 6). UV, λ_{m 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 [ν_s(NH₂) free]; 3381 s, 3330 s, sh , 3290 s, br, sh [ν_as(NH₂),

ν(NH) bon ded]; 3219 s, 3080 m , br, sh [ν_s(NH₂)]; 1706 s [ν(C=O) triazin e]; 1673 s, 1663 s, sh

[β_s(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 [β_s(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 [δ_s(Me),

urea]; 1365 m [δ_s(Me), triazin e]; 1310 s, br (am ide III, urea); 1121 m [β_s(NH₂), triazin e];

1086 m [δ⁻_as(Me)]; 791 s (rin g breath in g, triazin e); 714 m , vbr, [γ_s(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 (NH₂of 6); 6.52 br, 8 H (4 × NH₂of 7); 5.98 br s, 2 H (2 × NH of

7); 3.20 s, 3 H (CH₃of 6); 2.49 d, 6 H, J(CH₃,NH) = 4.6 (2 × CH₃of 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 (CH₃of 6), 26.52 (CH₃of 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

Collect. Czech. Chem. Commun. (Vol. 68) (2003)

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.),

R_F(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 (NH₂of 6); 3.20 s, 3 H (CH₃of 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 CH₃of 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 (CH₃of 6), 26.29 (CH₃of 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 procedure²^{3 1}H 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 1}H 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, R_F(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 (NH₂of 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 (NH₂of 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.), R_F0.36 (B, D₂). UV, λ_{m 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), ν_as(NH₂),

ν_s(NH₂)]; 1700 m , 1680 s, 1638 vs (am ide I); 1601 m , sh [β_s(NH₂)]; 1565 s, br, 1537 s, br

(am ide II); 1472 m , sh , 1451 m [δ⁺_as(Me) + ν(C=O)]; 1403 m [δ_s(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 ), R_F0.50 (B, D₁). UV, λ_{m 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 [δ_s(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.), R_F0.45 (B, D₁). UV, λ_{m 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 [ν_as(Me)]; 2890 w, sh , 2858 w, sh [ν_s(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 [δ_s(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, R_F(A) 0.58 (D₁). ¹H NMR (CDCl₃): th e sign als of NH an d NH₂groups

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

[ν_as(NH₂)]; 3174 m , br [ν_s(NH₂)]; 1724 s [ν(C=O)] (on deuteration sh ifted down by 1–2 cm ^–1);

1702 s, 1669 s, sh [ν(C=O) + β_s(NH₂)] (on deuteration in CCl₄a pron oun ced sh ift down an d

th e lower ban d disappears); 1653 s [β_s(NH₂)] (on deuteration in CCl₄th e ban d disappears);

1613 s [ν(C=N) + β(NH)] (on deuteration in CCl₄an 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 [ν_as(NH₂)]; 3191 m , br [ν_s(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) + β_s(NH₂)]; 1656 s, 1651 s, 1638 s [β_s(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, λ_{m 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 M_20a

+ H]⁺, 219 (87) [M_20a

+

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+ M_20a

+ Na]⁺, 459 (2) [2 M_20a

+ Na]⁺, 457 (7) [2 M₂+ H]⁺, 447 (14) [M₂

+

or 20b

M_20a

+ H]⁺, 437 (12) [2 M_20a

+ H]⁺, 251 (11) [M₂+ Na]⁺, 241 (13) [M_20a

or 20b

or 20b or 20b

Na]⁺, 229 (14) [M₂+ H]⁺, 219 (100) [M_20a

+ H]⁺, 113 (79) [aglycon e₂+ H]⁺, 103 (10)

or 20b

[aglycon e_20a

+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 e²⁸4 (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.), R_F0.58 (C, D₄), [α]_D+123.8 (c 0.3, water).

UV (MeOH), λ_{m ax}(log ε): 220 (4.27). CD (pH 6.93), λ_{m 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, R_F0.92 (B, D₃), [α]_D–190 (c 0.35, DMF). UV (EtOH),

λ_{m ax}(log ε): 221 (4.25). CD (MeOH–H₂O, 9:1), λ_{m 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)

Article Doi

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

DOI: 10.1135/cccc20030711

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Article abstract of DOI:10.1135/cccc20030711

Full text of DOI:10.1135/cccc20030711

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